1 00:00:06,950 --> 00:00:09,052 LOOKS LIKE WE HAVE A PRETTY GOOD 2 00:00:09,052 --> 00:00:12,022 TURNOUT AND HOPE NOT TOO MANY 3 00:00:12,022 --> 00:00:13,790 ARE STRUCK IN SECURITY AND GET 4 00:00:13,790 --> 00:00:14,658 GOING ON THE MEETING AND WELCOME 5 00:00:14,658 --> 00:00:15,192 BACK. 6 00:00:15,192 --> 00:00:16,226 WELCOME TO NEWCOMERS. 7 00:00:16,226 --> 00:00:19,129 I HOPE YOU SEE THAT WE ALL LOVE 8 00:00:19,129 --> 00:00:20,297 TO TALK ABOUT SCIENCE. 9 00:00:20,297 --> 00:00:21,665 THAT'S WHAT WE'RE HERE TO DO AND 10 00:00:21,665 --> 00:00:24,034 WELCOME ESPECIALLY TO THE 11 00:00:24,034 --> 00:00:25,035 VIDEOCAST MEMBERS ONLINE THAT 12 00:00:25,035 --> 00:00:34,778 COULDN'T MAKE IT TO THE MEETING. 13 00:00:34,778 --> 00:00:39,383 SO WE'RE HERE IS TO EVALUATE THE 14 00:00:39,383 --> 00:00:42,419 WILL HIV CENTER FOR STRUCTURAL 15 00:00:42,419 --> 00:00:44,321 BIOLOGY AND TALK ABOUT THE 16 00:00:44,321 --> 00:00:47,024 SCIENCE AND SEE WHAT CUTTING 17 00:00:47,024 --> 00:00:48,825 EDGE SCIENCE LOOKS LIKE AND WHAT 18 00:00:48,825 --> 00:00:52,996 LARGE GROUPS OF GOOD SCIENTISTS 19 00:00:52,996 --> 00:00:54,398 CAN GET DONE. 20 00:00:54,398 --> 00:00:55,966 SO, I DIDN'T NOTICE UNTIL I PUT 21 00:00:55,966 --> 00:00:57,401 THIS ON THE SLIDE TOGETHER THE 22 00:00:57,401 --> 00:01:03,173 OTHER DAY, WE'VE GOT THEM SET UP 23 00:01:03,173 --> 00:01:07,944 THREE A DAY. 24 00:01:07,944 --> 00:01:13,016 YOU I'M NOT SURE IF YOU'VE SEEN 25 00:01:13,016 --> 00:01:19,356 THIS BUT WE HAVE THREE ONE-HOUR 26 00:01:19,356 --> 00:01:22,993 SESSIONS AND SET THEM UP WEST TO 27 00:01:22,993 --> 00:01:24,795 EAST BUT THEN ALSO REALIZED I 28 00:01:24,795 --> 00:01:29,366 SET UP A GENDER BALANCE AND THIS 29 00:01:29,366 --> 00:01:33,003 MEETING HAS BEEN GOING ON 18 30 00:01:33,003 --> 00:01:37,040 YEARS THIS PROGRAM IS IN ITS 31 00:01:37,040 --> 00:01:47,584 18th YEAR AND BEEN AT NI AD AND 32 00:01:49,086 --> 00:01:50,387 THE RO1s AROUND GRANTS 33 00:01:50,387 --> 00:01:52,422 REPRESENTED IN THE ROOM AND IN 34 00:01:52,422 --> 00:01:54,057 THE BASIC SCIENCE PORTFOLIO 35 00:01:54,057 --> 00:01:56,693 REGARDING MOLECULAR AND 36 00:01:56,693 --> 00:02:02,466 STRUCTURAL BIOLOGY ARE KIND OF 37 00:02:02,466 --> 00:02:12,709 BOY 38 00:02:17,647 --> 00:02:17,881 B 39 00:02:17,881 --> 00:02:22,652 BOEYED AND TO THE CENTERED I 40 00:02:22,652 --> 00:02:25,555 WANT TO TALK ABOUT THIS IS 41 00:02:25,555 --> 00:02:27,724 SPONSORED BY THE BASIC SCIENCE 42 00:02:27,724 --> 00:02:28,024 PROGRAM. 43 00:02:28,024 --> 00:02:30,060 THIS IS BASIC SCIENCE WE'LL BE 44 00:02:30,060 --> 00:02:30,761 TALKING ABOUT TODAY. 45 00:02:30,761 --> 00:02:37,400 ORGANIZERS MYSELF AND UDI AND 46 00:02:37,400 --> 00:02:39,636 MODERATORS BRIGITA AND ROGER 47 00:02:39,636 --> 00:02:40,937 WILL TAKE OVER THE AFTERNOON 48 00:02:40,937 --> 00:02:47,844 SESSIONS. 49 00:02:47,844 --> 00:02:49,379 THE REASON I'M LOSING IT IS HERE 50 00:02:49,379 --> 00:02:50,947 IS BECAUSE I WAS GOING TO RUN A 51 00:02:50,947 --> 00:02:51,848 SCRIPT AND THEN FORGOT. 52 00:02:51,848 --> 00:02:58,288 SO I'M GOING TO GO BACK AND 53 00:02:58,288 --> 00:03:00,490 START BY THANKING OUR LEADERSHIP 54 00:03:00,490 --> 00:03:04,161 AND BRINGING TOGETHER TALENTED 55 00:03:04,161 --> 00:03:04,828 SCIENTISTS FROM DIVERSE FIELDS 56 00:03:04,828 --> 00:03:07,798 AND THE MAGIC THEY DO WHAT I WAS 57 00:03:07,798 --> 00:03:08,932 TRYING TO DESCRIBE POORLY BEFORE 58 00:03:08,932 --> 00:03:10,967 IS PUT TOGETHER THE 59 00:03:10,967 --> 00:03:12,035 COLLABORATIONS AND ANSWER BIG 60 00:03:12,035 --> 00:03:22,212 QUESTIONS. 61 00:03:22,445 --> 00:03:24,114 FIRST I WANT TO ACKNOWLEDGE THE 62 00:03:24,114 --> 00:03:24,948 PEOPLE THAT GOT OUT HERE. 63 00:03:24,948 --> 00:03:28,218 THIS IS A PICTURE FROM THE '90s 64 00:03:28,218 --> 00:03:32,022 ON THE NIH CAMPUS WHEN THE 65 00:03:32,022 --> 00:03:35,358 EPIDEMIC IN THE U.S. WAS 66 00:03:35,358 --> 00:03:38,595 PRIMARILY AMONG YOUNG URBAN 67 00:03:38,595 --> 00:03:40,430 WHITE GAY MEN AND THEY WERE 68 00:03:40,430 --> 00:03:42,032 DYING AND ANGRY THE GOVERNMENT 69 00:03:42,032 --> 00:03:44,134 WASN'T RESPONDING WELL. 70 00:03:44,134 --> 00:03:46,002 THERE'S A LOT OF FACES YOU 71 00:03:46,002 --> 00:03:47,771 SHOULD RECOGNIZE UP HERE AND 72 00:03:47,771 --> 00:03:51,374 THREE IMPORTANT LEADERS IN THIS 73 00:03:51,374 --> 00:03:55,645 ACTIVISM WERE PETER STALEY, 74 00:03:55,645 --> 00:03:58,281 LARRY KRAMER, FAMOUSLY WROTE IN 75 00:03:58,281 --> 00:04:01,918 THE VILLAGE VOICE AND MARTIN 76 00:04:01,918 --> 00:04:03,453 DELANEY AND THESE WITH OTHERS 77 00:04:03,453 --> 00:04:04,921 THAT WERE AFFECTED BY THE 78 00:04:04,921 --> 00:04:06,289 EPIDEMIC AT THE TIME CAME TO THE 79 00:04:06,289 --> 00:04:08,291 NIH AND PROTESTED. 80 00:04:08,291 --> 00:04:10,694 YOU AGAIN RECOGNIZE SOME OF THE 81 00:04:10,694 --> 00:04:11,394 FACES. 82 00:04:11,394 --> 00:04:13,763 I THINK DR. FAUCI IS BACK HERE 83 00:04:13,763 --> 00:04:22,706 AND HIS WILL EFFAGY BEING BURNED 84 00:04:22,706 --> 00:04:24,541 AND DR. FAUCI EMBRACED THE 85 00:04:24,541 --> 00:04:26,576 IDEALS OF THE ACTIVIST 86 00:04:26,576 --> 00:04:26,877 COMMUNITIES. 87 00:04:26,877 --> 00:04:28,979 THEY WANTED ACCESS TO CARE AND 88 00:04:28,979 --> 00:04:30,480 LIFE-SAVING MEDICINES AND CHANGE 89 00:04:30,480 --> 00:04:36,052 THE WAY THERAPIES WERE TESTED 90 00:04:36,052 --> 00:04:40,690 AND TREAT ED AND THIS GUY, 91 00:04:40,690 --> 00:04:42,058 PRESIDENT BUSH ASKED DR. FAUCI 92 00:04:42,058 --> 00:04:47,197 FOR ADVICE ON WHAT TO DO ABOUT 93 00:04:47,197 --> 00:04:51,201 THE AIDS PANDEMIC AND 94 00:04:51,201 --> 00:04:52,836 RECOMMENDED THE CONGRESSIONALLY 95 00:04:52,836 --> 00:04:56,006 MANDATED SET ASIDE FOR HIV 96 00:04:56,006 --> 00:04:59,542 FUNDING AND ESTABLISH NEXT 97 00:04:59,542 --> 00:05:00,877 OFFICE OF AIDS RESEARCH. 98 00:05:00,877 --> 00:05:03,146 THAT FUNDING STILL STANDS 30 99 00:05:03,146 --> 00:05:07,784 YEARS LATER AROUND $5 BILLION A 100 00:05:07,784 --> 00:05:08,985 YEAR FOR INTRAMURAL AND 101 00:05:08,985 --> 00:05:15,358 EXTRAMURAL IT NIH PROGRAMS AND 102 00:05:15,358 --> 00:05:21,998 IT'S CHANGED THE PANDEMIC WE 103 00:05:21,998 --> 00:05:23,833 REALIZE IT'S A WORLDWIDE 104 00:05:23,833 --> 00:05:25,468 PANDEMIC HITTING MIDDLE AND 105 00:05:25,468 --> 00:05:26,503 LOW-INCOME COUNTRIES AT THIS 106 00:05:26,503 --> 00:05:26,703 TIME. 107 00:05:26,703 --> 00:05:28,071 THE PROBLEM IS PEOPLE INFECTED 108 00:05:28,071 --> 00:05:29,973 WITH HIV WHO HAVE ACCESS TO CARE 109 00:05:29,973 --> 00:05:31,975 CAN BE HEALTHY. 110 00:05:31,975 --> 00:05:35,345 WE HAVE OVER 30 APPROVED 111 00:05:35,345 --> 00:05:36,579 MEDICATIONS THAT REVERSE AND 112 00:05:36,579 --> 00:05:37,447 REDUCE VIRAL LOAD. 113 00:05:37,447 --> 00:05:39,349 IF YOU TAKE THEM IN COMBINATION 114 00:05:39,349 --> 00:05:42,052 YOU BECOME UNDETECTABLE WHICH 115 00:05:42,052 --> 00:05:46,323 MEANS YOU'RE BOTH FREE FROM HIV 116 00:05:46,323 --> 00:05:49,092 DISEASE AND SYMPTOMS AND YOU'RE 117 00:05:49,092 --> 00:05:49,926 UNTRANSMISSIBLE. 118 00:05:49,926 --> 00:05:51,695 YOU WON'T TRANSMIT THE VIRUS TO 119 00:05:51,695 --> 00:05:52,062 OTHERS. 120 00:05:52,062 --> 00:05:54,030 THE OTHER THING IS YOU MIGHT 121 00:05:54,030 --> 00:05:58,601 NOTICE THE DIVERSITY OF THE HIV 122 00:05:58,601 --> 00:06:00,070 COMMUNITY IS GREATLY WILL 123 00:06:00,070 --> 00:06:04,374 INCREASED AND ENHANCE. 124 00:06:04,374 --> 00:06:06,576 SO THE PANDEMIC STILL HERE BUT 125 00:06:06,576 --> 00:06:07,777 FORTUNATELY WE HAVE ACCESS TO 126 00:06:07,777 --> 00:06:09,179 CARE FOR A LOT OF PEOPLE. 127 00:06:09,179 --> 00:06:10,914 THERE'S ABOUT $40 MILLION PEOPLE 128 00:06:10,914 --> 00:06:18,054 GIVE OR TAKE INFECTED WORLDWIDE 129 00:06:18,054 --> 00:06:22,392 AND THERE ARE ABOUT 30 MILLION 130 00:06:22,392 --> 00:06:24,327 UNDER CARE TO RETROVIRAL THERAPY 131 00:06:24,327 --> 00:06:29,265 BUT STILL ABOUT 10 MILLION LEFT. 132 00:06:29,265 --> 00:06:32,969 SO WE NEED OUR COMMUNITY MORE 133 00:06:32,969 --> 00:06:36,072 THAN EVER AND TO COMMUNICATE TO 134 00:06:36,072 --> 00:06:39,442 US AND COMMUNICATE OUT TO THEIR 135 00:06:39,442 --> 00:06:41,077 IT COMMUNITY TO BRING THEM INTO 136 00:06:41,077 --> 00:06:47,650 EDUCATION AND CARE. 137 00:06:47,650 --> 00:06:52,389 IT BRINGS ME TO THIS. 138 00:06:52,389 --> 00:06:56,993 DR. MARAZZO PUT OUT A STATEMENT 139 00:06:56,993 --> 00:06:59,429 LAST WEEK THE INCLUSIVE 140 00:06:59,429 --> 00:07:06,469 FIRST-PERSON USE LANGUAGE IN ALL 141 00:07:06,469 --> 00:07:08,371 NIAID SPONSORED EVENTS AND THE 142 00:07:08,371 --> 00:07:09,439 COMMUNITY SAID THEY DON'T WANT 143 00:07:09,439 --> 00:07:11,041 TO BE CALLED PEOPLE INFECTED BUT 144 00:07:11,041 --> 00:07:19,082 PEOPLE LIVING WITH HIV AND WE'RE 145 00:07:19,082 --> 00:07:21,051 NOT GOING TO POLICE THE LANGUAGE 146 00:07:21,051 --> 00:07:23,620 BUT ASK EVERYBODY BE RESPECTFUL 147 00:07:23,620 --> 00:07:28,925 AND UNDERSTAND THAT IT'S AN 148 00:07:28,925 --> 00:07:31,261 IMPORTANT ASPECT OF OUR LANGUAGE 149 00:07:31,261 --> 00:07:32,495 THAT WE COMMUNICATE TO OTHERS. 150 00:07:32,495 --> 00:07:35,098 WE INVITE ALL THE COMMUNITY INTO 151 00:07:35,098 --> 00:07:36,766 OUR TALKS AND PRESENTATIONS FROM 152 00:07:36,766 --> 00:07:41,004 BASIC SCIENCE ALL THE WAY TO 153 00:07:41,004 --> 00:07:42,705 CLINICAL AND TRANSLATIONAL WORK. 154 00:07:42,705 --> 00:07:45,542 AND WE ASK -- I THINK IF WE GET 155 00:07:45,542 --> 00:07:48,978 THROUGH THE FIRST TWO -- IT DOES 156 00:07:48,978 --> 00:07:51,748 FEEL UNCOMFORTABLE, CLUMSY AND 157 00:07:51,748 --> 00:07:53,483 DIFFICULT AT TIMES TO CHANGE. 158 00:07:53,483 --> 00:07:55,552 I'M SURE I'VE MADE THIS MISTAKE 159 00:07:55,552 --> 00:07:57,053 ALREADY IN THIS TALK AND SOME 160 00:07:57,053 --> 00:07:58,955 MAY HAVE NOTICED THAT BECAUSE WE 161 00:07:58,955 --> 00:08:00,023 DO. 162 00:08:00,023 --> 00:08:01,458 IT'S HARD TO CHANGE THE WAY YOU 163 00:08:01,458 --> 00:08:03,026 TALK BUT WE THINK THIS WAY. 164 00:08:03,026 --> 00:08:07,097 SO THE BIGGEST ONE I THINK IS 165 00:08:07,097 --> 00:08:08,798 PERSON-FIRST LANGUAGE AND PEOPLE 166 00:08:08,798 --> 00:08:12,035 LIVING WITH HIV INSTEAD OF HIV 167 00:08:12,035 --> 00:08:13,436 INFECTED PEOPLE. 168 00:08:13,436 --> 00:08:16,139 PEOPLE THAT INJECT DRUGS AND 169 00:08:16,139 --> 00:08:17,941 IT'S A FLIP DRUGS AND IT'S 170 00:08:17,941 --> 00:08:19,142 SIMPLE AND RESPECTFUL. 171 00:08:19,142 --> 00:08:24,114 ANOTHER HOT BUTTON ARE SUBJECTS 172 00:08:24,114 --> 00:08:26,282 AND PATIENTS AND COMMUNITY WOULD 173 00:08:26,282 --> 00:08:27,450 PREFERS TO BE PARTICIPANT OR 174 00:08:27,450 --> 00:08:27,717 VOLUNTEER. 175 00:08:27,717 --> 00:08:29,786 THIS IS ALSO TRICKY BECAUSE 176 00:08:29,786 --> 00:08:32,989 HUMAN SUBJECTS IS WRITTEN INTO 177 00:08:32,989 --> 00:08:38,194 OUR CONTRACTS AT NIH NOT ALL 178 00:08:38,194 --> 00:08:39,429 THESE WILL CHANGE RIGHT AWAY BUT 179 00:08:39,429 --> 00:08:41,998 WE ASK TO BE RESPECTFUL AND YOU 180 00:08:41,998 --> 00:08:43,399 CAN READ THE REST AND THERE'S A 181 00:08:43,399 --> 00:08:45,101 LOT OF INFORMATION. 182 00:08:45,101 --> 00:08:47,670 I PUT DOWN THREE WILL ONES THAT 183 00:08:47,670 --> 00:08:52,909 IMPACTED ME A LOT OVER THE IT 30 184 00:08:52,909 --> 00:08:53,943 OR MORE YEARS AND ENCOURAGE YOU 185 00:08:53,943 --> 00:08:55,812 TO LEARN A HISTORY TO LEARN 186 00:08:55,812 --> 00:08:56,980 ABOUT THE IMPORTANCE OF 187 00:08:56,980 --> 00:09:00,283 COMMUNITY IN EVERYTHING WE DO. 188 00:09:00,283 --> 00:09:01,251 SO DETAILS. 189 00:09:01,251 --> 00:09:09,259 I HOPE EVERYBODY KNOWS FOR LUNCH 190 00:09:09,259 --> 00:09:12,996 WILL THE CAFETERIA IS OPEN AND 191 00:09:12,996 --> 00:09:13,997 DON'T RECOMMEND WALKING ACROSS 192 00:09:13,997 --> 00:09:15,198 THE CAMPUS AND IT'S COOL IN HERE 193 00:09:15,198 --> 00:09:16,633 BUT HOPE IT WILL WARM UP WITH 194 00:09:16,633 --> 00:09:21,004 THE BODIES AND HAVE THIS CHOP 195 00:09:21,004 --> 00:09:22,572 OPTION THIS YEAR DELIVERED TO 196 00:09:22,572 --> 00:09:24,807 THE AREA IN FRONT OF THE 197 00:09:24,807 --> 00:09:26,042 AUDITORIUM AND YOU CAN ORDER 198 00:09:26,042 --> 00:09:27,610 THOSE UP TO 9:30. 199 00:09:27,610 --> 00:09:28,378 YOU HAVE A LITTLE BIT OF TIME 200 00:09:28,378 --> 00:09:33,149 LEFT FOR TODAY. 201 00:09:33,149 --> 00:09:34,150 AGAIN THE LAYOUT OF THE MEETING 202 00:09:34,150 --> 00:09:35,485 I DON'T HAVE THE AGENDA IN FRONT 203 00:09:35,485 --> 00:09:36,419 OF ME. 204 00:09:36,419 --> 00:09:43,059 WE'LL DO THREE ONE-OUR SECT -- 205 00:09:43,059 --> 00:09:45,628 ONE-HOUR SESSIONS AND 206 00:09:45,628 --> 00:09:46,462 PRESENTATIONS AND Q&A AND BREAKS 207 00:09:46,462 --> 00:09:48,431 IN THERE AT THE END OF THE THIRD 208 00:09:48,431 --> 00:09:50,867 WE'LL HAVE A LONG LUNCH AND 209 00:09:50,867 --> 00:09:51,501 POSTERS. 210 00:09:51,501 --> 00:09:53,069 WE HAVE 135 POSTERS THIS YEAR. 211 00:09:53,069 --> 00:09:54,571 THAT'S MORE THAN I'VE SEEN EVER. 212 00:09:54,571 --> 00:09:56,973 WE SPLIT THEM INTO TWO DAYS TO 213 00:09:56,973 --> 00:10:00,977 GIVE RELIEF TO OUR PRESENTERS 214 00:10:00,977 --> 00:10:03,079 AND ALLOWS EVERYONE TO GO TO 215 00:10:03,079 --> 00:10:04,247 EVERYONE ELSE'S POSTERS. 216 00:10:04,247 --> 00:10:07,083 I ENCOURAGE EVERYONE TO VIEW THE 217 00:10:07,083 --> 00:10:07,317 POSTERS. 218 00:10:07,317 --> 00:10:09,519 THIS IS REALLY EXCITING SCIENCE 219 00:10:09,519 --> 00:10:11,788 AND WHAT'S GOING TO BE HAPPENING 220 00:10:11,788 --> 00:10:15,491 IN THE NEXT 10 YEARS IN THIS 221 00:10:15,491 --> 00:10:16,693 CENTER PROJECTS. 222 00:10:16,693 --> 00:10:21,664 SO WILL AGAIN WE APPRECIATE THAT 223 00:10:21,664 --> 00:10:22,832 THERE'S AS MANY SENIOR AND VERY 224 00:10:22,832 --> 00:10:25,101 JUNIOR PEOPLE AT THE MEETING AND 225 00:10:25,101 --> 00:10:28,871 LOVE THE MIX AND HOPE WE CAN 226 00:10:28,871 --> 00:10:29,872 SUPPORT OUR JUNIOR FOLKS YOU, 227 00:10:29,872 --> 00:10:32,375 LOOK AT THEIR POSTERS, ASK 228 00:10:32,375 --> 00:10:34,244 QUESTIONS AND ENJOY. 229 00:10:34,244 --> 00:10:36,145 AND THEN IN THE AFTERNOON AS 230 00:10:36,145 --> 00:10:39,649 USUAL WE'VE SET UP SOME ABSTRACT 231 00:10:39,649 --> 00:10:42,085 SELECTED TALKS AND WE'LL RUN IT 232 00:10:42,085 --> 00:10:44,087 AS IT GOES AND TRY TO STAY ON 233 00:10:44,087 --> 00:10:44,921 TIME. 234 00:10:44,921 --> 00:10:48,124 YOU GUYS HAVE TWO MINUTES, 235 00:10:48,124 --> 00:10:48,691 ENJOY, EVERYBODY. 236 00:10:48,691 --> 00:10:48,992 237 00:10:48,992 --> 00:10:59,168 [APPLAUSE] 238 00:11:28,531 --> 00:11:30,133 >> OKAY. 239 00:11:30,133 --> 00:11:31,434 GOOD MORNING, EVERYONE. 240 00:11:31,434 --> 00:11:32,902 I'M WES SUNDQUIST AND IT'S MY 241 00:11:32,902 --> 00:11:34,370 PRIVILEGE TO INTRODUCE AND GIVE 242 00:11:34,370 --> 00:11:36,639 THE FIRST TALK FOR THE CHEETAH 243 00:11:36,639 --> 00:11:36,873 CENTER. 244 00:11:36,873 --> 00:11:40,109 LIKE ALL THE CENTERS, OUR 245 00:11:40,109 --> 00:11:41,311 SUCCESS IS REALLY PREDICATED ON 246 00:11:41,311 --> 00:11:41,911 A GREAT GROUP OF PEOPLE. 247 00:11:41,911 --> 00:11:44,914 NIS IS 248 00:11:47,216 --> 00:11:49,852 THIS IS A PICTURE THE ONE-DAY 249 00:11:49,852 --> 00:11:51,387 MEETING RUN BY TRAINEES. 250 00:11:51,387 --> 00:11:52,789 THE CENTER'S ORGANIZED AROUND 251 00:11:52,789 --> 00:11:53,323 THREE PROJECTS. 252 00:11:53,323 --> 00:11:55,558 THE FIRST IS AIMED AT 253 00:11:55,558 --> 00:11:58,761 UNDERSTANDING THE FIRST HALF OF 254 00:11:58,761 --> 00:12:00,129 THE VIRAL LIFE CYCLE IN 255 00:12:00,129 --> 00:12:03,132 MOLECULAR DETAIL AND THE SECOND 256 00:12:03,132 --> 00:12:04,967 AT HOW CELLS CAN DEFEND 257 00:12:04,967 --> 00:12:06,135 THEMSELVES AGAINST HIV AND THE 258 00:12:06,135 --> 00:12:07,804 THIRD PROJECT IS AIMED AT 259 00:12:07,804 --> 00:12:10,473 OBSERVING AND MANIPULATING VIRAL 260 00:12:10,473 --> 00:12:13,376 DYNAMICS ACROSS THE LARGE RANGE 261 00:12:13,376 --> 00:12:15,545 OF SPATIAL AND TEMPORAL SCALES. 262 00:12:15,545 --> 00:12:17,213 YOU'LL HEAR FROM THREE GROUPS OF 263 00:12:17,213 --> 00:12:18,014 PEOPLE FROM OUR CENTER. 264 00:12:18,014 --> 00:12:20,316 I'LL TELL YOU ABOUT OUR EFFORTS 265 00:12:20,316 --> 00:12:23,453 THAT ARE AIMED AT DEVELOPING A 266 00:12:23,453 --> 00:12:26,155 CELL-FREE SYSTEM WITH PURE 267 00:12:26,155 --> 00:12:27,457 RE-AGENTS TO DO REVERSE 268 00:12:27,457 --> 00:12:28,591 TRANSCRIPTION AND INTEGRATION. 269 00:12:28,591 --> 00:12:31,027 THEN YOU'LL HEAR FROM THOMAS 270 00:12:31,027 --> 00:12:32,028 SCHWARTZ AND THEY'RE LOVELY WORK 271 00:12:32,028 --> 00:12:34,097 AIMED AT UNDERSTANDING HOW NANO 272 00:12:34,097 --> 00:12:36,032 BODIES CAN HELP CONTROL THE 273 00:12:36,032 --> 00:12:37,333 STRUCTURE AND FUNCTION OF THE 274 00:12:37,333 --> 00:12:41,371 CAPSID AND THEN JEREMY LUBEN 275 00:12:41,371 --> 00:12:43,639 WILL TALK ABOUT THEIR DISCOVERY 276 00:12:43,639 --> 00:12:46,943 OF A MOLECULE FOR CELLS TO 277 00:12:46,943 --> 00:12:49,212 SENSED UNSPLICED HIV RNA. 278 00:12:49,212 --> 00:12:52,348 I'M JUMP INTO MY PART OF THE 279 00:12:52,348 --> 00:12:52,548 TALK. 280 00:12:52,548 --> 00:12:54,584 THIS IS IT THE FIRST HALF OF THE 281 00:12:54,584 --> 00:12:55,785 VIRAL LIFE CYCLE. 282 00:12:55,785 --> 00:12:57,620 I WANT TO MAKE A COUPLE POINTS 283 00:12:57,620 --> 00:12:58,554 ABOUT IMPORTANT TRANSFORMATIONS 284 00:12:58,554 --> 00:13:01,190 THAT HAVE TO OCCUR. 285 00:13:01,190 --> 00:13:02,992 OF COURSE THE VIRAL RNA SINGLE 286 00:13:02,992 --> 00:13:05,361 STRANDED HAS TO BE CONVERTED TO 287 00:13:05,361 --> 00:13:07,063 DOUBLE-STRANDED DNA COPY. 288 00:13:07,063 --> 00:13:09,799 AND THE IT DOUBLE-STRANDED DNA 289 00:13:09,799 --> 00:13:10,933 COPY HAS TO BREACH THE CAPSID TO 290 00:13:10,933 --> 00:13:12,101 GET OUT. 291 00:13:12,101 --> 00:13:13,169 THAT'S THE SECOND TRANSFORMATION 292 00:13:13,169 --> 00:13:15,938 AND THE THIRD IS INTEGRATION 293 00:13:15,938 --> 00:13:19,175 INTO THE HOST CHROMOSOME. 294 00:13:19,175 --> 00:13:20,676 IF WE'RE GOING TO RECONSTITUTE 295 00:13:20,676 --> 00:13:22,612 THOSE ARE THE STEPS. 296 00:13:22,612 --> 00:13:24,147 AND WORK A LOT OF IT BY PEOPLE 297 00:13:24,147 --> 00:13:27,183 HERE IN THE AUDIENCE OVER THE 298 00:13:27,183 --> 00:13:29,886 LAST FEW YEARS HAS TAUGHT US THE 299 00:13:29,886 --> 00:13:31,387 TRANSFORMATION ARE OCCURRING 300 00:13:31,387 --> 00:13:33,723 DEEP IN THE CELL LARGELY IN THE 301 00:13:33,723 --> 00:13:35,258 NUCLEUS AND THAT'S GREAT 302 00:13:35,258 --> 00:13:36,492 PROGRESS BUT MEANS IT'S HARD TO 303 00:13:36,492 --> 00:13:37,927 STUDY BECAUSE SINGLE VARIANTS 304 00:13:37,927 --> 00:13:40,997 ARE UNCODING AND REPLICATING IN 305 00:13:40,997 --> 00:13:41,764 THE NUCLEUS AND THAT'S PART OF 306 00:13:41,764 --> 00:13:43,733 THE MOTIVATION FOR US TO DEVELOP 307 00:13:43,733 --> 00:13:46,736 A CELL-FREE SYSTEM TO STUDY 308 00:13:46,736 --> 00:13:48,137 THESE PROCESSES IN MOLECULAR AND 309 00:13:48,137 --> 00:13:51,574 STRUCTURAL DETAIL IN A BULK 310 00:13:51,574 --> 00:13:51,808 EXAMINE. 311 00:13:51,808 --> 00:13:53,376 SINCE I'M THE FIRST SPEAKER I'LL 312 00:13:53,376 --> 00:13:56,913 GIVE A BRIEF PRIMER ON THE CORE 313 00:13:56,913 --> 00:13:57,246 PARTICLE. 314 00:13:57,246 --> 00:14:00,149 THE OUTER SHELL IS CALLED THE 315 00:14:00,149 --> 00:14:02,985 CAPSID AND COMPOSED OF ABOUT 250 316 00:14:02,985 --> 00:14:13,529 COPIES OF THE CA HEXAMER AND 12 317 00:14:14,430 --> 00:14:16,999 COPIES OF THE DEXAMER. 318 00:14:16,999 --> 00:14:25,842 AND THE HEXAMER HAS BASIC 319 00:14:25,842 --> 00:14:28,978 RESIDUES REPULSING TOGETHER AND 320 00:14:28,978 --> 00:14:31,214 TWO LAYERS EXCEPT COMBINE THE 321 00:14:31,214 --> 00:14:34,083 PHOSPHATE WHICH NEUTRALIZES 322 00:14:34,083 --> 00:14:35,151 THEM. 323 00:14:35,151 --> 00:14:40,857 THAT'S STABILIZES THE HEXAMER 324 00:14:40,857 --> 00:14:44,126 AND CAN DO CORE PARTICLES WITH 325 00:14:44,126 --> 00:14:45,194 CAPSIDS AND A LAB DISCOVERED THE 326 00:14:45,194 --> 00:14:48,331 CENTER OF THE HEXAMER HAS A 327 00:14:48,331 --> 00:14:49,932 SPECIAL PROPERTY OF BEING ABLE 328 00:14:49,932 --> 00:14:52,235 TO PASS NUCLEOTIDES IN AND OUT 329 00:14:52,235 --> 00:14:52,869 OF THE CAPSID. 330 00:14:52,869 --> 00:14:54,904 NOW WE CAN THINK OF REVERSE 331 00:14:54,904 --> 00:14:56,205 TRANSCRIPTION OF TAKING PLACE 332 00:14:56,205 --> 00:14:59,809 INSIDE A CLOSED CONTAINER. 333 00:14:59,809 --> 00:15:04,981 THIS IS A REMINDER THE CORE 334 00:15:04,981 --> 00:15:08,985 PARTICLE INCLUDES THE VIRAL 335 00:15:08,985 --> 00:15:12,121 REVERSE TRANSCRIPTASE AND TWO 336 00:15:12,121 --> 00:15:14,290 COPIES OF THE SINGLE STRANDED 337 00:15:14,290 --> 00:15:16,392 RNA AND THOUSANDS OF THE NC 338 00:15:16,392 --> 00:15:16,626 PROTEIN. 339 00:15:16,626 --> 00:15:19,262 SO A FEW YEARS AGO WE REPORTED 340 00:15:19,262 --> 00:15:22,365 WE WERE ABLE TO DO REVERSE 341 00:15:22,365 --> 00:15:23,933 TRANSCRIPTION AND INTEGRATION IN 342 00:15:23,933 --> 00:15:26,002 A CELL FREE SYSTEM AND THAT'S 343 00:15:26,002 --> 00:15:28,971 WHAT IT LOOKED LIKE AND START 344 00:15:28,971 --> 00:15:39,415 WITH PURE VIRAL PARTICLES. 345 00:15:42,385 --> 00:15:44,754 AND THE PEPTIDE GIVES ACCESS TO 346 00:15:44,754 --> 00:15:48,424 THE CORE PARTICLE AND IP6 AND 347 00:15:48,424 --> 00:15:49,992 NTPs THE CORE PARTICLE IS STABLE 348 00:15:49,992 --> 00:15:54,864 AND IN THE PRESENCE OF DNTPs 349 00:15:54,864 --> 00:15:55,598 ENDOGENOUS REVERSE TRANSCRIPTION 350 00:15:55,598 --> 00:15:57,900 STARTS AND CAN BE EFFICIENT TO 351 00:15:57,900 --> 00:15:58,834 REPLICATE THE ENTIRE GENOME. 352 00:15:58,834 --> 00:16:03,773 THEN IF WE HAVE A TARGET DNA 353 00:16:03,773 --> 00:16:06,742 PRESENT LIKE A PLASMID WE 354 00:16:06,742 --> 00:16:08,978 OBSERVE INTEGRATION BUT IT 355 00:16:08,978 --> 00:16:16,619 IMPLIES THE REQUIREMENT FOR HOST 356 00:16:16,619 --> 00:16:16,852 FACTORS. 357 00:16:16,852 --> 00:16:19,021 IT AND THERE WERE IMPORTANT 358 00:16:19,021 --> 00:16:20,656 CONTRIBUTIONS ON A NUMBER OF 359 00:16:20,656 --> 00:16:22,825 FRONTS BUT IMPORTANTLY IN OUR 360 00:16:22,825 --> 00:16:27,697 UNDERSTANDING OF INTEGRATION 361 00:16:27,697 --> 00:16:30,166 SITE SELECTION AND OTHERS HAVE 362 00:16:30,166 --> 00:16:31,434 SPEARHEADED THE ELEMENTS AND 363 00:16:31,434 --> 00:16:38,975 I'LL SHOW SOME LATER IN MY TALK. 364 00:16:38,975 --> 00:16:47,249 WE CONCLUDED IT ENDOGENOUS 365 00:16:47,249 --> 00:16:48,985 TRANSCRIPTION WAS REQUIRED TO 366 00:16:48,985 --> 00:16:50,186 GET EFFICIENT REVERSE 367 00:16:50,186 --> 00:16:50,920 TRANSCRIPTION IMPLYING THE 368 00:16:50,920 --> 00:16:52,355 CAPSID WAS AN IMPORTANT PART OF 369 00:16:52,355 --> 00:16:54,156 THE REPLICATION COMPLEX. 370 00:16:54,156 --> 00:16:59,729 AND AS I TOLD YOU WE OBSERVED 371 00:16:59,729 --> 00:17:00,363 INTEGRATION BUT REQUIRED EXTRACT 372 00:17:00,363 --> 00:17:03,199 AND THEREFORE HOST FACTORS AND 373 00:17:03,199 --> 00:17:05,067 SO YOU INTEGRATION THAT OCCURRED 374 00:17:05,067 --> 00:17:06,435 IN THE SEQUENCE PREFERENCES. 375 00:17:06,435 --> 00:17:08,504 IT TOOK US A WHILE BUT WE 376 00:17:08,504 --> 00:17:10,506 FIGURED OUT HOW TO QUANTIFY THE 377 00:17:10,506 --> 00:17:11,574 INTEGRATION EFFICIENCY AND WHEN 378 00:17:11,574 --> 00:17:14,877 WE DID THAT THE NUMBER WAS A 379 00:17:14,877 --> 00:17:16,979 LITTLE BIT EMBARRASSING. 380 00:17:16,979 --> 00:17:18,648 SO OVER ALL THE EFFICIENCY WAS 381 00:17:18,648 --> 00:17:18,914 .03%. 382 00:17:18,914 --> 00:17:22,251 WE PUT IN 10,000 CORES AND GOT 383 00:17:22,251 --> 00:17:24,987 THREE TO REPLICATE COMPLETELY 384 00:17:24,987 --> 00:17:26,188 AND INTEGRATE. 385 00:17:26,188 --> 00:17:27,990 SINCE WE'RE INTERESTED IN USING 386 00:17:27,990 --> 00:17:30,159 THIS TO WATCH THE PROCESS IN 387 00:17:30,159 --> 00:17:32,395 STRUCTURAL STUDY WE FELT WE 388 00:17:32,395 --> 00:17:33,062 NEEDED TO DO BETTERS ORE ELSE WE 389 00:17:33,062 --> 00:17:34,630 WOULDN'T KNOW FOR SURE WE WERE 390 00:17:34,630 --> 00:17:36,165 LOOKING AT PARTICLES THAT WERE 391 00:17:36,165 --> 00:17:42,638 ON PATHWAY. 392 00:17:42,638 --> 00:17:45,007 THEY OPTIMIZED THE BUFFERS I 393 00:17:45,007 --> 00:17:48,177 WON'T TALK ABOUT BUT HAS BEEN 394 00:17:48,177 --> 00:17:53,949 ABLE TO REDUCE CONTAMINATING 395 00:17:53,949 --> 00:17:56,952 EXOSOMES AND NUCLEIC ACIDS AND 396 00:17:56,952 --> 00:17:58,988 USING EVERYTHING NOT INSIDE THE 397 00:17:58,988 --> 00:18:03,959 VARIANT AND AN OLD TRICK TO GET 398 00:18:03,959 --> 00:18:05,628 RID OF EXOSOMES BY TREATING AND 399 00:18:05,628 --> 00:18:07,129 WHEN HE DOES THAT THIS IS OUR 400 00:18:07,129 --> 00:18:10,266 OLD PREP AND A MARKER FOR 401 00:18:10,266 --> 00:18:16,639 EXOSOMES AND WE NOW HAVE EXOSOME 402 00:18:16,639 --> 00:18:19,642 FREE PREPARATIONS THIS IS A 403 00:18:19,642 --> 00:18:21,544 NUCLEIC ACID AND STARTING WITH 404 00:18:21,544 --> 00:18:23,546 PURER VIRAL PARTICLES. 405 00:18:23,546 --> 00:18:27,817 MEANWHILE DEVON CHRISTIANSON WAS 406 00:18:27,817 --> 00:18:29,218 INVESTIGATING HOW WE COULD GET 407 00:18:29,218 --> 00:18:32,621 CORES FROM THE VIRUS TO DO WITH 408 00:18:32,621 --> 00:18:38,728 PURE CORE PREPARATIONS AND USES 409 00:18:38,728 --> 00:18:42,064 MELTON BUT MAKES BIGGER HOLES IN 410 00:18:42,064 --> 00:18:46,802 THE MEMBRANE AND IT WILL AT 411 00:18:46,802 --> 00:18:49,271 ABOUT THIS TIME THEY WERE ABLE 412 00:18:49,271 --> 00:18:53,509 TO PURIFY CAPSID ASSEMBLY TUBES 413 00:18:53,509 --> 00:18:55,077 ON GEL FILTRATION COLUMNS IF 414 00:18:55,077 --> 00:18:58,147 THEY DID THAT IN THE PRESENCE OF 415 00:18:58,147 --> 00:18:58,447 IP6. 416 00:18:58,447 --> 00:19:02,084 WE THOUGHT WE'D TRY THAT WITH AU 417 00:19:02,084 --> 00:19:10,426 THEN AUTHENTIC CORES AND CAN DO 418 00:19:10,426 --> 00:19:12,194 THIS WITH SUFFICIENT IP6 419 00:19:12,194 --> 00:19:13,496 PRESENT. 420 00:19:13,496 --> 00:19:14,930 THIS PEAK NUMBER 2 IS THE PEAK 421 00:19:14,930 --> 00:19:15,898 IN WHICH THE CORES COME OUT. 422 00:19:15,898 --> 00:19:20,970 YOU CAN SEE IT CONTAINS AALL THE 423 00:19:20,970 --> 00:19:22,371 EXPECTED PROTEINS AND IT'S QUITE 424 00:19:22,371 --> 00:19:24,440 PURE IN TERMS OF PROTEIN CONTENT 425 00:19:24,440 --> 00:19:26,175 AND WHEN YOU LOOK AT THAT YOU 426 00:19:26,175 --> 00:19:28,911 CAN SEE IT CONTAINS IN FACT CORE 427 00:19:28,911 --> 00:19:29,845 PARTICLES. 428 00:19:29,845 --> 00:19:33,816 THE ABILITY TO MAKE LARGE 429 00:19:33,816 --> 00:19:34,950 QUANTITIES OF CORE PARTICLES 430 00:19:34,950 --> 00:19:42,458 WILL BE IMPORTANT FOR IMAGING. 431 00:19:42,458 --> 00:19:46,195 AND THEY CAN NOW LITERALLY IMAGE 432 00:19:46,195 --> 00:19:48,998 THOUSANDS AND EVEN TENS OF 433 00:19:48,998 --> 00:19:50,733 THOUSANDS OF VIRAL CORE 434 00:19:50,733 --> 00:19:53,602 PARTICLES BY CRYO EM AND TREAT 435 00:19:53,602 --> 00:20:02,978 THE LATTICE TO GET SUB 3 436 00:20:02,978 --> 00:20:07,483 RESOLUTION AND ALSO WORKS FOR 437 00:20:07,483 --> 00:20:08,584 LIGANDS. 438 00:20:08,584 --> 00:20:15,958 THEY ADDED THE CAPSID INHIBITOR 439 00:20:15,958 --> 00:20:19,228 AND OBSERVED THAT THE LATTICE IS 440 00:20:19,228 --> 00:20:20,996 FLATTENED AND THE SITES THAT 441 00:20:20,996 --> 00:20:22,565 WERE PENTAMERS ARE STILL MISSING 442 00:20:22,565 --> 00:20:26,268 AND STILL HEXAMERS AND CAN USE 443 00:20:26,268 --> 00:20:28,204 THE SINGLE PARTICLE TRICK TO END 444 00:20:28,204 --> 00:20:31,040 UP WITH RECONSTRUCTION OF THE 445 00:20:31,040 --> 00:20:35,611 CAPSID HEXAMER AND YOU CAN SEE 446 00:20:35,611 --> 00:20:38,981 WILL LENACAPAVIR AND INTERPRET 447 00:20:38,981 --> 00:20:41,083 IT IN MOLECULAR DETAIL. 448 00:20:41,083 --> 00:20:43,385 OUR LAB'S BEEN WORKING ON CAPSID 449 00:20:43,385 --> 00:20:44,987 30 YEARS AND I WOND HAVE 450 00:20:44,987 --> 00:20:46,121 BELIEVED IF YOU YOU WOULD HAVE 451 00:20:46,121 --> 00:20:51,193 TOLD ME IT WOULD BE POSSIBLE FOR 452 00:20:51,193 --> 00:21:01,470 THE FIELD AND GET THIS 453 00:21:01,470 --> 00:21:03,873 RESOLUTION AND THERE'S INHIBITOR 454 00:21:03,873 --> 00:21:07,743 YOU MAY HAVE SEEN JUST THIS WEEK 455 00:21:07,743 --> 00:21:18,120 GILEAD ANNOUNCED THAT 456 00:21:19,588 --> 00:21:22,057 LENACAPAVIR IS PROTECTING 457 00:21:22,057 --> 00:21:22,758 AFRICAN AMERICAN WOMEN AND 458 00:21:22,758 --> 00:21:23,726 THERE'S REVERSE TRANSCRIPTION 459 00:21:23,726 --> 00:21:24,193 AND INTEGRATION. 460 00:21:24,193 --> 00:21:29,098 I WOULDN'T HAVE KNOWN THAT WAS 461 00:21:29,098 --> 00:21:30,766 POSSIBLE BUT IT'S AN EXCITING 462 00:21:30,766 --> 00:21:32,201 TIME TO BE WORKING ON CORE 463 00:21:32,201 --> 00:21:32,468 PARTICLES. 464 00:21:32,468 --> 00:21:34,703 THIS REMINDS ME TO TELL YOU 465 00:21:34,703 --> 00:21:36,205 THERE ARE MANY STEPS IN THE 466 00:21:36,205 --> 00:21:38,540 VIRAL REPLICATION GOING FROM A 467 00:21:38,540 --> 00:21:40,542 SINGLE STRANDED RNA TO DOUBLE 468 00:21:40,542 --> 00:21:43,012 STRANDED DNA AND CAN MEASURE 469 00:21:43,012 --> 00:21:44,513 DIFFERENT STEPS USING DIFFERENT 470 00:21:44,513 --> 00:21:47,182 PCR PRIMERS TO MEASURE THE 471 00:21:47,182 --> 00:21:48,284 INITIATION OF REVERSE 472 00:21:48,284 --> 00:21:49,351 TRANSCRIPTION AND MEASURE LATE 473 00:21:49,351 --> 00:21:52,588 DOUBLE STRANDED DNA PRODUCTION 474 00:21:52,588 --> 00:21:54,223 AFTER SECONDS-STRAND TRANSFER 475 00:21:54,223 --> 00:21:55,891 AND MEASURE INTEGRATION. 476 00:21:55,891 --> 00:22:03,399 WE DO ALL OF THAT QUANTITATIVELY 477 00:22:03,399 --> 00:22:10,306 USING DROPLET PCRs AND THE LAB 478 00:22:10,306 --> 00:22:11,974 DID A SIMILAR EXPERIMENT 479 00:22:11,974 --> 00:22:14,343 STARTING WITH PURE CORES AND 480 00:22:14,343 --> 00:22:16,378 THIS IS THE NUMBER OF DNA COPIES 481 00:22:16,378 --> 00:22:19,315 PER CORE AND IT'S QUITE GOOD FOR 482 00:22:19,315 --> 00:22:20,983 INITIATION OF REVERSE 483 00:22:20,983 --> 00:22:24,053 TRANSCRIPTION AND LATE ERT. 484 00:22:24,053 --> 00:22:28,958 DEVON STARTED TO FRACTIONATE OUR 485 00:22:28,958 --> 00:22:32,528 CELL AND THIS STEP DOESN'T 486 00:22:32,528 --> 00:22:34,129 REQUIRE EXTRACT BUT YOU CAN SEE 487 00:22:34,129 --> 00:22:35,564 WE ONLY SEE INTEGRATION IN THE 488 00:22:35,564 --> 00:22:37,066 PRESENCE OF NUCLEAR EXTRACT. 489 00:22:37,066 --> 00:22:40,669 SO IT'S NUCLEAR FACTORS THAT ARE 490 00:22:40,669 --> 00:22:41,070 REQUIRED FOR THIS. 491 00:22:41,070 --> 00:22:42,204 AND YOU'LL ALSO SEE BECAUSE OF 492 00:22:42,204 --> 00:22:44,540 THESE IMPROVEMENTS WE'VE 493 00:22:44,540 --> 00:22:45,741 INCREASED EFFICIENCY ABOUT 100 494 00:22:45,741 --> 00:22:48,210 FOLD SO NOW IT'S NOT SO 495 00:22:48,210 --> 00:22:57,519 EMBARRASSING ANYMORE. 496 00:22:57,519 --> 00:23:00,990 WE'D LOVE TO KNOW ALL THE 497 00:23:00,990 --> 00:23:04,426 FACTORS AND I WANT TO HIGHLIGHT 498 00:23:04,426 --> 00:23:07,062 A FEW THINGS. 499 00:23:07,062 --> 00:23:08,330 THE INTEGRASE PROTEIN INTEGRATES 500 00:23:08,330 --> 00:23:11,734 BINDING DOMAIN AND THE VIRUS USE 501 00:23:11,734 --> 00:23:13,302 TO TETHER TO CHROMATIN. 502 00:23:13,302 --> 00:23:19,241 AND WE HAVE A STRUCTURE FROM THE 503 00:23:19,241 --> 00:23:22,011 LAB AND KNOW WHAT THAT LOOKS 504 00:23:22,011 --> 00:23:23,112 LIKE AND IDENTIFIED WHAT KNOCKS 505 00:23:23,112 --> 00:23:25,547 OUT THE INTERACTION AND THE 506 00:23:25,547 --> 00:23:32,988 VIRUS IS USING THAT TO TETHER 507 00:23:32,988 --> 00:23:35,357 THE INTOSOME TO CHROMATIN AND 508 00:23:35,357 --> 00:23:45,367 THEY'RE TRI METHYLATED ON 509 00:23:45,367 --> 00:23:48,237 LYESENE 36 AND YOU CAN SEE THE 510 00:23:48,237 --> 00:23:51,774 EFFICIENT REACTION AND WE EXPECT 511 00:23:51,774 --> 00:23:54,643 ONE DNA COPY FOR CORE AND FOR 512 00:23:54,643 --> 00:23:58,113 THE FIRST TIME IN THE PURE 513 00:23:58,113 --> 00:24:00,449 RECOMBINANT SYSTEM WE CAN 514 00:24:00,449 --> 00:24:03,152 MEASURE INTEGRATION AND DO THAT 515 00:24:03,152 --> 00:24:06,255 IN THE PRESENCE OF CHROMATINIZED 516 00:24:06,255 --> 00:24:07,656 DNA AND THIS IS MIXED CHROMATIN 517 00:24:07,656 --> 00:24:09,725 AND YOU CAN SEE WE CAN KNOW GET 518 00:24:09,725 --> 00:24:12,261 UP TO ABOUT 10% OVERALL 519 00:24:12,261 --> 00:24:15,164 REPLICATION AND INTEGRATION 520 00:24:15,164 --> 00:24:20,436 EFFICIENCY AND THAT REQUIRES AN 521 00:24:20,436 --> 00:24:23,806 ACTIVE IT-LEDGF MOLECULE WITHOUT 522 00:24:23,806 --> 00:24:24,106 INTEGRATION. 523 00:24:24,106 --> 00:24:26,041 THIS NEXT SLIDE IS A RECENT 524 00:24:26,041 --> 00:24:27,076 OBSERVATION AND A SURPRISE TO US 525 00:24:27,076 --> 00:24:30,179 AND THE OBSERVATION IS THAT THE 526 00:24:30,179 --> 00:24:32,781 INTEGRATION STEP GOES WELL AT 4 527 00:24:32,781 --> 00:24:33,415 DEGREES CENTIGRADE. 528 00:24:33,415 --> 00:24:35,751 WE CAN DO OUR NORMAL EXPERIMENT 529 00:24:35,751 --> 00:24:37,986 THIS IS A 10 HOUR INCUBATION AND 530 00:24:37,986 --> 00:24:39,888 GET DOUBLE-STRANDED DNA AND THEN 531 00:24:39,888 --> 00:24:43,859 TAKE THAT SAMPLE AND EITHER 532 00:24:43,859 --> 00:24:47,563 LEAVE IT AT 37 FOR 10 MORE HOURS 533 00:24:47,563 --> 00:24:49,698 OR SHIFT IT TO 4 DEGREES. 534 00:24:49,698 --> 00:24:55,337 WE CAN STOP INTEGRATION WHEREVER 535 00:24:55,337 --> 00:25:01,110 WE WANT BY ADDING WILL 536 00:25:01,110 --> 00:25:03,278 REGTAGLAVIRE AND IN THE FIRST 537 00:25:03,278 --> 00:25:04,980 CONDITION HE GOES FOR 10 HOURS 538 00:25:04,980 --> 00:25:08,984 AND 37 AND SHIFTS TO FOUR 539 00:25:08,984 --> 00:25:16,959 DEGREES FOR ANOTHER 10 HOURS. 540 00:25:16,959 --> 00:25:20,462 YOU SEE THE SLIGHT INCREASE IN 541 00:25:20,462 --> 00:25:21,763 ERT AND TO BE EXPECTED AT 37. 542 00:25:21,763 --> 00:25:24,900 THE REAL SURPRISE IS WE ONLY 543 00:25:24,900 --> 00:25:26,068 OBSERVE INTEGRATION ESSENTIALLY 544 00:25:26,068 --> 00:25:27,970 UNDER THE 4 DEGREE CONDITIONS. 545 00:25:27,970 --> 00:25:29,705 THAT MEANS WE'RE DOING 546 00:25:29,705 --> 00:25:30,105 INTEGRATION. 547 00:25:30,105 --> 00:25:31,607 THERE'S SOME STEP BETWEEN THE 548 00:25:31,607 --> 00:25:32,708 COMPLETION OF REVERSE 549 00:25:32,708 --> 00:25:33,642 TRANSCRIPTION AND INTEGRATION 550 00:25:33,642 --> 00:25:35,611 THAT IS PROMOTED BY 4 DEGREES. 551 00:25:35,611 --> 00:25:38,046 I'M HAPPY TO SPECULATE THAT IN 552 00:25:38,046 --> 00:25:40,149 QUESTIONS IF YOU WANT BUT WE 553 00:25:40,149 --> 00:25:40,782 DON'T COMPLETELY UNDERSTAND IT 554 00:25:40,782 --> 00:25:40,949 YET. 555 00:25:40,949 --> 00:25:44,386 THE FINAL THING I WANT TO TELL 556 00:25:44,386 --> 00:25:45,621 YOU TO MAP THE SEQUENCE AND 557 00:25:45,621 --> 00:25:47,322 THAT'S WHAT THIS IS, WE'RE NOW 558 00:25:47,322 --> 00:25:48,991 EFFICIENT ENOUGH WE DON'T HAVE 559 00:25:48,991 --> 00:25:51,960 TO USE PCR, WE CAN DIRECTLY 560 00:25:51,960 --> 00:25:52,861 SEQUENCE ALL THE INTEGRATION 561 00:25:52,861 --> 00:25:56,899 SITE AND WHEN WE DO THAT WE SEE 562 00:25:56,899 --> 00:25:58,600 THE EXPECTED PALINDROME 563 00:25:58,600 --> 00:25:59,935 PREFERENCE AND THAT MATCHES THE 564 00:25:59,935 --> 00:26:02,471 SEQUENCE THAT WE HAVE PREVIOUSLY 565 00:26:02,471 --> 00:26:04,973 SEEN IN OUR CELL EXTRACTS AND 566 00:26:04,973 --> 00:26:07,242 OTHERS HAVE SEEN IN INFECTED 567 00:26:07,242 --> 00:26:09,678 CELLS AND PATIENTS. 568 00:26:09,678 --> 00:26:11,213 THERE WAS AN INTERESTING 569 00:26:11,213 --> 00:26:16,385 OBSERVATION THE PALINDROME IS 570 00:26:16,385 --> 00:26:18,086 EMBEDDED WITHIN A LARGER 571 00:26:18,086 --> 00:26:22,457 PALINDROME AND SEE IT ON THE 572 00:26:22,457 --> 00:26:23,792 INTEGRATION SITE AND SO FORTH. 573 00:26:23,792 --> 00:26:28,630 WE THINK THAT CAN ONLY BE 574 00:26:28,630 --> 00:26:34,169 EXPLAINED THE INTOSOME SEES 50 575 00:26:34,169 --> 00:26:36,271 BASE PAIRS AND THAT'S OF 576 00:26:36,271 --> 00:26:39,775 INTEREST FROM THE STRUCTURES 577 00:26:39,775 --> 00:26:41,743 THAT HAVE COME UP FOR 578 00:26:41,743 --> 00:26:42,077 LENTIVIRUSES. 579 00:26:42,077 --> 00:26:46,048 SO JUST TO FINISH, WE NOW HAVE 580 00:26:46,048 --> 00:26:46,915 QUITE EFFICIENT ERT. 581 00:26:46,915 --> 00:26:48,750 IT GOES WITH PURE CORES. 582 00:26:48,750 --> 00:26:50,385 THE PURE CORES ARE STILL VISIBLE 583 00:26:50,385 --> 00:26:51,753 AT THE END OF THE REACTION. 584 00:26:51,753 --> 00:26:53,722 WE THINK EVERYTHING'S TAKING 585 00:26:53,722 --> 00:26:58,860 PLACE INSIDE AN INTACT CAPSID 586 00:26:58,860 --> 00:27:05,334 AND USE LEGGF BUT HAS A LARGE 587 00:27:05,334 --> 00:27:06,335 PALINDROMIC PIECE OF DNA AND 588 00:27:06,335 --> 00:27:08,503 INTEGRATION IS STRONGLY FAVORED 589 00:27:08,503 --> 00:27:10,072 AT 4 AND OUR OVER ALL YIELDS ARE 590 00:27:10,072 --> 00:27:11,506 NOW ABOUT 10%. 591 00:27:11,506 --> 00:27:12,808 WITH THAT I'LL FINISH BY 592 00:27:12,808 --> 00:27:13,642 THANKING THE PEOPLE ONCE MORE 593 00:27:13,642 --> 00:27:16,979 WHO DID ALL THE WORK I TALKED 594 00:27:16,979 --> 00:27:17,179 ABOUT. 595 00:27:17,179 --> 00:27:27,322 THANKS. 596 00:27:50,679 --> 00:27:51,647 OF 597 00:27:55,150 --> 00:27:56,418 >> OKAY. 598 00:27:56,418 --> 00:27:56,618 HELLO. 599 00:27:56,618 --> 00:27:59,588 MY NAME IS THOMAS SCHWARTZ. 600 00:27:59,588 --> 00:28:02,791 I'M ALSO AT THE CENTER AND I'LL 601 00:28:02,791 --> 00:28:05,160 TELL YOU TODAY ABOUT NANO BODIES 602 00:28:05,160 --> 00:28:10,265 AGAINST CAPSIDS WE GENERATED IN 603 00:28:10,265 --> 00:28:10,599 THE LAB. 604 00:28:10,599 --> 00:28:13,802 THE MOTIVATION FOR THIS IS NANO 605 00:28:13,802 --> 00:28:17,005 BODIES THE CAPSID WOULD BE A 606 00:28:17,005 --> 00:28:19,174 USEFUL TOOL SIMPLY BECAUSE NANO 607 00:28:19,174 --> 00:28:20,942 BODIES ARE WONDERFUL SMALL 608 00:28:20,942 --> 00:28:22,577 PROTEINS THAT TYPICALLY CAN HAVE 609 00:28:22,577 --> 00:28:25,247 VERY HIGH AFFINITY AND WE WANTED 610 00:28:25,247 --> 00:28:28,116 TO GENERATE THOSE TO TRACK 611 00:28:28,116 --> 00:28:34,056 CAPSIDS AND TO LABEL IT OR 612 00:28:34,056 --> 00:28:36,258 ANCHOR IT AT CERTAIN POSITIONS 613 00:28:36,258 --> 00:28:42,297 IN THE PASSAGE THROUGH THE CELL. 614 00:28:42,297 --> 00:28:44,499 AND WE INTRODUCED THE HIV 615 00:28:44,499 --> 00:28:44,733 CAPSID. 616 00:28:44,733 --> 00:28:47,102 SO ALL WE NEED TO KNOW FOR THIS 617 00:28:47,102 --> 00:28:50,572 PURPOSE IS IF YOU WANT TO FIND 618 00:28:50,572 --> 00:28:52,174 AN ANTIBODY THAT BINDS TO THE 619 00:28:52,174 --> 00:28:58,113 CAPSID IT NEEDS TO BIND TO THE 620 00:28:58,113 --> 00:29:04,986 CA PROTEIN AND THE MONOMER IS 621 00:29:04,986 --> 00:29:14,663 ORGANIZED IN PENPENTAMERS AND 622 00:29:14,663 --> 00:29:14,996 HEXAMERS. 623 00:29:14,996 --> 00:29:18,967 AND THE CAPSID HAS A SMALL 624 00:29:18,967 --> 00:29:20,268 PORTION OF THE PORTION TO BIND 625 00:29:20,268 --> 00:29:24,306 TO NORD TO WORK. 626 00:29:24,306 --> 00:29:32,681 -- IN ORDER TO WORK. 627 00:29:32,681 --> 00:29:35,817 AND THE ONES PUBLISHED ARE 628 00:29:35,817 --> 00:29:45,861 INCOMPATIBLE WITH THE CAPSID. 629 00:29:45,861 --> 00:29:48,697 AND SOME BIND TO THE HEXAMER AND 630 00:29:48,697 --> 00:29:51,133 AREN'T OF USE TO US. 631 00:29:51,133 --> 00:30:00,275 AND WE TEAMED UP IN GERMANY AND 632 00:30:00,275 --> 00:30:03,011 COLLABORATED WITH VARIOUS THINGS 633 00:30:03,011 --> 00:30:11,920 AND PRODUCED NANOBODIES IN THE 634 00:30:11,920 --> 00:30:13,155 ALPACA FARM AND WE THOUGHT LET'S 635 00:30:13,155 --> 00:30:19,428 TRY THIS. 636 00:30:19,428 --> 00:30:21,430 THEY HAD ALREADY IMMUNIZED THEM 637 00:30:21,430 --> 00:30:23,899 BEFORE WE STARTED AND THOUGHT WE 638 00:30:23,899 --> 00:30:28,003 CAN SCREEN AGAINST THE HEXAMER. 639 00:30:28,003 --> 00:30:34,176 WE DID THIS AND GOT A LIBRARY OF 640 00:30:34,176 --> 00:30:38,113 13 DIFFERENT NANO BODIED AND 641 00:30:38,113 --> 00:30:40,382 SEQUENCE TO FIVE GROUPS. 642 00:30:40,382 --> 00:30:48,857 WE TESTED THESE NANOBODIES AND 643 00:30:48,857 --> 00:30:56,965 SEE THEY ALL BIND WITH HIGH 644 00:30:56,965 --> 00:30:57,799 AFFINITY. 645 00:30:57,799 --> 00:31:05,540 AND THIS HAS ONE THAT'S ALMOST 646 00:31:05,540 --> 00:31:10,212 NEGLIGIBLE AND WE DID THIS ASSAY 647 00:31:10,212 --> 00:31:20,755 WHERE WE INCUBATED THEM WITH THE 648 00:31:21,656 --> 00:31:25,293 MONOMER AND SUGGESTED A 1:1 649 00:31:25,293 --> 00:31:26,795 BINDING AND WE CAN SEE THEY 650 00:31:26,795 --> 00:31:29,331 DON'T ALL BIND THE SAME WAY 651 00:31:29,331 --> 00:31:31,533 BECAUSE SOME HAVE THIS 652 00:31:31,533 --> 00:31:36,004 INTERESTING BEHAVIOR WHEN THEY 653 00:31:36,004 --> 00:31:44,379 BIND TO THE MONOMER IT SHIFTS 654 00:31:44,379 --> 00:31:48,083 SUGGESTING THEY ASSEMBLE THE CA 655 00:31:48,083 --> 00:31:49,885 PRESUMABLY INTO A HEXAMER AND 656 00:31:49,885 --> 00:31:51,820 DIFFERENT IN THE BINDING AND 657 00:31:51,820 --> 00:31:52,988 STRUCTURE AND WE WANT TO KNOW 658 00:31:52,988 --> 00:32:00,295 THE ATOMIC DETAILS OR RESIDUE 659 00:32:00,295 --> 00:32:07,702 DETAILS. 660 00:32:07,702 --> 00:32:11,206 NANOBODIES AND THE STRONGEST 661 00:32:11,206 --> 00:32:14,242 BINDER WAS CRYO EM AND THE OTHER 662 00:32:14,242 --> 00:32:18,947 CRYSTALS GREW QUITE NICELY WITH 663 00:32:18,947 --> 00:32:20,715 GOOD RESOLUTION AND WHAT WE CAN 664 00:32:20,715 --> 00:32:23,118 SEE FROM THIS IS THEY ACTUALLY 665 00:32:23,118 --> 00:32:25,487 ALL BIND TO THE INTERNAL DOMAIN 666 00:32:25,487 --> 00:32:26,788 BUT IN DIFFERENT WAYS AS YOU CAN 667 00:32:26,788 --> 00:32:30,292 SEE MOST DRAMATICALLY HERE AND 668 00:32:30,292 --> 00:32:31,660 THESE TWO CAN BIND TOGETHER AND 669 00:32:31,660 --> 00:32:36,298 EVEN HAVE A CRYSTAL STRUCTURE OF 670 00:32:36,298 --> 00:32:37,666 BOTH OF THEM BOUND. 671 00:32:37,666 --> 00:32:38,199 ALL RIGHT. 672 00:32:38,199 --> 00:32:43,872 SO IT GIVES US DETAILS AND HOW 673 00:32:43,872 --> 00:32:48,276 DOES THIS RELATE TO THE 674 00:32:48,276 --> 00:32:49,711 PHENOMENA WE SAW? 675 00:32:49,711 --> 00:32:53,682 WE CAN NICELY EXPLAIN WHY SOME 676 00:32:53,682 --> 00:32:56,751 BIND MONOMER AND SOME BIND AND 677 00:32:56,751 --> 00:32:58,019 FORCE THE ASSEMBLY. 678 00:32:58,019 --> 00:33:04,359 THE ONES THAT REALLY BIND 1:1 679 00:33:04,359 --> 00:33:06,294 BIND JUST ONE CA AND HAVE A 680 00:33:06,294 --> 00:33:08,697 BINDING SITE AND WHAT YOU CAN 681 00:33:08,697 --> 00:33:10,665 APPRECIATE HERE IS THAT THE 682 00:33:10,665 --> 00:33:13,668 BINDING SITE IS PRETTY MUCH 683 00:33:13,668 --> 00:33:13,969 OVERLAPPING. 684 00:33:13,969 --> 00:33:17,138 THEY ARE COMPETITIVE BINDERS. 685 00:33:17,138 --> 00:33:19,474 BUT REALLY HOW THEY RECOGNIZE 686 00:33:19,474 --> 00:33:22,277 THIS IS QUITE SIGNIFICANTLY 687 00:33:22,277 --> 00:33:22,978 DIFFERENT. 688 00:33:22,978 --> 00:33:24,179 AND WHAT HAPPENS IN THE OTHER 689 00:33:24,179 --> 00:33:24,479 CASE? 690 00:33:24,479 --> 00:33:27,582 WHAT HAPPENS IN THE CASE WHERE 691 00:33:27,582 --> 00:33:28,984 WE SEE THE SHIFT INFILTRATION IS 692 00:33:28,984 --> 00:33:32,988 HERE THE BINDING SITE IS 693 00:33:32,988 --> 00:33:34,856 ACTUALLY COMPOSED OF THE TWO 694 00:33:34,856 --> 00:33:36,625 NEIGHBORING CAs AND BRINGS THE 695 00:33:36,625 --> 00:33:40,996 TWO TOGETHER AND STABILIZES THE 696 00:33:40,996 --> 00:33:43,064 HEXAMER. 697 00:33:43,064 --> 00:33:45,333 ALL RIGHT. 698 00:33:45,333 --> 00:33:48,136 SO THAT'S ALL GOOD PRELIMINARY 699 00:33:48,136 --> 00:33:51,306 DATA SUGGESTING THESE NANOBODIES 700 00:33:51,306 --> 00:33:54,309 BIND HEXAMERS OR SHOWING THEY 701 00:33:54,309 --> 00:33:56,344 BIND HEXAMERS BUT WE WANT IT 702 00:33:56,344 --> 00:33:58,179 THEM TO BIND CAPSIDS. 703 00:33:58,179 --> 00:34:04,352 WE FIRST CHECK THESE IN SILICO 704 00:34:04,352 --> 00:34:13,261 AND IMPOSED WHAT THEY THE CA. 705 00:34:13,261 --> 00:34:18,400 -- CAPSID WOULD LOOK LIKE AND 706 00:34:18,400 --> 00:34:21,002 THESE TWO THERE'S NO CLASH WE 707 00:34:21,002 --> 00:34:23,972 CAN SEE AND SUGGESTS THEY SHOULD 708 00:34:23,972 --> 00:34:25,006 BE COMPATIBLE WITH BINDING TO 709 00:34:25,006 --> 00:34:29,878 THE CAPSID AND WE SEE CLASH WITH 710 00:34:29,878 --> 00:34:31,212 A NEIGHBORING ANTIBODY 711 00:34:31,212 --> 00:34:36,317 SELF-CLASH OR CLASH OF THE 712 00:34:36,317 --> 00:34:37,686 NEIGHBORING IT CAPSID PROGRAM 713 00:34:37,686 --> 00:34:39,988 AND LATTICE CLASH AND THESE ARE 714 00:34:39,988 --> 00:34:42,691 CASES WHERE THEY WERE IN THE 715 00:34:42,691 --> 00:34:47,429 FIELD OF THE CAPSID OR BIND WITH 716 00:34:47,429 --> 00:34:48,997 STABILITY OR WE WOULD SEE WILL 717 00:34:48,997 --> 00:34:53,001 THE SUBBINDING AND TESTED THIS 718 00:34:53,001 --> 00:34:58,740 AGAIN IN AN ASSAY WHERE WE 719 00:34:58,740 --> 00:35:04,979 STABILIZE AND FORM CRPs AND THEN 720 00:35:04,979 --> 00:35:09,617 LOOKED AND SAW THE TWO PRIME 721 00:35:09,617 --> 00:35:12,987 ANTIBODIES AOA AND AO5 WITH THE 722 00:35:12,987 --> 00:35:15,490 TWO CLPs AND THE OTHER THREE 723 00:35:15,490 --> 00:35:20,662 ALSO BIND BUT YOU CAN CLEARLY 724 00:35:20,662 --> 00:35:27,936 SEE WILL THEY'RE DIFFERENT AND 725 00:35:27,936 --> 00:35:31,072 CONTROL ANTIBODIES DO NOT AS 726 00:35:31,072 --> 00:35:31,339 EXPECTED. 727 00:35:31,339 --> 00:35:38,046 THEN WE WANTED TO LOOK AT THIS 728 00:35:38,046 --> 00:35:41,282 STRUCTURE AS WELL AND ASSEMBLED 729 00:35:41,282 --> 00:35:43,885 WITH THE RESPECTIVE NANOBODIES 730 00:35:43,885 --> 00:35:47,489 AND MADE TWO DIFFERENT CRYO EM 731 00:35:47,489 --> 00:35:50,058 STRUCTURES ONE OF THE STRONGEST 732 00:35:50,058 --> 00:35:54,329 BINDER AS IT BINDS A SINGLE CA 733 00:35:54,329 --> 00:35:56,998 YOU CAN DO THIS IN THE CONTEXT 734 00:35:56,998 --> 00:36:01,936 OF THE CLP AND CAN DECORATE THE 735 00:36:01,936 --> 00:36:05,774 PENTAMER AS WELL AS THE HEXAMER 736 00:36:05,774 --> 00:36:08,076 AND EVERY CA SEEMS TO BE 737 00:36:08,076 --> 00:36:09,177 OCCUPIED BY THE NANOBODY. 738 00:36:09,177 --> 00:36:14,682 IF YOU DO THIS WITH PO5 WHICH 739 00:36:14,682 --> 00:36:16,751 BINDS BETWEEN THE TWO CA MONOMER 740 00:36:16,751 --> 00:36:19,854 AND HEXAMER AND BINDS AND ALSO 741 00:36:19,854 --> 00:36:22,957 DOES NOT BIND TO PENTAMER 742 00:36:22,957 --> 00:36:25,727 BECAUSE IN THE PENTAMER IT 743 00:36:25,727 --> 00:36:29,030 CANNOT PROPERLY BIND BUT IT CAN 744 00:36:29,030 --> 00:36:31,032 DECORATE THE CAPSID. 745 00:36:31,032 --> 00:36:32,500 ALL RIGHT. 746 00:36:32,500 --> 00:36:35,036 SO NOW I SAID SOMETHING IN THE 747 00:36:35,036 --> 00:36:38,540 TITLE ABOUT USING THESE 748 00:36:38,540 --> 00:36:39,374 NANOBODIES AS A THERAPEUTIC. 749 00:36:39,374 --> 00:36:45,713 WHAT IS THE IDEA HERE? 750 00:36:45,713 --> 00:36:48,983 THE INTERESTING PART ABOUT THE 751 00:36:48,983 --> 00:36:50,518 NANOBODIES IS THEY INTERESTINGLY 752 00:36:50,518 --> 00:36:52,554 BIND CLOSE AND SOME OVERLAPPING 753 00:36:52,554 --> 00:36:58,793 FASHION WITH THE BINDING SITE ON 754 00:36:58,793 --> 00:37:04,966 C 755 00:37:04,966 --> 00:37:09,404 CA AWE CAN SEE IT BINDS AND 756 00:37:09,404 --> 00:37:11,873 BLOCKED IF IT COINCUBATE WITH 757 00:37:11,873 --> 00:37:14,475 THE DIFFERENT ANTIBODIES. 758 00:37:14,475 --> 00:37:16,110 WHY IS THIS IMPORTANT? 759 00:37:16,110 --> 00:37:18,346 THE FG BINDING IS IMPORTANT IN 760 00:37:18,346 --> 00:37:22,517 THE IT HIV LIFE CYCLE AS THE 761 00:37:22,517 --> 00:37:24,552 CAPSID TRAVELS SO THE CYTOPLASM 762 00:37:24,552 --> 00:37:28,957 AND THE NUCLEUS IT HAS TO PASS 763 00:37:28,957 --> 00:37:32,961 THE NUCLEAR CORE COMPLEX AND 764 00:37:32,961 --> 00:37:35,530 THAT CHANNEL IS ESSENTIALLY A 765 00:37:35,530 --> 00:37:39,200 CLOUD FULL OF FGs AND WHAT IT'S 766 00:37:39,200 --> 00:37:42,303 ENGAGED WITH. 767 00:37:42,303 --> 00:37:43,404 SO WE BASICALLY SHOWED EARLIER 768 00:37:43,404 --> 00:37:47,876 WE PUBLISHED EARLIER THIS YEAR 769 00:37:47,876 --> 00:37:50,979 TOGETHER WITH A GROUP IN BACK TO 770 00:37:50,979 --> 00:37:58,086 BACK PUBLICATION HOW THE IS 771 00:37:58,086 --> 00:37:59,587 ASSEMBLED CAPSID CAN GO THROUGH 772 00:37:59,587 --> 00:38:01,589 THE COMPLEX AND THE CORE OF THE 773 00:38:01,589 --> 00:38:04,459 NUCLEAR CORE COMPLEX IS FILLED 774 00:38:04,459 --> 00:38:08,663 WITH THIS THIN MESH WORK OF 775 00:38:08,663 --> 00:38:17,005 PROTEINS RICH IN FG REPEATS AND 776 00:38:17,005 --> 00:38:20,842 CAN PASS AND THE KEY ASSAY AWAY 777 00:38:20,842 --> 00:38:26,214 USED HERE WAS AN FG HYDROGEL 778 00:38:26,214 --> 00:38:28,449 ASSAY DONE IN THIS LAB AND THEY 779 00:38:28,449 --> 00:38:31,452 CAN MIMIC WHAT HAPPENS IN THE 780 00:38:31,452 --> 00:38:33,955 NUCLEAR CORE COMPLEX IN A SMALL 781 00:38:33,955 --> 00:38:36,991 HYDRO SHELL AND YOU CAN TAKE FG 782 00:38:36,991 --> 00:38:41,429 PEPTIDES OR PROTEINS AND FORM A 783 00:38:41,429 --> 00:38:43,531 MICROFG DROPLETS WITH THE 784 00:38:43,531 --> 00:38:44,999 PROPERTIES OF THE NUCLEAR CORE 785 00:38:44,999 --> 00:38:46,968 COMPLEX MEANING CARGO THAT IS 786 00:38:46,968 --> 00:38:48,369 BOUND BY NUCLEAR TRANSPORT 787 00:38:48,369 --> 00:38:55,209 RECEPTOR CAN PENETRATE THE 788 00:38:55,209 --> 00:38:55,510 DROPLETS. 789 00:38:55,510 --> 00:38:58,179 IF YOU LABEL IT AND BIND IN WITH 790 00:38:58,179 --> 00:39:02,750 THE RECEPTOR IT GOES IN THE 791 00:39:02,750 --> 00:39:04,152 DROPLETS AND PAINT THEM GREEN 792 00:39:04,152 --> 00:39:06,054 WHERE IN EXCLUSION MODELS THEY 793 00:39:06,054 --> 00:39:07,355 STAY OUTSIDE. 794 00:39:07,355 --> 00:39:10,325 IN THE PAPER WE HAVE SHOWN THIS 795 00:39:10,325 --> 00:39:13,528 WITH CLPs WE ASSEMBLED FOR THE 796 00:39:13,528 --> 00:39:22,003 NANO METER SPHERES OR THE THOSE 797 00:39:22,003 --> 00:39:23,705 FILLED WITH THE IT MARKER AND 798 00:39:23,705 --> 00:39:28,343 YOU CAN SEE THE IMPORT OF THE OF 799 00:39:28,343 --> 00:39:30,945 THESE HYDROGELS AND IT GOES IN 800 00:39:30,945 --> 00:39:32,847 JUST A NUCLEAR TRANSPORT 801 00:39:32,847 --> 00:39:34,682 RECEPTOR AND NOW OF COURSE THE 802 00:39:34,682 --> 00:39:36,384 QUESTION IS WHAT DO THE 803 00:39:36,384 --> 00:39:36,918 NANOBODIES DO? 804 00:39:36,918 --> 00:39:39,754 NOW IF YOU DO THE EASY IN THE 805 00:39:39,754 --> 00:39:47,328 PRESENCE OF THESE -- ASSAY IN 806 00:39:47,328 --> 00:39:53,935 THE PRESENCE OF THE NANO MARKERS 807 00:39:53,935 --> 00:39:56,604 AND THEY'RE FILLED WITH GFP AND 808 00:39:56,604 --> 00:40:00,008 IF YOU CONTROL OUT GET THE GFP 809 00:40:00,008 --> 00:40:02,777 SIGNAL INSIDE THE DROPLETS AND 810 00:40:02,777 --> 00:40:04,112 COME IN WITH THE DIFFERENT 811 00:40:04,112 --> 00:40:05,446 NANOBODIES AND YOU CAN SEE 812 00:40:05,446 --> 00:40:07,415 BASICALLY IN ALL OF THEM IT'S 813 00:40:07,415 --> 00:40:08,983 COMPLETELY BLANK AND THEY BLOCK 814 00:40:08,983 --> 00:40:11,519 THE IMPORT INTO THE NUCLEAR CORE 815 00:40:11,519 --> 00:40:15,289 AND IN A CONTROL EXPERIMENT WITH 816 00:40:15,289 --> 00:40:18,059 A POINT MUTATION THAT CANNOT 817 00:40:18,059 --> 00:40:20,495 BIND THE CAPSID ANYMORE THEN OF 818 00:40:20,495 --> 00:40:24,365 COURSE YOU CAN RESCUE. 819 00:40:24,365 --> 00:40:28,136 SO THEY COULD WORK IN PRINCIPLE 820 00:40:28,136 --> 00:40:31,773 AS A COMPETITOR FOR NUCLEAR CORE 821 00:40:31,773 --> 00:40:34,475 IMPORT BUT OF COURSE WE ALSO 822 00:40:34,475 --> 00:40:36,277 KNOW THERE IS THIS STABILITY 823 00:40:36,277 --> 00:40:40,181 ISSUE IF YOU BIND SOMETHING INTO 824 00:40:40,181 --> 00:40:42,917 CAPSID THAT AFFECTS POTENTIAL 825 00:40:42,917 --> 00:40:48,389 THE CAPSID STABILITY MOST 826 00:40:48,389 --> 00:40:55,129 FAMOUSLY SHOWN BY THE 827 00:40:55,129 --> 00:40:57,632 LENACAPAVIR AND WANTED TO TEST 828 00:40:57,632 --> 00:41:00,968 WITH THE STABILITY OF CAPSIDS 829 00:41:00,968 --> 00:41:11,512 AND AND THIS IS THE ST. LOUIS IS 830 00:41:13,047 --> 00:41:18,386 ASSAY WHERE WE TAKE PURIFIED 831 00:41:18,386 --> 00:41:23,257 CAPSIDS THAT UNDER GO EN DODGE 832 00:41:23,257 --> 00:41:26,160 NEWS REVERSE TRANSCRIPTION -- 833 00:41:26,160 --> 00:41:27,728 ENDOGENOUS REVERSE TRANSCRIPTION 834 00:41:27,728 --> 00:41:32,400 AND ASK HOW STABLE IT IS IF WE 835 00:41:32,400 --> 00:41:35,403 COME IN WITH NANOBODIES OR 836 00:41:35,403 --> 00:41:38,172 LENACAPAVIR AND COLLECT FOR THE 837 00:41:38,172 --> 00:41:40,975 RELEASE OF DOUBLE-STRANDED DNA 838 00:41:40,975 --> 00:41:51,519 AND CONVERT ATP AND GTP TO CGAMP 839 00:41:52,553 --> 00:41:58,159 AND LYDIA DID THIS WONDERFUL 840 00:41:58,159 --> 00:42:08,169 EXPERIMENT IN THE LAB. 841 00:42:08,169 --> 00:42:10,638 YOU CAN SEE HOW THE CAPSID IS DE 842 00:42:10,638 --> 00:42:14,408 STABILIZED IN A CONCENTRATION 843 00:42:14,408 --> 00:42:16,077 DEPENDENT MANNER. 844 00:42:16,077 --> 00:42:17,478 IF WE DO THIS WITH DIFFERENT 845 00:42:17,478 --> 00:42:20,481 ANTIBODIES, SOME HAVE A SMALL 846 00:42:20,481 --> 00:42:20,715 EFFECT. 847 00:42:20,715 --> 00:42:23,551 OUR PREFERRED BINDER HAS NO 848 00:42:23,551 --> 00:42:25,620 EFFECT AND THEN WE HAVE ONE WITH 849 00:42:25,620 --> 00:42:27,121 A STRONGER EFFECT SO WHEN WE 850 00:42:27,121 --> 00:42:28,956 LOOK AT THIS WE HAD THE 851 00:42:28,956 --> 00:42:31,392 STRUCTURE AND DID SOME STRUCTURE 852 00:42:31,392 --> 00:42:34,629 GAZING AND MODIFIED THIS AND NOW 853 00:42:34,629 --> 00:42:38,466 HAVE A NANOBODY THAT REALLY IS 854 00:42:38,466 --> 00:42:39,967 BASICALLY AS STRONG AND 855 00:42:39,967 --> 00:42:43,371 DESTABILIZING AS LENACAPAVIR IS. 856 00:42:43,371 --> 00:42:49,510 AND LAST BUT NOT LEAST, WE 857 00:42:49,510 --> 00:42:52,980 TESTED THESE NANOBODIES CAN 858 00:42:52,980 --> 00:43:01,689 RESTRICT SO WE TURNED TO JEREMY 859 00:43:01,689 --> 00:43:05,359 LUBEN'S LAB AND ASKED IN AN 860 00:43:05,359 --> 00:43:06,761 INFECTION ASSAY WHETHER THESE 861 00:43:06,761 --> 00:43:09,564 NANOBODIES COULD RESTRICT AND SO 862 00:43:09,564 --> 00:43:13,401 IN THIS ASSAY OF GFP IS 863 00:43:13,401 --> 00:43:17,138 BASICALLY A READ OUT FOR 864 00:43:17,138 --> 00:43:19,774 INFECTION. 865 00:43:19,774 --> 00:43:26,681 AND YOU CAN LOOK AND GET A 866 00:43:26,681 --> 00:43:28,316 CERTAIN SIGNAL WITH A KNOWN 867 00:43:28,316 --> 00:43:33,654 RESTRICTION FACTOR YOU CAN GET A 868 00:43:33,654 --> 00:43:36,424 REDUCTION IN INTEGRATION AND IF 869 00:43:36,424 --> 00:43:39,727 YOU DO THE SAME ASSAY WITH OUR 870 00:43:39,727 --> 00:43:41,395 MODIFIED NANO BODY THEN THE 871 00:43:41,395 --> 00:43:43,397 INFECTION GOES EVEN FURTHER DOWN 872 00:43:43,397 --> 00:43:47,835 AND IF YOU TAKE A SINGLE POINT 873 00:43:47,835 --> 00:43:48,636 MUTATION WITH THE INTERACTION 874 00:43:48,636 --> 00:43:58,212 WE'RE BACK TO CONTROL. 875 00:43:58,212 --> 00:43:59,614 [RINGING] 876 00:43:59,614 --> 00:44:01,549 >> SORRY. 877 00:44:01,549 --> 00:44:01,916 [LAUGHTER] 878 00:44:01,916 --> 00:44:04,418 SO THEY CLEARLY OPERATE IN A 879 00:44:04,418 --> 00:44:05,720 RESTRICT ITCH MANNER. 880 00:44:05,720 --> 00:44:10,491 SO IN SUMMARY WE'VE DEVELOPED A 881 00:44:10,491 --> 00:44:12,026 NUMBER OF NANOBODIES. 882 00:44:12,026 --> 00:44:14,729 SOME WE CAN NICELY SAY CAN BIND 883 00:44:14,729 --> 00:44:16,664 THE CAPSID WITH VERY HIGH 884 00:44:16,664 --> 00:44:19,467 AFFINITY AND DON'T SEEM TO 885 00:44:19,467 --> 00:44:21,302 INTERFERE WITH THE STABILITY AND 886 00:44:21,302 --> 00:44:23,838 USE THEM IN MARKERS AND TRACERS 887 00:44:23,838 --> 00:44:30,411 AND WE HAVE ENGINEERED IN A WAY 888 00:44:30,411 --> 00:44:35,850 THAT DESTROY THE CAPSID AND CAN 889 00:44:35,850 --> 00:44:37,251 BE USED IN A THERAPEUTIC WAY. 890 00:44:37,251 --> 00:44:38,753 I'LL END WITH MY 891 00:44:38,753 --> 00:44:39,453 ACKNOWLEDGEMENTS. 892 00:44:39,453 --> 00:44:41,389 I TOLD YOU ABOUT OUR WONDERFUL 893 00:44:41,389 --> 00:44:41,756 COLLABORATIONS. 894 00:44:41,756 --> 00:44:46,527 THIS WORK IS PRIMARILY DONE BY A 895 00:44:46,527 --> 00:44:48,029 FANTASTIC POSTDOC IN MY LAB WHO 896 00:44:48,029 --> 00:44:53,901 HAS DONE ALL THE STRUCTURES AND 897 00:44:53,901 --> 00:44:57,004 ASSAYS AND NICK SWANSON A CRYO 898 00:44:57,004 --> 00:44:58,372 EM EXPERT AND WAS HEAVILY 899 00:44:58,372 --> 00:45:02,109 INVOLVED IN THAT ASPECT OF THE 900 00:45:02,109 --> 00:45:04,979 PROJECT. 901 00:45:04,979 --> 00:45:14,689 THANK YOU. 902 00:45:14,689 --> 00:45:20,628 IT 903 00:46:13,347 --> 00:46:16,984 >> THERE'S A COLLABORATION OF 904 00:46:16,984 --> 00:46:17,985 THE CHEETAH CENTER. 905 00:46:17,985 --> 00:46:19,353 IT'S AN INCREDIBLE PRIVILEGE TO 906 00:46:19,353 --> 00:46:20,821 BE HERE WITH YOU TODAY. 907 00:46:20,821 --> 00:46:24,425 AND I'M GOING TO TELL YOU A 908 00:46:24,425 --> 00:46:30,097 STORY ABOUT DETECTION OF THE 909 00:46:30,097 --> 00:46:32,600 UNSPLICED RNA BY MD85. 910 00:46:32,600 --> 00:46:35,970 BRIEFLY I'M GOING TO REMIND YOU 911 00:46:35,970 --> 00:46:38,873 THAT -- MDA5. 912 00:46:38,873 --> 00:46:40,708 I'M GOING TO REMIND YOU THERE'S 913 00:46:40,708 --> 00:46:43,778 30 YEARS OF RESEARCH LARGELY 914 00:46:43,778 --> 00:46:45,980 FUNDED BY A.I., I'D SAY TO 915 00:46:45,980 --> 00:46:48,449 IDENTIFY AND CHARACTERIZE 916 00:46:48,449 --> 00:46:54,455 RESTRICTION FACTORS OF HIV-1 AND 917 00:46:54,455 --> 00:47:00,161 IN PARALLEL FACTORS THAT COUNTER 918 00:47:00,161 --> 00:47:10,638 ACT AND AND CHEETAH AND THE 919 00:47:12,506 --> 00:47:15,609 OTHER CENTERS HAVE BEEN 920 00:47:15,609 --> 00:47:19,547 CHARACTERIZING THESE LARGELY 921 00:47:19,547 --> 00:47:21,315 PROTEIN-PROTEIN INTERACTIONS FOR 922 00:47:21,315 --> 00:47:24,251 SOME IN THE FIELD. 923 00:47:24,251 --> 00:47:26,520 IN FACT, SIGNATURES OF POSITIVE 924 00:47:26,520 --> 00:47:27,788 SELECTION ARE CONSIDERED A 925 00:47:27,788 --> 00:47:28,789 DEFINING FEATURE OF RESTRICTION 926 00:47:28,789 --> 00:47:36,831 FACTORS. 927 00:47:36,831 --> 00:47:40,734 SO CHEETAH HAS BEEN INVOLVED IN 928 00:47:40,734 --> 00:47:42,937 HOW THEY INTERACT WITH VIRAL 929 00:47:42,937 --> 00:47:45,906 PROTEINS AND HOW THEY COUNTER 930 00:47:45,906 --> 00:47:47,341 ACT AND HIGHLIGHT A COUPLE 931 00:47:47,341 --> 00:47:48,375 PEOPLE IN OUR CENTER. 932 00:47:48,375 --> 00:47:50,778 THESE TWO VERY TALENTED PEOPLE 933 00:47:50,778 --> 00:47:54,482 CAN TURN THEIR HEADS COMPLETELY 934 00:47:54,482 --> 00:47:59,253 180 DEGREES BACKWARDS. 935 00:47:59,253 --> 00:48:00,955 WE'VE HEARD A BIT ABOUT JARED 936 00:48:00,955 --> 00:48:04,959 WORK AND MOLLY AND SIMILARLY AS 937 00:48:04,959 --> 00:48:09,530 A GIANT IN OUR CENTER SCREENING 938 00:48:09,530 --> 00:48:12,466 AND IDENTIFYING NEW HOST 939 00:48:12,466 --> 00:48:21,308 FACTORS. 940 00:48:21,308 --> 00:48:22,610 WE HAVE THESE SENIOR MEMBERS 941 00:48:22,610 --> 00:48:23,511 WELL KNOWN IN OUR COMMUNITY. 942 00:48:23,511 --> 00:48:26,814 THEY HAVE A LARGE NUMBER OF 943 00:48:26,814 --> 00:48:28,983 ABSTRACTS THAT WILL BE APPEARING 944 00:48:28,983 --> 00:48:36,290 AT THE MEETING. 945 00:48:36,290 --> 00:48:38,492 AT THE SAME TIME FOR A NUMBER OF 946 00:48:38,492 --> 00:48:39,960 YEARS WE'VE BEEN WONDERING 947 00:48:39,960 --> 00:48:45,566 WHETHER THE CELL HAS THE ABILITY 948 00:48:45,566 --> 00:48:52,273 TO DISTINGUISH BETWEEN 949 00:48:52,273 --> 00:49:00,080 RETROVIRAL RNA AND GENERIC mRNAs 950 00:49:00,080 --> 00:49:08,088 ARE CAPPED AND SAME OF 951 00:49:08,088 --> 00:49:10,024 RETROVIRAL RETROVIRAL RNAs AND 952 00:49:10,024 --> 00:49:14,328 THEY HAVE UNSPLICED FORMS OF THE 953 00:49:14,328 --> 00:49:18,499 RNA THAT HAVE TO GET TO THE 954 00:49:18,499 --> 00:49:26,206 CYTOPLASM AS A GENOMIC RNA AND 955 00:49:26,206 --> 00:49:34,248 ENCODE STRUCTURAL PROTEINS WE'VE 956 00:49:34,248 --> 00:49:36,984 WONDERED WHETHER THE CELLS HAS 957 00:49:36,984 --> 00:49:38,285 TOOLS TO DISTINGUISH BETWEEN THE 958 00:49:38,285 --> 00:49:38,986 RNAs. 959 00:49:38,986 --> 00:49:42,489 OVER A NUMBER OF YEARS MY LAB 960 00:49:42,489 --> 00:49:44,291 HAS BEEN FOCUSSING ON TWO 961 00:49:44,291 --> 00:49:46,327 PROJECTING WHERE WE THINK WE 962 00:49:46,327 --> 00:49:50,764 HAVE CONVINCING EFFORTS THAT 963 00:49:50,764 --> 00:49:52,266 RETAINED UNSPLICED FORMS OF 964 00:49:52,266 --> 00:50:00,407 THESE ELEMENTS ARE DETECTED AS A 965 00:50:00,407 --> 00:50:01,709 DANGER SIGNAL AND CONTINUAL SAN 966 00:50:01,709 --> 00:50:05,412 FRANCISCO THEY'VE SEEN A SIMILAR 967 00:50:05,412 --> 00:50:09,016 PATTERN IN CRYPTO CAUCUS WHERE 968 00:50:09,016 --> 00:50:12,219 THEY DETECTED AND THE PATHOGENIC 969 00:50:12,219 --> 00:50:12,486 FUNGUS. 970 00:50:12,486 --> 00:50:16,590 ONE PROJECT WE HAVE WHICH I 971 00:50:16,590 --> 00:50:20,995 WON'T TALK ABOUT CONCERNS CORE 972 00:50:20,995 --> 00:50:24,398 MUCH A CAUSING AN EPIDEMIC IN 973 00:50:24,398 --> 00:50:26,233 KUALAS ALONG THE EAST COAST OF 974 00:50:26,233 --> 00:50:27,434 AUSTRALIA AND WE'RE INTERESTED 975 00:50:27,434 --> 00:50:30,671 BECAUSE IT GOES IN GERM LINE AND 976 00:50:30,671 --> 00:50:38,912 IT'S ENDODGENIZING AND THEY HAVE 977 00:50:38,912 --> 00:50:47,221 AN ADAPTIVE IMMUNE CELL THE 978 00:50:47,221 --> 00:50:49,390 PIRNA SYSTEM AND THE INITIAL 979 00:50:49,390 --> 00:50:52,726 EVENT INVOLVES DETECTION OF THE 980 00:50:52,726 --> 00:50:54,495 UNSPLICED RNA AND THE SPLICED 981 00:50:54,495 --> 00:51:03,003 STRIPS SEEM TO BE IGNORED. 982 00:51:03,003 --> 00:51:09,076 I'LL GET TO THE DETECTION OF THE 983 00:51:09,076 --> 00:51:16,684 UNSPLICED RNA AND ONE MADE BY 984 00:51:16,684 --> 00:51:20,220 RALPH STEINMAN WHO DISCOVERED 985 00:51:20,220 --> 00:51:24,491 THEM AND NAMED THEM DENDRITIC 986 00:51:24,491 --> 00:51:25,125 CELLS. 987 00:51:25,125 --> 00:51:28,729 NOW I'M CRASHING. 988 00:51:28,729 --> 00:51:29,430 AH, OKAY. 989 00:51:29,430 --> 00:51:31,331 SO THIS PROJECT BEGINS WITH A 990 00:51:31,331 --> 00:51:33,600 GRADUATE STUDENT, SHAWN, WHO SET 991 00:51:33,600 --> 00:51:38,038 UP THE SYSTEM MAKING DENDRITIC 992 00:51:38,038 --> 00:51:39,540 CELLS FROM BLOOD AND THE DATA IS 993 00:51:39,540 --> 00:51:42,476 FROM PRIMARY HUMAN BLOOD WHERE 994 00:51:42,476 --> 00:51:45,746 YOU GENERATE AND ISOLATE 995 00:51:45,746 --> 00:51:47,781 MONOCYTES FROM BLOOD. 996 00:51:47,781 --> 00:51:51,118 SHAWN DEVELOPED A PROTOCOL WITH 997 00:51:51,118 --> 00:51:57,624 HOMEMADE HUMAN PRODUCED IL4 AND 998 00:51:57,624 --> 00:52:00,127 IT HUMAN SERUM AND WHEN YOU 999 00:52:00,127 --> 00:52:01,729 CHALLENGE WITH HIV-1 IN THIS 1000 00:52:01,729 --> 00:52:03,564 CASE FOR ALL THE ASSAYS I'LL 1001 00:52:03,564 --> 00:52:07,034 SHOW YOU WE'RE USING A SINGLE 1002 00:52:07,034 --> 00:52:08,969 CYCLE VIRUS WITH REPORTER GENE 1003 00:52:08,969 --> 00:52:13,240 AND YOU GET MATURATION AND OUT 1004 00:52:13,240 --> 00:52:16,877 CAN LOOK LIKE THIS AND THIS 1005 00:52:16,877 --> 00:52:19,613 COMPARES THE RESPONSE OF THIS 1006 00:52:19,613 --> 00:52:24,351 ESSENTIALLY FULL-LENGTH HIV 1007 00:52:24,351 --> 00:52:26,386 VECTOR WITH A MINIMAL LENGTHY 1008 00:52:26,386 --> 00:52:28,956 VECTORS AND THEY GET ACTIVATED 1009 00:52:28,956 --> 00:52:37,331 BY THE VIRUS AND NOT THE VECTOR 1010 00:52:37,331 --> 00:52:40,667 AND WHEN YOU DO RNA SEQ YOU CAN 1011 00:52:40,667 --> 00:52:42,503 SEE PATTERNS THAT LOOK LIKE A 1012 00:52:42,503 --> 00:52:45,105 TYPE 1 INTERFERON RESPONSE. 1013 00:52:45,105 --> 00:52:46,473 SO SEAN FOR HIS THESIS PROJECT 1014 00:52:46,473 --> 00:52:52,813 WANTED TO FIGURE OUT WHAT WAS 1015 00:52:52,813 --> 00:52:53,814 BEING DETECTED. 1016 00:52:53,814 --> 00:52:56,650 AND TRANSCRIPTION WAS BLOCKED 1017 00:52:56,650 --> 00:53:00,254 WITH MUTANTS AND DRUGS AND THEY 1018 00:53:00,254 --> 00:53:04,458 WOULD PREVENT TYPE 1 INTERFERON 1019 00:53:04,458 --> 00:53:06,627 INDUCTION. 1020 00:53:06,627 --> 00:53:08,428 TRANSCRIPTION WAS REQUIRED. 1021 00:53:08,428 --> 00:53:12,499 TAD WAS NOT REQUIRED AND YOU CAN 1022 00:53:12,499 --> 00:53:16,570 USE A HETEROLOGOUS SYSTEM TO 1023 00:53:16,570 --> 00:53:21,742 PRODUCE THE FULL LENGTH RNA. 1024 00:53:21,742 --> 00:53:26,880 THE ONLY REQUIRED WAS REF AND HE 1025 00:53:26,880 --> 00:53:34,421 FOUND LEPTOMYCIN WOULD BLOCK 1026 00:53:34,421 --> 00:53:40,661 WITH THE HIV CHALLENGE BUT THE 1027 00:53:40,661 --> 00:53:42,529 VIRUS REPLICATES IN THE 1028 00:53:42,529 --> 00:53:47,601 CYTOPLASM DIDN'T SEEM TO CARE 1029 00:53:47,601 --> 00:53:49,002 AND THESE EXPERIMENTS CONCLUDED 1030 00:53:49,002 --> 00:53:57,010 THE UNSPLICED RNA IS LIKELY THE 1031 00:53:57,010 --> 00:54:01,215 DANGER SIGNAL AND SHOWED REM 1032 00:54:01,215 --> 00:54:02,883 CRM1 WAS REQUIRED AND WILL THEN 1033 00:54:02,883 --> 00:54:06,119 THEY WANTED TO IDENTIFY WHAT THE 1034 00:54:06,119 --> 00:54:08,989 RECEPTOR WAS AND THEY MODIFIED 1035 00:54:08,989 --> 00:54:09,823 THE PROTOCOL A LITTLE BIT SO 1036 00:54:09,823 --> 00:54:12,492 WHEN THE MONOCYTES ARE FIRST 1037 00:54:12,492 --> 00:54:21,134 ISOLATED HE CHALLENGES WITH A 1038 00:54:21,134 --> 00:54:23,170 THAT DELIVERED A MICRORNA TO 1039 00:54:23,170 --> 00:54:27,107 KNOCK DOWN GENES OF INTEREST. 1040 00:54:27,107 --> 00:54:31,612 HE MAKES DCs AND ASKS WHETHER 1041 00:54:31,612 --> 00:54:33,180 THE KNOCK DOWN HAS AN EFFECT. 1042 00:54:33,180 --> 00:54:38,785 HE AND SHAWN PICKED A NUMBER OF 1043 00:54:38,785 --> 00:54:40,787 CANDIDA 1044 00:54:40,787 --> 00:54:44,224 CANDIDATES THERE ARE ADDITIONAL 1045 00:54:44,224 --> 00:54:45,993 ONES BUT THIS COMES FROM RNA 1046 00:54:45,993 --> 00:54:51,431 BINDING PROTEINS WE TESTED AND 1047 00:54:51,431 --> 00:55:01,642 OF THE AND XPL1 PREVENTED TYPE 1 1048 00:55:01,642 --> 00:55:02,676 INTERFERON INDUCTION. 1049 00:55:02,676 --> 00:55:11,551 AND THE GENE THAT ENCODES MDA5 1050 00:55:11,551 --> 00:55:14,855 AND IN CONTRAST THE DDX58 WHICH 1051 00:55:14,855 --> 00:55:17,858 ENCODES DID NOT HAVE AN EFFECT. 1052 00:55:17,858 --> 00:55:19,760 AND SO THIS IS JUST A LITTLE BIT 1053 00:55:19,760 --> 00:55:21,128 MORE DETAIL. 1054 00:55:21,128 --> 00:55:24,531 EACH DOT IS AN INDIVIDUAL BLOOD 1055 00:55:24,531 --> 00:55:24,998 DONOR. 1056 00:55:24,998 --> 00:55:28,335 YOU PUT HI ON A CONTROL, 1057 00:55:28,335 --> 00:55:32,973 KNOCKOUT DOWN AND GET ACTIVATION 1058 00:55:32,973 --> 00:55:36,510 AND YOU KNOCK DOWN REGI IT DOES 1059 00:55:36,510 --> 00:55:40,981 NOT PREDICT AND KNOCK DOWN MDA5 1060 00:55:40,981 --> 00:55:41,515 YOU PREVENT IT. 1061 00:55:41,515 --> 00:55:47,120 AND THERE WAS AN ALL IN ONE 1062 00:55:47,120 --> 00:55:47,988 SINGLE TRANSCRIPT VECTOR THAT 1063 00:55:47,988 --> 00:55:50,557 DOES A KNOCK DOWN AND RESCUE 1064 00:55:50,557 --> 00:55:56,196 WITH THE CDNA OR CDNA ENCODING 1065 00:55:56,196 --> 00:55:58,198 MDA5 WITH A MUTATION. 1066 00:55:58,198 --> 00:55:59,800 SO IT'S NOT TARGETED BY THE 1067 00:55:59,800 --> 00:56:01,068 KNOCK DOWN. 1068 00:56:01,068 --> 00:56:03,403 YOU CAN SEE BY WESTERN BLOT IF 1069 00:56:03,403 --> 00:56:07,908 YOU KNOCK DOWN MDA5, IFIH1 1070 00:56:07,908 --> 00:56:11,345 PROTEIN YOU LOSE THE PROTEIN AND 1071 00:56:11,345 --> 00:56:12,713 CAN PUT IT BACK IN THE CONTEXT 1072 00:56:12,713 --> 00:56:17,884 OF THE KNOCK DOWNS. 1073 00:56:17,884 --> 00:56:22,055 AND THE ISG15 TRACKS WITH THIS. 1074 00:56:22,055 --> 00:56:24,124 INDEED IT'S NOT AN OFF TARGET 1075 00:56:24,124 --> 00:56:27,627 EFFECT BUT SEEMS THE MDA5 1076 00:56:27,627 --> 00:56:29,262 PROTEIN IS REQUIRED. 1077 00:56:29,262 --> 00:56:32,199 AND IN KNOCK DOWN OF MDA5 1078 00:56:32,199 --> 00:56:34,001 EXPRESSES THE INDUCTION OF THE 1079 00:56:34,001 --> 00:56:36,036 MAJORITY OF THE ISGs. 1080 00:56:36,036 --> 00:56:37,471 WE'RE VERY INTERESTED IN WHAT 1081 00:56:37,471 --> 00:56:40,774 THE OTHER GENES ARE THAT ARE 1082 00:56:40,774 --> 00:56:41,875 HANGING AROUND WHETHER IT'S JUST 1083 00:56:41,875 --> 00:56:44,711 THIS IS A KNOCK DOWN AND NOT A 1084 00:56:44,711 --> 00:56:48,515 KNOCKOUT OR WHETHER THERE ARE 1085 00:56:48,515 --> 00:56:51,084 OTHER PATHWAYS INVOLVED. 1086 00:56:51,084 --> 00:56:53,854 SO WE WANTED TO SEE IF WE CAN 1087 00:56:53,854 --> 00:56:55,922 DETECT DIRECT INTERACTION AND 1088 00:56:55,922 --> 00:56:57,958 SHE CHALLENGED BETWEEN MDA5 AND 1089 00:56:57,958 --> 00:57:01,561 THE RNA AND CROSS LINKED WITH 1090 00:57:01,561 --> 00:57:07,134 FORMALDEHYDE AND DID IPs AND DID 1091 00:57:07,134 --> 00:57:10,137 PRIMERS FOR SPLICED AND 1092 00:57:10,137 --> 00:57:12,239 UNSPLICED RNAs. 1093 00:57:12,239 --> 00:57:21,314 SHE CAN PULL OUT RIGGI HERE OR 1094 00:57:21,314 --> 00:57:27,087 MDA5 TO UNSPLICED RNA PRIMERS OR 1095 00:57:27,087 --> 00:57:32,993 SPLICED PRODUCTS AND WHEN SHE 1096 00:57:32,993 --> 00:57:42,536 QUANTITATED THESE BY RTQPCR 1097 00:57:42,536 --> 00:57:48,375 FINDS RNAs SPLICED AND IF SHE 1098 00:57:48,375 --> 00:57:49,943 PULLS WITH RIGGI SHE DOESN'T SEE 1099 00:57:49,943 --> 00:57:50,577 THE SAME. 1100 00:57:50,577 --> 00:57:51,711 THERE'S A NUMBER OF OTHER 1101 00:57:51,711 --> 00:57:54,514 CONTROLS SHE'S DONE. 1102 00:57:54,514 --> 00:57:58,251 SO WE THINK THE UNSPLICED RNA 1103 00:57:58,251 --> 00:58:00,220 SEEMS TO BE THE PATH AND GOING 1104 00:58:00,220 --> 00:58:01,221 FORWARD WE'RE INTERESTED IN WHAT 1105 00:58:01,221 --> 00:58:03,023 THE STRUCTURAL REQUIREMENTS ARE 1106 00:58:03,023 --> 00:58:08,028 FOR RECOGNITION OF THE UNSPLICED 1107 00:58:08,028 --> 00:58:09,996 RNA WITH ASSAYS LIKE THESE AND 1108 00:58:09,996 --> 00:58:15,936 AS PART OF CHEETAH WE'RE HOPING 1109 00:58:15,936 --> 00:58:16,837 TO DEVELOP AN IN VITRO SYSTEM 1110 00:58:16,837 --> 00:58:20,173 USING PROTEINS PRODUCED BY HEIDI 1111 00:58:20,173 --> 00:58:23,376 AND CHRIS IN SALT LAKE CITY AND 1112 00:58:23,376 --> 00:58:28,949 MARY HAS SOME AMAZING PREPS OF 1113 00:58:28,949 --> 00:58:33,019 FULL LENGTH IN VITRO TRANSCRIBE 1114 00:58:33,019 --> 00:58:34,955 HIVs PUT IN THE SYSTEM. 1115 00:58:34,955 --> 00:58:39,092 THESE ARE THE FOLKS IN MY LAB 1116 00:58:39,092 --> 00:58:46,166 WHO PRODUCED THE DATA I SHOWED 1117 00:58:46,166 --> 00:58:46,333 YOU. 1118 00:58:46,333 --> 00:58:52,272 THESE GRADUATED AND THOMAS HAS 1119 00:58:52,272 --> 00:58:54,608 AN ABSTRACT AND WE HAVE A BRAND 1120 00:58:54,608 --> 00:58:57,077 NEW STUDENT HERE. 1121 00:58:57,077 --> 00:59:07,320 SO THANK YOU. 1122 00:59:35,582 --> 00:59:46,026 >> I DON'T THINK YOU'RE LIVE. 1123 00:59:53,567 --> 00:59:55,869 >> CONGRATULATIONS. 1124 00:59:55,869 --> 00:59:56,970 BEAUTIFUL PRESENTATION. 1125 00:59:56,970 --> 01:00:01,208 FANTASTIC DATA. 1126 01:00:01,208 --> 01:00:06,580 ONE QUESTION ABOUT THE ASSAYS 1127 01:00:06,580 --> 01:00:08,815 AND COMPARING TO LENACAPAVIR AND 1128 01:00:08,815 --> 01:00:12,152 IN THAT ASSAY IT SEEMS 1129 01:00:12,152 --> 01:00:14,621 LENACAPAVIR WAS WORKING HIGHLY 1130 01:00:14,621 --> 01:00:16,756 EFFECTIVE. 1131 01:00:16,756 --> 01:00:20,994 10 NANO MOLAR AND MAYBE I MISSED 1132 01:00:20,994 --> 01:00:21,695 IT. 1133 01:00:21,695 --> 01:00:26,433 10 NANO MOLAR IS MORE THAN WE'D 1134 01:00:26,433 --> 01:00:30,170 EXPECT FOR LENACAPAVIR AND 1135 01:00:30,170 --> 01:00:36,309 WONDER IF YOU HAVE COMMENTS FOR 1136 01:00:36,309 --> 01:00:36,509 THAT. 1137 01:00:36,509 --> 01:00:44,751 >> I SHARE ON THE EXACT ASSAY 1138 01:00:44,751 --> 01:00:50,423 MEAN AND WE LOOK AT IT AS A 1139 01:00:50,423 --> 01:00:55,428 COMPARISON TO THE NANO BODY AND 1140 01:00:55,428 --> 01:00:55,862 COMPARISON. 1141 01:00:55,862 --> 01:00:58,431 >> IT'S WORTH ASKING JARED THE 1142 01:00:58,431 --> 01:01:00,734 QUESTION BUT THEY SEE A BIPHASIC 1143 01:01:00,734 --> 01:01:04,638 BEHAVIOR AND IF YOU GET HIGH 1144 01:01:04,638 --> 01:01:08,908 ENOUGH FOR REVERSE TRANSCRIPTION 1145 01:01:08,908 --> 01:01:13,113 BUT IT'S ABOUT 10 NANOMOLE AR. 1146 01:01:13,113 --> 01:01:14,514 >> GREAT TALKS. 1147 01:01:14,514 --> 01:01:18,952 I'M FROM THE DANA FAR BER 1148 01:01:18,952 --> 01:01:20,787 INSTITUTE AND WE HAVE YOU IN THE 1149 01:01:20,787 --> 01:01:23,256 DID CLARIFY WHETHER THAT FULLY 1150 01:01:23,256 --> 01:01:29,296 REPLACES THE NEED FOR NUCLEAR 1151 01:01:29,296 --> 01:01:29,529 EXTRACT. 1152 01:01:29,529 --> 01:01:31,598 >> YEAH, WE GET HIGHER 1153 01:01:31,598 --> 01:01:33,333 EFFICIENCIES SO IN THAT SENSE IT 1154 01:01:33,333 --> 01:01:35,735 REPLACES IT BUT I THINK THERE 1155 01:01:35,735 --> 01:01:39,639 ARE ADDITIONAL FACTORS IN OUR 1156 01:01:39,639 --> 01:01:40,974 EXTRACT THAT I THINK IT ACCOUNTS 1157 01:01:40,974 --> 01:01:43,043 FOR THE 4 DEGREES AND WE'RE 1158 01:01:43,043 --> 01:01:44,978 MISSING AN ENCODING FACTOR BUT 1159 01:01:44,978 --> 01:01:48,982 BECAUSE WE CAN DRIVE THE 1160 01:01:48,982 --> 01:01:56,656 REACTION WITH LEGF IT REPLACES 1161 01:01:56,656 --> 01:01:59,759 IT COMPLETELY BUT I DON'T THINK 1162 01:01:59,759 --> 01:02:03,363 WE HAVE PRECONSTITUTED 1163 01:02:03,363 --> 01:02:04,964 EVERYTHING YESTERDAY. 1164 01:02:04,964 --> 01:02:07,567 >> WONDERING ABOUT COLD 1165 01:02:07,567 --> 01:02:07,867 TEMPERATURE. 1166 01:02:07,867 --> 01:02:10,437 CAN TO YOU PONTIFICATE MORE? 1167 01:02:10,437 --> 01:02:12,572 >> I THINK AND MAYBE OTHERS 1168 01:02:12,572 --> 01:02:16,443 THINK THE SAME SOME EVIDENCE 1169 01:02:16,443 --> 01:02:18,578 THAT IT'S NOT JUST ENOUGH FOR 1170 01:02:18,578 --> 01:02:23,350 THE CAPSID TO FALL A PART BUT 1171 01:02:23,350 --> 01:02:25,518 ONE STEP IS FAVORED REACTION 1172 01:02:25,518 --> 01:02:28,154 WHERE THE EQUILIBRIUM IS BEING 1173 01:02:28,154 --> 01:02:30,256 DRIVEN AT 4 DEGREES BUT WE 1174 01:02:30,256 --> 01:02:32,425 HONESTLY DON'T KNOW THAT. 1175 01:02:32,425 --> 01:02:34,227 >> IT'S CALLED BREWED COFFEE. 1176 01:02:34,227 --> 01:02:36,563 >> AND IN THE CELL THERE'S SOME 1177 01:02:36,563 --> 01:02:37,797 PROTEIN COMPLEX OR INTERACTION. 1178 01:02:37,797 --> 01:02:38,965 >> YES. 1179 01:02:38,965 --> 01:02:40,133 WE SEE LESS OF A TEMPERATURE 1180 01:02:40,133 --> 01:02:44,938 DEPENDENCE WHEN WE USE THE 1181 01:02:44,938 --> 01:02:46,840 NUCLEAR VERSUS THE LEGF BUT 1182 01:02:46,840 --> 01:02:48,775 HAVEN'T REALLY NAILED IT YET. 1183 01:02:48,775 --> 01:02:52,979 >> AND THOMAS IN TERMS OF THE 1184 01:02:52,979 --> 01:02:56,583 WILL CELL INFECTION OF THE 1185 01:02:56,583 --> 01:03:02,522 PRESENCE OF THE ANTIBODIES OR 1186 01:03:02,522 --> 01:03:08,561 HAVE YOU INFECTED WITH N74D? 1187 01:03:08,561 --> 01:03:09,329 >> 1188 01:03:09,329 --> 01:03:10,997 >> THE LATTER NOT YET. 1189 01:03:10,997 --> 01:03:15,268 THESE ARE BRAND NEW. 1190 01:03:15,268 --> 01:03:15,969 WE'RE DOING SPREADING. 1191 01:03:15,969 --> 01:03:25,745 SPREADING INFECTIONS WE CAN'T -- 1192 01:03:25,745 --> 01:03:26,913 CAN SEE EVERYTHING. 1193 01:03:26,913 --> 01:03:31,251 IT'S SPECTACULAR. 1194 01:03:31,251 --> 01:03:32,185 >> THANK YOU. 1195 01:03:32,185 --> 01:03:42,729 GREAT TALKS, REALLY IMPRESSIVE. 1196 01:03:42,962 --> 01:03:44,497 I HAVE A QUESTION. 1197 01:03:44,497 --> 01:03:49,436 THIS ENHANCED THE INTEGRATION 1198 01:03:49,436 --> 01:03:52,005 FOR ENCHROMATINIZED CONTEXT BUT 1199 01:03:52,005 --> 01:03:54,407 ON THE LEFT ID LOOKED LIKE NAKED 1200 01:03:54,407 --> 01:03:59,646 DNA AND LOOKED BETTER THAN 1201 01:03:59,646 --> 01:04:01,648 BEFORE. 1202 01:04:01,648 --> 01:04:06,619 >> IT STIMULATES BETTER ON 1203 01:04:06,619 --> 01:04:08,922 CHROMATIN AND HAS THESE A.T. 1204 01:04:08,922 --> 01:04:12,592 HOOKS AND THINK IT COULD BE A 1205 01:04:12,592 --> 01:04:13,326 DNA BINDING ELEMENT. 1206 01:04:13,326 --> 01:04:15,028 >> SO IT'S NOT JUST CHROMATIN. 1207 01:04:15,028 --> 01:04:15,895 >> THAT'S CORRECT. 1208 01:04:15,895 --> 01:04:18,198 IT'S BETTER ON CHROMATIN BUT 1209 01:04:18,198 --> 01:04:23,369 STIMULATES IN MAKING DNA. 1210 01:04:23,369 --> 01:04:27,307 >> THEN MY LAST QUESTION TO 1211 01:04:27,307 --> 01:04:29,008 JEREMY DOES MDA5 HAVE AN EFFECT 1212 01:04:29,008 --> 01:04:32,979 IN SPREADING INFECTIONS IN 1213 01:04:32,979 --> 01:04:35,014 DENDRITIC CELLS? 1214 01:04:35,014 --> 01:04:40,353 >> MDA5? 1215 01:04:40,353 --> 01:04:50,530 >> RIGHT. 1216 01:04:56,870 --> 01:04:59,806 >> SHAWN SPREAD INFECTIONS IN 1217 01:04:59,806 --> 01:05:01,941 THE POSITIVE T CELLS IN CASES OF 1218 01:05:01,941 --> 01:05:02,542 ACTIVATION. 1219 01:05:02,542 --> 01:05:05,812 THIS BRA WE KNEW ABOUT MDA35. 1220 01:05:05,812 --> 01:05:15,855 FAFRZ SPREADING INFECTION AND -- 1221 01:05:15,855 --> 01:05:17,690 IN TERMS OF SPREADING CELLS WE 1222 01:05:17,690 --> 01:05:19,325 DON'T HAVE MEANINGFUL DATA YET. 1223 01:05:19,325 --> 01:05:20,660 >> OKAY. 1224 01:05:20,660 --> 01:05:21,060 >> GOOD QUESTION. 1225 01:05:21,060 --> 01:05:30,570 >> THANK YOU. 1226 01:05:30,570 --> 01:05:31,704 >> ADDING ON WITH THE QUESTION 1227 01:05:31,704 --> 01:05:33,506 IS THERE A TIME TO ADD AT 10 1228 01:05:33,506 --> 01:05:33,773 HOURS? 1229 01:05:33,773 --> 01:05:40,513 >> WE ADD IT FROM THE START. 1230 01:05:40,513 --> 01:05:42,315 I BET DEVON HAS DONE THAT AND 1231 01:05:42,315 --> 01:05:44,317 THINK IT WOULD BE CORRELATED 1232 01:05:44,317 --> 01:05:46,786 WITH THE INTEGRATION TIME BUT 1233 01:05:46,786 --> 01:05:48,888 DON'T REMEMBER FOR SURE. 1234 01:05:48,888 --> 01:05:50,390 DEVON CHRISTIANSON'S HERE AND 1235 01:05:50,390 --> 01:05:53,092 HAS A POSTER SO YOU SHOULD ASK. 1236 01:05:53,092 --> 01:05:54,594 >> IF YOU INCREASE TEMPERATURE 1237 01:05:54,594 --> 01:05:56,996 AT A CERTAIN POINT DOES IT 1238 01:05:56,996 --> 01:05:58,331 INCREASE INTEGRATION? 1239 01:05:58,331 --> 01:06:00,166 >> THE TEMPERATURE DEPENDENCE IN 1240 01:06:00,166 --> 01:06:03,069 THE NUCLEAR EXTRACT IS LESS 1241 01:06:03,069 --> 01:06:03,503 DRAMATIC. 1242 01:06:03,503 --> 01:06:04,904 THERE'S STILL SOME TEMPERATURE 1243 01:06:04,904 --> 01:06:09,876 DEPENDENCE AND WITH THE LEGF YOU 1244 01:06:09,876 --> 01:06:12,245 CAN GO TO 72 HOURS AND KEEPS 1245 01:06:12,245 --> 01:06:12,779 GETTING BETTER AND BETTER. 1246 01:06:12,779 --> 01:06:16,049 >> THANK YOU. 1247 01:06:16,049 --> 01:06:17,350 >> KELLY LEE, UNIVERSITY OF 1248 01:06:17,350 --> 01:06:17,750 WASHINGTON. 1249 01:06:17,750 --> 01:06:23,089 THE CORES THAT ARE ICE LAID ARE 1250 01:06:23,089 --> 01:06:23,656 BEAUTIFUL. 1251 01:06:23,656 --> 01:06:27,293 CAN YOU SEE REVERSE 1252 01:06:27,293 --> 01:06:28,294 TRANSCRIPTASE OR RNA OR 1253 01:06:28,294 --> 01:06:28,561 INTEGRASE? 1254 01:06:28,561 --> 01:06:36,603 >> I THINK OWEN AND BARBI ARE 1255 01:06:36,603 --> 01:06:39,906 LOOKING AT THAT BUT THAT'S A 1256 01:06:39,906 --> 01:06:40,106 GOAL. 1257 01:06:40,106 --> 01:06:47,246 >> MIKE SOMMERS AT UNBC. 1258 01:06:47,246 --> 01:06:49,782 >> I KNOW THAT. 1259 01:06:49,782 --> 01:06:52,986 >> I THINK IT'S INTERESTING YOU 1260 01:06:52,986 --> 01:06:55,221 HAVE THE PRIMARY AT THE 1261 01:06:55,221 --> 01:06:57,724 PALINDROME AND PALINDROME 1262 01:06:57,724 --> 01:07:00,159 STRUCTURE DNA CAN BE SENSITIVE 1263 01:07:00,159 --> 01:07:05,765 TO TEMPERATURE A HAIR PIN VERSUS 1264 01:07:05,765 --> 01:07:06,366 DUPLEX. 1265 01:07:06,366 --> 01:07:08,534 HAVE YOU THOUGHT ABOUT THAT AS A 1266 01:07:08,534 --> 01:07:08,835 POSSIBILITY? 1267 01:07:08,835 --> 01:07:12,505 >> THEY WERE PUSHING US TO 1268 01:07:12,505 --> 01:07:13,906 REMIND US IT WAS TEMPERATURE 1269 01:07:13,906 --> 01:07:18,244 SENSITIVE AND THAT'S POSSIBLE 1270 01:07:18,244 --> 01:07:25,051 AND THE INTOSOME IS TWOFOLD 1271 01:07:25,051 --> 01:07:26,719 SYMMETRIC AND THE SURPRISE IS 1272 01:07:26,719 --> 01:07:28,955 IT'S SUCH A BIG FOOTPRINT THAT 1273 01:07:28,955 --> 01:07:30,590 GIVES CONSENSUS. 1274 01:07:30,590 --> 01:07:35,028 YEAH. 1275 01:07:35,028 --> 01:07:36,929 >> I'M ALICE FROM THE UNIVERSITY 1276 01:07:36,929 --> 01:07:38,665 OF MICHIGAN. 1277 01:07:38,665 --> 01:07:39,332 HI, WES. 1278 01:07:39,332 --> 01:07:42,335 SO I KNOW I'VE ASKED THIS 1279 01:07:42,335 --> 01:07:44,470 QUESTION OF YOU AND JARED BEFORE 1280 01:07:44,470 --> 01:07:47,006 AND WENT FAST SO I MAY HAVE 1281 01:07:47,006 --> 01:07:49,308 MISSED IT BUT SHOWED THE PROTEIN 1282 01:07:49,308 --> 01:07:50,710 GELS SHOWED NO PROTEASE IN THE 1283 01:07:50,710 --> 01:07:50,910 CORES? 1284 01:07:50,910 --> 01:07:54,447 >> THAT'S A GOOD QUESTION. 1285 01:07:54,447 --> 01:07:57,817 >> I'M NOT SURE WE LOOKED 1286 01:07:57,817 --> 01:07:59,118 CAREFULLY ENOUGH. 1287 01:07:59,118 --> 01:08:02,188 IT'S NOT -- THAT WAS A PRO STAIN 1288 01:08:02,188 --> 01:08:07,560 SO THAT'S LIKE THE STOCHIOMETRIC 1289 01:08:07,560 --> 01:08:09,562 AND WOULD WANT TO DO THAT IN A 1290 01:08:09,562 --> 01:08:10,029 METRIC. 1291 01:08:10,029 --> 01:08:12,231 >> I'M POINTING THIS OUT TO 1292 01:08:12,231 --> 01:08:16,969 EVERYBODY THAT'S POTENTIALLY A 1293 01:08:16,969 --> 01:08:21,441 COOL QUESTION HOW THERE WOULD BE 1294 01:08:21,441 --> 01:08:22,241 INSIDE THE CAPSID. 1295 01:08:22,241 --> 01:08:28,448 >> WE DON'T SEE IT AS A 1296 01:08:28,448 --> 01:08:29,782 PROMINENT PROTEIN. 1297 01:08:29,782 --> 01:08:31,250 THAT'S TRUE. 1298 01:08:31,250 --> 01:08:33,453 >> I'M LOUISA FROM THE 1299 01:08:33,453 --> 01:08:35,188 UNIVERSITY OF MINNESOTA. 1300 01:08:35,188 --> 01:08:37,924 ABOUT THE MDA5 DETECTION HAVE 1301 01:08:37,924 --> 01:08:43,196 YOU CHECKED OTHER CELL LINES 1302 01:08:43,196 --> 01:08:50,036 THAT ARE. 1303 01:08:50,036 --> 01:08:52,171 METRICS TO FOR THE CELL. 1304 01:08:52,171 --> 01:08:56,509 >> MDA5 SPECIFICALLY WE ONLY 1305 01:08:56,509 --> 01:08:58,044 LOOKED AT PRIMARY MYELOID CELLS 1306 01:08:58,044 --> 01:09:01,948 SO DCs AND MACRO PHAGES. 1307 01:09:01,948 --> 01:09:03,750 I KNOW SOME HAVE LOOKED AT SOME 1308 01:09:03,750 --> 01:09:04,984 CELL LINES AND SEEN THE SAME 1309 01:09:04,984 --> 01:09:13,159 THING. 1310 01:09:13,159 --> 01:09:15,261 WE'VE LOOKED AT INNATE 1311 01:09:15,261 --> 01:09:20,099 ACTIVATION OF OTHER CELL TYPES. 1312 01:09:20,099 --> 01:09:22,502 WE HAVE SOUGHT EYE TRANSFORMED 1313 01:09:22,502 --> 01:09:24,337 CELL LINE DO THE BIO CHEMICAL 1314 01:09:24,337 --> 01:09:26,439 EXPERIMENTS AND SO FAR WE'VE 1315 01:09:26,439 --> 01:09:28,441 BEEN UNSUCCESSFUL FINDING THE 1316 01:09:28,441 --> 01:09:31,410 CELL LINE THAT BEHAVES THE WAY 1317 01:09:31,410 --> 01:09:35,882 WE LIKE SO WE COULD DO THE BIO 1318 01:09:35,882 --> 01:09:37,283 CHEMISTRY EASIER TO REMIND YOU 1319 01:09:37,283 --> 01:09:39,852 EVERYTHING I SHOWED YOU IS 1320 01:09:39,852 --> 01:09:42,255 PRIMARY BLOOD CELL DERIVED FROM 1321 01:09:42,255 --> 01:09:42,922 A DIFFERENT DONOR AND IT'S A LOT 1322 01:09:42,922 --> 01:09:53,065 OF WORK. 1323 01:09:57,203 --> 01:09:59,639 >> MY QUESTION IS FOR WES. 1324 01:09:59,639 --> 01:10:01,974 I WONDER IF YOU CAN COMMENT MORE 1325 01:10:01,974 --> 01:10:04,911 WHY DO YOU THINK THERE'S AN 1326 01:10:04,911 --> 01:10:12,351 EXPANDED SEQUENCE SPECIFICITY 1327 01:10:12,351 --> 01:10:15,555 AND WHY IT WASN'T OBSERVED IN 1328 01:10:15,555 --> 01:10:16,255 PREVIOUS STUDIES? 1329 01:10:16,255 --> 01:10:17,390 >> THIS IS BRAND NEW DATA IN 1330 01:10:17,390 --> 01:10:18,324 WHAT WE OBSERVED. 1331 01:10:18,324 --> 01:10:20,560 I DIDN'T MAKE IT CLEAR BUT 1332 01:10:20,560 --> 01:10:26,032 THAT'S NOT THE CHROMATINIZED 1333 01:10:26,032 --> 01:10:27,200 SEQUENCE. 1334 01:10:27,200 --> 01:10:29,402 IT'S POSSIBLE THE LEGF IS SO 1335 01:10:29,402 --> 01:10:31,137 STRONG YOU DON'T SEE THE REST OF 1336 01:10:31,137 --> 01:10:33,406 THE SEQUENCE SIGNAL SO THIS IS 1337 01:10:33,406 --> 01:10:36,642 LINEAR -- WELL, NAKED DNA. 1338 01:10:36,642 --> 01:10:37,643 I THINK IT'S POSSIBLE. 1339 01:10:37,643 --> 01:10:39,245 I WAS TRYING TO FIGURE OUT 1340 01:10:39,245 --> 01:10:41,914 WHETHER YOU THINK THE STRUCTURE 1341 01:10:41,914 --> 01:10:44,283 COULD COVER 50 BASE PAIRS BUT 1342 01:10:44,283 --> 01:10:45,985 THAT'S WHAT WE OBSERVED BUT IT'S 1343 01:10:45,985 --> 01:10:47,854 ON NAKED DNA. 1344 01:10:47,854 --> 01:10:48,287 >> THANK YOU. 1345 01:10:48,287 --> 01:10:50,423 THAT MAKES SENSE. 1346 01:10:50,423 --> 01:10:58,698 IT COULD ALSO BE THE BIPHYSICAL 1347 01:10:58,698 --> 01:11:00,967 STRUCTURE AND IT ONLY COVERS 1348 01:11:00,967 --> 01:11:02,935 FIVE OR SIX NUCLEOTIDES SO COULD 1349 01:11:02,935 --> 01:11:07,974 BE THE BENABILITY OF THE DNA. 1350 01:11:07,974 --> 01:11:10,776 >> AGREE BUT WHEN I LOOK AT YOUR 1351 01:11:10,776 --> 01:11:13,479 STRUCTURE THERE WERE EMPTY 1352 01:11:13,479 --> 01:11:14,614 SEQUENCE THAT LOOK LIKE THEY 1353 01:11:14,614 --> 01:11:18,284 COULD BE BOUND TO DNA AND NOT 1354 01:11:18,284 --> 01:11:20,620 NOT IN YOUR STRUCTURE. 1355 01:11:20,620 --> 01:11:23,456 >> WE CAN DISCUSS FURTHER. 1356 01:11:23,456 --> 01:11:24,991 >> LESLIE FROM PENN STATE 1357 01:11:24,991 --> 01:11:26,292 COLLEGE OF MEDICINE. 1358 01:11:26,292 --> 01:11:28,961 A QUESTION FOR YOU, IF I 1359 01:11:28,961 --> 01:11:32,832 UNDERSTAND CORRECTLY YOU USED 1360 01:11:32,832 --> 01:11:33,733 CMNCA BUT DIDN'T MENTION IF 1361 01:11:33,733 --> 01:11:36,002 ANTIBODY WAS DETECTED OR THE 1362 01:11:36,002 --> 01:11:36,936 FULL LENGTH GAG PROTEIN. 1363 01:11:36,936 --> 01:11:39,071 DID YOU LOOK FOR THAT? 1364 01:11:39,071 --> 01:11:41,974 >> NO, WE DIDN'T SCREEN FOR IT 1365 01:11:41,974 --> 01:11:43,943 BUT THE WAY IT CAME ABOUT IS 1366 01:11:43,943 --> 01:11:47,647 THEY HAD THE LIBRARY AND THEN WE 1367 01:11:47,647 --> 01:11:49,749 WERE INTERESTED IN HEXAMER AND 1368 01:11:49,749 --> 01:11:52,418 CAPSID SO THAT'S WHAT WE 1369 01:11:52,418 --> 01:11:52,919 SCREENED. 1370 01:11:52,919 --> 01:11:54,654 >> THERE COULD BE HIDDEN GEMS IN 1371 01:11:54,654 --> 01:11:54,954 THERE. 1372 01:11:54,954 --> 01:11:58,925 >> THERE COULD BE. 1373 01:11:58,925 --> 01:12:00,993 >> THE SO-CALLED PALINDROME HAS 1374 01:12:00,993 --> 01:12:02,728 BEEN REPORTED BY A COUPLE 1375 01:12:02,728 --> 01:12:02,962 GROUPS. 1376 01:12:02,962 --> 01:12:06,165 FOR EXAMPLE, GO BACK AND LOOK AT 1377 01:12:06,165 --> 01:12:08,968 SOME WORK AND THERE'S A 1378 01:12:08,968 --> 01:12:10,736 SECONDARY PIECE OF INFORMATION 1379 01:12:10,736 --> 01:12:11,837 THAT'S USEFUL AND THAT'S 1380 01:12:11,837 --> 01:12:14,607 ESSENTIALLY A PALINDROME AND TWO 1381 01:12:14,607 --> 01:12:15,508 HALVES. 1382 01:12:15,508 --> 01:12:17,743 THE INDIVIDUAL SITES ARE NOT 1383 01:12:17,743 --> 01:12:18,044 PALINDROMIC. 1384 01:12:18,044 --> 01:12:20,780 >> I DIDN'T REALIZE THE EXTENDED 1385 01:12:20,780 --> 01:12:21,213 FLANKING SEQUENCE. 1386 01:12:21,213 --> 01:12:23,349 >> IT'S BEEN REPORTED AS 1387 01:12:23,349 --> 01:12:24,350 APPROXIMATELY 100 BASE PAIRS. 1388 01:12:24,350 --> 01:12:24,684 >> THANK YOU. 1389 01:12:24,684 --> 01:12:25,751 I DIDN'T KNOW THAT. 1390 01:12:25,751 --> 01:12:28,988 I SHOULD HAVE REFERENCED THAT. 1391 01:12:28,988 --> 01:12:39,131 THANKS. 1392 01:13:12,331 --> 01:13:12,965 FIELD LIKE WES' INTEGRATION 1393 01:13:12,965 --> 01:13:23,142 CONDITION. 1394 01:14:24,036 --> 01:14:24,236 1395 01:14:24,236 --> 01:14:25,805 >> IN THE PROJECTION BOOTH CAN 1396 01:14:25,805 --> 01:14:28,941 WE GET SLIDES UP? 1397 01:14:28,941 --> 01:14:29,208 THANK YOU. 1398 01:14:29,208 --> 01:14:32,144 >> OKAY. 1399 01:14:32,144 --> 01:14:33,946 GOOD MORNING, EVERYBODY. 1400 01:14:33,946 --> 01:14:39,385 SO B-HIVE STANDS FOR BEHAVIOR OF 1401 01:14:39,385 --> 01:14:40,352 HIV IN ENVIRONMENT ENVIRONMENTS 1402 01:14:40,352 --> 01:14:42,688 AND ONE OF THE CENTERS OF 1403 01:14:42,688 --> 01:14:44,123 SEVERAL OF US HAVE BEEN SINCE 1404 01:14:44,123 --> 01:14:46,392 EARLY ON AND MORE THAN A QUARTER 1405 01:14:46,392 --> 01:14:47,860 CENTURY SINCE THE EVOLUTION OF 1406 01:14:47,860 --> 01:14:52,998 ALL THESE CENTERS HAVE SUPPORTED 1407 01:14:52,998 --> 01:14:54,767 HIV STRUCTURAL BIOLOGY. 1408 01:14:54,767 --> 01:15:00,706 AND B-HIVE IS CO-DIRECTED BY 1409 01:15:00,706 --> 01:15:01,941 BRUCE CORBETT AND MYSELF AT 1410 01:15:01,941 --> 01:15:03,008 EMORY UNDERSTAND AND THIS YEAR 1411 01:15:03,008 --> 01:15:03,876 IS MY TURN. 1412 01:15:03,876 --> 01:15:05,711 LAST YEAR WAS BRUCE. 1413 01:15:05,711 --> 01:15:09,882 AND NEXT YEAR AGAIN BRUCE. 1414 01:15:09,882 --> 01:15:14,553 SO COMPRISED WE HAVE A CENTER 1415 01:15:14,553 --> 01:15:16,989 THAT HAS APPROXIMATELY 31 1416 01:15:16,989 --> 01:15:18,691 INVESTIGATORS THAT COME FROM 16 1417 01:15:18,691 --> 01:15:20,359 DIFFERENT INSTITUTIONS 1418 01:15:20,359 --> 01:15:23,195 THROUGHOUT THE WORLD. 1419 01:15:23,195 --> 01:15:26,298 AND THEN THIS FORMAT HAS ENABLED 1420 01:15:26,298 --> 01:15:29,001 INCREDIBLE OPPORTUNITIES FOR 1421 01:15:29,001 --> 01:15:29,602 COLLABORATIONS THROUGHOUT THE 1422 01:15:29,602 --> 01:15:31,036 YEARS. 1423 01:15:31,036 --> 01:15:34,907 AND WE MEET TWICE A YEAR IN FACE 1424 01:15:34,907 --> 01:15:37,309 TO FACE MEETINGS AND HERE'S THE 1425 01:15:37,309 --> 01:15:39,078 LATEST IN FEBRUARY '79 AND MEET 1426 01:15:39,078 --> 01:15:40,980 AGAIN IN A COUPLE DAYS FROM 1427 01:15:40,980 --> 01:15:42,481 TODAY. 1428 01:15:42,481 --> 01:15:48,587 AND WE HAD ABOUT 100 PEOPLE THAT 1429 01:15:48,587 --> 01:15:48,888 ATTENDED. 1430 01:15:48,888 --> 01:15:52,191 THE STRUCTURE OF THE CENTER IS 1431 01:15:52,191 --> 01:15:52,992 BASED IN THREE SCIENTIFIC 1432 01:15:52,992 --> 01:15:53,325 PROJECT. 1433 01:15:53,325 --> 01:15:55,761 THE FIRST STUDIES THE DYNAMICS 1434 01:15:55,761 --> 01:15:58,497 OF HIV CORE AND SPECIFICALLY THE 1435 01:15:58,497 --> 01:16:00,699 INTERACTIONS OF CAPSIDS WITH 1436 01:16:00,699 --> 01:16:03,135 SMALL MOLECULE AND WITH HOST 1437 01:16:03,135 --> 01:16:03,369 FACTORS. 1438 01:16:03,369 --> 01:16:05,371 THE SECOND PROJECT STUDY THE 1439 01:16:05,371 --> 01:16:06,705 DYNAMICS OF HIV NUCLEAR 1440 01:16:06,705 --> 01:16:08,207 INTERACTIONS. 1441 01:16:08,207 --> 01:16:11,610 AND THEN AGAIN WE STUDY HOST 1442 01:16:11,610 --> 01:16:12,711 FACTORS THERE AND HOW THEY 1443 01:16:12,711 --> 01:16:16,448 AFFECT EVENTS IN THE NUCLEUS AS 1444 01:16:16,448 --> 01:16:18,450 WELL AS NUCLEIC ACID HIV 1445 01:16:18,450 --> 01:16:20,286 INTERACTION AND THIRD IS LATER 1446 01:16:20,286 --> 01:16:23,122 STAGES OF THE INFECTION, 1447 01:16:23,122 --> 01:16:24,523 PACKAGING AND ASSEMBLY. 1448 01:16:24,523 --> 01:16:26,692 WE ALSO HAVE FIVE CORES. 1449 01:16:26,692 --> 01:16:32,831 THE FIRST IS LED BY GREG BOOTH 1450 01:16:32,831 --> 01:16:35,568 AND STEPHAN AND THE SECOND IS ON 1451 01:16:35,568 --> 01:16:39,205 STRUCTURAL BIOLOGY AND DIMITRY 1452 01:16:39,205 --> 01:16:44,643 AND ROB DICK AND THEN BIO 1453 01:16:44,643 --> 01:16:48,681 PHYSICS WITH GREG AND CORINNE 1454 01:16:48,681 --> 01:16:55,888 AND DNA SEQUENCING WITH WILL 1455 01:16:55,888 --> 01:16:57,056 DEVELOPMENT AND OUTREACH. 1456 01:16:57,056 --> 01:16:58,757 IN TERMS OF THE ADMINISTRATION 1457 01:16:58,757 --> 01:17:00,693 IT'S RUN BY THE EXECUTIVE 1458 01:17:00,693 --> 01:17:06,699 COMMITTEE IN ADDITION TO THE TWO 1459 01:17:06,699 --> 01:17:11,103 CO-DIRECTORS WE HAVE TWO NIH 1460 01:17:11,103 --> 01:17:16,108 COLLEAGUES RACIAL AND ERIC FREED 1461 01:17:16,108 --> 01:17:18,677 AND CHRISTIAN WHO WORKS ON THE 1462 01:17:18,677 --> 01:17:21,947 NUTS AND BOLTS OF EVERYDAY 1463 01:17:21,947 --> 01:17:22,248 OPERATIONS. 1464 01:17:22,248 --> 01:17:27,753 WE'RE HONORED TO HAVE GREAT 1465 01:17:27,753 --> 01:17:29,922 SCIENTIFIC ADVISORY GROUP AND 1466 01:17:29,922 --> 01:17:31,957 CAROL CARTER AND SHOUT OUT TO 1467 01:17:31,957 --> 01:17:36,495 HER SHE BECAME A MEMBER OF THE 1468 01:17:36,495 --> 01:17:39,298 NATIONAL ACADEMIES THIS YEAR AND 1469 01:17:39,298 --> 01:17:43,102 DOUG RICHMOND. 1470 01:17:43,102 --> 01:17:50,576 WE HAVE TWO PROGRAMS AND WE WORK 1471 01:17:50,576 --> 01:17:56,982 ON ARTIFICIAL INTELLIGENCE AND 1472 01:17:56,982 --> 01:18:02,721 MEGAN KEN AND GUOCUHN JIANG 1473 01:18:02,721 --> 01:18:06,225 WORKING ON HIV LATENCY RESERVOIR 1474 01:18:06,225 --> 01:18:10,562 IN THE HUMAN BRAIN AND MYELOID 1475 01:18:10,562 --> 01:18:12,631 CELLS. 1476 01:18:12,631 --> 01:18:18,570 PREVIOUS CDP VEEP -- EVEN AFTER 1477 01:18:18,570 --> 01:18:20,172 FUNDING WAS FINISHED THEY 1478 01:18:20,172 --> 01:18:28,914 CONTINUED TO PARTICIPATE IN ALL 1479 01:18:28,914 --> 01:18:30,683 COLLABORATION WITH OTHER MEMBERS 1480 01:18:30,683 --> 01:18:35,387 AND SOPHI HARVEY AND DANIEL FROM 1481 01:18:35,387 --> 01:18:36,689 OHIO STATE UNIVERSITY AND THE 1482 01:18:36,689 --> 01:18:40,125 UNIVERSITY OF MINNESOTA. 1483 01:18:40,125 --> 01:18:42,695 LAST YEAR WE'VE BEEN BUSY. 1484 01:18:42,695 --> 01:18:44,763 SEVERAL PUBLICATIONS. 1485 01:18:44,763 --> 01:18:46,632 ONE INTERESTING NEW THING WE 1486 01:18:46,632 --> 01:18:51,403 HAVE IN THE WEBSITE WHERE WE 1487 01:18:51,403 --> 01:18:53,205 HIGHLIGHT SOME HIGH IMPACT 1488 01:18:53,205 --> 01:18:56,442 PUBLICATIONS AND IN ADDITION TO 1489 01:18:56,442 --> 01:18:58,977 THE SCIENCE WE HIGHLIGHT THE 1490 01:18:58,977 --> 01:19:00,679 POST-DOCTORAL FELLOW IN THIS 1491 01:19:00,679 --> 01:19:02,681 CASE WE HAVE INTERVIEWS AND 1492 01:19:02,681 --> 01:19:03,949 DISCUSSION OF THE SCIENTIFIC 1493 01:19:03,949 --> 01:19:07,119 TRAJECTORY AND CAREER PATH WHERE 1494 01:19:07,119 --> 01:19:08,954 THEY COME FROM AND WHERE THEY'RE 1495 01:19:08,954 --> 01:19:15,094 GOING. 1496 01:19:15,094 --> 01:19:17,096 AND THERE'S CONTINUED OUTREACH 1497 01:19:17,096 --> 01:19:20,666 AND TRAINING EFFORTS AND DAVID 1498 01:19:20,666 --> 01:19:22,368 GOODSELL CONTINUED TO WORK ON 1499 01:19:22,368 --> 01:19:26,905 THE PDP WEBSITE AND WE HAVE 1500 01:19:26,905 --> 01:19:28,540 MENTORING AND TEACHING IN PERSON 1501 01:19:28,540 --> 01:19:29,975 COMPUTATIONAL METHODS AND 1502 01:19:29,975 --> 01:19:30,743 WORKSHOP. 1503 01:19:30,743 --> 01:19:36,749 THIS TIME IT WAS DELIVERED BY 1504 01:19:36,749 --> 01:19:40,986 STE 1505 01:19:40,986 --> 01:19:48,761 STEFAN FORLY AND GREG BOOTH AND 1506 01:19:48,761 --> 01:19:51,930 WE HAVE A PROGRAM IN SAN DIEGO 1507 01:19:51,930 --> 01:19:53,332 FOR UNDER REPRESENTED MINORITY 1508 01:19:53,332 --> 01:19:55,968 STUDENTS AND TO PROMOTE 1509 01:19:55,968 --> 01:19:56,902 DEVELOPMENT OF SCIENTIFIC 1510 01:19:56,902 --> 01:20:00,739 INTEREST AND ACTIVE 1511 01:20:00,739 --> 01:20:01,907 PARTICIPATION IN S.T.E.M. 1512 01:20:01,907 --> 01:20:04,576 THERE'S ABOUT 30 POSTERS OUT 1513 01:20:04,576 --> 01:20:06,745 THERE AND THERE'S ENTHUSIASTIC 1514 01:20:06,745 --> 01:20:09,681 YOUNG PEOPLE PRESENTING THEM AND 1515 01:20:09,681 --> 01:20:12,751 WE APPRECIATE ALSO IF YOU STOP 1516 01:20:12,751 --> 01:20:17,022 BY TO ENCOURAGE THEM AND LEARN 1517 01:20:17,022 --> 01:20:20,826 ABOUT WHAT THEY DO. 1518 01:20:20,826 --> 01:20:24,696 IS FINALLY THE PRESENTATIONS 1519 01:20:24,696 --> 01:20:28,834 FROM ON THE LONG TRANSCRIPTION 1520 01:20:28,834 --> 01:20:33,005 PRODUCTS FOR HIV ENCODING AND 1521 01:20:33,005 --> 01:20:35,441 THE SECOND IS LENACAPAVIR BASED 1522 01:20:35,441 --> 01:20:38,977 AND DELIVER BY KAREN AND ROBERT 1523 01:20:38,977 --> 01:20:43,182 TAG TEAM AND THE LENACAPAVIR 1524 01:20:43,182 --> 01:20:44,983 WILL WOULD BE BY KAREN. 1525 01:20:44,983 --> 01:20:50,556 ON THE M 6I MUTATIONS AND 1526 01:20:50,556 --> 01:20:52,991 COMPOUNDS THAT WORK WELL AGAINST 1527 01:20:52,991 --> 01:20:57,463 IT. 1528 01:20:57,463 --> 01:21:00,999 ROB IS GOING TO PRESENT WITH 1529 01:21:00,999 --> 01:21:11,510 IMAGERY LATTICE AND GREG VOTH 1530 01:21:15,581 --> 01:21:16,782 WILL PRESENT ON COMPUTER 1531 01:21:16,782 --> 01:21:17,216 MODELLING. 1532 01:21:17,216 --> 01:21:18,951 WITH THAT WE'RE READY FOR THE 1533 01:21:18,951 --> 01:21:21,787 FIRST SPRINGS. 1534 01:21:21,787 --> 01:21:32,164 -- FIRST PRESENTATION. 1535 01:21:41,807 --> 01:21:42,441 >> OKAY. 1536 01:21:42,441 --> 01:21:44,209 THANK YOU VERY MUCH, STEPHAN. 1537 01:21:44,209 --> 01:21:46,445 IT'S AN HONOR TO HAVE THE 1538 01:21:46,445 --> 01:21:49,715 OPPORTUNITY TO TALK TO YOU ABOUT 1539 01:21:49,715 --> 01:21:51,817 OUR RECENT WORK. 1540 01:21:51,817 --> 01:21:56,989 SO THE TRADITIONAL VIEW OF HIV 1541 01:21:56,989 --> 01:21:58,690 REPLICATION UNTIL RECENTLY -- 1542 01:21:58,690 --> 01:22:02,628 I'M HAVING TROUBLE SEEING THE 1543 01:22:02,628 --> 01:22:02,861 POINTER. 1544 01:22:02,861 --> 01:22:06,532 THE CODING IN THE CYTOPLASM IN 1545 01:22:06,532 --> 01:22:07,599 COORDINATION WITH REVERSE 1546 01:22:07,599 --> 01:22:09,768 TRANSCRIPTION AT THE NUCLEAR 1547 01:22:09,768 --> 01:22:11,136 ENVELOPE DURING IMPORT. 1548 01:22:11,136 --> 01:22:12,771 ENCODING HAS BEEN TRADITIONALLY 1549 01:22:12,771 --> 01:22:16,241 DIFFICULT TO STUDY BECAUSE 1550 01:22:16,241 --> 01:22:18,544 QUANTITATIVE ASSAYS TO DETERMINE 1551 01:22:18,544 --> 01:22:20,078 CAPSID PROTEIN AMOUNTS 1552 01:22:20,078 --> 01:22:22,114 ASSOCIATED WITH COMPLEXES WERE 1553 01:22:22,114 --> 01:22:23,949 NOT UNTIL RECENTLY AVAILABLE. 1554 01:22:23,949 --> 01:22:26,919 VERY FEW CORES THAT ENTERED THE 1555 01:22:26,919 --> 01:22:29,021 CELLS LEAD TO PRODUCTIVE 1556 01:22:29,021 --> 01:22:31,223 INFECTION AND BIO CHEMICAL 1557 01:22:31,223 --> 01:22:32,891 ASSAYS THAT DETERMINE THE 1558 01:22:32,891 --> 01:22:37,896 PROPERTIES OF THE POPULATION OF 1559 01:22:37,896 --> 01:22:38,664 VIRUSES LEAD TO INFORMATION 1560 01:22:38,664 --> 01:22:40,299 ABOUT POST THE PARTICLES THAT 1561 01:22:40,299 --> 01:22:50,542 ARE DEFECTIVE. 1562 01:22:52,244 --> 01:22:57,382 IN 2020 WE STATED REVERSE 1563 01:22:57,382 --> 01:22:58,250 TRANSCRIPTION OCCURS WITHIN THE 1564 01:22:58,250 --> 01:23:00,786 INTACT CAPSID AND COMPLETED IN 1565 01:23:00,786 --> 01:23:02,955 THE NUCLEAR. 1566 01:23:02,955 --> 01:23:12,798 THE VIRAL CORES LOSE THEIR 1567 01:23:12,798 --> 01:23:16,034 INTEGRITY AND SEVERAL STUDY IN 1568 01:23:16,034 --> 01:23:17,502 THE LAST FEW YEARS HAVE STRONGLY 1569 01:23:17,502 --> 01:23:20,739 SUPPORTED VARIOUS ASPECTS OF 1570 01:23:20,739 --> 01:23:26,745 THIS MODEL SUCH AS INTACT CORES 1571 01:23:26,745 --> 01:23:30,015 ENTERING THE NUCLEUS AND THE 1572 01:23:30,015 --> 01:23:32,651 NEED FOR CAPSIDS TO INTERACT TO 1573 01:23:32,651 --> 01:23:33,352 ENTER THE NUCLEUS. 1574 01:23:33,352 --> 01:23:39,424 SO A KEY FEATURE OF THIS MODEL 1575 01:23:39,424 --> 01:23:43,061 IS THAT INFECTIOUS VIRAL CORES 1576 01:23:43,061 --> 01:23:45,063 ENCODE IN THE NUCLEUS AND WANTED 1577 01:23:45,063 --> 01:23:50,702 TO LOOK AT THE MOLECULAR 1578 01:23:50,702 --> 01:23:52,971 TRIGGER. 1579 01:23:52,971 --> 01:23:57,943 RYAN BURDIC VISUALIZED THE 1580 01:23:57,943 --> 01:23:59,277 CODING AND LABELLED THE PROTEINS 1581 01:23:59,277 --> 01:24:02,314 AND INFECTS THEM AND ALLOWS THE 1582 01:24:02,314 --> 01:24:04,549 CORE TO ENTER THE NUCLEUS AND 1583 01:24:04,549 --> 01:24:06,785 DID TIME LAPSED IMAGING TO 1584 01:24:06,785 --> 01:24:09,588 DETERMINE THE FATE OF THE CORES. 1585 01:24:09,588 --> 01:24:13,291 AND THIS LABELLING METHOD HAS 1586 01:24:13,291 --> 01:24:14,826 BEEN CHARACTERIZED AND LESS THAN 1587 01:24:14,826 --> 01:24:19,431 A TWO-FOLD EFFECT ON VIRUS 1588 01:24:19,431 --> 01:24:20,098 INFECTIVITY AND FOR THOSE 1589 01:24:20,098 --> 01:24:22,768 INTERESTED IN THIS METHOD GO TO 1590 01:24:22,768 --> 01:24:26,204 THE POSTER NUMBER 56. 1591 01:24:26,204 --> 01:24:28,273 AND HE'LL HAVE MORE DETAILS 1592 01:24:28,273 --> 01:24:30,842 ABOUT THE CHARACTERIZATION. 1593 01:24:30,842 --> 01:24:36,815 SO RYAN OBSERVED PARTICLES THAT 1594 01:24:36,815 --> 01:24:38,050 EITHER DISAPPEARED IN THE 1595 01:24:38,050 --> 01:24:40,686 OBSERVATION TIME OF 24 HOURS AND 1596 01:24:40,686 --> 01:24:44,423 LOSS OF THE GFPC SIGNAL 1597 01:24:44,423 --> 01:24:46,558 INDICATES CAPSID DISASSEMBLY AND 1598 01:24:46,558 --> 01:24:48,794 PARTICLES DID NOT ENCODE AND 1599 01:24:48,794 --> 01:24:49,761 STAYED IN THE END OF THE 1600 01:24:49,761 --> 01:24:52,998 OBSERVATION PERIOD. 1601 01:24:52,998 --> 01:24:54,566 HE QUANTIFIED THE EFFICIENCY AND 1602 01:24:54,566 --> 01:24:56,768 ABOUT 60% OF THE CORES IN THE IT 1603 01:24:56,768 --> 01:24:58,737 NUCLEUS ENCODE WITH MEDIAN TIME 1604 01:24:58,737 --> 01:25:00,806 OF ABOUT 10.6 HOURS AFTER 1605 01:25:00,806 --> 01:25:03,408 INFECTION. 1606 01:25:03,408 --> 01:25:08,013 HOWEVER, IF YOU BLOCK REVERSE 1607 01:25:08,013 --> 01:25:10,649 TRANSCRIPTION OR USE THE 1608 01:25:10,649 --> 01:25:12,117 CATALYTIC SITE MUTANT THE 1609 01:25:12,117 --> 01:25:13,618 EFFICIENCY DROPS TO A BASELINE 1610 01:25:13,618 --> 01:25:16,054 OF ABOUT 13% AND THE KINETICS 1611 01:25:16,054 --> 01:25:17,989 ARE SLOWER AND OCCUR WITHIN THE 1612 01:25:17,989 --> 01:25:18,790 MEDIAN TIME OF ABOUT 16, 17 1613 01:25:18,790 --> 01:25:24,996 HOURS. 1614 01:25:24,996 --> 01:25:27,532 IMPORTANTLY IF WE ADD IT WILL 1615 01:25:27,532 --> 01:25:30,702 AFTER IB INFECTION 75% OF THE 1616 01:25:30,702 --> 01:25:41,146 LATE PRODUCTS HAVE BEEN 1617 01:25:41,580 --> 01:25:44,449 SYNTHESIZED AND TELLS US IT MUST 1618 01:25:44,449 --> 01:25:47,552 BE SYNTHESIZED TO TRIGGER 1619 01:25:47,552 --> 01:25:50,222 EFFICIENT ENCODING. 1620 01:25:50,222 --> 01:25:51,990 NEXT WE WANTED TO WE WANTED TO 1621 01:25:51,990 --> 01:25:53,592 LOOK AT THE RELATIONSHIP BETWEEN 1622 01:25:53,592 --> 01:26:01,600 KINETICS OF UNCODING AND REVERSE 1623 01:26:01,600 --> 01:26:05,003 TRANSCRIPTION AND ADDS THE 1624 01:26:05,003 --> 01:26:08,573 CAPSID WILL INHIBITOR PF74 AND 1625 01:26:08,573 --> 01:26:10,742 LOOKS AMOUNT THE LOSS OF 1626 01:26:10,742 --> 01:26:12,811 SENSITIVITY OF PARTICLES AND IT 1627 01:26:12,811 --> 01:26:15,313 OCCURS WHEN ENCODING HAS TAKEN 1628 01:26:15,313 --> 01:26:15,514 PLACE. 1629 01:26:15,514 --> 01:26:17,048 RYAN ADDED THE THERAPY AT THE 1630 01:26:17,048 --> 01:26:20,485 BEGINNING OF THE INFECTION TO 1631 01:26:20,485 --> 01:26:22,721 INHIBIT REVERSE TRANSCRIPTION 1632 01:26:22,721 --> 01:26:24,256 INITIATION AND WASHED IT OUT TWO 1633 01:26:24,256 --> 01:26:27,726 HOURS LATER THAT ALLOWED RT TO 1634 01:26:27,726 --> 01:26:28,994 PROCEED. 1635 01:26:28,994 --> 01:26:31,229 AND THEN ASKED WHAT HAPPENS TO 1636 01:26:31,229 --> 01:26:31,496 ENCODING. 1637 01:26:31,496 --> 01:26:32,964 HE FINDS IF YOU DELAY THE 1638 01:26:32,964 --> 01:26:40,972 REVERSE TRANSCRIPTION BY TWO 1639 01:26:40,972 --> 01:26:44,943 HOURS AND SAME DELAY WAS 1640 01:26:44,943 --> 01:26:47,179 OBSERVED AND INDICATES A 1641 01:26:47,179 --> 01:26:49,681 RELATIONSHIP BETWEEN KINETICS OF 1642 01:26:49,681 --> 01:26:51,850 THE REVERSE TRANSCRIPTION AND 1643 01:26:51,850 --> 01:26:52,117 ENCODING. 1644 01:26:52,117 --> 01:26:54,753 NEXT WE WANTED TO ASK THE EFFECT 1645 01:26:54,753 --> 01:27:00,826 OF THE GENOME SIZE ON ENCODING 1646 01:27:00,826 --> 01:27:08,967 EFFICIENCY WE LOOK AT THE 1647 01:27:08,967 --> 01:27:10,535 VECTORS WITH THE ELEMENTS 1648 01:27:10,535 --> 01:27:14,406 REQUIRED FOR PACK ANDING AND 1649 01:27:14,406 --> 01:27:15,807 REVERSE TRANSCRIPTION. 1650 01:27:15,807 --> 01:27:17,642 THEY'RE TRANSFERRED WITH THE 1651 01:27:17,642 --> 01:27:20,445 CAPSID AND WILD TYPE AND 1652 01:27:20,445 --> 01:27:21,413 ENCODING EFFICIENCY WAS 1653 01:27:21,413 --> 01:27:25,050 DETERMINED IN THE IMAGING ASSAY 1654 01:27:25,050 --> 01:27:27,752 AND THE ENCODING EFFICIENCY 1655 01:27:27,752 --> 01:27:32,090 CORRELATES WITH GENOME SIZE AND 1656 01:27:32,090 --> 01:27:35,160 AS IT APPROACH APPROACHES THE 1657 01:27:35,160 --> 01:27:36,862 EFFICIENCY REACHES THE MAXIMUM 1658 01:27:36,862 --> 01:27:41,533 OF 60% IN ADDITION THE MINIMUM 1659 01:27:41,533 --> 01:27:44,135 GENOME SIZE IS REQUIRED FOR 1660 01:27:44,135 --> 01:27:48,006 ENCODING SINCE GENOMES LESS THAN 1661 01:27:48,006 --> 01:27:55,046 6.5GV DID NOT ENCODE MORE 1662 01:27:55,046 --> 01:27:55,680 EFFICI 1663 01:27:55,680 --> 01:27:56,281 EFFICIENTLY THAN THOSE THAT 1664 01:27:56,281 --> 01:27:59,851 CONTAINED NO GENOME AT ALL AND 1665 01:27:59,851 --> 01:28:01,486 THEY WERE REVERSE TRANSCRIBED 1666 01:28:01,486 --> 01:28:03,288 INDICATING THE SMALL GENOMES THE 1667 01:28:03,288 --> 01:28:08,960 FAILURES ARE NOT DUE TO THEIR 1668 01:28:08,960 --> 01:28:12,430 FAILURE TO REVERSE TRANSCRIBE 1669 01:28:12,430 --> 01:28:16,968 THEIR DNA AND WANTED TO SEE IF 1670 01:28:16,968 --> 01:28:19,271 THEY'RE CAPABLE OF INTEGRATION 1671 01:28:19,271 --> 01:28:20,872 AND GENE EXPRESSION AND THERE 1672 01:28:20,872 --> 01:28:24,809 WAS A DIFFERENT CONSTRUCT THAT 1673 01:28:24,809 --> 01:28:30,749 CONTAINED MOSTLY HIV SEQUENCES 1674 01:28:30,749 --> 01:28:35,453 THAT EXPRESSED GFP IN THE 1675 01:28:35,453 --> 01:28:37,956 PROTEIN AND FULL LENGTH THAT 1676 01:28:37,956 --> 01:28:48,466 ALSO EXPRESSED GFP AND FOUND 1677 01:28:48,867 --> 01:28:52,203 SHORT VECTORS WERE EFFICIENTLY 1678 01:28:52,203 --> 01:28:52,971 REVERSE TRANSCRIBED AND 1679 01:28:52,971 --> 01:28:56,374 TRANSPORT THE INTO THE NUCLEUS 1680 01:28:56,374 --> 01:29:00,278 AND WERE EFFICIENT OF ENCODING 1681 01:29:00,278 --> 01:29:04,983 AND DIDN'T ENCODE MORE THAN THE 1682 01:29:04,983 --> 01:29:08,954 GENOME CONTROL AND IT WAS LOW 1683 01:29:08,954 --> 01:29:10,555 COMPARED TO THE CONTROL VECTOR 1684 01:29:10,555 --> 01:29:11,957 WE THINK BECAUSE THE DNA WAS 1685 01:29:11,957 --> 01:29:15,327 TRAPPED INSIDE THE CORES THAT 1686 01:29:15,327 --> 01:29:16,628 ARE INTACT BECAUSE THEY DIDN'T 1687 01:29:16,628 --> 01:29:16,861 ENCODE. 1688 01:29:16,861 --> 01:29:19,998 WE WANTED TO ASK CAN WE RELEASE 1689 01:29:19,998 --> 01:29:28,073 THE SHORT DNA WITH CAPSID 1690 01:29:28,073 --> 01:29:35,246 INHIBITORS AND CAN IT EXPRESS 1691 01:29:35,246 --> 01:29:40,952 THE REPORTER GENE AND HE FOUND 1692 01:29:40,952 --> 01:29:45,991 THAT WHEN YOU SIS ASSEMBLE -- 1693 01:29:45,991 --> 01:29:51,096 DISASSEMBLE THE CORES THE DNA IS 1694 01:29:51,096 --> 01:29:51,363 DEGRADED. 1695 01:29:51,363 --> 01:29:53,698 MOST THE DNAs IS DEGRADED AND 1696 01:29:53,698 --> 01:29:54,566 SOME SURVIVES. 1697 01:29:54,566 --> 01:30:04,776 SO THE QUESTION IS DOES THE 1698 01:30:04,776 --> 01:30:07,712 SURVIVING DNA EXPRESS AND 1699 01:30:07,712 --> 01:30:09,147 INTEGRATES WITH THE T ONE HALF 1700 01:30:09,147 --> 01:30:16,187 OF 77 MINUTES AFTER TRIGGERING 1701 01:30:16,187 --> 01:30:18,323 ENCODING AND THE DNA THAT 1702 01:30:18,323 --> 01:30:24,829 SURVIVES IS INTEGRATED AS 1703 01:30:24,829 --> 01:30:29,000 EFFICIENTLY WITHOUT THE PF74 1704 01:30:29,000 --> 01:30:29,267 TREATMENT. 1705 01:30:29,267 --> 01:30:39,811 IN ADDITION -- IF WE TREAT WITH 1706 01:30:42,914 --> 01:30:46,351 THE LENACAPAVIR 10 HOURS LATER 1707 01:30:46,351 --> 01:30:48,853 AND ASK HOW MANY CELLS ARE 1708 01:30:48,853 --> 01:30:52,991 EXPRESSING GFP WE FIND COMPARED 1709 01:30:52,991 --> 01:30:59,731 TO THE UNTREATED CELLS ADDING 1710 01:30:59,731 --> 01:31:03,768 PF74 INCREASES THE EXPRESSING 1711 01:31:03,768 --> 01:31:08,139 CELLS SO IT MEANS THE SHORT DNAs 1712 01:31:08,139 --> 01:31:12,210 CAN INTEGRATE AND EXPRESS THE 1713 01:31:12,210 --> 01:31:12,477 REPORTER. 1714 01:31:12,477 --> 01:31:14,946 TO SUMMARIZE MOST SHORT DNA 1715 01:31:14,946 --> 01:31:16,981 REMAINS TRAPPED IN THE CORES AND 1716 01:31:16,981 --> 01:31:24,823 IT'S NOT DEGRADED AFTER PF74 1717 01:31:24,823 --> 01:31:26,691 INDUCED CAPSID AND CAN EXPRESS 1718 01:31:26,691 --> 01:31:27,859 THE GFP REPORTING. 1719 01:31:27,859 --> 01:31:30,829 NEXT WE WANTED TO LOOK AT THE 1720 01:31:30,829 --> 01:31:33,064 ROLE OF CAPSID STABILITY IN 1721 01:31:33,064 --> 01:31:33,631 ENCODING. 1722 01:31:33,631 --> 01:31:39,771 WE ENVISIONED THE DNA THAT IS 1723 01:31:39,771 --> 01:31:40,972 SYNTHESIZED CREATES AN OUTWARD 1724 01:31:40,972 --> 01:31:42,841 PRESSURE I'LL SAY MORE ABOUT 1725 01:31:42,841 --> 01:31:44,976 THAT LATER AND THE OUTWARD 1726 01:31:44,976 --> 01:31:48,613 PRESSURE IS COUNTER ACTED BY THE 1727 01:31:48,613 --> 01:31:53,017 INWARD PRESSURE CREATED BY THE 1728 01:31:53,017 --> 01:31:54,185 CAPSID STABILITY. 1729 01:31:54,185 --> 01:31:56,287 YOU LOOK AT THE CAPSID STABILITY 1730 01:31:56,287 --> 01:32:04,996 WE MANIPULATED THE CORE BINDING 1731 01:32:04,996 --> 01:32:08,900 TO AND INCREASES THE STABILITY 1732 01:32:08,900 --> 01:32:12,904 OF THE CA MUTANT AND THE BINDING 1733 01:32:12,904 --> 01:32:16,641 HAS MANY AFFECTS ON IT POWER 1734 01:32:16,641 --> 01:32:19,744 REPCATION AND PROTECT THE VIRAL 1735 01:32:19,744 --> 01:32:24,616 CORES AND SLOWS THE KINETIC OF 1736 01:32:24,616 --> 01:32:28,987 IMPORT AND INCREASES CAPSID 1737 01:32:28,987 --> 01:32:37,428 STABILITY. 1738 01:32:37,428 --> 01:32:39,030 THEY'D BE FOUND IT WAS MORE 1739 01:32:39,030 --> 01:32:40,165 EFFICIENT THAN THE CONTROL 1740 01:32:40,165 --> 01:32:40,532 VECTOR. 1741 01:32:40,532 --> 01:32:44,969 SIMILARLY IF THE CELLS WERE 1742 01:32:44,969 --> 01:32:47,472 TREATED TO INHIBIT AND THIS 1743 01:32:47,472 --> 01:32:48,873 EFFICIENCY WAS INCREASED 1744 01:32:48,873 --> 01:32:59,284 COMPARED TO THE CONTROL. 1745 01:33:04,622 --> 01:33:06,991 AND WE DON'T GET ENCODING IN THE 1746 01:33:06,991 --> 01:33:09,961 ABSENCE OF THE BINDING. 1747 01:33:09,961 --> 01:33:11,329 THEREFORE LOW CAPSID STABILITY 1748 01:33:11,329 --> 01:33:16,367 INFLUENCES THE EFFICIENCY OF 1749 01:33:16,367 --> 01:33:18,970 ENCODING AND SYNTHESIS OF DOUBLE 1750 01:33:18,970 --> 01:33:20,271 STRAND DNA REMAINING A TRIGGER 1751 01:33:20,271 --> 01:33:21,139 FOR ENCODING. 1752 01:33:21,139 --> 01:33:24,342 NEXT WE WANTED TO LOOK AT THE 1753 01:33:24,342 --> 01:33:26,477 EFFECT S OF CYCLOPHILIN BINDING 1754 01:33:26,477 --> 01:33:30,848 ON THE KINETICS OF REVERSE 1755 01:33:30,848 --> 01:33:35,920 TRANSCRIPTION ENCODING AND WE 1756 01:33:35,920 --> 01:33:41,192 COMPARED THE KINETICS IN THE 1757 01:33:41,192 --> 01:33:42,594 TREATED CELLS AND FOUND REVERSE 1758 01:33:42,594 --> 01:33:44,596 TRANSCRIPTION IS COMPLETED AT 1759 01:33:44,596 --> 01:33:47,699 ABOUT THE SAME TIME IN THE CELLS 1760 01:33:47,699 --> 01:33:50,101 HOWEVER, THE ENCODING OCCURS 1761 01:33:50,101 --> 01:33:54,205 MUCH FASTER IN THE CSA TREATED 1762 01:33:54,205 --> 01:33:54,672 CELLS. 1763 01:33:54,672 --> 01:33:56,941 TO SUMMARIZE IN THE PRESENCE OF 1764 01:33:56,941 --> 01:34:00,845 BINDING THE CAPSIDS ARE MORE 1765 01:34:00,845 --> 01:34:02,847 STABLE AND ENCODING OCCURS TWO 1766 01:34:02,847 --> 01:34:04,349 AND A HALF HOURS AFTER REVERSE 1767 01:34:04,349 --> 01:34:06,784 TRANSCRIPTION IS COMPLETED AND 1768 01:34:06,784 --> 01:34:08,419 BINDING ENSURES ENCODING OCCURS 1769 01:34:08,419 --> 01:34:10,088 AFTER COMPLETION OF THE REVERSE 1770 01:34:10,088 --> 01:34:10,421 TRANSCRIPTION. 1771 01:34:10,421 --> 01:34:20,598 WHEN THE IT CODING OCCURS FASTER 1772 01:34:20,598 --> 01:34:23,134 WHEN THEY'RE UNSTABLE AND SAME 1773 01:34:23,134 --> 01:34:24,836 AS REVERSE COMPLETION AND 1774 01:34:24,836 --> 01:34:28,973 ENCODING STILL REQUIRES REVERSE 1775 01:34:28,973 --> 01:34:29,807 TRANSCRIPTION INDICATING THE 1776 01:34:29,807 --> 01:34:34,145 PRODUCT IS A MAJOR TRIGGER FOR 1777 01:34:34,145 --> 01:34:35,213 ENCODING. 1778 01:34:35,213 --> 01:34:40,318 FINALLY WE'RE COLLABORATING WITH 1779 01:34:40,318 --> 01:34:45,823 MARK WILLIAMS AND THE LAB STO 1780 01:34:45,823 --> 01:34:48,026 UNDERSTAND HOW SYNTHESIS 1781 01:34:48,026 --> 01:34:49,961 TRIGGERS ENCODING. 1782 01:34:49,961 --> 01:34:52,230 BASED ON PREVIOUS STUDIES WE 1783 01:34:52,230 --> 01:34:55,600 THINK IT'S MORE RIGID AND 1784 01:34:55,600 --> 01:34:56,801 RESISTS COMPRESSION AND 1785 01:34:56,801 --> 01:34:58,303 THEREFORE IT THE LONG STRANDED 1786 01:34:58,303 --> 01:35:02,073 DNA INSIDE THE CAPSID INSERTS 1787 01:35:02,073 --> 01:35:05,009 INTERNAL PRESSURE ON THE CAPSID 1788 01:35:05,009 --> 01:35:08,513 AND HOWEVER, THE DOUBLE STRANDED 1789 01:35:08,513 --> 01:35:10,548 DNA IS EXPRESSED OR CONDENSED BY 1790 01:35:10,548 --> 01:35:14,919 THE HIV NUCLEAR CAPSID PROTEIN. 1791 01:35:14,919 --> 01:35:16,988 THIS ATOMIC FORCE MICROSCOPY 1792 01:35:16,988 --> 01:35:22,627 PERFORMED BY MIKE MORRIS SHOWED 1793 01:35:22,627 --> 01:35:26,064 A 7.5KB DOUBLE STRAND DNA 1794 01:35:26,064 --> 01:35:31,602 COMPARED TO THE CONTROL IS 1795 01:35:31,602 --> 01:35:33,004 SIGNIFICANTLY CONDENSED WHEN NC 1796 01:35:33,004 --> 01:35:36,808 IS ADDED IN A CONCENTRATION 1797 01:35:36,808 --> 01:35:40,745 DEPENDENT MANNER AS THERE'S MORE 1798 01:35:40,745 --> 01:35:41,079 CONDENSATION. 1799 01:35:41,079 --> 01:35:44,549 IN THE SECOND EXPERIMENT HE 1800 01:35:44,549 --> 01:35:50,188 ADDED 16 NUCLEOTIDE FRAGMENTS TO 1801 01:35:50,188 --> 01:35:53,891 MIMIC POWER GENOME DEGRADATION 1802 01:35:53,891 --> 01:35:59,597 THAT MAY BE REVERSED INSIDE THE 1803 01:35:59,597 --> 01:36:06,871 CORE AND FOUND IF WE ADD THE RNA 1804 01:36:06,871 --> 01:36:09,440 FRAGMENTS THEY RESULT IN LESS. 1805 01:36:09,440 --> 01:36:12,877 DNA CONDENSATION AND BASED ON 1806 01:36:12,877 --> 01:36:15,980 THE OBSERVATIONS AND SOME 1807 01:36:15,980 --> 01:36:17,014 INFORMATION OF REVERSE 1808 01:36:17,014 --> 01:36:18,916 TRANSCRIPTION WE THINK THE 1809 01:36:18,916 --> 01:36:22,920 CAPSID IS INCREASED BY 150 TO 1810 01:36:22,920 --> 01:36:25,189 200% AND THE AMOUNT OF NC 1811 01:36:25,189 --> 01:36:27,392 AVAILABLE IN THE CAPSID MAY NOT 1812 01:36:27,392 --> 01:36:30,862 BE EFFICIENCY TO FULLY CONDENSE 1813 01:36:30,862 --> 01:36:33,698 THE DNA AND AS A RESULT THE FULL 1814 01:36:33,698 --> 01:36:36,901 LENGTH STRANDED DNA DOES NOT 1815 01:36:36,901 --> 01:36:38,870 REMAIN CONDENSED AND THIS 1816 01:36:38,870 --> 01:36:41,038 INCREASES THE PRESSURE INSIDE 1817 01:36:41,038 --> 01:36:46,577 THE CORE LEADING TO ENCODING. 1818 01:36:46,577 --> 01:36:50,615 TO SUMMARIZE THE LONG BINDED DNA 1819 01:36:50,615 --> 01:36:55,386 IS REQUIRE TO INDUCE EFFICIENCY 1820 01:36:55,386 --> 01:36:57,755 ENCODING AND THAT DO NO REDUCE 1821 01:36:57,755 --> 01:37:00,892 DEFISH IN THE ENCODING AND COULD 1822 01:37:00,892 --> 01:37:04,695 BE RELEASED WITH PF74 AND MOST 1823 01:37:04,695 --> 01:37:08,699 THE DNA IS DEGRADED AND THE DNA 1824 01:37:08,699 --> 01:37:11,202 THAT SURVIVES IS CAPABLE OF 1825 01:37:11,202 --> 01:37:14,539 REPORTER GENE EXPRESSION. 1826 01:37:14,539 --> 01:37:18,910 WE THINK OVER ALL IT BALANCES 1827 01:37:18,910 --> 01:37:21,446 THE PRESSURE WITH CAPSID 1828 01:37:21,446 --> 01:37:24,916 STABILITY AND ENSURES THE 1829 01:37:24,916 --> 01:37:26,451 SYNTHESIS IS COMPLETED IN THE 1830 01:37:26,451 --> 01:37:28,986 INTACT CAPSID AND IT CAN ONLY 1831 01:37:28,986 --> 01:37:30,888 OCCUR AFTER COMPLETION OF 1832 01:37:30,888 --> 01:37:35,226 REVERSE TRANSCRIPTION. 1833 01:37:35,226 --> 01:37:40,298 I WANT TO THANK RYAN BURRDOCK 1834 01:37:40,298 --> 01:37:42,967 WHO CONDUCTED MOST OF THE 1835 01:37:42,967 --> 01:37:49,440 SCIENCES AND MY COLLABORATORS 1836 01:37:49,440 --> 01:37:53,110 AND WILL OTHERS. 1837 01:37:53,110 --> 01:38:03,421 THANK YOU SO MUCH. 1838 01:38:36,988 --> 01:38:46,998 >> 1839 01:39:25,536 --> 01:39:26,370 >> GOOD MORNING. 1840 01:39:26,370 --> 01:39:28,973 ROB AND I WILL PRESENT OUR 1841 01:39:28,973 --> 01:39:31,676 RECENT RESULTS ON ONGOING CAPSID 1842 01:39:31,676 --> 01:39:32,977 RELATED PROJECTS BUT FIRST I 1843 01:39:32,977 --> 01:39:37,748 WANT TO MENTION THAT WE HAVE 1844 01:39:37,748 --> 01:39:38,616 SEVERAL COLLEAGUES IN THE B-HIVE 1845 01:39:38,616 --> 01:39:42,887 CENTER THAT WORK ON VARIOUS 1846 01:39:42,887 --> 01:39:44,422 ASPECTS OF CAPSID AND I DON'T 1847 01:39:44,422 --> 01:39:45,423 HAVE TIME TO RECOGNIZE THEM ALL 1848 01:39:45,423 --> 01:39:48,225 TODAY BUT YOU SEE THE PEOPLE WHO 1849 01:39:48,225 --> 01:39:51,762 WORKED DIRECTLY ON THE PROJECTS 1850 01:39:51,762 --> 01:39:53,965 YOU WILL HEAR ABOUT TODAY AND 1851 01:39:53,965 --> 01:39:58,169 ENCOURAGE YOU TO VISIT OUR 1852 01:39:58,169 --> 01:40:00,638 TRAINEES WHO HAVE RELATED CAPSID 1853 01:40:00,638 --> 01:40:03,708 POSTERS TOMORROW RELATED TO THIS 1854 01:40:03,708 --> 01:40:03,908 TALK. 1855 01:40:03,908 --> 01:40:09,880 SO AS I WAS INTRODUCED BY THE 1856 01:40:09,880 --> 01:40:14,118 CHEETAH CENTER EARLIER, 1857 01:40:14,118 --> 01:40:16,387 LENACAPAVIR TARGETS VALIDATING 1858 01:40:16,387 --> 01:40:19,690 CAPSID AS A THERAPEUTIC TARGET 1859 01:40:19,690 --> 01:40:22,193 AND POTENT AND A LONG ACTING 1860 01:40:22,193 --> 01:40:25,763 FORMATION AND MANY GROUPS OVER 1861 01:40:25,763 --> 01:40:30,067 THE YEARS DEVELOPING LENACAPAVIR 1862 01:40:30,067 --> 01:40:32,570 AND OTHER ANTIVIRALS AND 1863 01:40:32,570 --> 01:40:34,672 APOLOGIES FOR NOT CAPTURING 1864 01:40:34,672 --> 01:40:36,974 EVERYBODY HERE FOR TIME 1865 01:40:36,974 --> 01:40:39,143 CONSTRAINTS AND IT BIND THE 1866 01:40:39,143 --> 01:40:42,947 CAPSID CORE SHOWN IN RED AND IN 1867 01:40:42,947 --> 01:40:45,983 DOING SO IT BLOCKS NUCLEAR 1868 01:40:45,983 --> 01:40:47,118 TRANSPORT BECAUSE IT COMPETES OR 1869 01:40:47,118 --> 01:40:50,554 BLOCKS BINDING OF NUCLEAR AND 1870 01:40:50,554 --> 01:40:52,990 OTHER HOST FACTORS NEEDED FOR 1871 01:40:52,990 --> 01:40:54,959 NUCLEAR TRANSPORT. 1872 01:40:54,959 --> 01:40:56,394 IT'S ALSO KNOWN TO EFFECT 1873 01:40:56,394 --> 01:40:58,029 ASSEMBLY IN MATURATION AND AT 1874 01:40:58,029 --> 01:40:59,764 THE BEGINNING OF THE TALK I'M 1875 01:40:59,764 --> 01:41:04,435 GOING TO TALK ABOUT RESISTANCE 1876 01:41:04,435 --> 01:41:06,671 MUTANTS THAT AFFECT LENACAPAVIR 1877 01:41:06,671 --> 01:41:08,039 ABILITY TO BLOCK TRANS ORDER 1878 01:41:08,039 --> 01:41:10,541 AND ROB WILL TALK ABOUT THE 1879 01:41:10,541 --> 01:41:12,777 IMMATURE CAPSID LATTICE. 1880 01:41:12,777 --> 01:41:15,112 THOUGH IT'S BEEN A BREAKTHROUGH 1881 01:41:15,112 --> 01:41:17,815 IN THE CLINIC, RESISTANCE 1882 01:41:17,815 --> 01:41:18,749 MUTATIONS HAVE BEEN IDENTIFIED 1883 01:41:18,749 --> 01:41:20,584 IN VITRO AND IN THE CLINIC. 1884 01:41:20,584 --> 01:41:24,955 AMONG THEM M66I IS THE MOST 1885 01:41:24,955 --> 01:41:26,891 DEVASTATING CAUSING HIGH 1886 01:41:26,891 --> 01:41:27,958 RESISTANCE TO LENACAPAVIR. 1887 01:41:27,958 --> 01:41:30,828 FIRST IT WAS IDENTIFIED IN VITRO 1888 01:41:30,828 --> 01:41:32,463 AND THEN ALSO SEEN IN THE CLINIC 1889 01:41:32,463 --> 01:41:35,032 WITH OTHER DRUG RESISTANCE 1890 01:41:35,032 --> 01:41:35,299 MUTATIONS. 1891 01:41:35,299 --> 01:41:39,670 SO THE BIG QUESTION IS HOW CAN 1892 01:41:39,670 --> 01:41:40,237 LENACAPAVIR RESISTANCE BE 1893 01:41:40,237 --> 01:41:41,338 ADDRESSED? 1894 01:41:41,338 --> 01:41:46,744 FROM DECADES OF RESEARCH ON 1895 01:41:46,744 --> 01:41:48,946 REVERSE TRANSCRIPTASE WE KNOW 1896 01:41:48,946 --> 01:41:50,848 COMBINATIONS HAVE BEEN 1897 01:41:50,848 --> 01:41:52,817 SUCCESSFUL TREATMENTS AND 1898 01:41:52,817 --> 01:41:54,518 SUCCESSFUL STRATEGIES FOR 1899 01:41:54,518 --> 01:41:56,554 OVERCOMING DRUG RESISTANT 1900 01:41:56,554 --> 01:41:58,723 MUTATIONS. 1901 01:41:58,723 --> 01:42:00,958 TRADITIONALLY COMBINATIONS USE 1902 01:42:00,958 --> 01:42:03,594 DIFFERENT MOLECULES THAT TARGET 1903 01:42:03,594 --> 01:42:07,131 DIFFERENT TARGETS IN THE LIFE 1904 01:42:07,131 --> 01:42:07,631 CYCLE. 1905 01:42:07,631 --> 01:42:12,403 MOST OF THOSE ARE REVERSE 1906 01:42:12,403 --> 01:42:18,476 TRANSCRIPTASE AND PROTEASE ARE 1907 01:42:18,476 --> 01:42:19,210 USING COMBINATION WITH EACH 1908 01:42:19,210 --> 01:42:20,644 OTHER. 1909 01:42:20,644 --> 01:42:23,781 AND MOST OF THEM HAVE ONE SINGLE 1910 01:42:23,781 --> 01:42:27,752 BINDING SITE PER ENZYME BUT IF 1911 01:42:27,752 --> 01:42:30,387 YOU THINK IN TERM OF THE CAPSID 1912 01:42:30,387 --> 01:42:32,323 CORE YOU HAVE SO MANY BINDING 1913 01:42:32,323 --> 01:42:35,559 SITES, 1500 AND WE KNOW FROM THE 1914 01:42:35,559 --> 01:42:36,527 STRUCTURE OF THE CAPSID CORE 1915 01:42:36,527 --> 01:42:38,562 THAT THESE ARE ALL LIKELY TO BE 1916 01:42:38,562 --> 01:42:39,096 DIFFERENT. 1917 01:42:39,096 --> 01:42:42,800 WE ASKED THE QUESTION CAN 1918 01:42:42,800 --> 01:42:45,069 LENACAPAVIR BE COMBINED WITH 1919 01:42:45,069 --> 01:42:47,671 OTHER ANTIVIRALS? 1920 01:42:47,671 --> 01:42:51,075 WE USED A TECHNIQUE IN 1921 01:42:51,075 --> 01:42:52,977 COLLABORATION WITH SOPHI HARVEY 1922 01:42:52,977 --> 01:42:55,279 AT OHIO STATE UNIVERSITY. 1923 01:42:55,279 --> 01:42:59,784 SOPHI BEING A FORMER CVPs AND 1924 01:42:59,784 --> 01:43:10,327 USED NATIVE MASS SPECTOMETRY AND 1925 01:43:13,030 --> 01:43:20,971 USED A COMPOUND PREVIOUSLY 1926 01:43:20,971 --> 01:43:31,515 IDENTIFIED 57D WE COULD STE THE 1927 01:43:40,291 --> 01:43:42,960 AND WHAT WOULD SEE WHAT WAS 1928 01:43:42,960 --> 01:43:44,628 BINDING AND THE NEW WAY TO LOOK 1929 01:43:44,628 --> 01:43:47,331 AT THE MOLECULE BOUND TO THE 1930 01:43:47,331 --> 01:43:49,266 SAME SITE WITHIN ONE CAPSID 1931 01:43:49,266 --> 01:43:49,667 HEXAMER. 1932 01:43:49,667 --> 01:43:51,836 TO FURTHER UNDERSTAND IF WE CAN 1933 01:43:51,836 --> 01:43:56,707 USE DIFFERENT FG MOLECULES IN 1934 01:43:56,707 --> 01:43:59,243 TERMS OF USING COMBINATIONS WE 1935 01:43:59,243 --> 01:44:01,946 DID CELL-BASED ANTIVIRAL ASSAYS 1936 01:44:01,946 --> 01:44:03,981 AND TITRATED DIFFERENT 1937 01:44:03,981 --> 01:44:05,516 CONCENTRATIONS OF LENACAPAVIR 1938 01:44:05,516 --> 01:44:09,353 AND 1261 AND FOUND THEY'RE NOT 1939 01:44:09,353 --> 01:44:09,787 ANTAGONISTIC. 1940 01:44:09,787 --> 01:44:11,522 THEY'RE AT LEAST ADDITIVE AT 1941 01:44:11,522 --> 01:44:14,258 SOME CONCENTRATIONS. 1942 01:44:14,258 --> 01:44:16,961 THIS IS PROOF OF PRINCIPLE WE 1943 01:44:16,961 --> 01:44:19,396 CAN USE COMBINATIONS OF 1944 01:44:19,396 --> 01:44:25,236 MOLECULES THAT TARGET THE FG 1945 01:44:25,236 --> 01:44:27,171 BINDING SITE AS FEASIBLE. 1946 01:44:27,171 --> 01:44:29,974 TO FURTHER ADDRESS THIS WE HAVE 1947 01:44:29,974 --> 01:44:33,377 BEEN WORKING TO DO A 1948 01:44:33,377 --> 01:44:37,248 STRUCTURE-RELATIONSHIP ACTIVITY 1949 01:44:37,248 --> 01:44:37,615 STUDY. 1950 01:44:37,615 --> 01:44:40,251 WE EXAMINED SEVERAL CRYSTAL 1951 01:44:40,251 --> 01:44:44,421 STRUCTURES, BIO PHYSICAL AND 1952 01:44:44,421 --> 01:44:46,156 VIROLOGICAL DATA TO 1953 01:44:46,156 --> 01:44:47,124 STRATEGICALLY COMBINED COMPOUNDS 1954 01:44:47,124 --> 01:44:48,492 TO BIND THE I6 MUTATION WITH THE 1955 01:44:48,492 --> 01:44:51,762 GOAL OF OVERCOMING THE 1956 01:44:51,762 --> 01:44:54,431 LENACAPAVIR RESISTANCE AND USING 1957 01:44:54,431 --> 01:44:55,666 THESE IN THE FUTURE IN 1958 01:44:55,666 --> 01:44:56,767 COMBINATION WITH LENACAPAVIR AND 1959 01:44:56,767 --> 01:45:01,005 THIS IS WORK DONE WITH THE 1960 01:45:01,005 --> 01:45:05,276 UNIVERSITY OF MINNESOTA. 1961 01:45:05,276 --> 01:45:08,145 SO WE INITIALLY TESTED THE 1962 01:45:08,145 --> 01:45:11,048 COMPOUNDS IN THERMAL SHIFT 1963 01:45:11,048 --> 01:45:13,851 ASSAYS AND THIS PRESENCE 1964 01:45:13,851 --> 01:45:14,919 LENACAPAVIR LOSES BINDING 1965 01:45:14,919 --> 01:45:18,088 SIGNIFICANTLY AND WHEN WE TEST 1966 01:45:18,088 --> 01:45:19,823 OUR NEW COMPOUNDS WE HAVE 1967 01:45:19,823 --> 01:45:22,726 THEY'RE BINDING BETTER TO M66I 1968 01:45:22,726 --> 01:45:26,230 THAN TO WILD TYPE YOU CAN SEE BY 1969 01:45:26,230 --> 01:45:28,198 THE THIRD COLUMN AND THE 1970 01:45:28,198 --> 01:45:29,099 DIFFERENCE IN BINDING BETWEEN 1971 01:45:29,099 --> 01:45:33,003 THE M66I AND THE WILD TYPE. 1972 01:45:33,003 --> 01:45:35,673 TO FURTHER UNDERSTAND THIS 1973 01:45:35,673 --> 01:45:40,978 BINDING, WE ALSO DID BY LAYER 1974 01:45:40,978 --> 01:45:42,980 ASSAY TO UNDERSTAND THE BINDING 1975 01:45:42,980 --> 01:45:44,782 KINETICS AND I'M SHOWING THE 1976 01:45:44,782 --> 01:45:48,986 FULL CHANGE OF THE MUTATION 1977 01:45:48,986 --> 01:45:52,990 COMPARED TO THE WILD TYPE. 1978 01:45:52,990 --> 01:45:56,961 AS EXPECTED. 1979 01:45:56,961 --> 01:46:00,664 LENACAPAVIR BINDING THE KV 1980 01:46:00,664 --> 01:46:02,766 INCREASES AND IN THE PRESENCE OF 1981 01:46:02,766 --> 01:46:05,736 THE COMPOUNDS THERE'S STRONGER 1982 01:46:05,736 --> 01:46:09,106 BINDING TO M66I CAPSID HEXAMER. 1983 01:46:09,106 --> 01:46:13,110 AND WE TESTED THEM IN CELL-BASED 1984 01:46:13,110 --> 01:46:15,512 ANTIVIRAL ASSAYS USING FULLY 1985 01:46:15,512 --> 01:46:16,280 INFECTIOUS VIRUS. 1986 01:46:16,280 --> 01:46:18,716 AGAIN FOR SIMPLICITY AND TIME 1987 01:46:18,716 --> 01:46:20,217 SAKE I'M SHOWING THE FULL CHANGE 1988 01:46:20,217 --> 01:46:23,187 IN M66I COMPARED TO THE WILD 1989 01:46:23,187 --> 01:46:24,588 TYPE VIRUS. 1990 01:46:24,588 --> 01:46:26,390 SO LENACAPAVIR AS PREVIOUSLY 1991 01:46:26,390 --> 01:46:30,995 REPORTED IS HIGHLY RESISTANT AND 1992 01:46:30,995 --> 01:46:32,863 DETRIMENTAL TO LENACAPAVIR 1993 01:46:32,863 --> 01:46:38,402 INHIBITION WITH THE COMPOUNDS. 1994 01:46:38,402 --> 01:46:44,441 IN SOME CASES THEY INHIBIT M66I 1995 01:46:44,441 --> 01:46:47,111 BETTER THAN WILD TYPE. 1996 01:46:47,111 --> 01:46:50,280 AND ADDITIONALLY WE USE OUR 1997 01:46:50,280 --> 01:46:53,317 NATIVE FULL LENGTH CAPSID 1998 01:46:53,317 --> 01:46:57,421 CONSTRUCT WITH ATLANTIC OR M66I 1999 01:46:57,421 --> 01:46:58,088 MUTATION. 2000 01:46:58,088 --> 01:47:00,924 UNFORTUNATELY I CANNOT SHOW THE 2001 01:47:00,924 --> 01:47:02,826 COMPOUNDS TODAY BECAUSE OF 2002 01:47:02,826 --> 01:47:04,361 INTELLECTUAL PROPERTY CONCERNS 2003 01:47:04,361 --> 01:47:06,096 BUT WE HAVE STRUCTURES AVAILABLE 2004 01:47:06,096 --> 01:47:10,300 AND WE'RE USING THOSE FOR FUTURE 2005 01:47:10,300 --> 01:47:10,534 DESIGN. 2006 01:47:10,534 --> 01:47:12,069 QUITE HIGH RESOLUTION STRUCTURES 2007 01:47:12,069 --> 01:47:12,703 PROVIDING A LOT OF INFORMATION 2008 01:47:12,703 --> 01:47:15,105 ON HOW THE COMPOUNDS ARE BINDING 2009 01:47:15,105 --> 01:47:16,974 AND GIVING INSIGHTS INTO HOW WE 2010 01:47:16,974 --> 01:47:20,677 CAN OVERCOME RESISTANCE. 2011 01:47:20,677 --> 01:47:23,313 AND IN THE FUTURE BECAUSE THESE 2012 01:47:23,313 --> 01:47:24,515 COMPOUNDS -- I FORGOT TO MENTION 2013 01:47:24,515 --> 01:47:26,850 ON THE PREVIOUS SLIDE, THEY'RE 2014 01:47:26,850 --> 01:47:30,721 NOT QUITE AS POTENT IN WILD TYPE 2015 01:47:30,721 --> 01:47:32,289 AS LENACAPAVIR. 2016 01:47:32,289 --> 01:47:37,494 SO WE'RE IMPROVING IN THE NEXT 2017 01:47:37,494 --> 01:47:38,195 ROUND OF DESIGN. 2018 01:47:38,195 --> 01:47:42,366 WE ARE TRYING TO IMPROVE THE 2019 01:47:42,366 --> 01:47:43,400 POTENCY AS WELL AS TRYING TO 2020 01:47:43,400 --> 01:47:46,370 TEST AGAINST OTHER MUTATIONS AND 2021 01:47:46,370 --> 01:47:49,073 SEE IF WE CAN DEVELOP COMPOUNDS 2022 01:47:49,073 --> 01:47:51,141 THAT WILL BE EFFECTIVE OR ACTIVE 2023 01:47:51,141 --> 01:47:53,277 AGAINST SEVERAL MUTATIONS. 2024 01:47:53,277 --> 01:47:56,847 AND I ALSO ENCOURAGE YOU TO SEE 2025 01:47:56,847 --> 01:47:59,316 MY COLLEAGUE CHRIS' POSTER 2026 01:47:59,316 --> 01:48:01,885 NUMBER 127 RELATED TO THE CRYO 2027 01:48:01,885 --> 01:48:05,989 EM STRUCTURE WITH M66I. 2028 01:48:05,989 --> 01:48:12,329 AND SO JUST IN SUMMARY, WE 2029 01:48:12,329 --> 01:48:14,331 STRATEGICALLY DESIGN COMPOUNDS 2030 01:48:14,331 --> 01:48:16,200 SUCCESSFULLY IN THE FIRST ROUND 2031 01:48:16,200 --> 01:48:18,869 OF COMPOUNDS UNDER THE NEW 2032 01:48:18,869 --> 01:48:23,040 DESIGN THAT WE HAVE THAT CAN BE 2033 01:48:23,040 --> 01:48:27,144 EFFECTIVE AGAINST M66I COMPARED 2034 01:48:27,144 --> 01:48:28,979 TO WILD TYPE VIRUS AND FUTURE 2035 01:48:28,979 --> 01:48:32,983 STUDIES WILL FOCUS ON IMPROVING 2036 01:48:32,983 --> 01:48:36,620 POTENCY AND DESIGN TOWARDS 2037 01:48:36,620 --> 01:48:37,588 HOPEFULLY POTENTIAL STUDIES WITH 2038 01:48:37,588 --> 01:48:40,190 LENACAPAVIR AND WITH THAT I'LL 2039 01:48:40,190 --> 01:48:48,966 TURN THE MIC OVER TO ROB. 2040 01:48:48,966 --> 01:48:53,437 >> THANK YOU, KAREN. 2041 01:48:53,437 --> 01:48:57,975 SO WE'VE BEEN USING A METHOD IN 2042 01:48:57,975 --> 01:48:59,209 THE LAB CALLED TEMPLATING METHOD 2043 01:48:59,209 --> 01:49:02,412 TO DETERMINE THE STRUCTURE 2044 01:49:02,412 --> 01:49:05,415 MATURE HIV LATTICE UNDER A 2045 01:49:05,415 --> 01:49:06,917 NUMBER OF CONDITIONS AND WE'VE 2046 01:49:06,917 --> 01:49:08,418 BEEN ABLE TO LEVERAGE THIS TO 2047 01:49:08,418 --> 01:49:11,722 STUDY SOME OF THE EFFECTS OF THE 2048 01:49:11,722 --> 01:49:11,989 MUTATIONS. 2049 01:49:11,989 --> 01:49:17,728 HERE'S AN EXAMPLE OF WHERE THE 2050 01:49:17,728 --> 01:49:19,730 STRUCTURE OF THE M66I MUTATION 2051 01:49:19,730 --> 01:49:20,697 AND ABOUT WHAT WAS INTERESTING 2052 01:49:20,697 --> 01:49:21,632 IN THE CONTEXT OF THE STRUCTURE 2053 01:49:21,632 --> 01:49:24,601 WAS THE LARGEST CHANGE WE SAW IN 2054 01:49:24,601 --> 01:49:35,145 M66I WAS THE POSITION OF THE K70 2055 01:49:42,519 --> 01:49:44,988 LYESENE AND USED CRYO EM TO 2056 01:49:44,988 --> 01:49:48,225 DETERMINE THE STRUCTURE OF THE 2057 01:49:48,225 --> 01:49:54,731 IMMATURE LATTICE AND ADDED 2058 01:49:54,731 --> 01:49:57,601 LENACAPAVIR AND FIND IT BINDS 2059 01:49:57,601 --> 01:50:00,971 WELL TO THE IMMATURE LATTICE IN 2060 01:50:00,971 --> 01:50:03,574 VITRO AND WONDERED IF WE CAN 2061 01:50:03,574 --> 01:50:07,077 FIND THESE IN CELLS TREATED WITH 2062 01:50:07,077 --> 01:50:11,381 LENACAPAVIR AND THESE ARE 2063 01:50:11,381 --> 01:50:16,987 RELEASED CELLS AT 10 NANO MOLAR 2064 01:50:16,987 --> 01:50:18,121 LENACAPAVIR WE SEE MATURE 2065 01:50:18,121 --> 01:50:19,856 LATTICE AND THEN NO LONGER 2066 01:50:19,856 --> 01:50:24,294 DETECT AND ALL THE LATTICE IS 2067 01:50:24,294 --> 01:50:24,528 MATURE. 2068 01:50:24,528 --> 01:50:28,765 THE MATURE LATTICE IS IN THE 2069 01:50:28,765 --> 01:50:31,868 CONTEXT OF FULL LENGTH GAG AND 2070 01:50:31,868 --> 01:50:41,278 IN THE CONTEXT OF 50 NANO MOLAR 2071 01:50:41,278 --> 01:50:46,550 LENACAPAVIR SO ALL LATTICE DI 2072 01:50:46,550 --> 01:50:49,786 DETECTED IS IMMATURE AND HERE 2073 01:50:49,786 --> 01:50:52,422 YOU CAN SEE THE PROXIMITY OF THE 2074 01:50:52,422 --> 01:50:56,693 LAYER TO THE PLASMA MEMBRANE AND 2075 01:50:56,693 --> 01:50:58,262 SEE LENACAPAVIR BOUND WELL AND 2076 01:50:58,262 --> 01:51:00,964 DO NOT SEE THE FORMATION OF THE 2077 01:51:00,964 --> 01:51:02,399 CLASSIC AND TERMINAL BETA TERM 2078 01:51:02,399 --> 01:51:04,034 SO THAT PROTEIN IS EXTENDED UP 2079 01:51:04,034 --> 01:51:08,972 TOWARDS THE MEMBRANE. 2080 01:51:08,972 --> 01:51:12,442 IN COLLABORATION WITH DANIEL A 2081 01:51:12,442 --> 01:51:15,579 NUMBER OF LENACAPAVIR ANALOGS 2082 01:51:15,579 --> 01:51:20,517 HAVE BEEN MADE AND TESTED. 2083 01:51:20,517 --> 01:51:26,790 AND MIA VISITED MY LAB AND 2084 01:51:26,790 --> 01:51:28,759 LEARNED OUR IN VITRO IS ASSEMBLY 2085 01:51:28,759 --> 01:51:31,094 REACTION AND DONE THIS IN THE 2086 01:51:31,094 --> 01:51:35,065 CONTEXT OF THE ANALOG AND WE 2087 01:51:35,065 --> 01:51:36,700 HAVE DONE PRELIMINARY CRYO EM 2088 01:51:36,700 --> 01:51:38,268 SCREENING AND THIS LOOKS LIKE 2089 01:51:38,268 --> 01:51:41,672 WE'RE ABLE TO COLLECT NICE DATA 2090 01:51:41,672 --> 01:51:42,739 SETS. 2091 01:51:42,739 --> 01:51:44,741 ONE OF THE PRELIMINARY DATA SETS 2092 01:51:44,741 --> 01:51:45,876 WITH WAS THE MOLECULE AND HERE'S 2093 01:51:45,876 --> 01:51:48,979 AN EXAMPLE HOW WE CAN SEE THE 2094 01:51:48,979 --> 01:51:51,848 DETAILED CHANGE BETWEEN THE 2095 01:51:51,848 --> 01:51:53,684 DENSITY BETWEEN THE LENACAPAVIR 2096 01:51:53,684 --> 01:51:56,119 ANALOG AND IF WE ZOOM IN THAT'S 2097 01:51:56,119 --> 01:51:58,622 THE CHANGE IN THE MOLECULE 2098 01:51:58,622 --> 01:52:01,958 MISSING THE MT AND YOU SEE THE 2099 01:52:01,958 --> 01:52:03,293 CRYO EM DENSITY NOT PRESENT FOR 2100 01:52:03,293 --> 01:52:04,494 THAT PART OF THE MOLECULE. 2101 01:52:04,494 --> 01:52:06,830 WE CAN CAPTURE THE DETAILS OF 2102 01:52:06,830 --> 01:52:08,899 THESE MOLECULES AND THE BINDING. 2103 01:52:08,899 --> 01:52:12,969 AND THIS IS NOW BEING DONE FOR 2104 01:52:12,969 --> 01:52:16,773 BOTH IMMATURE AND MATURE 2105 01:52:16,773 --> 01:52:17,774 LATTICES IN THE CONTEXT OF 2106 01:52:17,774 --> 01:52:18,442 MUTATIONS. 2107 01:52:18,442 --> 01:52:20,977 WITH THAT I'D LIKE TO THANK 2108 01:52:20,977 --> 01:52:23,814 CURRENT AND PAST LAB MEMBERS AS 2109 01:52:23,814 --> 01:52:28,985 WELL AS THE GRADUATE STUDENT 2110 01:52:28,985 --> 01:52:33,023 WITH THE PRIMERATIZATION OF THE 2111 01:52:33,023 --> 01:52:33,290 MOLECULE. 2112 01:52:33,290 --> 01:52:43,467 THANK YOU. 2113 01:53:40,457 --> 01:53:44,961 >> OKAY. 2114 01:53:44,961 --> 01:53:48,432 I'M GOING TO TALK ABOUT B-HIVE 2115 01:53:48,432 --> 01:53:50,634 AND IF I FREEZE UP IT'S BECAUSE 2116 01:53:50,634 --> 01:53:53,103 I FORGOT MY PARKA WHILE IT'S 100 2117 01:53:53,103 --> 01:53:56,973 OUT SIDE AND I'LL TALK ABOUT 2118 01:53:56,973 --> 01:53:58,875 CAPSID ASSEMBLY AND THE OTHER 2119 01:53:58,875 --> 01:54:00,377 HAS TO DO WITH NUCLEAR IMPORT. 2120 01:54:00,377 --> 01:54:04,981 IN MY GROUP WE SPECIALIZED FOR 2121 01:54:04,981 --> 01:54:07,651 ABOUT 20 YEARS IN THE CORES 2122 01:54:07,651 --> 01:54:09,619 GRAIN MODELLING AND TAKE 2123 01:54:09,619 --> 01:54:10,921 STRUCTURE INTERACTIONS AND 2124 01:54:10,921 --> 01:54:13,123 SIMPLIFY THEM TO GET A 2125 01:54:13,123 --> 01:54:15,592 COMPUTATIONALLY EFFICIENT 2126 01:54:15,592 --> 01:54:15,892 METHODOLOGY. 2127 01:54:15,892 --> 01:54:18,995 FOR EXAMPLE, ON THE CAPSID IF 2128 01:54:18,995 --> 01:54:22,265 YOU WANT TO SIMULATE JUST A 2129 01:54:22,265 --> 01:54:28,972 SINGLE CAPSID IT'S CLOSE TO 100 2130 01:54:28,972 --> 01:54:31,074 BILLION ATOMS AND DEMANDING AND 2131 01:54:31,074 --> 01:54:32,442 YOU HAVE TO DO THE METHODS WE 2132 01:54:32,442 --> 01:54:32,809 HAVE DEVELOPED. 2133 01:54:32,809 --> 01:54:36,213 I WANT TO TELL YOU ABOUT THE IP6 2134 01:54:36,213 --> 01:54:40,250 STORY AND I THINK IT GOES BACK 2135 01:54:40,250 --> 01:54:42,719 TO THIS PAPER FIRST YOU LOOK AT 2136 01:54:42,719 --> 01:54:43,687 THE IN VITRO EXPERIMENTS IF YOU 2137 01:54:43,687 --> 01:54:47,257 DON'T HAVE IP6 YOU TEND TO GET 2138 01:54:47,257 --> 01:54:48,959 LOTS OF TUBULES. 2139 01:54:48,959 --> 01:54:52,128 IF YOU ADD A BIT OF IP6 YOU 2140 01:54:52,128 --> 01:54:56,132 START GETTING CIGARS AND YOU CAN 2141 01:54:56,132 --> 01:54:58,101 START TO SEE CORES AND SMALLER 2142 01:54:58,101 --> 01:54:59,536 STRUCTURE AND THE QUESTION IS 2143 01:54:59,536 --> 01:55:01,071 HOW DOES THAT HAPPEN? 2144 01:55:01,071 --> 01:55:04,641 WELL, WE WERE ABLE TO TRANSLATE 2145 01:55:04,641 --> 01:55:06,676 THIS INFORMATION INTO THE CORES 2146 01:55:06,676 --> 01:55:10,547 GRAIN MODELS AND IF YOU DO AT 2147 01:55:10,547 --> 01:55:16,586 ATOMISTIC CALCULATIONS IT BINDS 2148 01:55:16,586 --> 01:55:18,388 BETTER THAN TO HEXAMERS AND THIS 2149 01:55:18,388 --> 01:55:21,625 IS CRITICAL. 2150 01:55:21,625 --> 01:55:23,660 I'LL SHOW YOU IS FROM A PAPER 2151 01:55:23,660 --> 01:55:25,896 YOU MAY NOT HAVE READ THE 2152 01:55:25,896 --> 01:55:26,396 SUPPORTING INFORMATION. 2153 01:55:26,396 --> 01:55:27,964 THE SUPPORTING INFORMATION HAS 2154 01:55:27,964 --> 01:55:30,333 MOVIES IN IT BUT WHAT I'LL SHOW 2155 01:55:30,333 --> 01:55:33,003 YOU IS TIME AND THIS IS THE SIZE 2156 01:55:33,003 --> 01:55:37,007 OF THE ASSEMBLY UP TO 1,000 THE 2157 01:55:37,007 --> 01:55:40,911 GRAY HASHING IS OVER MULTIPLE 2158 01:55:40,911 --> 01:55:43,113 TRAJECTORIES AND AN INITIAL SEED 2159 01:55:43,113 --> 01:55:44,748 YOU'LL SEE A CRITICAL GROWTH OF 2160 01:55:44,748 --> 01:55:48,151 A SHEET AND THE ROLE OF IP6 2161 01:55:48,151 --> 01:55:49,953 AROUND STABILIZING THAT FORMING 2162 01:55:49,953 --> 01:55:53,990 A CAP AND THEN A RAPID GROWTH OF 2163 01:55:53,990 --> 01:55:55,625 THE CAPSID AND THIS IS 2164 01:55:55,625 --> 01:55:55,926 PRODUCIBLE. 2165 01:55:55,926 --> 01:55:59,095 WHAT YOU WANT TO LOOK FOR IN THE 2166 01:55:59,095 --> 01:56:03,066 MOVIE IS THE IP6'S EARNING 2167 01:56:03,066 --> 01:56:08,605 SPHERES AND THE PENTAMERS AND 2168 01:56:08,605 --> 01:56:09,739 THIS IS THE ACTUAL SIMULATION 2169 01:56:09,739 --> 01:56:11,841 AND THIS IS TAKEN OUT OF THE 2170 01:56:11,841 --> 01:56:16,079 CONDENSED PHASE. 2171 01:56:16,079 --> 01:56:26,089 THE PENTAMERS COME AND GO AND 2172 01:56:26,089 --> 01:56:29,192 IP6 NAILS THEM AND WANTS TO GROW 2173 01:56:29,192 --> 01:56:31,161 A TUBULE BUT CAN'T AND YOU FORM 2174 01:56:31,161 --> 01:56:33,096 THE LONG BODY AND THEN CLOSE AND 2175 01:56:33,096 --> 01:56:34,764 THIS HAPPENS OVER AND OVER AGAIN 2176 01:56:34,764 --> 01:56:39,803 IN THE SIMULATIONS. 2177 01:56:39,803 --> 01:56:45,575 IF YOU LOOK AT THIS PLOT THERE'S 2178 01:56:45,575 --> 01:56:48,578 A FINICKY THING I'M STEPPING 2179 01:56:48,578 --> 01:56:49,813 ON -- OH, NO. 2180 01:56:49,813 --> 01:56:56,987 THAT'S THE LAST THING WE NEEDED. 2181 01:56:56,987 --> 01:57:07,097 SORRY. 2182 01:57:09,032 --> 01:57:09,199 OKAY. 2183 01:57:09,199 --> 01:57:10,800 THIS IS A VERY INTERESTING PLOT. 2184 01:57:10,800 --> 01:57:12,969 THIS IS IT TIME. 2185 01:57:12,969 --> 01:57:16,706 THIS IS ASSEMBLY SIZE AND LOOK 2186 01:57:16,706 --> 01:57:22,545 AT THE PENTAMERS THAT FORM AND 2187 01:57:22,545 --> 01:57:28,852 STABILIZE BY IP6 IS 2 OF 4 OF 12 2188 01:57:28,852 --> 01:57:31,021 AND THIS IS A KINETIC SIGNATURE 2189 01:57:31,021 --> 01:57:31,554 OF THE CAPSID GROWTH. 2190 01:57:31,554 --> 01:57:33,723 IF YOU LOOK AT A CONTROL 2191 01:57:33,723 --> 01:57:36,326 SIMULATION WHERE YOU DON'T HAVE 2192 01:57:36,326 --> 01:57:38,828 IP6 -- I'M GOING TO FORM TUBULES 2193 01:57:38,828 --> 01:57:40,230 OR MAYBE THEY'RE CAPPED A LITTLE 2194 01:57:40,230 --> 01:57:41,731 BIT. 2195 01:57:41,731 --> 01:57:43,667 YOU GET A LITTLE BIT OF THAT 2196 01:57:43,667 --> 01:57:44,601 SIGMOIDAL BEHAVIOR BUT NOT MUCH. 2197 01:57:44,601 --> 01:57:48,972 IT DOES NOT HAVE THE SAME 2198 01:57:48,972 --> 01:57:50,540 KINETIC SIGNATURE AND OF COURSE 2199 01:57:50,540 --> 01:57:51,207 KEEPS GROWING. 2200 01:57:51,207 --> 01:57:57,914 YOU CAN KEEP GROWING A TUBE -- 2201 01:57:57,914 --> 01:58:00,750 TUBULE AND PENTAMERS COME AS 2202 01:58:00,750 --> 01:58:02,218 DEFECTS IN THE LATTICE AND COME 2203 01:58:02,218 --> 01:58:03,386 AND HEAL AND THERE'S NOTHING 2204 01:58:03,386 --> 01:58:05,522 LIKE IP6 TO STABILIZE THEM AND 2205 01:58:05,522 --> 01:58:12,095 YOU'LL END UP WITH A TUBULE. 2206 01:58:12,095 --> 01:58:14,030 SO THIS IS FASCINATING IF YOU 2207 01:58:14,030 --> 01:58:17,667 THINK ABOUT IT FROM A PHYSICAL 2208 01:58:17,667 --> 01:58:18,768 POINT OF VIEW. 2209 01:58:18,768 --> 01:58:21,971 YOU GET THESE ARE JOHN BRIGGS 2210 01:58:21,971 --> 01:58:24,674 TOMOGRAMS WE INVERTED AND TURNED 2211 01:58:24,674 --> 01:58:28,978 INTO BACK MAPPING METHODS AND 2212 01:58:28,978 --> 01:58:31,581 MOLECULAR STRUCTURES AND YOU GET 2213 01:58:31,581 --> 01:58:34,017 THESE PLEAO MORPHIC CAPSIDS AND 2214 01:58:34,017 --> 01:58:35,685 SOMETIMES YOU GET THE PERFECT 2215 01:58:35,685 --> 01:58:39,155 ONE AND YOU CAN GET THESE PILLS 2216 01:58:39,155 --> 01:58:40,323 AND THEY ALL HAVE DIFFERENT 2217 01:58:40,323 --> 01:58:48,098 BEHAVIOR AND I'M LED TO CONCLUDE 2218 01:58:48,098 --> 01:58:52,102 THESE ARE KINETICALLY ARRESTED 2219 01:58:52,102 --> 01:58:58,174 AND WOULD HAVE TO BE A MINNINA 2220 01:58:58,174 --> 01:59:01,644 TO GET KINETICALLY ARRESTED. 2221 01:59:01,644 --> 01:59:04,013 THE COMPETITION BETWEEN 2222 01:59:04,013 --> 01:59:04,647 LENACAPAVIR AND IP6. 2223 01:59:04,647 --> 01:59:15,091 THE EXPERIMENTS APPEARED. 2224 01:59:21,297 --> 01:59:25,735 AND THIS IS HARD TO SEE. 2225 01:59:25,735 --> 01:59:32,008 WHAT HAPPENS IS THIS IS THE 1:1. 2226 01:59:32,008 --> 01:59:35,211 IP6 WOULD LOVE TO STABILIZE 2227 01:59:35,211 --> 01:59:39,015 PENTAMERS AND IT CAN'T. 2228 01:59:39,015 --> 01:59:44,354 THE LENACAPAVIR IS BINDING AND 2229 01:59:44,354 --> 01:59:48,992 BINDING THE HEXAMERIC 2230 01:59:48,992 --> 01:59:51,161 ENVIRONMENT AND DOES NOT SUCCEED 2231 01:59:51,161 --> 01:59:53,763 AND THIS BEGINS TO STABILIZE 2232 01:59:53,763 --> 01:59:55,165 PENTAMERS. 2233 01:59:55,165 --> 01:59:58,568 THIS SOMETHING DIFFERENT ABOUT 2234 01:59:58,568 --> 02:00:00,637 THE STIMULATION LARGER PIECES OF 2235 02:00:00,637 --> 02:00:02,705 HEXAMER FORM FROM THE CONDENSED 2236 02:00:02,705 --> 02:00:04,941 SIMULATION AND IP6 IS ABLE TO 2237 02:00:04,941 --> 02:00:06,109 HELP STITCH THEM TOGETHER AND 2238 02:00:06,109 --> 02:00:10,246 YOU END UP WITH A CAPSID. 2239 02:00:10,246 --> 02:00:13,783 WE HAVE BEEN ASKING OURSELVES 2240 02:00:13,783 --> 02:00:15,451 THIS IS INTERESTING FOR IN VITRO 2241 02:00:15,451 --> 02:00:18,488 ASSEMBLY BUT IN THE VIRUS YOU 2242 02:00:18,488 --> 02:00:22,158 NEED TO INTEGRATE THE RNP IN THE 2243 02:00:22,158 --> 02:00:22,425 STRUCTURE. 2244 02:00:22,425 --> 02:00:30,700 THESE ARE EXPLORATORY SIMULATION 2245 02:00:30,700 --> 02:00:32,335 AND THERE'S A MODEL IN THE 2246 02:00:32,335 --> 02:00:33,536 STRENGTH OF SIMULATION IS TO 2247 02:00:33,536 --> 02:00:35,839 TAKE MODELS AND DO EXPLORATION. 2248 02:00:35,839 --> 02:00:40,977 WHAT WE CAN DO IS MODULATE THIS 2249 02:00:40,977 --> 02:00:51,054 RNP STRUCTURE TO EXPRESS MORE OR 2250 02:00:51,054 --> 02:00:52,989 LESS IN THE STRUCTURE AND YOU 2251 02:00:52,989 --> 02:00:56,125 FIND IT'S SENSITIVE TO THE 2252 02:00:56,125 --> 02:01:02,866 AMOUNT ON THE SURFACE ON THE RNP 2253 02:01:02,866 --> 02:01:06,502 AND IF YOU HAVE RELATIVE FEW 2254 02:01:06,502 --> 02:01:07,203 YOU'LL HAVE WHAT I TOLD YOU 2255 02:01:07,203 --> 02:01:09,105 ABOUT AND IF YOU HAVE TOO MANY 2256 02:01:09,105 --> 02:01:11,574 YOU BEGIN TO MESS UP THE 2257 02:01:11,574 --> 02:01:11,975 CAPSIDS. 2258 02:01:11,975 --> 02:01:13,610 SOMETIMES YOU GET WEIRD CAPSID 2259 02:01:13,610 --> 02:01:14,043 LOOKING THINGS. 2260 02:01:14,043 --> 02:01:22,452 THIS IS VERY FASCINATIFASCINATI. 2261 02:01:22,452 --> 02:01:26,022 ARE AND THERE'S THIS RELEVANT TO 2262 02:01:26,022 --> 02:01:28,992 THE SWITCH AND THE OTHER IS FROM 2263 02:01:28,992 --> 02:01:34,764 A LABORATORY SHOWING THE 2264 02:01:34,764 --> 02:01:45,308 IMPORTANCE OF PENTAMERIC BINDING 2265 02:01:45,308 --> 02:01:46,776 AND WE UNDERSTAND THERE'S A 2266 02:01:46,776 --> 02:01:47,176 COUPLE ISSUES. 2267 02:01:47,176 --> 02:01:51,281 ONE IS OF THE CAPSID 2268 02:01:51,281 --> 02:01:53,016 INTO THE NUCLEAR CORE AND WHAT 2269 02:01:53,016 --> 02:01:56,819 INTERFERES AND WHAT HAPPENS TO 2270 02:01:56,819 --> 02:01:58,855 IT AND PASSING THROUGH THE 2271 02:01:58,855 --> 02:02:00,023 NUCLEAR BASKET AND GETTING INTO 2272 02:02:00,023 --> 02:02:00,356 THE NUCLEUS. 2273 02:02:00,356 --> 02:02:02,325 I'M GOING TO TALK ABOUT THE 2274 02:02:02,325 --> 02:02:02,625 FIRST THING. 2275 02:02:02,625 --> 02:02:05,628 WHAT WE WERE LUCKY TO HAVE WAS A 2276 02:02:05,628 --> 02:02:08,197 STRUCTURE OF THE NUCLEAR CORE. 2277 02:02:08,197 --> 02:02:09,666 THERE'S BEEN SEVERAL COME OUT 2278 02:02:09,666 --> 02:02:11,501 FROM BECK'S GROUP IS THE ONE WE 2279 02:02:11,501 --> 02:02:16,506 USE THE MOST AND ARE ABLE TO 2280 02:02:16,506 --> 02:02:18,741 PERFORM A BOTTOM-UP CORE SCREEN 2281 02:02:18,741 --> 02:02:20,176 MODEL AND THOUSANDS OF PROTEINS 2282 02:02:20,176 --> 02:02:22,111 YOU DON'T HAVE TO WORRY ABOUT 2283 02:02:22,111 --> 02:02:23,880 THE MATHEMATICS RIGHT NOW BUT 2284 02:02:23,880 --> 02:02:25,214 BELIEVE WE DO IT SYSTEMATICALLY 2285 02:02:25,214 --> 02:02:28,885 AND GET A NUCLEAR CORE MODEL. 2286 02:02:28,885 --> 02:02:30,853 AND WE CAN START SIMULATING 2287 02:02:30,853 --> 02:02:33,089 DOCKING OF CAPSIDS INTO THIS 2288 02:02:33,089 --> 02:02:41,297 NUCLEAR CORE. 2289 02:02:41,297 --> 02:02:43,232 YOU TOOK MOST OF MY TIME SO YOU 2290 02:02:43,232 --> 02:02:45,001 SHOULD CHILL FOR A MINUTE, OKAY. 2291 02:02:45,001 --> 02:02:48,738 SORRY. 2292 02:02:48,738 --> 02:02:50,940 YEAH, CHILL OUT LITERALLY. 2293 02:02:50,940 --> 02:02:52,875 SO WHAT YOU FIND IN THE NUCLEAR 2294 02:02:52,875 --> 02:02:54,544 CORE ENTRY I HAVE TO SHOW YOU 2295 02:02:54,544 --> 02:02:55,244 THIS MOVIE BECAUSE IT'S 2296 02:02:55,244 --> 02:03:00,416 IMPORTANT. 2297 02:03:00,416 --> 02:03:05,154 IT'S AN ELECTROSTATIC RATCHET 2298 02:03:05,154 --> 02:03:09,892 AND IT RACH ETS INTO THE NUCLEAR 2299 02:03:09,892 --> 02:03:11,928 IN A STOCHASTIC WAY AND DEPENDS 2300 02:03:11,928 --> 02:03:12,762 ON THE ORIENTATION. 2301 02:03:12,762 --> 02:03:18,901 IF YOU'RE ORIENTED THE WRONG WAY 2302 02:03:18,901 --> 02:03:23,039 YOU GET SPIT OUT THE PILL LOOKS 2303 02:03:23,039 --> 02:03:24,974 LIKE IT CAN GO THROUGH THE CORE 2304 02:03:24,974 --> 02:03:27,176 BUT HAS OTHER VULNERABILITIES TO 2305 02:03:27,176 --> 02:03:28,177 IT. 2306 02:03:28,177 --> 02:03:29,579 SOMETIMES YOU CRACK THE NUCLEAR 2307 02:03:29,579 --> 02:03:29,779 CORE. 2308 02:03:29,779 --> 02:03:32,281 THE PAPER JUST CAME OUT AND WE 2309 02:03:32,281 --> 02:03:34,417 SEE THESE NATURALLY IN OUR 2310 02:03:34,417 --> 02:03:34,717 SIMULATIONS. 2311 02:03:34,717 --> 02:03:39,088 THEY MAKE A BIG DEAL OUT OF IT. 2312 02:03:39,088 --> 02:03:43,659 BUT NOW, THE PUNCH LINE THIS 2313 02:03:43,659 --> 02:03:44,994 SHOWS HOW IMPORTANT IT IS IN THE 2314 02:03:44,994 --> 02:03:45,328 ENTRY. 2315 02:03:45,328 --> 02:03:48,097 IT OBSCURES THE VIEW. 2316 02:03:48,097 --> 02:03:57,473 LET ME FINISH UP STEFAN. 2317 02:03:57,473 --> 02:04:02,111 THE CAPSID CAN COMPRESS AND 2318 02:04:02,111 --> 02:04:02,378 CONTRACT. 2319 02:04:02,378 --> 02:04:05,214 IT CAN OBSERVE PUNISHMENT AND 2320 02:04:05,214 --> 02:04:10,053 MAKE THE ENTROPY GO UP SO MINUS 2321 02:04:10,053 --> 02:04:13,256 TS GOES UP AND ENERGY GOES DOWN 2322 02:04:13,256 --> 02:04:16,292 AND HAS AN ABILITY TO WITHSTAND 2323 02:04:16,292 --> 02:04:20,763 THE NUCLEAR CORE ENTRY AS IT'S 2324 02:04:20,763 --> 02:04:24,534 GETTING SQUEEZED AND WHEN YOU 2325 02:04:24,534 --> 02:04:26,736 COVER IN LENACAPAVIR IT LIKES TO 2326 02:04:26,736 --> 02:04:28,971 FIND DISORDERED REGIONS. 2327 02:04:28,971 --> 02:04:31,874 I DON'T MEAN TOTALLY DISORDERED, 2328 02:04:31,874 --> 02:04:42,385 I MEAN DISTORTED REGIONS AND 2329 02:04:45,221 --> 02:04:49,826 REGIDIFIES IT AND IT WILL 2330 02:04:49,826 --> 02:04:50,560 RUPTURE. 2331 02:04:50,560 --> 02:04:53,396 THIS IS A MOVIE. 2332 02:04:53,396 --> 02:04:55,231 WE LOVE THEM AND WORK HARD TO 2333 02:04:55,231 --> 02:04:58,167 GET THEM. 2334 02:04:58,167 --> 02:05:04,140 THIS WILL RUPTURE BUT THAT 2335 02:05:04,140 --> 02:05:05,408 PERTURBATION PROPAGATES RAPIDLY 2336 02:05:05,408 --> 02:05:08,644 UP HERE AND YOU'LL START TO SEE 2337 02:05:08,644 --> 02:05:09,912 PENTAMERS FAIL AND BREAK A PART 2338 02:05:09,912 --> 02:05:13,049 AND WHAT'S FASCINATING IS I'LL 2339 02:05:13,049 --> 02:05:20,823 CALL IT ALOESTERY AND IT GOES 2340 02:05:20,823 --> 02:05:21,190 QUICKLY. 2341 02:05:21,190 --> 02:05:22,925 AND YOU BEGIN TO SEE THIS AND 2342 02:05:22,925 --> 02:05:27,396 THEN WE BEGIN TO SEE A SEAM 2343 02:05:27,396 --> 02:05:30,833 BREAK UP AND WAY FROM THE 2344 02:05:30,833 --> 02:05:32,702 PENTAMER AND SEEMS TO MY EYE 2345 02:05:32,702 --> 02:05:35,004 TRACK WHERE LENACAPAVIR IS 2346 02:05:35,004 --> 02:05:38,107 BINDING AND SEE IT RUPTURE. 2347 02:05:38,107 --> 02:05:40,209 SO WE GOT BEAUTIFUL EXPERIMENTS 2348 02:05:40,209 --> 02:05:45,815 THAT SHOW CAPSID DOCKED IN THE 2349 02:05:45,815 --> 02:05:49,018 NUCLEAR CORE. 2350 02:05:49,018 --> 02:05:52,989 IT'S ALWAYS A BUMMER TO BE AT 2351 02:05:52,989 --> 02:05:56,859 THE END OF THE GRAVY TRAIN. 2352 02:05:56,859 --> 02:05:58,294 SO DNA RESULTS CONFIRM THAT. 2353 02:05:58,294 --> 02:06:00,096 SO ANYWAY, THE LAST THING IS 2354 02:06:00,096 --> 02:06:10,573 WE'RE CURRENTLY WORKING ON 2355 02:06:10,773 --> 02:06:12,975 CYCLOPHILIN A AND I WILL THANK 2356 02:06:12,975 --> 02:06:14,177 THE CREW WHO DID THIS AND 2357 02:06:14,177 --> 02:06:15,077 ENCOURAGE YOU TO GO TO THEIR 2358 02:06:15,077 --> 02:06:25,454 POSTER AND THAT'S IT. 2359 02:06:29,058 --> 02:06:30,560 >> REALLY GREAT TALKS. 2360 02:06:30,560 --> 02:06:32,595 AND I'M NOT SURE YOU'RE THE BEST 2361 02:06:32,595 --> 02:06:33,963 PERSON BUT THIS WAS MOST RELATED 2362 02:06:33,963 --> 02:06:35,431 TO YOUR TALK AND MIGHT BE BETTER 2363 02:06:35,431 --> 02:06:37,934 PEOPLE IN THE AUDIENCE TO 2364 02:06:37,934 --> 02:06:38,868 ADDRESS THIS AS WELL. 2365 02:06:38,868 --> 02:06:40,570 WHAT IS KNOWN ABOUT THE FUNCTION 2366 02:06:40,570 --> 02:06:46,008 OF AN RNA GENOME BEING PACKAGED 2367 02:06:46,008 --> 02:06:47,910 AND THE SIZE OF THE CORE THAT IS 2368 02:06:47,910 --> 02:06:50,713 THEN BUILT AROUND THAT RNA OR 2369 02:06:50,713 --> 02:06:52,381 THE RNA THAT'S NOT THERE BECAUSE 2370 02:06:52,381 --> 02:06:55,284 AN IMPORTANT DENOMINATOR IN YOUR 2371 02:06:55,284 --> 02:06:57,687 ARGUMENT IS THAT YOU HAVE A 2372 02:06:57,687 --> 02:06:59,522 SMALLER DNA SO THAT DOESN'T 2373 02:06:59,522 --> 02:07:00,523 BREAK IT. 2374 02:07:00,523 --> 02:07:02,391 YOU'RE ASSUMING THAT THE CAPSID 2375 02:07:02,391 --> 02:07:04,594 IS THE SAME SIZE ACROSS ALL 2376 02:07:04,594 --> 02:07:05,828 THOSE DIFFERENT GENOMES. 2377 02:07:05,828 --> 02:07:07,296 HAVE YOU LOOKED AT THAT 2378 02:07:07,296 --> 02:07:10,733 DIRECTLY? 2379 02:07:10,733 --> 02:07:13,269 >> NO, WE HAVE NOT. 2380 02:07:13,269 --> 02:07:16,305 BUT WE GET ESSENTIALLY NORMAL 2381 02:07:16,305 --> 02:07:19,275 SIZE CONES EVEN IN THE ABSENCE 2382 02:07:19,275 --> 02:07:21,410 OF PACKAGEABLE GENOMIC RNA. 2383 02:07:21,410 --> 02:07:24,213 SO WE DON'T THINK THAT GENOME 2384 02:07:24,213 --> 02:07:24,981 SIZE MAKES MUCH DIFFERENCE TO 2385 02:07:24,981 --> 02:07:27,516 THE SIZE OF THE CAPSID. 2386 02:07:27,516 --> 02:07:31,087 IT'S JUST DESIGN TO ASSEMBLE 2387 02:07:31,087 --> 02:07:32,989 INTO WHATEVER IT LOOKS LIKE. 2388 02:07:32,989 --> 02:07:35,291 AND THAT'S MY VIEW. 2389 02:07:35,291 --> 02:07:36,792 OTHERS MAY COMMENT. 2390 02:07:36,792 --> 02:07:37,960 >> I THINK THAT'S CONSISTENT 2391 02:07:37,960 --> 02:07:38,527 WITH YOUR DATA. 2392 02:07:38,527 --> 02:07:43,299 IT COULD BE INTERESTING TO LOOK 2393 02:07:43,299 --> 02:07:45,635 DIRECTLY. 2394 02:07:45,635 --> 02:07:47,236 AND SECOND PART IS THAT UNDER 2395 02:07:47,236 --> 02:07:48,404 SOME CONDITIONS WHERE YOU 2396 02:07:48,404 --> 02:07:51,207 MANIPULATED THE POSITIONS OF 2397 02:07:51,207 --> 02:07:58,314 ENCODING EITHER BY P90A OR 2398 02:07:58,314 --> 02:07:59,382 CYCLOPHILIN KNOCKOUT WE AND 2399 02:07:59,382 --> 02:08:04,987 OTHERS PUBLISHED IT ALTERS SITE 2400 02:08:04,987 --> 02:08:08,190 WHERE THE INTEGRATION TAKES 2401 02:08:08,190 --> 02:08:09,358 PLACE HAVE YOU LOOK AT 2402 02:08:09,358 --> 02:08:10,993 INTEGRATION SITES FOR YOUR 2403 02:08:10,993 --> 02:08:11,294 GENOMES? 2404 02:08:11,294 --> 02:08:14,430 WE'RE STARTING TO LOOK AT THAT 2405 02:08:14,430 --> 02:08:16,132 AND RYAN'S GOING TO TALK ABOUT 2406 02:08:16,132 --> 02:08:17,266 THAT IN THE B-HIVE MEETING IN A 2407 02:08:17,266 --> 02:08:18,134 COUPLE DAYS. 2408 02:08:18,134 --> 02:08:21,804 HE PRESENTED SOME OF IT AT COLD 2409 02:08:21,804 --> 02:08:26,175 SPRING HARBOR THIS YEAR. 2410 02:08:26,175 --> 02:08:28,511 INTERESTINGLY THE SHORT VECTORS 2411 02:08:28,511 --> 02:08:32,982 IF YOU BLOW THEM UP WITH 2412 02:08:32,982 --> 02:08:35,851 LENACAPAVIR IT ALTERS THE SITES 2413 02:08:35,851 --> 02:08:36,986 OF INTEGRATION WHICH WE THINK IS 2414 02:08:36,986 --> 02:08:40,589 INTERESTING. 2415 02:08:40,589 --> 02:08:51,133 >> SO THIS QUESTION IS FOR VINAY 2416 02:08:53,669 --> 02:08:54,103 AND KAREN. 2417 02:08:54,103 --> 02:08:55,604 I'M WITH STONY BROOK. 2418 02:08:55,604 --> 02:08:58,240 THEY'RE KNOWN TO PACKAGE A LOT 2419 02:08:58,240 --> 02:09:00,976 OF RNA DO YOU THINK PERHAPS 2420 02:09:00,976 --> 02:09:02,345 EVOLUTIONARY OR WHATEVER THAT IS 2421 02:09:02,345 --> 02:09:04,046 ACTUALLY PLAYING AN IMPORTANT 2422 02:09:04,046 --> 02:09:05,314 ROLE IN THE STABILIZATION 2423 02:09:05,314 --> 02:09:09,952 FUNCTION? 2424 02:09:09,952 --> 02:09:12,988 >> STABILIZATION OF THE CORE? 2425 02:09:12,988 --> 02:09:17,293 >> YEAH. 2426 02:09:17,293 --> 02:09:18,994 >> I THINK RNA PLAYS A CRITICAL 2427 02:09:18,994 --> 02:09:20,596 ROLE IN THE ASSEMBLY. 2428 02:09:20,596 --> 02:09:24,500 IN ABSENCE OF THE GENOMIC RNA 2429 02:09:24,500 --> 02:09:28,304 THEY PACKAGE RANDOM RNAs BUT I 2430 02:09:28,304 --> 02:09:33,976 THINK HAVING RNA IS CRITICAL FOR 2431 02:09:33,976 --> 02:09:36,212 FORMING A PROPERLY SHAPED CAPSID 2432 02:09:36,212 --> 02:09:38,314 WITH THE RIGHT STABILITY. 2433 02:09:38,314 --> 02:09:39,815 THAT'S WHAT I THINK. 2434 02:09:39,815 --> 02:09:41,717 >> I'M DRAWN TO THE QUESTION 2435 02:09:41,717 --> 02:09:43,919 BECAUSE OTHER VIRUSES, 2436 02:09:43,919 --> 02:09:47,757 NON-RETROS WILL PACKAGE A LOT OF 2437 02:09:47,757 --> 02:09:49,291 SMALL RNAs AND WONDERING IF YOU 2438 02:09:49,291 --> 02:09:52,962 HAVE AN ACROSS THE BOARD MOTION 2439 02:09:52,962 --> 02:09:54,463 OF THEIR ROLE. 2440 02:09:54,463 --> 02:09:55,064 THANK YOU. 2441 02:09:55,064 --> 02:09:56,332 >> THANK YOU. 2442 02:09:56,332 --> 02:10:00,970 >> SO FOR KAREN, SO YOU SHOWED A 2443 02:10:00,970 --> 02:10:06,108 DISTRIBUTION OF WHEN YOU WOULD 2444 02:10:06,108 --> 02:10:08,310 USE LEN IN COMBINATION WITH 2445 02:10:08,310 --> 02:10:10,679 OTHER MOLECULES YOU GET A 2446 02:10:10,679 --> 02:10:11,113 DISTRIBUTION. 2447 02:10:11,113 --> 02:10:14,216 SO DO YOU THINK YOU MAY BE ABLE 2448 02:10:14,216 --> 02:10:17,686 TO DESIGN EXPERIMENTS THAT WOULD 2449 02:10:17,686 --> 02:10:20,990 ACTUALLY TARGET WHAT THE 2450 02:10:20,990 --> 02:10:23,859 FREQUENCY WILL BE FREQUENCY SO 2451 02:10:23,859 --> 02:10:26,562 YOU'LL CONTROL HIGH OR LOW 2452 02:10:26,562 --> 02:10:27,530 FREQUENCY A LOT OF LEN VERSUS A 2453 02:10:27,530 --> 02:10:29,398 LITTLE OF THE OTHER AND VICE 2454 02:10:29,398 --> 02:10:29,932 VERSA. 2455 02:10:29,932 --> 02:10:31,700 >> YES, I THINK WE CAN 2456 02:10:31,700 --> 02:10:33,502 DEFINITELY DO THAT. 2457 02:10:33,502 --> 02:10:35,471 I THINK WE CAN -- YEAH, WE CAN 2458 02:10:35,471 --> 02:10:36,038 DO THAT. 2459 02:10:36,038 --> 02:10:39,141 I'M SORRY, THE VIROLOGY 2460 02:10:39,141 --> 02:10:41,010 EXPERIMENTS WERE DONE BY OTHERS 2461 02:10:41,010 --> 02:10:43,846 IN THE LAB SO I'M NOT AS 2462 02:10:43,846 --> 02:10:45,014 FAMILIAR BUT YES, IT SHOULD BE 2463 02:10:45,014 --> 02:10:46,282 ABLE TO BE DONE. 2464 02:10:46,282 --> 02:10:50,953 >> I HAVE A QUESTION FOR VINAY. 2465 02:10:50,953 --> 02:10:57,026 DO YOU THINK YOUR EXTENDS TO 2466 02:10:57,026 --> 02:10:58,727 RETROVIRAL VECTORS IN GENERAL 2467 02:10:58,727 --> 02:11:01,297 BECAUSE IN MY EXPERIENCE THEY'RE 2468 02:11:01,297 --> 02:11:04,667 OFTEN BETTER IF THEY HAVE NO 2469 02:11:04,667 --> 02:11:08,103 INSERT, IN OTHER WORDS, THEY'RE 2470 02:11:08,103 --> 02:11:10,306 PRETTY SHORT THAN INSERT 2471 02:11:10,306 --> 02:11:12,808 CONTAINING VECTORS. 2472 02:11:12,808 --> 02:11:14,577 >> YEAH, THAT'S A VERY 2473 02:11:14,577 --> 02:11:17,012 INTERESTING QUESTION. 2474 02:11:17,012 --> 02:11:21,417 THE GENOMES FOR HIV THE LIMIT OF 2475 02:11:21,417 --> 02:11:25,020 THE SIZE THAT WE'RE TALKING 2476 02:11:25,020 --> 02:11:28,991 ABOUT THE 2. 8, 3.5KV ARE ALL 2477 02:11:28,991 --> 02:11:34,063 THE ELEMENTS WITHOUT INSERTS IN 2478 02:11:34,063 --> 02:11:38,133 THEM. 2479 02:11:38,133 --> 02:11:41,837 I THINK MOST RETROVIRAL ARE 2480 02:11:41,837 --> 02:11:43,973 LIKELY TO BE THIS SIZE AND 2481 02:11:43,973 --> 02:11:47,243 SECONDLY, THE SHORTER GENOMES 2482 02:11:47,243 --> 02:11:49,011 SEEM TO REVERSE TRANSCRIBE MORE 2483 02:11:49,011 --> 02:11:50,646 EFFICIENTLY THAN THE LARGER 2484 02:11:50,646 --> 02:11:50,846 ONES. 2485 02:11:50,846 --> 02:11:54,049 THOUGH THEY MAY LOSE SOME OF 2486 02:11:54,049 --> 02:11:55,985 EFFICIENCY IN ENCODING THEY MAY 2487 02:11:55,985 --> 02:11:59,688 MAKE UP FOR IT BY ENHANCING 2488 02:11:59,688 --> 02:12:00,689 THEIR ABILITY TO COMPLETE 2489 02:12:00,689 --> 02:12:04,026 REVERSE TRANSCRIPTION. 2490 02:12:04,026 --> 02:12:07,329 SO IT MAY BE I THINK A BALANCE 2491 02:12:07,329 --> 02:12:07,830 OF THOSE TWO PROCESSES. 2492 02:12:07,830 --> 02:12:14,069 >> THANKS. 2493 02:12:14,069 --> 02:12:14,570 >> SORRY. 2494 02:12:14,570 --> 02:12:17,873 I LOVE THE IDEA OF THE MINIMAL 2495 02:12:17,873 --> 02:12:18,440 SIZE. 2496 02:12:18,440 --> 02:12:20,943 WE SPENT YEARS TRYING TO MAKE 2497 02:12:20,943 --> 02:12:24,079 TINY, TINY VECTORS AND WE SEEM 2498 02:12:24,079 --> 02:12:26,115 TO HIT A LIMIT NEAR YOUR DATA 2499 02:12:26,115 --> 02:12:27,683 OFFERS AN EXPLANATION. 2500 02:12:27,683 --> 02:12:33,055 ALSO WONDER CERTAIN ELEMENTS 2501 02:12:33,055 --> 02:12:35,991 LIKE THE WOOD CHOP WPRE ELEMENTS 2502 02:12:35,991 --> 02:12:39,595 CAN HELP THE ENCODING. 2503 02:12:39,595 --> 02:12:42,298 BIG CHUNKS OF DNA THAT RESULT. 2504 02:12:42,298 --> 02:12:45,000 I WAS CURIOUS, HAVE YOU IN YOUR 2505 02:12:45,000 --> 02:12:48,003 EXPERIMENTS HAVE YOU LOOKED 2506 02:12:48,003 --> 02:12:49,572 DIRECTLY AT WHETHER CYCLOPHILIN 2507 02:12:49,572 --> 02:12:51,874 A IS BINDING TO THE CORE IN 2508 02:12:51,874 --> 02:12:53,008 CYTOPLASM IN TRACKING WITH THE 2509 02:12:53,008 --> 02:12:55,511 CORE INTO THE NUCLEUS WITH THE 2510 02:12:55,511 --> 02:12:57,446 SAME MOLECULES REMAINING 2511 02:12:57,446 --> 02:12:58,147 ATTACHED? 2512 02:12:58,147 --> 02:13:00,616 >> YOU KNOW, THAT'S A GREAT 2513 02:13:00,616 --> 02:13:03,085 QUESTION. 2514 02:13:03,085 --> 02:13:04,420 WE TRIED A LITTLE BIT WITH 2515 02:13:04,420 --> 02:13:09,992 THEFUL CADS RED. 2516 02:13:09,992 --> 02:13:14,396 WE THINK THE CYCLOPHILIN IS 2517 02:13:14,396 --> 02:13:15,831 REMOVED DURING THE PROCESS AND 2518 02:13:15,831 --> 02:13:17,833 YOU MAY HAVE A BETTER IDEA BIS 2519 02:13:17,833 --> 02:13:21,937 THIS BUT I WONDER WHETHER THE 2520 02:13:21,937 --> 02:13:24,506 CYCLOPHILIN A CAN GET IMPORTED 2521 02:13:24,506 --> 02:13:27,009 AND REBIND THE CAPSIDS ONCE THEY 2522 02:13:27,009 --> 02:13:28,277 GO IN AND THAT'S ONE OF THE 2523 02:13:28,277 --> 02:13:32,781 THINGS WE'RE THINKING ABOUT BUT 2524 02:13:32,781 --> 02:13:34,350 WE HAVEN'T DIRECTLY VISUALIZED 2525 02:13:34,350 --> 02:13:35,484 BOUND TO THE CORE AND NUCLEUS. 2526 02:13:35,484 --> 02:13:39,221 WE HAVE TO DO THAT. 2527 02:13:39,221 --> 02:13:41,390 >> I HAD A QUESTION FOR GREG. 2528 02:13:41,390 --> 02:13:43,025 DO YOU THINK YOU CAN START TO 2529 02:13:43,025 --> 02:13:45,628 ADDRESS ALL THE CAPSID SHAPES 2530 02:13:45,628 --> 02:13:48,297 YOU SEE OF MODELLING OF WHICH 2531 02:13:48,297 --> 02:13:50,599 WILL GENERATE PRODUCTIVE 2532 02:13:50,599 --> 02:13:50,966 INFECTION? 2533 02:13:50,966 --> 02:13:53,268 >> THAT'S THE IDEA. 2534 02:13:53,268 --> 02:13:55,337 WE LOOKED AT WHICH ONES MAY BE 2535 02:13:55,337 --> 02:13:56,572 ABLE TO DOCK WELL IN THE NUCLEAR 2536 02:13:56,572 --> 02:13:57,106 CORE. 2537 02:13:57,106 --> 02:14:00,542 WE HAVE OTHER ELEMENTS BUT 2538 02:14:00,542 --> 02:14:03,612 THAT'S A GOAL, I THINK IN HOW 2539 02:14:03,612 --> 02:14:06,215 IMPORTANT THAT PERFECT CONE OR 2540 02:14:06,215 --> 02:14:07,349 NEARLY PERFECT CONE WE'VE BEEN 2541 02:14:07,349 --> 02:14:09,418 LOOKING AT SINCE 1999. 2542 02:14:09,418 --> 02:14:13,322 SO FAR IT LOOKS IMPORTANT TO 2543 02:14:13,322 --> 02:14:14,990 DOCK PROPERLY. 2544 02:14:14,990 --> 02:14:17,826 THIS PILL SHAPED THINGS AND IT'S 2545 02:14:17,826 --> 02:14:18,994 FRAGILE AND BREAKS A PART 2546 02:14:18,994 --> 02:14:19,228 EASILY. 2547 02:14:19,228 --> 02:14:19,995 YEAH. 2548 02:14:19,995 --> 02:14:22,064 >> THANK YOU. 2549 02:14:22,064 --> 02:14:38,224 [APPLAUSE] 2550 02:14:38,224 --> 02:14:43,029 >> WE'LL BE UP NEXT AND 2551 02:14:43,029 --> 02:14:48,601 DR. KROGAN WILL KICK IT OFF. 2552 02:14:48,601 --> 02:14:50,703 >> IT'S A GREAT PLEASURE TO GIVE 2553 02:14:50,703 --> 02:14:57,210 YOU AN UPDATE ON THE HARC CENTER 2554 02:14:57,210 --> 02:14:59,812 STAND FOR HIV ACCESSORY AND 2555 02:14:59,812 --> 02:15:00,446 REGULATORY COMPLEX. 2556 02:15:00,446 --> 02:15:04,117 WE HAVE 18 P.I.s PRIMARILY BASED 2557 02:15:04,117 --> 02:15:06,152 IN CALIFORNIA. 2558 02:15:06,152 --> 02:15:11,324 13 P.I.s ARE AT UCSF, GLADSTONE 2559 02:15:11,324 --> 02:15:13,126 AND BERKELEY AND WE INCLUDED THE 2560 02:15:13,126 --> 02:15:18,598 HUTCH UP THE COST AND LILLY AND 2561 02:15:18,598 --> 02:15:22,635 MICHAEL IN SEATTLE, JUD AT 2562 02:15:22,635 --> 02:15:27,640 NORTHWEST AND NORTH CAROLINA. 2563 02:15:27,640 --> 02:15:34,147 IN OUR MORE RECENT CARNATION WE 2564 02:15:34,147 --> 02:15:38,518 HAVE STRUCTURE AND EVOLUTION ON 2565 02:15:38,518 --> 02:15:40,219 DEGRADATION AND EVOLUTION AND IN 2566 02:15:40,219 --> 02:15:41,287 THE JUST RESTRICTION BUT 2567 02:15:41,287 --> 02:15:42,121 PACKAGING. 2568 02:15:42,121 --> 02:15:43,656 WE'RE LOOKING AT PACKAGING WITH 2569 02:15:43,656 --> 02:15:46,125 RESPECT TO THIS INTERACTION IN 2570 02:15:46,125 --> 02:15:47,527 THE COMPLEX. 2571 02:15:47,527 --> 02:15:49,495 PROJECT 2 IS REGULATION OF HIV 2572 02:15:49,495 --> 02:15:52,332 TRANSCRIPTION AND LATENCY AND 2573 02:15:52,332 --> 02:15:54,133 WE'RE LOOKING AT TRANSCRIPTION 2574 02:15:54,133 --> 02:15:57,970 AND DIFFERENT TRANSCRIPTIONAL 2575 02:15:57,970 --> 02:15:58,271 FACTORS. 2576 02:15:58,271 --> 02:16:00,206 AIM 2 LOOK AT EXPORT. 2577 02:16:00,206 --> 02:16:03,910 THE STUDY OF REV AND AIM 3 2578 02:16:03,910 --> 02:16:08,014 LATENCY SO BIG FORCE ON 2579 02:16:08,014 --> 02:16:09,549 CHROMATIN MODELLING AND PROJECT 2580 02:16:09,549 --> 02:16:11,884 3 IS GENETICS. 2581 02:16:11,884 --> 02:16:16,389 COHORTS INVOLVED AND 2582 02:16:16,389 --> 02:16:18,091 EVOLUTIONARY GENETICS LED BY 2583 02:16:18,091 --> 02:16:22,161 ALEX AND TRYING TO PUSH CRISPR 2584 02:16:22,161 --> 02:16:23,830 TECHNOLOGY AND MACROPHAGES 2585 02:16:23,830 --> 02:16:25,298 LOOKING AT DIFFERENT HOST 2586 02:16:25,298 --> 02:16:27,233 FACTORS WITH RESPECT TO POOLED 2587 02:16:27,233 --> 02:16:28,401 APPROACHES AND RATE APPROACHES 2588 02:16:28,401 --> 02:16:29,369 AND SOME PROGRESS ON THAT FRONT 2589 02:16:29,369 --> 02:16:30,870 TODAY. 2590 02:16:30,870 --> 02:16:33,239 AND IF YOU KNOW HARC YOU KNOW AT 2591 02:16:33,239 --> 02:16:35,208 TO THE HART OF HARC IS 2592 02:16:35,208 --> 02:16:35,508 TECHNOLOGY. 2593 02:16:35,508 --> 02:16:42,148 AND WE HAVE FOUR CORES THAT 2594 02:16:42,148 --> 02:16:44,650 WE'VE STITCHED TOGETHER THE 2595 02:16:44,650 --> 02:16:46,352 HERE'S THE CHARACTERS ASSOCIATED 2596 02:16:46,352 --> 02:16:47,854 WITH EACH ONE OF THE CORES. 2597 02:16:47,854 --> 02:16:50,189 FOR ME PERSONALLY IT'S BEEN SO 2598 02:16:50,189 --> 02:16:54,127 INTERESTING TO SEE HOW THE 2599 02:16:54,127 --> 02:16:58,197 TECHNOLOGY HAS BEEN INTEGRATED 2600 02:16:58,197 --> 02:17:01,267 TOGETHER AND PROVIDING 2601 02:17:01,267 --> 02:17:04,036 UNPRECEDENTED INSIGHT TO HIV 2602 02:17:04,036 --> 02:17:05,972 BIOLOGY AND PROTEIN-PROTEIN 2603 02:17:05,972 --> 02:17:10,143 INTERACTIONS AND COMPLEXES AND 2604 02:17:10,143 --> 02:17:13,546 NOW THAT A.I.s BEING WOVEN IN 2605 02:17:13,546 --> 02:17:15,715 I'M EXCITED TO SEE THE BIOLOGY. 2606 02:17:15,715 --> 02:17:18,418 HIGHLIGHTS IN THE LAST YEAR 2607 02:17:18,418 --> 02:17:21,654 WE'RE WORKED ON TARGETS MANY 2608 02:17:21,654 --> 02:17:21,888 YEAR. 2609 02:17:21,888 --> 02:17:28,161 AND THERE WAS A STRUCTURE ON THE 2610 02:17:28,161 --> 02:17:32,598 COMPLEX WITH RNA LED BY JOHN 2611 02:17:32,598 --> 02:17:35,401 GROSS AND A KEY DISCOVERY IS RNA 2612 02:17:35,401 --> 02:17:37,270 IS A KEY GLUE WITH REGARD TO 2613 02:17:37,270 --> 02:17:38,404 THIS COMPLEX. 2614 02:17:38,404 --> 02:17:40,807 THIS HAS BEEN PUBLISHED RECENTLY 2615 02:17:40,807 --> 02:17:46,913 AND HERE WE'RE LOOKING AT THE 2616 02:17:46,913 --> 02:17:53,853 COMPLEX THAT I THINK THESE ARE 2617 02:17:53,853 --> 02:17:55,087 WHY THEY'RE IMPORTANT AND THE 2618 02:17:55,087 --> 02:17:57,523 CENTERS ALLOW FOR RUNWAY AND THE 2619 02:17:57,523 --> 02:17:58,624 COLLABORATIVE INFRASTRUCTURE TO 2620 02:17:58,624 --> 02:18:00,026 SOLVE THESE IMPORTANT PROBLEMS. 2621 02:18:00,026 --> 02:18:01,160 AND THESE ARE LONG STANDING 2622 02:18:01,160 --> 02:18:01,894 PROBLEMS. 2623 02:18:01,894 --> 02:18:04,597 WE STARTED WORKING ON THIS 2624 02:18:04,597 --> 02:18:07,233 COMPLEX 17 YEARS AGO WHEN THESE 2625 02:18:07,233 --> 02:18:12,738 WERE INSTEAD OF U54s P50s AND I 2626 02:18:12,738 --> 02:18:14,140 COMPLEX ALEN'S WORKED ON OVER 20 2627 02:18:14,140 --> 02:18:14,941 YEARS. 2628 02:18:14,941 --> 02:18:16,676 TO ME WE NEED THE PROGRAMS TO 2629 02:18:16,676 --> 02:18:18,044 BRING COLLABORATORS TOGETHER TO 2630 02:18:18,044 --> 02:18:19,178 SOLVE IMPORTANT PROBLEMS LIKE 2631 02:18:19,178 --> 02:18:19,679 THIS. 2632 02:18:19,679 --> 02:18:20,746 WE HAVE OTHER BREAKTHROUGHS HERE 2633 02:18:20,746 --> 02:18:23,249 OVER THE LAST YEAR. 2634 02:18:23,249 --> 02:18:26,118 WE SHOWED THE HOST FACTOR 2635 02:18:26,118 --> 02:18:28,654 REQUIRED FOR HIV TRANSCRIPTION 2636 02:18:28,654 --> 02:18:31,824 AND LATENCY AND IDENTIFIED HIV 2637 02:18:31,824 --> 02:18:34,393 REGULATOR AND CD4 POSITIVE T 2638 02:18:34,393 --> 02:18:39,298 CELLS AND IT F1 INVOLVED IN HIV 2639 02:18:39,298 --> 02:18:40,666 LATENCY. 2640 02:18:40,666 --> 02:18:42,168 WHAT YOU'LL HEAR TODAY IS 2641 02:18:42,168 --> 02:18:46,172 FURTHER WORK WE'RE CARRYING OUT 2642 02:18:46,172 --> 02:18:48,975 LOOKING AT THE COMPLEXES FROM 2643 02:18:48,975 --> 02:18:51,210 CAROLYN AND MICHAEL EMERMAN'S 2644 02:18:51,210 --> 02:18:53,746 LAB LOOKING AT THE EVOLUTIONARY 2645 02:18:53,746 --> 02:18:55,214 CONSTRAINTS OF THE KEY 2646 02:18:55,214 --> 02:18:58,150 CONNECTION AND WE'LL ALSO HEAR 2647 02:18:58,150 --> 02:19:01,988 FROM WILLIAM TARGETING 2648 02:19:01,988 --> 02:19:03,022 TRANSCRIPTIONAL ELONGATION 2649 02:19:03,022 --> 02:19:04,023 LOOKING AT THE COMPLEX AND THESE 2650 02:19:04,023 --> 02:19:10,162 ARE CONNECTIONS THE HARC CENTER 2651 02:19:10,162 --> 02:19:14,166 UNCOVERED AND WE'RE SHOWING 2652 02:19:14,166 --> 02:19:18,905 TARGETING PROMOTES HIV-1 LATENCY 2653 02:19:18,905 --> 02:19:20,339 REVERSAL. 2654 02:19:20,339 --> 02:19:24,577 WE MADE BREAKTHROUGHS 2655 02:19:24,577 --> 02:19:27,346 TECHNOLOGICALLY INCLUDING WILL 2656 02:19:27,346 --> 02:19:28,948 KAS 9 T CELLS AND TISSUES. 2657 02:19:28,948 --> 02:19:31,717 WE HAVE SOMETHING WE ARE ABOUT 2658 02:19:31,717 --> 02:19:34,654 TO ANNOUNCE TIED WITH THE HARC 2659 02:19:34,654 --> 02:19:35,555 CENTER CALLED THE READY CENTER 2660 02:19:35,555 --> 02:19:37,757 AND WE'RE DEVELOPING A PIPELINE 2661 02:19:37,757 --> 02:19:41,494 WHERE WE CAN CRISPR MANIPULATE 2662 02:19:41,494 --> 02:19:43,296 IPSC CELLS PUTTING IN MUTATIONS 2663 02:19:43,296 --> 02:19:45,765 AN TAGS AND DIFFERENTIATING TO 2664 02:19:45,765 --> 02:19:48,167 PRIMARY T CELLS AND PRIMARY 2665 02:19:48,167 --> 02:19:50,136 MACROPHAGES PURIFYING THE 2666 02:19:50,136 --> 02:19:52,605 COMPLEXES AND LOOKING AT AT MASS 2667 02:19:52,605 --> 02:19:57,376 SPECT AND CRYO EM AND ET AND 2668 02:19:57,376 --> 02:20:01,914 FOCUSSED ALMOST EXCLUSIVELY IN 2669 02:20:01,914 --> 02:20:03,916 CRYO ET IN THE BAY AREA. 2670 02:20:03,916 --> 02:20:06,919 AND A SCIENTIST THAT WORKS WITH 2671 02:20:06,919 --> 02:20:14,894 MYSELF UJJWAL WILL TELL YOU 2672 02:20:14,894 --> 02:20:24,503 ABOUT THE FIRST CD4 IN T CELLS 2673 02:20:24,503 --> 02:20:25,905 AND THERE'S BIOLOGY UNCOVERED 2674 02:20:25,905 --> 02:20:26,739 HE'LL TELL YOU ABOUT. 2675 02:20:26,739 --> 02:20:28,507 I WANT TO TELL YOU ABOUT OUR 2676 02:20:28,507 --> 02:20:30,443 EFFORTS LOOKING AT ALPHA FOLD. 2677 02:20:30,443 --> 02:20:33,212 EVERYONE IS USING IT NOWADAYS 2678 02:20:33,212 --> 02:20:34,780 AND FOR SOMEONE LIKE MYSELF THAT 2679 02:20:34,780 --> 02:20:37,149 WORKED ON PROTEIN-PROTEIN 2680 02:20:37,149 --> 02:20:40,152 INTERACTIONS FOR DECADES IT'S 2681 02:20:40,152 --> 02:20:40,686 REVOLUTIONIZING THE WORK. 2682 02:20:40,686 --> 02:20:43,055 SO AS YOU KNOW WE'VE BEEN 2683 02:20:43,055 --> 02:20:46,158 HEAVILY INVOLVED IN GENERATING 2684 02:20:46,158 --> 02:20:48,461 PROTEIN-PROTEIN DATA SETS. 2685 02:20:48,461 --> 02:20:50,863 THE FIRST WAS IN HIV AND WE HAVE 2686 02:20:50,863 --> 02:20:52,431 EXTRACTED FROM THAT MAP AS IN 2687 02:20:52,431 --> 02:20:53,966 OTHERS AND USED WHAT WE LEARNED 2688 02:20:53,966 --> 02:20:56,569 IN HIV AND APPLIED IT TO OTHER 2689 02:20:56,569 --> 02:20:57,570 PATHOGENS OVER THE LAST MANY 2690 02:20:57,570 --> 02:20:58,571 YEARS BUT THE CHALLENGE IS YOU 2691 02:20:58,571 --> 02:21:04,310 LOOK AT THE MAPS AND SAY WHICH 2692 02:21:04,310 --> 02:21:05,978 HOST FACTORS SHOULD WE BE 2693 02:21:05,978 --> 02:21:08,047 LOOKING AT AND LOOKING AT WHAT 2694 02:21:08,047 --> 02:21:10,716 IS TOUCHING THE PROTEIN OF 2695 02:21:10,716 --> 02:21:14,153 INTEREST THAT IS RELEVANT. 2696 02:21:14,153 --> 02:21:18,991 WE SUBJECT THIS TO ALPHA FOLD TO 2697 02:21:18,991 --> 02:21:22,128 IDENTIFY THE 101 OF THE 2698 02:21:22,128 --> 02:21:29,869 INTERACTIONS DIRECTLY TOUCHING. 2699 02:21:29,869 --> 02:21:38,878 BE AND OF AND NOW WE'RE MARCHING 2700 02:21:38,878 --> 02:21:40,613 THROUGH THE HUNDREDS OF 2701 02:21:40,613 --> 02:21:42,148 CONNECTIONS AND THEY'RE HIGHLY 2702 02:21:42,148 --> 02:21:44,350 ENRICHED FOR BEING RESTRACTABLE 2703 02:21:44,350 --> 02:21:45,384 AND THERE'S A NUMBER -- 2704 02:21:45,384 --> 02:21:48,220 RETRACTIBLE AND THERE'S 2705 02:21:48,220 --> 02:21:49,989 STRUCTURES WE'RE CIRCLING BACK 2706 02:21:49,989 --> 02:21:51,857 ON AND ONE THING WE'RE CARRYING 2707 02:21:51,857 --> 02:21:55,261 OUT IS TAKING THE 15 PROTEINS 2708 02:21:55,261 --> 02:21:57,396 DEPENDING HOW YOU CARVE IT UP 2709 02:21:57,396 --> 02:22:00,332 AND LOOK AT ALL 20,000 HUMAN 2710 02:22:00,332 --> 02:22:01,267 PROTEINS USING ALPHA FOLD. 2711 02:22:01,267 --> 02:22:04,103 WE'LL SEE IF THE JUICE IS WORTH 2712 02:22:04,103 --> 02:22:04,403 THE SQUEEZE. 2713 02:22:04,403 --> 02:22:05,971 IT'S NOT CHEAP COMPUTATIONALLY 2714 02:22:05,971 --> 02:22:08,441 AND THIS IS DATA WE'LL RELEASE 2715 02:22:08,441 --> 02:22:12,812 TO THE COMMUNITY AND I THINK 2716 02:22:12,812 --> 02:22:14,947 THERE WILL BE NOVEL INSIGHTS WE 2717 02:22:14,947 --> 02:22:16,282 CAN LOOK INTO IN THE FUTURE. 2718 02:22:16,282 --> 02:22:18,117 WITH THE HARC DEVELOPMENTAL 2719 02:22:18,117 --> 02:22:19,885 AWARDS WE HAVE GREAT YOUNG 2720 02:22:19,885 --> 02:22:22,121 SCIENTISTS AND WE HAVE JEANNETT 2721 02:22:22,121 --> 02:22:25,891 NOW A PROFESSOR AT UCSF LOOKING 2722 02:22:25,891 --> 02:22:28,661 AT MODELLING TO HOOK AT HIV 2723 02:22:28,661 --> 02:22:31,831 HUMAN INTERACTIONS. 2724 02:22:31,831 --> 02:22:38,137 AND MEDHI IS LOOKING AT 2725 02:22:38,137 --> 02:22:41,073 IMPACTING SIGNALLING IN 2726 02:22:41,073 --> 02:22:42,808 POLYNEUOPATHY IN PATIENTS 2727 02:22:42,808 --> 02:22:46,078 INFECTED WITH HIV AND AMANDA 2728 02:22:46,078 --> 02:22:50,883 LOOKING AT CD4 POSITIVE T CELLS 2729 02:22:50,883 --> 02:23:01,527 AND ERICA SANCHEZ AND WE HAVE 2730 02:23:02,928 --> 02:23:06,098 JACQUELINE AND SHE'S LOOKING AT 2731 02:23:06,098 --> 02:23:07,633 EPIGENETIC AND PROTEIN EXCHANGES 2732 02:23:07,633 --> 02:23:10,703 IN HIV GENES AND HOST GENES WITH 2733 02:23:10,703 --> 02:23:13,539 AN INTERESTING COHORT. 2734 02:23:13,539 --> 02:23:15,875 SO PLEASE FOLLOW THESE PEOPLES' 2735 02:23:15,875 --> 02:23:18,144 WORKS IN THE CONTEXT OF THE HARC 2736 02:23:18,144 --> 02:23:22,148 CENTER AND TWO SCIENTIST AWARDS 2737 02:23:22,148 --> 02:23:26,152 LOOKING AT A LIGASE WE FOUND 2738 02:23:26,152 --> 02:23:28,587 INTERACTING WITH VIF AND 2739 02:23:28,587 --> 02:23:29,421 STRUCTURIZING THAT CONNECTION 2740 02:23:29,421 --> 02:23:31,690 AND MY GROUP IS TRYING TO SET UP 2741 02:23:31,690 --> 02:23:34,160 THIS PIPELINE PUR FIGE CON NEXT 2742 02:23:34,160 --> 02:23:38,164 INTO CELLS ED WITH HIV AND GO TO 2743 02:23:38,164 --> 02:23:40,800 CRYO EM AND MASS SPEC TOM METRY. 2744 02:23:40,800 --> 02:23:44,436 WE HAD ROBUST OUTREACH OVER THE 2745 02:23:44,436 --> 02:23:45,404 LAST YEAR AND ALMOST EVERYTHING 2746 02:23:45,404 --> 02:23:48,774 IN PERSON NOW WHICH IS GREAT TO 2747 02:23:48,774 --> 02:23:52,545 SEE AND AND WE HAVE AN 2748 02:23:52,545 --> 02:23:53,712 INTERNSHIP PROGRAM HIGHLY 2749 02:23:53,712 --> 02:23:55,114 REPRESENTATIVE OF THE FIVE 2750 02:23:55,114 --> 02:24:01,754 SPEAKERS NINE WERE AIATEDD -- 2751 02:24:01,754 --> 02:24:04,223 AFFILIATED AND WE RECENTLY 2752 02:24:04,223 --> 02:24:06,258 SOLVED THE STRUCTURE OF RIV RRE. 2753 02:24:06,258 --> 02:24:09,094 WE WENT BACK AND TALK TO HIM 2754 02:24:09,094 --> 02:24:16,535 ABOUT A 1989 PAPER WHERE HE 2755 02:24:16,535 --> 02:24:19,004 SHOWED RIF INVOLVED AND I WAS IN 2756 02:24:19,004 --> 02:24:19,872 ELEMENTARY SCHOOL WHEN THE PAPER 2757 02:24:19,872 --> 02:24:22,842 CAME OUT AND I EVIDENTLY READ IT 2758 02:24:22,842 --> 02:24:27,913 IN 8th GRADE AND I CA CAN SAY 2759 02:24:27,913 --> 02:24:29,682 THIS AND THAT'S THE LAST TIME A 2760 02:24:29,682 --> 02:24:34,153 CANADIAN WON THE STANLEY CUP SO 2761 02:24:34,153 --> 02:24:39,291 I ENCOURAGE YOU TO LOOK AT THIS 2762 02:24:39,291 --> 02:24:45,164 AND THIS IS IN COLLABORATION 2763 02:24:45,164 --> 02:24:46,131 WITH INFECTIOUS DISEASE AND 2764 02:24:46,131 --> 02:24:47,132 RECOGNIZE CHARACTERS HERE AND 2765 02:24:47,132 --> 02:24:49,602 THIS WAS INITIATED WITH A VISIT 2766 02:24:49,602 --> 02:24:51,704 WE HAD BACK IN 2019 AND WE HAVE 2767 02:24:51,704 --> 02:24:56,609 A TWO-DAY SYMPOSIUM WHERE A 2768 02:24:56,609 --> 02:24:58,711 NUMBER OF AFRICAN TRAINEES 2769 02:24:58,711 --> 02:25:01,413 AROUND WORKSHOPS WITH GRANT 2770 02:25:01,413 --> 02:25:04,383 WRITING AND MICROBIOLOGY AND 2771 02:25:04,383 --> 02:25:07,620 CLOTH AND SITE VISIT IN CLONALAL 2772 02:25:07,620 --> 02:25:14,059 WITH THE EPICENTER OF EBOLA AND 2773 02:25:14,059 --> 02:25:18,130 CARS COV2 AND HAVE A PODCAST SO 2774 02:25:18,130 --> 02:25:19,865 ENCOURAGE YOU TO LOOK AT THE 2775 02:25:19,865 --> 02:25:20,165 INFORMATION. 2776 02:25:20,165 --> 02:25:25,704 LOOK ONLINE AND REACH OUT TO US 2777 02:25:25,704 --> 02:25:26,639 IF YOU'RE INTERESTED. 2778 02:25:26,639 --> 02:25:30,676 THE MOST IMPORTANT STLIEDZ -- 2779 02:25:30,676 --> 02:25:35,114 SLIDES ARE THOSE INVOLVED AND 2780 02:25:35,114 --> 02:25:37,449 THEY'RE ALL HERE ALONG WITH THE 2781 02:25:37,449 --> 02:25:38,784 WORK ON THE HEART CENTERS. 2782 02:25:38,784 --> 02:25:44,556 HERE'S A PICK TOUGHER US AT THE 2783 02:25:44,556 --> 02:25:45,891 PRESIDIO RECENTLY TWO MONTHS AGO 2784 02:25:45,891 --> 02:25:49,862 AND IT'S GREAT WE HAVE SUCH A 2785 02:25:49,862 --> 02:25:52,464 FANTASTIC IT SIB AND I WANT TO 2786 02:25:52,464 --> 02:25:53,666 THANK THEM FOR THEIR HELP AND 2787 02:25:53,666 --> 02:25:54,366 INTEREST OVER THE YEARS. 2788 02:25:54,366 --> 02:25:56,201 WE HAVE A NUMBER OF POSTERS OVER 2789 02:25:56,201 --> 02:25:58,003 THE NEXT COUPLE DAYS. 2790 02:25:58,003 --> 02:26:02,141 I ENCOURAGE YOU TO LOOK AT WITH 2791 02:26:02,141 --> 02:26:04,243 ONGOING PROGRESS AND THINGS 2792 02:26:04,243 --> 02:26:07,246 DOESN'T DISCUSS HAPPENING AT THE 2793 02:26:07,246 --> 02:26:09,415 HARC CENTER AND WE'RE HEAVILY 2794 02:26:09,415 --> 02:26:10,849 EMPHASIZING TRAINEES AND HAVE 2795 02:26:10,849 --> 02:26:11,650 THREE GREAT TALKS. 2796 02:26:11,650 --> 02:26:19,692 THE FIRST IS FROM CAROLINE 2797 02:26:19,692 --> 02:26:22,895 STRAIN SPECIFIC DIFFERENCES IN 2798 02:26:22,895 --> 02:26:26,131 HIV-1 MUTATIONAL TOLERANCE IN 2799 02:26:26,131 --> 02:26:36,575 THE CONTEXT OF APOEOF BC3 2800 02:26:38,544 --> 02:26:38,777 RESTRICTION. 2801 02:26:38,777 --> 02:26:49,254 >> I'LL BE TALKING ABOUT MY 2802 02:26:49,722 --> 02:26:54,126 DISSERTATION WORK IN THE VIF 2803 02:26:54,126 --> 02:26:54,593 PROTEIN. 2804 02:26:54,593 --> 02:27:00,532 WE KNOW THE PROTEINS LIKE A3G 2805 02:27:00,532 --> 02:27:03,969 ACT AS ANTIVIRAL TRANSCRIPTION 2806 02:27:03,969 --> 02:27:07,873 FACTORS AND THIS IS SO AG3 CAN 2807 02:27:07,873 --> 02:27:11,543 GET PACKAGED INTO THESE VIRONES. 2808 02:27:11,543 --> 02:27:14,279 THEN A3G IS DELIVERED TO THE 2809 02:27:14,279 --> 02:27:16,582 TARGET CELL WHERE IT INTRODUCES 2810 02:27:16,582 --> 02:27:20,119 MUTATIONS TO THE VIRAL GENOME 2811 02:27:20,119 --> 02:27:21,954 GOING THROUGH REVERSE 2812 02:27:21,954 --> 02:27:22,287 TRANSCRIPTION. 2813 02:27:22,287 --> 02:27:25,758 THE SPECIFIC MOTIF AG3 MUTATES 2814 02:27:25,758 --> 02:27:29,528 IN IS PREMATURE STOP CODES AND 2815 02:27:29,528 --> 02:27:31,230 OTHER MUTATIONS WHICH CULMINATES 2816 02:27:31,230 --> 02:27:34,400 IN SUCCESSFUL VIRAL INFECTION. 2817 02:27:34,400 --> 02:27:36,268 BUT THIS IS NOT WHAT HAPPENS IN 2818 02:27:36,268 --> 02:27:38,070 THE TYPICAL HIV INFECTION. 2819 02:27:38,070 --> 02:27:41,640 AND THAT'S BECAUSE OF THE LENTI 2820 02:27:41,640 --> 02:27:47,012 VIRAL VIF PROTEIN BINDS A3G AND 2821 02:27:47,012 --> 02:27:51,016 PREVENTS IT FROM GETTING 2822 02:27:51,016 --> 02:27:53,352 PACKAGED TO GO THROUGH MUTATION 2823 02:27:53,352 --> 02:27:57,756 WITHOUT THESE BEING INTRODUCED. 2824 02:27:57,756 --> 02:28:03,195 AND VIF BINDS AND THE EXACT 2825 02:28:03,195 --> 02:28:05,731 INTERFACE WAS A MYSTERY IN THE 2826 02:28:05,731 --> 02:28:07,199 FIELD FOR SOME TIME WHICH IS WHY 2827 02:28:07,199 --> 02:28:10,169 IT WAS EXCITING WHEN JOHN GROSS 2828 02:28:10,169 --> 02:28:13,705 FROM UCSF HIS LAB SOLVED THE 2829 02:28:13,705 --> 02:28:16,742 CRYO EM STRUCTURE OF THE VIF IN 2830 02:28:16,742 --> 02:28:19,478 GREEN BOUND TO FULL LENGTH GENE 2831 02:28:19,478 --> 02:28:22,481 IN BLUE AND I WAS LUCKY TO GET 2832 02:28:22,481 --> 02:28:24,516 TO BE A PART OF THE STORY THEY 2833 02:28:24,516 --> 02:28:26,218 TOLD AND I'M SUMMARIZE A COUPLE 2834 02:28:26,218 --> 02:28:28,420 KEY FINDINGS. 2835 02:28:28,420 --> 02:28:32,524 FIRST OF COURSE WE PARSED OUT 2836 02:28:32,524 --> 02:28:34,693 THE EXACT MOLECULAR PLAYERS AT 2837 02:28:34,693 --> 02:28:38,163 THE INTERFACE AND WHICH VIF 2838 02:28:38,163 --> 02:28:40,232 RESIDUE INTERACTING DIRECTLY AND 2839 02:28:40,232 --> 02:28:42,134 WE LEARNED THE INTERFACE IS 2840 02:28:42,134 --> 02:28:42,634 SPECIES SPECIFIC. 2841 02:28:42,634 --> 02:28:45,637 AND THEN THE BIGGER SURPRISE IS 2842 02:28:45,637 --> 02:28:48,974 THAT RNA IS BEING USED AS A 2843 02:28:48,974 --> 02:28:53,946 MOLECULAR GLUE TO BRING THESE 2844 02:28:53,946 --> 02:28:54,913 PROTEINS TOGETHER. 2845 02:28:54,913 --> 02:28:57,616 AND WHAT I'LL TALK ABOUT TODAY 2846 02:28:57,616 --> 02:29:00,586 IS AIMED AT TRYING TO 2847 02:29:00,586 --> 02:29:02,154 COMPREHENSIVELY CHARACTERIZE THE 2848 02:29:02,154 --> 02:29:07,459 MUTATIONAL CONSTRAINT IN AG3. 2849 02:29:07,459 --> 02:29:09,328 SO A GOOD WAY OF ANSWERING THESE 2850 02:29:09,328 --> 02:29:12,264 KINDS OF QUESTIONS IS USING A 2851 02:29:12,264 --> 02:29:13,165 TECHNOLOGY CALLED MUTATIONAL 2852 02:29:13,165 --> 02:29:15,267 SCANNING OR DMS FOR SHORT. 2853 02:29:15,267 --> 02:29:16,902 AND WHAT THIS ALLOWS US TO DO IS 2854 02:29:16,902 --> 02:29:19,204 ASSAY THE EFFECTS OF ALL OF 2855 02:29:19,204 --> 02:29:21,707 AMINO ACID MUTATIONS IN A 2856 02:29:21,707 --> 02:29:22,407 PROTEIN SEQUENCE OF INTEREST IN 2857 02:29:22,407 --> 02:29:24,643 A SINGLE EXPERIMENT. 2858 02:29:24,643 --> 02:29:26,145 SO THE WAY THIS WORKS IS YOU 2859 02:29:26,145 --> 02:29:30,082 TAKE THE PROTEIN SEQUENCE IN MY 2860 02:29:30,082 --> 02:29:33,285 CASE THE VIF SEQUENCE AND PULL 2861 02:29:33,285 --> 02:29:35,621 THEM IN EQUAL OPPORTUNITIES AND 2862 02:29:35,621 --> 02:29:38,157 IT BECOMES YOUR DMS LIBRARY. 2863 02:29:38,157 --> 02:29:44,296 I LIGATED MINE TO MAKE INFECTION 2864 02:29:44,296 --> 02:29:46,865 HOUSE VIRUS WITH THE LIBRARY OF 2865 02:29:46,865 --> 02:29:47,132 VARIANTS. 2866 02:29:47,132 --> 02:29:48,734 THE NEXT IS TO HAVE A SELECTION 2867 02:29:48,734 --> 02:29:51,103 THAT ALLOWS US TO PARS OUT 2868 02:29:51,103 --> 02:29:53,405 NON-FUNCTIONAL VERSUS FUNCTIONAL 2869 02:29:53,405 --> 02:29:53,705 VARIANTS. 2870 02:29:53,705 --> 02:29:56,742 TO DO THIS, I FIRST INFECT SUB 2871 02:29:56,742 --> 02:30:00,345 T1 CELLS AT A LOW MOI AND 2872 02:30:00,345 --> 02:30:02,147 THEY'RE EXPRESSING AG3 AT 2873 02:30:02,147 --> 02:30:03,715 PHYSIOLOGICAL LEVELS. 2874 02:30:03,715 --> 02:30:06,151 BECAUSE WE KNOW AG3 HAS TO BE 2875 02:30:06,151 --> 02:30:08,253 PACKAGED AND DELIVERED TO HAVE 2876 02:30:08,253 --> 02:30:09,621 ANY SORT OF ANTIVIRAL PHENOTYPE 2877 02:30:09,621 --> 02:30:12,824 WE THEN TAKE THAT FIRST ROUND 2878 02:30:12,824 --> 02:30:16,762 AND DO A SECOND INFECTION AND 2879 02:30:16,762 --> 02:30:20,299 IT'S THE SUPER NATENT OFF THE 2880 02:30:20,299 --> 02:30:23,168 SECOND ROUND THAT'S FUNCTIONAL 2881 02:30:23,168 --> 02:30:25,537 VARIANCE. 2882 02:30:25,537 --> 02:30:34,913 WE CAN SEQUENCE THE AND USE THIS 2883 02:30:34,913 --> 02:30:39,151 TO GENERATE LOG ENRICHMENT 2884 02:30:39,151 --> 02:30:40,118 RATIOS FOR THE EFFECT OF 2885 02:30:40,118 --> 02:30:40,352 FITNESS. 2886 02:30:40,352 --> 02:30:42,988 AS A CONTROL IN PARALLEL TO MY 2887 02:30:42,988 --> 02:30:46,024 FULL SIZED LIBRARIES I PASSAGED 2888 02:30:46,024 --> 02:30:47,259 A MINI LIBRARY WHICH IS A 2889 02:30:47,259 --> 02:30:51,263 LIBRARY ONLY AT A SINGLE SITE IN 2890 02:30:51,263 --> 02:30:53,765 VIF POSITIONED Y40 WHICH WE 2891 02:30:53,765 --> 02:30:55,300 UNDERSTAND FROM THE STRUCTURE IS 2892 02:30:55,300 --> 02:30:58,136 IMPORTANT IN FACILITATING THE 2893 02:30:58,136 --> 02:31:00,138 INTERACTION WITH RNA. 2894 02:31:00,138 --> 02:31:04,309 ON THE Y AXIS WE LOOK AT THE 2895 02:31:04,309 --> 02:31:06,144 PERCENT OF CODONS AND ON THE 2896 02:31:06,144 --> 02:31:10,148 INPUT AND PRE-SELECTION AND 2897 02:31:10,148 --> 02:31:12,284 FIRST SUPER NATENT AND THIS 2898 02:31:12,284 --> 02:31:14,152 SHOWS THE SELECTION IS HAPPENING 2899 02:31:14,152 --> 02:31:17,589 BETWEEN THE PRE AND POST 2900 02:31:17,589 --> 02:31:18,857 SELECTION SUPER NATENTS WHICH IS 2901 02:31:18,857 --> 02:31:21,860 WHAT WE EXPECT OF AG3 2902 02:31:21,860 --> 02:31:22,160 RESTRICTION. 2903 02:31:22,160 --> 02:31:25,264 SO NOW LOOKING AT THE FULL SIZE 2904 02:31:25,264 --> 02:31:26,131 LIBRARY WE CAN VISUALIZE THE 2905 02:31:26,131 --> 02:31:28,400 DATA IN A NUMBER OF WAYS BUT 2906 02:31:28,400 --> 02:31:30,168 WHAT WE'RE LOOKING AT HERE IS 2907 02:31:30,168 --> 02:31:32,037 THE SITE LEVEL WHERE I'VE TAKEN 2908 02:31:32,037 --> 02:31:34,740 THE ENRICHMENT RATIO OF ALL 2909 02:31:34,740 --> 02:31:36,275 VARIANTS AND AVERAGED THEM TO 2910 02:31:36,275 --> 02:31:38,944 GIVE US AN IDEA OF WHAT THE OVER 2911 02:31:38,944 --> 02:31:41,613 ALL CONSTRAINT AT EACH SITE IS. 2912 02:31:41,613 --> 02:31:44,716 SO WE HAVE THE SITES ON THE X 2913 02:31:44,716 --> 02:31:49,588 AND LOG ENRICHMENT ON THE Y WITH 2914 02:31:49,588 --> 02:31:51,590 ANYTHING BELOW ZERO MEANING 2915 02:31:51,590 --> 02:31:53,492 DEPLETED MEANING THE SITE IS 2916 02:31:53,492 --> 02:31:54,126 PROBABLY HEAVILY CONSTRAINED BUT 2917 02:31:54,126 --> 02:31:57,796 THE VALUE IS ABOVE 0 IT MAY HAVE 2918 02:31:57,796 --> 02:32:01,366 MANY VARIANTS IMPROVING THE 2919 02:32:01,366 --> 02:32:03,168 FUNCTION IN A3G RESTRICTION. 2920 02:32:03,168 --> 02:32:04,569 TO TRY TO UNDERSTAND THIS 2921 02:32:04,569 --> 02:32:06,838 BETTER, I WANTED TO HIGHLIGHT 2922 02:32:06,838 --> 02:32:08,006 SOME SITES WE CHARACTERIZED VEE 2923 02:32:08,006 --> 02:32:09,641 JA THE STRUCTURE. 2924 02:32:09,641 --> 02:32:12,644 SO ANY SITE THAT HAS THIS 2925 02:32:12,644 --> 02:32:15,347 TRIANGLE WAS INTERACTING WITH 2926 02:32:15,347 --> 02:32:19,151 RNA THEN WE HAD A COUPLE THAT 2927 02:32:19,151 --> 02:32:21,520 INTERACTED WITH RNA WITH A G3 2928 02:32:21,520 --> 02:32:22,120 THE BLUE FILL. 2929 02:32:22,120 --> 02:32:24,323 FOR THIS TALK I WANTED TO FOCUS 2930 02:32:24,323 --> 02:32:25,824 ON THE SITES IN THE IT 2931 02:32:25,824 --> 02:32:26,091 INTERFACE. 2932 02:32:26,091 --> 02:32:30,128 THE ONES CIRCLES WHICH ARE ONLY 2933 02:32:30,128 --> 02:32:36,868 INTERACTING WITH AG3 AND NOT 2934 02:32:36,868 --> 02:32:37,035 RNA. 2935 02:32:37,035 --> 02:32:41,773 WE HAVE SITE W70 AND Q803. 2936 02:32:41,773 --> 02:32:44,142 I WANT TO HIGHLIGHT THE SITE 2937 02:32:44,142 --> 02:32:47,179 Q803 BECAUSE IT WAS ONE OF 70 2938 02:32:47,179 --> 02:32:50,582 SITES WITH AN ENRICHMENT RATIO 2939 02:32:50,582 --> 02:32:52,751 ABOVE 0 WHICH WAS SUPER 2940 02:32:52,751 --> 02:32:56,455 INTERESTING TO BECAUSE IT'S AN 2941 02:32:56,455 --> 02:32:58,323 IMPORTANT ARMS RACE INTERFACE 2942 02:32:58,323 --> 02:32:58,757 SITE. 2943 02:32:58,757 --> 02:33:00,792 TO TAKE ONE STEP CLOSER FROM THE 2944 02:33:00,792 --> 02:33:02,127 SITE LEVEL ANALYSIS WE JUST 2945 02:33:02,127 --> 02:33:03,829 LOOKED AT WE CAN LOOK AT HOW 2946 02:33:03,829 --> 02:33:06,164 EACH OF THE INDIVIDUAL VARIANTS 2947 02:33:06,164 --> 02:33:08,266 IN THE LIBRARY THAT PASSED OUR 2948 02:33:08,266 --> 02:33:08,867 COVERAGE THRESHOLDS IN THE 2949 02:33:08,867 --> 02:33:10,769 PRESELECTION POOL HOW EACH OF 2950 02:33:10,769 --> 02:33:14,906 THOSE FARES AT THE SPECIFIC SITE 2951 02:33:14,906 --> 02:33:16,074 A IT 83. 2952 02:33:16,074 --> 02:33:19,644 ONE WAY I FOUND HELPFUL TO 2953 02:33:19,644 --> 02:33:20,345 CONTEXTUALIZE THE INFORMATION 2954 02:33:20,345 --> 02:33:24,416 WAS TO LOOK AT THE SEQUENCE 2955 02:33:24,416 --> 02:33:26,585 FORMATION BOTH AMONG HIV SUB 2956 02:33:26,585 --> 02:33:29,755 TYPES AND THE SUB CURSER AND 2957 02:33:29,755 --> 02:33:31,857 AMONG BROAD SIVs. 2958 02:33:31,857 --> 02:33:37,429 THE REASON IT'S HELPFUL IS IN 2959 02:33:37,429 --> 02:33:41,233 NATURAL IN HIV AND CBC IT'S Q 2960 02:33:41,233 --> 02:33:42,167 AND IN THE WILD TYPE IN THE 2961 02:33:42,167 --> 02:33:44,069 LIBRARY IS A Q I WANTED TO LOOK 2962 02:33:44,069 --> 02:33:47,706 AT HOW THE H FARED AND IT 2963 02:33:47,706 --> 02:33:50,008 BEHAVES AS WELL AS WILD TYPE 2964 02:33:50,008 --> 02:33:51,109 WHICH MAKES SENSE. 2965 02:33:51,109 --> 02:33:53,211 FURTHERMORE IF WE LOOK AT HOW 2966 02:33:53,211 --> 02:33:54,146 THE Y VARIANT FARED THIS IS 2967 02:33:54,146 --> 02:33:55,547 IMPORTANT BECAUSE WE KNOW IT'S 2968 02:33:55,547 --> 02:33:58,784 ONE OF THE CHANGES THAT HAD TO 2969 02:33:58,784 --> 02:34:05,457 HAPPEN IN SIV VIF AND FACILITATE 2970 02:34:05,457 --> 02:34:08,226 THE TRANSMISSION IN OLD WORLD 2971 02:34:08,226 --> 02:34:12,164 MONKEYS AND SEE THE Y RES 2972 02:34:12,164 --> 02:34:12,431 DEPLETED. 2973 02:34:12,431 --> 02:34:14,833 WHAT WAS SURPRISING WAS THERE 2974 02:34:14,833 --> 02:34:16,535 WERE A NUMBER RESIDUES IF 2975 02:34:16,535 --> 02:34:18,170 ENRICHED IN THE LIBRARY NEVER 2976 02:34:18,170 --> 02:34:19,805 SAMPLED IN NATURAL PERFECT. 2977 02:34:19,805 --> 02:34:22,607 ONE OF WHICH IS THE IS VARIANT 2978 02:34:22,607 --> 02:34:27,345 WHICH WAS CHARACTERIZED IN IN 2979 02:34:27,345 --> 02:34:34,119 VIVO AND CONFERRED RESISTANCE. 2980 02:34:34,119 --> 02:34:39,624 LOG ENRICHMENT RATIOS IS ONE WAY 2981 02:34:39,624 --> 02:34:41,359 OF CHARACTERIZING BUT THE SECOND 2982 02:34:41,359 --> 02:34:43,762 CAME TO US IN THINKING HOW IN 2983 02:34:43,762 --> 02:34:45,330 PROVIRUSES AMPLIFIED FROM 2984 02:34:45,330 --> 02:34:47,365 INDIVIDUALS LIVING WITH HIV WE 2985 02:34:47,365 --> 02:34:50,135 OFTEN SEE THESE MUTATIONS 2986 02:34:50,135 --> 02:34:52,771 CHARAC 2987 02:34:52,771 --> 02:34:55,207 CHARACTERISTIC OF AG3 ACTIVITY. 2988 02:34:55,207 --> 02:34:57,075 CAN WE MEASURE THE NUMBER OF 2989 02:34:57,075 --> 02:34:59,611 WILL MUTATIONS IN EACH DMS 2990 02:34:59,611 --> 02:35:01,947 VARIANT TO GIVE US A SECONDARY 2991 02:35:01,947 --> 02:35:03,782 WAY OF CHARACTERIZING THE 2992 02:35:03,782 --> 02:35:04,716 EFFECTS OF THE VARIANT ON OVER 2993 02:35:04,716 --> 02:35:05,217 ALL FITNESS. 2994 02:35:05,217 --> 02:35:06,885 SO WHAT WE'RE LOOKING AT HERE IS 2995 02:35:06,885 --> 02:35:10,021 EACH ONE OF THE POINTS IS A 2996 02:35:10,021 --> 02:35:11,756 SPECIFIC VARIANT AT A SPECIFIC 2997 02:35:11,756 --> 02:35:13,758 SITE WITH THE LOG ENRICHMENT 2998 02:35:13,758 --> 02:35:15,560 RATIO ON THE Y AND THE 2999 02:35:15,560 --> 02:35:17,562 PERCENTAGE OF THREE OR MORE 3000 02:35:17,562 --> 02:35:22,367 MUTATIONS ON THE Y AXIS. AND THE 3001 02:35:22,367 --> 02:35:22,968 OF THREE OR MORE MUTATIONS ON 3002 02:35:22,968 --> 02:35:23,435 THE Y AXIS.X AND THE 3003 02:35:23,435 --> 02:35:25,070 PERCENTAGE OF THREE OR MORE 3004 02:35:25,070 --> 02:35:25,570 MUTATIONS ON THE Y AXIS. 3005 02:35:25,570 --> 02:35:32,744 THE MORE IN ENRICHED IT IS THE 3006 02:35:32,744 --> 02:35:34,813 MORE THERE'S VARIANTS AND 3007 02:35:34,813 --> 02:35:37,816 PREVENTS IT FROM GETTING 3008 02:35:37,816 --> 02:35:38,083 PACKAGED. 3009 02:35:38,083 --> 02:35:39,417 IT'S INTERESTING TO LOOK AT THE 3010 02:35:39,417 --> 02:35:42,087 DATA BECAUSE WE CAN USE IT TO 3011 02:35:42,087 --> 02:35:44,723 HIGHLIGHT SPECIFIC VARIANTS. 3012 02:35:44,723 --> 02:35:49,528 ALL THE RED DOTS DOTS ARE 3013 02:35:49,528 --> 02:35:50,262 CYSTEINE VARIANTS AND HERE ON 3014 02:35:50,262 --> 02:35:53,532 THE PLOT ENRICHED AND HAVING A 3015 02:35:53,532 --> 02:35:55,433 VERY SMALL AMOUNT OF WILL READS 3016 02:35:55,433 --> 02:35:58,169 WITH THIS VARIANT HAVING THREE 3017 02:35:58,169 --> 02:36:00,105 OR MORE MUTATIONS. 3018 02:36:00,105 --> 02:36:01,339 I ALSO THOUGHT IT WAS CURIOUS 3019 02:36:01,339 --> 02:36:03,875 THERE WERE A NUMBER OF SITES 3020 02:36:03,875 --> 02:36:08,547 WITH SIS CYSTEINE VARIANTS THAT 3021 02:36:08,547 --> 02:36:10,448 FIT THE PROFILE. 3022 02:36:10,448 --> 02:36:12,250 I WAS ABLE TO LOOK AT THE 3023 02:36:12,250 --> 02:36:19,057 STRUCTURE WHERE WE SEE VIF AND 3024 02:36:19,057 --> 02:36:21,092 GREEN AND BLUE AND THEY'RE 3025 02:36:21,092 --> 02:36:22,794 LOCALIZED IN RED AND WE THOUGHT 3026 02:36:22,794 --> 02:36:24,262 IT WAS INTERESTING THAT A LOT OF 3027 02:36:24,262 --> 02:36:27,232 THESE SITES ARE LOCALIZED TO THE 3028 02:36:27,232 --> 02:36:28,366 INTERFACE WHICH MAY INDICATE TO 3029 02:36:28,366 --> 02:36:32,203 US THE INTRODUCTION OF A 3030 02:36:32,203 --> 02:36:33,672 CYSTEINE BROADLY WOULD IMPROVE 3031 02:36:33,672 --> 02:36:38,143 THE BINDING IN RESTRICTION OF 3032 02:36:38,143 --> 02:36:39,444 AG3. 3033 02:36:39,444 --> 02:36:42,914 AND SO UP CONCLUSION WE'VE SHOWN 3034 02:36:42,914 --> 02:36:45,984 THAT CHALLENGING DMS LIBRARIES 3035 02:36:45,984 --> 02:36:46,985 REVEAL MUTATIONS THAT IMPROVE 3036 02:36:46,985 --> 02:36:48,486 THE FUNCTION THAT MAY NOT HAVE 3037 02:36:48,486 --> 02:36:49,588 BEEN SAMPLED IN NATURE BEFORE. 3038 02:36:49,588 --> 02:36:52,724 WE'RE CURRENTLY WORKING ON 3039 02:36:52,724 --> 02:36:54,626 VALIDATING THAT Q83C VARIANT AS 3040 02:36:54,626 --> 02:36:55,627 WELL AS OTHERS TO TRY AND 3041 02:36:55,627 --> 02:36:57,195 UNDERSTAND THE TRADE-OFFS AND 3042 02:36:57,195 --> 02:36:59,598 WHY THESE ARE NOT BEEN SAMPLED 3043 02:36:59,598 --> 02:37:00,932 IN NATURE AND THOUGH I ONLY 3044 02:37:00,932 --> 02:37:06,137 SHOWED DATA WITH ONE LIBRARY AND 3045 02:37:06,137 --> 02:37:11,876 ONE AVA VECTS WE HAVE DONE THIS 3046 02:37:11,876 --> 02:37:13,878 MULTIPLE STRAINS AND HAVING THE 3047 02:37:13,878 --> 02:37:14,946 STRUCTURAL INFORMATION MAKES IT 3048 02:37:14,946 --> 02:37:16,114 MORE POWERFUL TO GIVE VALUABLE 3049 02:37:16,114 --> 02:37:16,781 CONTEXT TO TRY TO UNDERSTAND THE 3050 02:37:16,781 --> 02:37:19,250 DATA. 3051 02:37:19,250 --> 02:37:22,120 AND FINALLY SOME NEWER FACET 3052 02:37:22,120 --> 02:37:24,055 WE'RE EXCITED ABOUT IS THAT WILL 3053 02:37:24,055 --> 02:37:26,725 WILL ENRICHMENT RATIOS AND IT 3054 02:37:26,725 --> 02:37:28,159 MUTATIONS GIVE US A SECONDARY 3055 02:37:28,159 --> 02:37:29,861 WAY TO CHARACTERIZE THE FITNESS 3056 02:37:29,861 --> 02:37:31,529 EFFECT OF THESE VARIANTS. 3057 02:37:31,529 --> 02:37:33,398 AND THANK YOU TO MY LAB AND THE 3058 02:37:33,398 --> 02:37:34,799 HARC CENTER AND I THINK WILLIAM 3059 02:37:34,799 --> 02:37:45,010 IS UP NEXT. 3060 02:37:49,114 --> 02:37:52,550 >> HI, EVERYONE. 3061 02:37:52,550 --> 02:37:55,153 I'M WILL CISNEROS I JUST FINISH 3062 02:37:55,153 --> 02:38:00,525 YOU HAD UP I Ph.D. AT NORTHWEST 3063 02:38:00,525 --> 02:38:06,131 UNIVERSITY AND GIVE WILL GIVE A 3064 02:38:06,131 --> 02:38:09,801 BRIEF VIGNETTE AND IT WILL OB ON 3065 02:38:09,801 --> 02:38:14,172 TARGETING TRANSCRIPTION IN 3066 02:38:14,172 --> 02:38:19,878 ELONGATION IN HHS LATENCY 3067 02:38:19,878 --> 02:38:20,779 REVE 3068 02:38:20,779 --> 02:38:21,046 REVERSAL. 3069 02:38:21,046 --> 02:38:23,448 THE CELLS PRODUCE VIRUS AND 3070 02:38:23,448 --> 02:38:27,018 PRESENT ANTIGEN AND CAN UNDER GO 3071 02:38:27,018 --> 02:38:30,155 CELL DEATH THROUGH PERIPHERAL 3072 02:38:30,155 --> 02:38:32,424 CYTOTOXIC QUALITIES OR INNATE 3073 02:38:32,424 --> 02:38:34,159 IMMUNE FUNCTION AND THE CELLS 3074 02:38:34,159 --> 02:38:38,797 CAN ENTER A LATENT STATE 3075 02:38:38,797 --> 02:38:40,331 ESTABLISHING THE RESERVOIR AND 3076 02:38:40,331 --> 02:38:43,034 ANOTHER INTERESTING DETAIL IS 3077 02:38:43,034 --> 02:38:46,171 HIV SOMETIMES CAN ENTER ALREADY 3078 02:38:46,171 --> 02:38:47,939 LATENTLY INFECTED CELLS AND HIDE 3079 02:38:47,939 --> 02:38:49,541 FROM THE IMMUNE SYSTEM. 3080 02:38:49,541 --> 02:38:54,145 THE LATENT RESERVOIR IS A MAJOR 3081 02:38:54,145 --> 02:38:56,214 OBSTA 3082 02:38:56,214 --> 02:38:57,348 OBSTACLE TOWARDS FINDING A 3083 02:38:57,348 --> 02:38:58,550 FUNCTIONAL THERAPY AND IF YOU 3084 02:38:58,550 --> 02:39:01,753 TRACK THE KINETICS OF A PATIENT, 3085 02:39:01,753 --> 02:39:03,688 ONE PATIENT UNDER GOES ANTRI 3086 02:39:03,688 --> 02:39:06,725 VIRAL THERAPY AND ONCE THE LOAD 3087 02:39:06,725 --> 02:39:12,664 IS SUPPRESSED THEY CAN FALL TO 3088 02:39:12,664 --> 02:39:18,169 UNDETECTABLE AND THE VIRUS CAN 3089 02:39:18,169 --> 02:39:21,673 REREBOUND -- REBOUND AND 3090 02:39:21,673 --> 02:39:23,508 STRATEGIES ARE NEEDED TOO DELAY 3091 02:39:23,508 --> 02:39:25,910 THE REBOUND OR TRANSITION AND 3092 02:39:25,910 --> 02:39:28,113 EVEN IN SUPPRESSED PATIENTS WHO 3093 02:39:28,113 --> 02:39:29,781 DON'T UNDER GO REBOUND THERE'S 3094 02:39:29,781 --> 02:39:34,152 ADDITIONAL RISK OF DEVELOPING 3095 02:39:34,152 --> 02:39:38,156 COMORBIDITIES MAYBE BY THE 3096 02:39:38,156 --> 02:39:40,959 PERSISTENT VIRAL REPLICATION. 3097 02:39:40,959 --> 02:39:43,294 SO FUTURE CURATIVE THERAPIES 3098 02:39:43,294 --> 02:39:46,164 LOOKED HOW TO TURN ON OR OFF THE 3099 02:39:46,164 --> 02:39:55,206 AN TA GOOD -- ANTIGENS AND 3100 02:39:55,206 --> 02:40:02,280 MOLECULES CAN HAVE PATHWAY TO 3101 02:40:02,280 --> 02:40:12,824 REACTIVATE THE VIRUS AND UNDER 3102 02:40:25,837 --> 02:40:27,172 EPIGENETICS AND AT EVERY STAGE 3103 02:40:27,172 --> 02:40:28,807 AND POST TRANSCRIPTION AND OUR 3104 02:40:28,807 --> 02:40:31,376 LAB HAS BEEN FOCUSSED ON 3105 02:40:31,376 --> 02:40:35,280 TRANSCRIPTIONAL ELONGATION 3106 02:40:35,280 --> 02:40:36,915 BLOCKS. 3107 02:40:36,915 --> 02:40:37,549 SIMPLISTIC TRANSCRIPTION FOR 3108 02:40:37,549 --> 02:40:42,153 THOSE UNFAMILIAR YOU HAVE 3109 02:40:42,153 --> 02:40:45,757 TRANSCRIPTION INITIATION WITH 3110 02:40:45,757 --> 02:40:49,928 SITES AND TRAVELS 20 TO 60 BASE 3111 02:40:49,928 --> 02:40:51,763 PAIRS IN THE PROMOTER AND 3112 02:40:51,763 --> 02:40:51,996 PAUSES. 3113 02:40:51,996 --> 02:40:53,932 AT THAT POINT YOU NEED 3114 02:40:53,932 --> 02:40:55,366 ADDITIONAL LICENSING THAT COMES 3115 02:40:55,366 --> 02:40:57,936 IN THE FORM OF TRANSCRIPTIONAL 3116 02:40:57,936 --> 02:40:59,337 ELONGATION WHICH I'LL DISCUSS 3117 02:40:59,337 --> 02:41:00,438 HERE FURTHER. 3118 02:41:00,438 --> 02:41:02,140 TRANSCRIPTIONAL ELONGATION IS 3119 02:41:02,140 --> 02:41:06,277 LICENSED AND THE PAUSING IS 3120 02:41:06,277 --> 02:41:15,720 RELIEVED AND IT CONTAINS A 3121 02:41:15,720 --> 02:41:17,622 CYCLIN AND IT DOESN'T KNOW WHERE 3122 02:41:17,622 --> 02:41:28,066 TO GO AND SHCHAPERONED AND AS MY 3123 02:41:28,066 --> 02:41:30,768 ARE FAMILIAR WITH THE VIRUS 3124 02:41:30,768 --> 02:41:32,170 ITSELF IS VERY SMART AND HAS 3125 02:41:32,170 --> 02:41:35,707 ENCODED A PROTEIN THAT CAN 3126 02:41:35,707 --> 02:41:37,141 SPECIFICALLY ENGAGE AND PROVIDE 3127 02:41:37,141 --> 02:41:38,543 THE LICENSING FOR ELONGATION 3128 02:41:38,543 --> 02:41:41,980 ALLOWING THE VIRUS TO CONTROL 3129 02:41:41,980 --> 02:41:50,054 ITS OWN REPLICATION. 3130 02:41:50,054 --> 02:41:53,491 P-TEFB IT WILL SIN MANY 3131 02:41:53,491 --> 02:41:55,393 COMPLEXES AND THERE'S BEEN 3132 02:41:55,393 --> 02:41:58,129 STRATEGIES TO RELEASE THEM FOR 3133 02:41:58,129 --> 02:42:01,266 LATENCY REVERSAL. 3134 02:42:01,266 --> 02:42:04,202 THE FIRST COMPLEX HAS NOT BEEN 3135 02:42:04,202 --> 02:42:05,370 SEEN AS A POTENTIAL RESERVOIR 3136 02:42:05,370 --> 02:42:07,772 TAR GET AND MEDIATED RELEASE AND 3137 02:42:07,772 --> 02:42:10,141 SUBSEQUENT RECRUITMENT TO VIRAL 3138 02:42:10,141 --> 02:42:11,442 INTEGRATION SITES. 3139 02:42:11,442 --> 02:42:16,481 TO EXPLORE THE IDEA THIS COULD 3140 02:42:16,481 --> 02:42:19,317 BE A POTENTIAL RESERVOIR WE HAD 3141 02:42:19,317 --> 02:42:20,885 TO ESTABLISH TWO GROUND TRUTHS 3142 02:42:20,885 --> 02:42:27,158 IT'S NOT NEEDED FOR HIV 3143 02:42:27,158 --> 02:42:33,097 TRANSCRIPTION AND CAN LEAVE THEM 3144 02:42:33,097 --> 02:42:33,631 INTACT FOR TRANSCRIPTION. 3145 02:42:33,631 --> 02:42:37,468 OUR LAB HAS A TOOLBOX TO 3146 02:42:37,468 --> 02:42:39,470 INTERROGATE FUNCTIONS IN PRIMARY 3147 02:42:39,470 --> 02:42:40,171 T CELLS. 3148 02:42:40,171 --> 02:42:44,309 FOR THIS PROJECT WE INITIATED A 3149 02:42:44,309 --> 02:42:46,744 MULTIPLEX CRISPR KAS 9 APPROACH 3150 02:42:46,744 --> 02:42:49,814 TARGETING EVERY SEC PROTEIN 3151 02:42:49,814 --> 02:42:51,649 ENCODING GENE. 3152 02:42:51,649 --> 02:42:57,522 WE WERE ABLE TO IT ACTIVATE CD4 3153 02:42:57,522 --> 02:43:01,726 T CELLS AND VALIDATE PROTEIN 3154 02:43:01,726 --> 02:43:03,528 KNOCKOUT AND IN PARALLEL 3155 02:43:03,528 --> 02:43:05,830 CHALLENGE THE CELLS WITH 3156 02:43:05,830 --> 02:43:07,231 REPLICATION COMPETENT VIRUS AND 3157 02:43:07,231 --> 02:43:09,334 LOOK AT CHANGES OF INFECTION. 3158 02:43:09,334 --> 02:43:12,870 FIRST WE'RE ABLE TO KNOCK OUT 3159 02:43:12,870 --> 02:43:14,105 EVERY PROTEIN WITHOUT MAJOR 3160 02:43:14,105 --> 02:43:16,174 VIABILITY DEFECTS WHICH IS 3161 02:43:16,174 --> 02:43:18,142 EXCITING AND ONCE WE VERIFIED 3162 02:43:18,142 --> 02:43:19,944 THE KNOCKOUT WE'RE ABLE TO 3163 02:43:19,944 --> 02:43:21,245 CHALLENGE THESE WITH VIRUS. 3164 02:43:21,245 --> 02:43:24,215 SO WHAT YOU'LL NOTICE ON THE Y 3165 02:43:24,215 --> 02:43:25,817 AXIS IS PERSON INFECTED CELLS TO 3166 02:43:25,817 --> 02:43:28,086 OUR NON-TARGETING CONTROL AND ON 3167 02:43:28,086 --> 02:43:30,154 THE X ARE DIFFERENT GENES TAR 3168 02:43:30,154 --> 02:43:37,295 GUESTED AND SEE WHEN WE KNOCKED 3169 02:43:37,295 --> 02:43:41,265 THESE OUT THEY'RE EXOTROPHIC AND 3170 02:43:41,265 --> 02:43:44,102 IF YOU KNOCK OUT THE REST OF THE 3171 02:43:44,102 --> 02:43:45,737 SUPER ELONGATION COMPLEX YOU 3172 02:43:45,737 --> 02:43:47,472 DON'T SEE A SIGNIFICANT EFFECT. 3173 02:43:47,472 --> 02:43:48,606 THAT'S THE FIRST GROUND TRUTH 3174 02:43:48,606 --> 02:43:54,012 MAYBE IT'S NOT NECESSARY FOR HIV 3175 02:43:54,012 --> 02:43:54,345 TRANSCRIPTION. 3176 02:43:54,345 --> 02:43:58,616 MAYBE PFB IS THE MOLECULE AND WE 3177 02:43:58,616 --> 02:43:59,350 PARTNERED AT NORTHWESTERN 3178 02:43:59,350 --> 02:44:01,319 UNIVERSITY TO DEVELOP A MOLECULE 3179 02:44:01,319 --> 02:44:04,222 THAT CAN DISRUPT THE SUPER 3180 02:44:04,222 --> 02:44:06,958 ELONGATION COMPLEX INTERACTION. 3181 02:44:06,958 --> 02:44:09,193 THE COMPOUND DEVELOPED KNOWN AS 3182 02:44:09,193 --> 02:44:15,533 KL-2 AND MIMICS THE BINDING 3183 02:44:15,533 --> 02:44:16,000 INTERFACE. 3184 02:44:16,000 --> 02:44:18,002 AND WE TOOK PRIMARY CD4 T CELLS 3185 02:44:18,002 --> 02:44:19,670 AND TREATED THEM WITH THE 3186 02:44:19,670 --> 02:44:20,772 COMPOUND AND CHALLENGED THEM 3187 02:44:20,772 --> 02:44:22,140 WITH THE COMPETENT VIRUS AND 3188 02:44:22,140 --> 02:44:27,245 LOOKED AT WHAT WOULD HAPPEN WITH 3189 02:44:27,245 --> 02:44:28,012 INFECTION. 3190 02:44:28,012 --> 02:44:31,916 WE WERE ABLE TO SEE INCREASED 3191 02:44:31,916 --> 02:44:34,152 INFECTION BETWEEN TREATED AND 3192 02:44:34,152 --> 02:44:38,156 YOU CAN DISRUPT THE INTERACTIONS 3193 02:44:38,156 --> 02:44:41,859 WITHOUT DISRUPTING PTFB 3194 02:44:41,859 --> 02:44:42,393 INTERACTIONS THEMSELVES. 3195 02:44:42,393 --> 02:44:45,096 THAT'S ACTIVE INFECTION. 3196 02:44:45,096 --> 02:44:47,031 THINGS DIFFER SIGNIFICANTLY IN 3197 02:44:47,031 --> 02:44:52,837 LATENCY AND WANTED TO TRY THIS 3198 02:44:52,837 --> 02:44:54,505 IN A LATENT MODEL AND LOOK AT IN 3199 02:44:54,505 --> 02:44:56,507 A PROVIRUS AND TREATED THE CELLS 3200 02:44:56,507 --> 02:45:01,245 WITH EITHER A PANEL OF LRAs ALL 3201 02:45:01,245 --> 02:45:05,616 VERY DIFFERENT SO JQ1 OR AGONIST 3202 02:45:05,616 --> 02:45:10,488 AND THE EXCITING NON-CANONICAL 3203 02:45:10,488 --> 02:45:12,990 KAPPA B AGONIST IN THE PRESENCE 3204 02:45:12,990 --> 02:45:14,058 OF KL-2. 3205 02:45:14,058 --> 02:45:18,896 WE DON'T SEE MUCH SINGLE AGENT 3206 02:45:18,896 --> 02:45:23,468 ACTIVITY OF K L-2 MAYBE A SMALL 3207 02:45:23,468 --> 02:45:24,035 BLIP BUT NOT STATISTICALLY 3208 02:45:24,035 --> 02:45:24,335 SIGNIFICANT. 3209 02:45:24,335 --> 02:45:26,137 WHAT YOU CAN SEE WITH OUR PANEL 3210 02:45:26,137 --> 02:45:29,574 OF LRAs IS SIGNIFICANT SYNERGY 3211 02:45:29,574 --> 02:45:34,145 ACROSS THE DIFFERENT ACTING 3212 02:45:34,145 --> 02:45:37,315 LRAs. 3213 02:45:37,315 --> 02:45:38,349 WE FURTHERMORE CALCULATED 3214 02:45:38,349 --> 02:45:42,153 SYNERGY AND PICKED OUT THE DATA 3215 02:45:42,153 --> 02:45:45,256 FROM IN COMBINATION WITH KL2 AND 3216 02:45:45,256 --> 02:45:48,659 SEE DOSE DEPENDENT SYNERGY WITH 3217 02:45:48,659 --> 02:45:51,329 A LOCKED CONCENTRATION OF A WITH 3218 02:45:51,329 --> 02:45:52,130 KL-2 IN COMBINATION. 3219 02:45:52,130 --> 02:45:54,432 SO TO LOOK DEEPER INTO THE 3220 02:45:54,432 --> 02:45:56,334 PROVIRUS ITSELF, WE WANTED TO 3221 02:45:56,334 --> 02:46:05,009 SEE WHAT IS ACTUALLY HAPPENING. 3222 02:46:05,009 --> 02:46:11,516 EITHER WITH PFT AND ON THE LEFT 3223 02:46:11,516 --> 02:46:14,452 IS THEP LITTLE AT THE 3224 02:46:14,452 --> 02:46:15,186 INTEGRATION SITE MAYBE SOME AT 3225 02:46:15,186 --> 02:46:16,954 THE PROMOTER. 3226 02:46:16,954 --> 02:46:22,193 WHEN YOU ADD KL-2 YOU GET MORE 3227 02:46:22,193 --> 02:46:28,566 BUT COMBINED YOU GET THIS HUGE 3228 02:46:28,566 --> 02:46:31,169 SYNERGISTIC EFFECT AND THE SAME 3229 02:46:31,169 --> 02:46:32,737 EFFECT WE CAN SEE WITH RNA POLL 3230 02:46:32,737 --> 02:46:34,472 2 AND SEE THE CONTROL LOOKED IN 3231 02:46:34,472 --> 02:46:37,842 AT THE PROMOTER RENALON. 3232 02:46:37,842 --> 02:46:39,977 WHEN KL-2 HAPPENS AROUND 3233 02:46:39,977 --> 02:46:44,081 THERE'LL BE FURTHER STUDIES WE 3234 02:46:44,081 --> 02:46:45,716 SEE RNA POL 2 STARTING TO 3235 02:46:45,716 --> 02:46:47,018 PROCEED BUT WEREN'T SEEING 3236 02:46:47,018 --> 02:46:48,352 PROTEIN EXPRESSION SO MAYBE YOU 3237 02:46:48,352 --> 02:46:53,424 NEED AN INDUCER NOT JUST 3238 02:46:53,424 --> 02:46:56,093 TRANSCRIPTIONAL ELONGATION AND 3239 02:46:56,093 --> 02:46:58,329 YOU SEE MORE PROTEIN EXPRESSION 3240 02:46:58,329 --> 02:47:01,299 WITH THE COMPOUND. 3241 02:47:01,299 --> 02:47:06,103 TOGETHER YOU SEE GREAT POL 3242 02:47:06,103 --> 02:47:07,305 OCCUPANCY ACROSS THE GENOME. 3243 02:47:07,305 --> 02:47:13,277 NEXT WE WANTED TO SEE IF IT IT'S 3244 02:47:13,277 --> 02:47:14,278 INTEGRATION SITE SPECIFIC. 3245 02:47:14,278 --> 02:47:15,646 WE CHOSE A COUPLE CLONES AND 3246 02:47:15,646 --> 02:47:19,083 HE'S ARE SOME WE TESTED 6.3 AND 3247 02:47:19,083 --> 02:47:21,819 11.1 WITH THE SAME PRO VIRUS AND 3248 02:47:21,819 --> 02:47:23,254 DIFFERENT INTEGRATION SITES AND 3249 02:47:23,254 --> 02:47:25,856 SEE SYNERGY WITH KL-2. 3250 02:47:25,856 --> 02:47:28,392 OBVIOUSLY IT'S STOCHASTIC AND I 3251 02:47:28,392 --> 02:47:33,431 THINK THAT AIDS IN SHOWING HOW 3252 02:47:33,431 --> 02:47:43,941 HARD THE LATENT RESERVOIR IS 3253 02:47:46,077 --> 02:47:48,246 HERE AND WE WANTED TO LOOK INTO 3254 02:47:48,246 --> 02:47:50,848 THE REAL DEAL PMBMs FROM 3255 02:47:50,848 --> 02:47:52,416 EXPRESSED PATIENTS LIVING WITH 3256 02:47:52,416 --> 02:47:52,583 HIV. 3257 02:47:52,583 --> 02:48:02,526 WE WORKED WITH STEVE WOLLINSKI 3258 02:48:02,526 --> 02:48:13,070 AND WE HAD UNDETECTABLE VIREMIA. 3259 02:48:21,846 --> 02:48:23,748 WE SAW VERY LITTLE RESULTS THERE 3260 02:48:23,748 --> 02:48:26,150 MAYBE A LITTLE OF A THREE-FOLD 3261 02:48:26,150 --> 02:48:33,190 INCREASE BUT WHEN WE LOOKED INTO 3262 02:48:33,190 --> 02:48:35,226 AZD35582 WE SEE A 54 FOLD 3263 02:48:35,226 --> 02:48:37,862 INCREASE OF GAG TRANSCRIPT IN 3264 02:48:37,862 --> 02:48:41,032 THIS CASE AND COMPARED TO OTHER 3265 02:48:41,032 --> 02:48:43,167 TRANSCRIPTIONAL ELONGATION 3266 02:48:43,167 --> 02:48:49,340 PROMOTERS AND IPI THE SAME AS 3267 02:48:49,340 --> 02:48:52,443 AZD582 WE SAW AN INCREASE AND 3268 02:48:52,443 --> 02:48:54,545 LEADS TO THIS IDEA THAT THEY'RE 3269 02:48:54,545 --> 02:48:57,515 NOT CREATED EQUAL AND THE 3270 02:48:57,515 --> 02:49:00,351 DIFFERENT RESERVOIRS CONTAIN 3271 02:49:00,351 --> 02:49:01,786 INNATE DIFFERENCES AND NEED TO 3272 02:49:01,786 --> 02:49:02,720 UNDERSTAND WHICH ONES ARE MORE 3273 02:49:02,720 --> 02:49:03,454 IMPORTANT THAN OTHERS. 3274 02:49:03,454 --> 02:49:07,525 IN CONCLUSION WE FUNCTIONALLY 3275 02:49:07,525 --> 02:49:08,793 VALIDATED THE RESERVOIR FOR 3276 02:49:08,793 --> 02:49:09,960 VIRAL TRANSCRIPTION SITES. 3277 02:49:09,960 --> 02:49:12,963 WE CLARIFIED THE ROLES OF THE 3278 02:49:12,963 --> 02:49:14,165 COMPLEX IN HIV TRANSCRIPTION AND 3279 02:49:14,165 --> 02:49:17,902 FURTHER DEMONSTRATED THAT THESE 3280 02:49:17,902 --> 02:49:21,272 RELEASERS ARE A POTENT AND 3281 02:49:21,272 --> 02:49:22,306 SYNERGISTIC STRATEGY. 3282 02:49:22,306 --> 02:49:26,043 THEY LOOK AT DIFFERENT PFBs AND 3283 02:49:26,043 --> 02:49:28,112 DETERMINE THE REGULATED GENES 3284 02:49:28,112 --> 02:49:30,648 AND MEDICINAL CHEMISTRY TO 3285 02:49:30,648 --> 02:49:31,582 IMPROVE OUR COMPOUND FOR IN VIVO 3286 02:49:31,582 --> 02:49:33,484 STUDIES AND I'D LIKE TO 3287 02:49:33,484 --> 02:49:35,853 ACKNOWLEDGE SOME FOLKS AND THE 3288 02:49:35,853 --> 02:49:41,592 HARC CENTER AND PASS THE MIC TO 3289 02:49:41,592 --> 02:49:46,097 UJJWAL. 3290 02:49:46,097 --> 02:49:47,031 >> HI, EVERYONE. 3291 02:49:47,031 --> 02:49:52,870 IT'S BEEN A FEW YEARS OF 3292 02:49:52,870 --> 02:49:53,137 DISCOVERY. 3293 02:49:53,137 --> 02:49:55,573 HOW IT TOOK AROUND 20 YEARS TO 3294 02:49:55,573 --> 02:49:56,841 DISCOVER THE FACTOR AGAINST HIV. 3295 02:49:56,841 --> 02:50:01,812 THIS WAS A BEAUTIFUL STUDY THAT 3296 02:50:01,812 --> 02:50:09,420 SHOWED IT THE RIF VIRUS WAS 3297 02:50:09,420 --> 02:50:11,021 AFFECTING CEM AND THE WAS 3298 02:50:11,021 --> 02:50:12,556 PERMISSIVE TO WILD TYPE 3299 02:50:12,556 --> 02:50:12,923 INFECTION. 3300 02:50:12,923 --> 02:50:16,327 THIS INDICATED IT CONTAINS 3301 02:50:16,327 --> 02:50:18,763 ANTIVIRAL HOST PROTEIN THAT 3302 02:50:18,763 --> 02:50:19,964 RESTRICTS INFECTION IF NOT 3303 02:50:19,964 --> 02:50:26,070 PRESENTED AND COUNTER ACTS THE 3304 02:50:26,070 --> 02:50:27,671 ANTIVIRAL FACTOR. 3305 02:50:27,671 --> 02:50:30,608 I WAS BLOWN AWAY BOUGHT IT 3306 02:50:30,608 --> 02:50:31,542 INDICATED IT'S AN IMPORTANT TO 3307 02:50:31,542 --> 02:50:33,778 DO HIV INFECTION STUDIES IN 3308 02:50:33,778 --> 02:50:36,414 DIFFERENT CELL TYPES AND THAT'S 3309 02:50:36,414 --> 02:50:39,417 WHAT THEY DID IN THE PAPER. 3310 02:50:39,417 --> 02:50:41,018 THEY TOOK A BUNCH OF PERMISSIVE 3311 02:50:41,018 --> 02:50:49,093 AND NON-PERMISSIVE CELL LINES 3312 02:50:49,093 --> 02:50:53,898 AND SHOWED THE NON-PERMISSIVE 3313 02:50:53,898 --> 02:50:56,267 CELL TYPES HAVE PROTEIN NOT IN 3314 02:50:56,267 --> 02:51:00,738 THE PERMISSIVE CELL LINES AND 3315 02:51:00,738 --> 02:51:05,042 LATER IDENTIFIED AS APOE 3G AND 3316 02:51:05,042 --> 02:51:07,011 IF YOU PERFORM KNOCKOUT STUDIES 3317 02:51:07,011 --> 02:51:10,147 IN CONDITIONS IN WHICH PROTEIN 3318 02:51:10,147 --> 02:51:12,750 IS NOT EVEN EXPRESSED YOU CAN'T 3319 02:51:12,750 --> 02:51:14,118 UNDERSTAND IT IN THE CONTEXT OF 3320 02:51:14,118 --> 02:51:14,485 HIV INFECTION. 3321 02:51:14,485 --> 02:51:19,557 WHAT WE DECIDED TO DO WAS DO TWO 3322 02:51:19,557 --> 02:51:22,359 GENOME WIDE CRISPR SCREENS IN 3323 02:51:22,359 --> 02:51:23,694 PRIMARY CELLS FROM MULTIPLE 3324 02:51:23,694 --> 02:51:23,994 DONORS. 3325 02:51:23,994 --> 02:51:28,699 IN ONE EXPERIMENT WE DID ALL 3326 02:51:28,699 --> 02:51:33,871 HUMAN GENES AND IN ANOTHER 3327 02:51:33,871 --> 02:51:35,039 INDEPENDENT GENES WE TOOK 3328 02:51:35,039 --> 02:51:38,142 EXPRESSING GENES AND INFECT THE 3329 02:51:38,142 --> 02:51:41,612 THOSE WITH REPLICATION 3330 02:51:41,612 --> 02:51:43,113 COMPETENCE HIV VIRUS. 3331 02:51:43,113 --> 02:51:47,651 BUT PERFORMING THIS TO 3332 02:51:47,651 --> 02:51:52,323 UNDERSTAND THE ROLE OF ALL HUMAN 3333 02:51:52,323 --> 02:51:53,858 GENES POSTINFECTION. 3334 02:51:53,858 --> 02:51:56,794 ON THE X AXIS IS THE LAWFUL 3335 02:51:56,794 --> 02:51:58,629 CHANGE IN HIV INFECTION AND WHAT 3336 02:51:58,629 --> 02:52:07,137 YOU SEE IS IN RED I'M SHOWING 3337 02:52:07,137 --> 02:52:11,709 TOP THE CD4 AND THIS TO ME 3338 02:52:11,709 --> 02:52:12,743 SHOWED THE SCREENS WORKED 3339 02:52:12,743 --> 02:52:18,115 CLEARLY WELL AND ALSO IDENTIFIED 3340 02:52:18,115 --> 02:52:21,685 FACTORS LIKE THIS ONE HERE AND 3341 02:52:21,685 --> 02:52:26,123 IN BLUE YOU SEE THE ANTIVIRAL 3342 02:52:26,123 --> 02:52:29,026 FACTORS IN THE SCREEN AND YOU 3343 02:52:29,026 --> 02:52:34,565 SEE THE LIST AND SIMILARLY FROM 3344 02:52:34,565 --> 02:52:35,799 AN ACTIVATION SCREEN AND 3345 02:52:35,799 --> 02:52:38,602 IDENTIFIED A NUMBER OF KNOWN AND 3346 02:52:38,602 --> 02:52:39,403 NOVEL ANTIVIRAL FACTORS. 3347 02:52:39,403 --> 02:52:41,505 WHAT I NOTICED WHEN I WAS 3348 02:52:41,505 --> 02:52:43,641 LOOKING AT THE RESULTS 3349 02:52:43,641 --> 02:52:48,045 ESPECIALLY FROM THE SCREEN WHEN 3350 02:52:48,045 --> 02:52:49,980 YOU OVER EXPRESS THE RECEPTORS 3351 02:52:49,980 --> 02:52:51,415 WE SEE AN INCREASE IN HIV 3352 02:52:51,415 --> 02:52:52,883 INFECTION. 3353 02:52:52,883 --> 02:52:55,119 SIMILAR WHEN WE OVEREXPRESS CD 3354 02:52:55,119 --> 02:52:57,154 48 PART OF THE SIGNALLING 3355 02:52:57,154 --> 02:53:00,524 COMPLEX YOU SEE AN INCREASE IN 3356 02:53:00,524 --> 02:53:02,126 ANTI-HIV INFECTION SHOWING SOME 3357 02:53:02,126 --> 02:53:04,161 IDENTIFIED MAY BE MODULATING HIV 3358 02:53:04,161 --> 02:53:05,162 INFECTION THROUGH REGULATING THE 3359 02:53:05,162 --> 02:53:06,964 B CELL STATE OR THROUGH CHANGING 3360 02:53:06,964 --> 02:53:10,501 THE LEVEL OF HIV RECEPTOR AND 3361 02:53:10,501 --> 02:53:14,138 NOT INTERACTING WITH THE VIRUS. 3362 02:53:14,138 --> 02:53:17,041 TO DELINEATE DIRECT EFFECTS FROM 3363 02:53:17,041 --> 02:53:21,645 HIV INFECTION WE TOOK A 3364 02:53:21,645 --> 02:53:22,913 SECONDARY LIBRARY OF THE GENES 3365 02:53:22,913 --> 02:53:25,549 TARGETING ONLY THE GENES SHOWN 3366 02:53:25,549 --> 02:53:28,485 IN COLOR HERE SO A FEW HUNDRED 3367 02:53:28,485 --> 02:53:32,323 FROM A GENOME WIDE SCREEN AND 3368 02:53:32,323 --> 02:53:33,457 DELETED THOSE IN A SEPARATE 3369 02:53:33,457 --> 02:53:35,192 EXPERIMENT. 3370 02:53:35,192 --> 02:53:38,462 THE IDEA WAS YOU DELETE OR OVER 3371 02:53:38,462 --> 02:53:41,065 EXPRESS THE FEW HUNDRED GENES 3372 02:53:41,065 --> 02:53:43,867 AND THEN DIVIDE YOUR CELLS INTO 3373 02:53:43,867 --> 02:53:49,840 DIFFERENT POOLS AND STAIN THE 3374 02:53:49,840 --> 02:53:53,110 CELLS WITH THE SELECT MARKERS 3375 02:53:53,110 --> 02:53:55,446 AND HIV VIRUS AND THIS WILL HELP 3376 02:53:55,446 --> 02:53:56,847 UNDERSTAND DIRECT EFFECTS AND 3377 02:53:56,847 --> 02:53:57,548 INDIRECT EFFECTS. 3378 02:53:57,548 --> 02:54:00,351 TO SHOW YOU A SNAPSHOT OF DATA 3379 02:54:00,351 --> 02:54:03,520 FROM THE CRISPR SCREENS WHAT YOU 3380 02:54:03,520 --> 02:54:09,426 SEE IS YOU SEE AN INCREASE 3381 02:54:09,426 --> 02:54:14,164 EXPRESSION OF AND THIS INCREASES 3382 02:54:14,164 --> 02:54:16,500 HIV INFECTION AND WHEN WHY YOU 3383 02:54:16,500 --> 02:54:20,704 OVEREXPRESS YOU SEE THE CD4 AND 3384 02:54:20,704 --> 02:54:22,139 INFECTION WITH CD4. 3385 02:54:22,139 --> 02:54:26,143 HOWEVER SOME FACTORS IDENTIFIED 3386 02:54:26,143 --> 02:54:28,278 IN THIS CASE HAD MORE COMPLEX 3387 02:54:28,278 --> 02:54:28,746 PHENOTYPE. 3388 02:54:28,746 --> 02:54:31,382 SO WHAT HAPPENS HERE IS WHEN YOU 3389 02:54:31,382 --> 02:54:34,151 OVEREXPRESS YOU SEE AN INCREASE 3390 02:54:34,151 --> 02:54:36,754 IN THE LEVELS BUT YOU SEE A 3391 02:54:36,754 --> 02:54:40,824 DECREASE IN CD4 LEVELS AND THEN 3392 02:54:40,824 --> 02:54:48,699 YOU SEE IT CAN SEE AN INCREASE 3393 02:54:48,699 --> 02:54:51,802 IN HIV AND INFECTION AND WE SEE 3394 02:54:51,802 --> 02:54:54,538 DIFFERENT VIRAL FACTORS. 3395 02:54:54,538 --> 02:54:56,240 WHEN WE OVER EXPRESS IT'S A GENE 3396 02:54:56,240 --> 02:54:59,076 THAT PRODUCES MUSIN. 3397 02:54:59,076 --> 02:55:01,311 YOU SEE CELLS ARE LESS ACTIVATED 3398 02:55:01,311 --> 02:55:04,081 BECAUSE THE LEVEL ARE DECREASED. 3399 02:55:04,081 --> 02:55:06,150 BUT AT THE SAME TIME YOU ALSO 3400 02:55:06,150 --> 02:55:08,018 GET DECREASED IN HIV INFECTION 3401 02:55:08,018 --> 02:55:09,887 SO CELLS ARE LESS ACTIVATED AND 3402 02:55:09,887 --> 02:55:15,626 GETTING LESS INFECTED WITH HIV. 3403 02:55:15,626 --> 02:55:19,830 WHEREAS SOME OTHER ANTIVIRAL 3404 02:55:19,830 --> 02:55:22,132 FACTORS WE SEE WHEN YOU 3405 02:55:22,132 --> 02:55:23,734 OVEREXPRESS THERE'S NO CHANGE IN 3406 02:55:23,734 --> 02:55:25,903 CD 25 LEVEL BUT YOU SEE A 3407 02:55:25,903 --> 02:55:30,674 DECREASE IN HIV INFECTION BOTH 3408 02:55:30,674 --> 02:55:34,878 THE WILD TYPE VIRUS AS WELL AS 3409 02:55:34,878 --> 02:55:38,148 FOR OTHER ANTIVIRAL FACTORS YOU 3410 02:55:38,148 --> 02:55:41,085 SEE THOUGH THEY CAN RESTRICT THE 3411 02:55:41,085 --> 02:55:44,354 VIRUS BUT NOTE THE INFECTION 3412 02:55:44,354 --> 02:55:47,091 INDICATING THESE FACTORS MAY BE 3413 02:55:47,091 --> 02:55:52,262 WORKING AT THE SITE OF HIV ENTRY 3414 02:55:52,262 --> 02:55:55,933 AND THE RESTRICTION GOES TO THE 3415 02:55:55,933 --> 02:56:00,804 HIV ENVELOPE AND WE HAVE 3416 02:56:00,804 --> 02:56:02,139 IDENTIFIED A NUMBER OF NEW 3417 02:56:02,139 --> 02:56:06,243 ANTIVIRAL FACTORS AND WANTED TO 3418 02:56:06,243 --> 02:56:07,611 SEE HOW STRONGLY THEY RESTRICT 3419 02:56:07,611 --> 02:56:08,746 THE HIV INFECTION. 3420 02:56:08,746 --> 02:56:14,151 WE DELETED OR OVER EXPRESSED 3421 02:56:14,151 --> 02:56:15,886 THESE FACTORS IN CELLS FROM 3422 02:56:15,886 --> 02:56:18,155 DONORS AND WHAT WE FOUND WAS 3423 02:56:18,155 --> 02:56:20,991 THAT HERE IN GRAY YOU SEE THE 3424 02:56:20,991 --> 02:56:24,328 PERCENT OF HIV INFECTION FROM A 3425 02:56:24,328 --> 02:56:27,598 CONTROLLED SENSE AND SEE WHEN WE 3426 02:56:27,598 --> 02:56:30,134 KNOCK THIS OUT YOU SEE AN 3427 02:56:30,134 --> 02:56:32,402 INCREASE INFECTION AND THESE 3428 02:56:32,402 --> 02:56:33,437 WERE IDENTIFIED AS ANTIVIRAL 3429 02:56:33,437 --> 02:56:37,174 FACTORS AND YOU SEE AN INCREASE 3430 02:56:37,174 --> 02:56:38,142 IN INFECTION. 3431 02:56:38,142 --> 02:56:48,685 BECAUSE WE WERE PERTURBING THE 3432 02:56:48,685 --> 02:56:52,523 GENES AND THIS MADE THE CELL 3433 02:56:52,523 --> 02:56:54,224 MORE ACTIVATED AND SEE THE 3434 02:56:54,224 --> 02:57:02,299 INCREASE IN HIV UP INFECTION AND 3435 02:57:02,299 --> 02:57:04,268 SAW THE ANTIVIRAL FACTORS TESTED 3436 02:57:04,268 --> 02:57:07,137 WERE SUPER STRONG IN CONTROLLING 3437 02:57:07,137 --> 02:57:08,572 HIV INFECTION. 3438 02:57:08,572 --> 02:57:10,140 AND WE GOT REALLY INTERESTED IN 3439 02:57:10,140 --> 02:57:13,410 SOME OF THE VECTORS HERE WHICH 3440 02:57:13,410 --> 02:57:15,612 COULD CONTROL HIV INFECTION FOR 3441 02:57:15,612 --> 02:57:15,879 MANY DAYS. 3442 02:57:15,879 --> 02:57:18,148 AND I'M GOING TO FIRST TALK 3443 02:57:18,148 --> 02:57:23,187 ABOUT SOME OF THESE WHICH WE 3444 02:57:23,187 --> 02:57:24,721 FOUND THROUGH PROTEIN 3445 02:57:24,721 --> 02:57:25,022 EXPRESSION. 3446 02:57:25,022 --> 02:57:28,792 SOME OF THESE, 50% OF THE 3447 02:57:28,792 --> 02:57:31,195 FACTORS IDENTIFIED IN THE 3448 02:57:31,195 --> 02:57:32,629 SCREENS ARE EXPRESSING D CELLS 3449 02:57:32,629 --> 02:57:35,132 AND THEY COME FROM A GAIN OF 3450 02:57:35,132 --> 02:57:36,834 FUNCTION SCREAM. 3451 02:57:36,834 --> 02:57:39,636 WE SAW THE PROTEASE INHIBITER IS 3452 02:57:39,636 --> 02:57:42,005 NOT EXPRESSED IN T CELLS. 3453 02:57:42,005 --> 02:57:45,142 HOWEVER, WHEN WE LOOK AT CELLS 3454 02:57:45,142 --> 02:57:48,879 AT SINGLE-CELL LEVEL WE FIND 3455 02:57:48,879 --> 02:57:52,249 THAT IT A SMALL TRACTION OF B 3456 02:57:52,249 --> 02:57:54,585 CELLS CAN EXPRESS PL16 AND SOME 3457 02:57:54,585 --> 02:57:58,989 CAN EXPRESS AT HIGH LEVELS. 3458 02:57:58,989 --> 02:58:02,125 THIS WAS INTERESTING. 3459 02:58:02,125 --> 02:58:03,760 THIS INDICATED MAYBE THERE MAY 3460 02:58:03,760 --> 02:58:07,898 BE CELLS IN THE BODY A SMALL 3461 02:58:07,898 --> 02:58:09,733 FRACTION WHICH ARE NATURALLY 3462 02:58:09,733 --> 02:58:14,771 RESISTANT TO HIV INFECTION. 3463 02:58:14,771 --> 02:58:17,241 WE WANTED TO UNDERSTAND HOW THIS 3464 02:58:17,241 --> 02:58:19,209 IS AFFECTING HIV INFECTION AND 3465 02:58:19,209 --> 02:58:20,944 WE STAINED THE CELLS TO SEE WHAT 3466 02:58:20,944 --> 02:58:21,845 PERCENTAGE OF THE CELLS 3467 02:58:21,845 --> 02:58:22,746 EXPRESSED THIS PROTEIN. 3468 02:58:22,746 --> 02:58:28,051 WHAT WE SAW WAS VERY 3469 02:58:28,051 --> 02:58:30,120 INTERESTING. 3470 02:58:30,120 --> 02:58:32,055 THEY EXPRESSED THE PROTEIN AND 3471 02:58:32,055 --> 02:58:34,024 HOWEVER, THE EXPRESSION GOES 3472 02:58:34,024 --> 02:58:35,158 DOWN WITH ACTIVATION. 3473 02:58:35,158 --> 02:58:36,760 IF YOU ARTIFICIALLY EXPRESS THIS 3474 02:58:36,760 --> 02:58:43,367 PROTEIN ON T CELLS WHAT YOU SEE 3475 02:58:43,367 --> 02:58:46,703 IS WHEN PI16 AT THE LEADS TO HIV 3476 02:58:46,703 --> 02:58:49,006 INFECTION AND IT COMES DOWN TO 3477 02:58:49,006 --> 02:58:50,941 1% WHEN YOU OVEREXPRESS THIS 3478 02:58:50,941 --> 02:58:52,576 PROTEIN IN T CELLS. 3479 02:58:52,576 --> 02:58:56,914 AND WE FOUND OUT THAT THE PI16 3480 02:58:56,914 --> 02:58:59,316 THE EXPRESSION DOESN'T AFFECT 3481 02:58:59,316 --> 02:59:02,152 BINDING TO THE CELL PHASE BUT 3482 02:59:02,152 --> 02:59:08,058 WHEN YOU OVEREXPRESS YOU WILL 3483 02:59:08,058 --> 02:59:09,192 PREVENT FUSION AND THE 3484 02:59:09,192 --> 02:59:10,894 OVEREXPRESSION PREVENTS VIRAL 3485 02:59:10,894 --> 02:59:14,164 ENTRY AND BY COLLABORATING WITH 3486 02:59:14,164 --> 02:59:18,502 THE PROTEOMICS CORE WE FOUND 3487 02:59:18,502 --> 02:59:22,172 PI16 INTERACTS WITH A LARGE 3488 02:59:22,172 --> 02:59:25,242 NUMBER OF HOST FACTORS AND FOR 3489 02:59:25,242 --> 02:59:28,111 EXAMPLE MOLECULES FOR SIGNALLING 3490 02:59:28,111 --> 02:59:29,780 AND THIS WAS A GREAT 3491 02:59:29,780 --> 02:59:32,282 COLLABORATION ON GENOMICS TO 3492 02:59:32,282 --> 02:59:35,018 UNDERSTAND HOW IT WAS AFFECTED 3493 02:59:35,018 --> 02:59:37,921 BY THE COHORT AND HOW IT WAS 3494 02:59:37,921 --> 02:59:39,756 WORKING. 3495 02:59:39,756 --> 02:59:42,159 THE QUESTION WAS THIS IS NOT 3496 02:59:42,159 --> 02:59:43,994 NORMALLY EXPRESSED T CELLS. 3497 02:59:43,994 --> 02:59:47,531 AND BY LOOKING AT THE DATA FROM 3498 02:59:47,531 --> 02:59:54,171 THOSE WITH ACUTE VIREMIA WE 3499 02:59:54,171 --> 02:59:59,910 FOUND THE T CELLS WITH HI 3500 02:59:59,910 --> 03:00:03,347 NEGATIVE HIGHER FRACTIONS OF 3501 03:00:03,347 --> 03:00:05,882 POSITIVE COMPARED TO IM POSITIVE 3502 03:00:05,882 --> 03:00:06,149 CELLS. 3503 03:00:06,149 --> 03:00:11,855 IT'S NOT WHAT IT LOOKS LIKE AND 3504 03:00:11,855 --> 03:00:17,194 CELLS THAT HAVE PI16 ARE MORE 3505 03:00:17,194 --> 03:00:23,200 RESISTANT TO INFECTION AND 3506 03:00:23,200 --> 03:00:25,402 IDENTIFIED THESE AND THE REASON 3507 03:00:25,402 --> 03:00:30,173 BEING PPA CAME UP IN BOTH 3508 03:00:30,173 --> 03:00:34,144 SCREENS AROUND STRONG ANTIVIRAL 3509 03:00:34,144 --> 03:00:39,249 FACTOR AND IT BELONGS TO THE 3510 03:00:39,249 --> 03:00:42,619 CYCLIN FAMILY OF PROTEINS AND 3511 03:00:42,619 --> 03:00:43,987 YOU SEE THERE'S AROUND 60% 3512 03:00:43,987 --> 03:00:45,088 SEQUENCE IDENTIFY AND WHEN YOU 3513 03:00:45,088 --> 03:00:48,191 LOOK AT THE STRUCTURE AND YOU 3514 03:00:48,191 --> 03:00:52,162 FIND OUT THAT IT CAN ACTUALLY 3515 03:00:52,162 --> 03:00:56,366 SUPER IMPROSE WITH PPIA AND 3516 03:00:56,366 --> 03:00:57,801 THERE'S STRONG POSITIVE 3517 03:00:57,801 --> 03:00:58,068 SELECTION. 3518 03:00:58,068 --> 03:01:02,139 IT'S POSSIBLE IT MAY BE 3519 03:01:02,139 --> 03:01:04,708 INTERACTING WITH CAPSID. 3520 03:01:04,708 --> 03:01:07,511 TO CONCLUDE I THINK WE HAVE 3521 03:01:07,511 --> 03:01:08,612 FOUND THINGS WHICH CAN AFFECT 3522 03:01:08,612 --> 03:01:10,147 THE HIV INFECTION IN MULTIPLE 3523 03:01:10,147 --> 03:01:16,386 STAGES AND WE NEED TO MAKE 3524 03:01:16,386 --> 03:01:18,188 FRIENDS TO STAY FOR FOM SIME IN 3525 03:01:18,188 --> 03:01:22,125 B-HIVE AND LOOK AT HOW THEY 3526 03:01:22,125 --> 03:01:22,325 WORK. 3527 03:01:22,325 --> 03:01:32,502 THANK YOU. 3528 03:01:37,007 --> 03:01:38,075 IT 3529 03:01:59,563 --> 03:02:01,331 >> GREAT TALKS, EVERYBODY. 3530 03:02:01,331 --> 03:02:03,633 I HAVE A MILLION QUESTIONS FOR 3531 03:02:03,633 --> 03:02:04,101 ALL OF YOU. 3532 03:02:04,101 --> 03:02:06,169 THE MOST RECENT I WANTED TO ASK 3533 03:02:06,169 --> 03:02:10,941 YOU THE SCREENS YOU'RE DOING 3534 03:02:10,941 --> 03:02:13,176 WITH EXPO TROPIC VIRUS WONDERING 3535 03:02:13,176 --> 03:02:17,214 IF YOU COULD COMMENT BETWEEN X4 3536 03:02:17,214 --> 03:02:22,519 AND R5. 3537 03:02:22,519 --> 03:02:26,089 CLEARLY IT'S TECHNICALLY HARDER 3538 03:02:26,089 --> 03:02:28,125 AND YOU TALKED ABOUT THE 3539 03:02:28,125 --> 03:02:28,525 DIFFERENT SUBSETS. 3540 03:02:28,525 --> 03:02:30,760 I'M CURIOUS, DO YOU HAVE SOME 3541 03:02:30,760 --> 03:02:31,361 COMMENTS ABOUT IT? 3542 03:02:31,361 --> 03:02:33,697 >> ABSOLUTELY. 3543 03:02:33,697 --> 03:02:38,135 SO WE INITIALLY STRUGGLED A LOT 3544 03:02:38,135 --> 03:02:39,936 TO GET HIGH INFECTION RATES OF 3545 03:02:39,936 --> 03:02:42,139 THE VIRUS TO DO THE SCREENS BUT 3546 03:02:42,139 --> 03:02:44,841 LATER WE WERE ABLE TO DO THAT. 3547 03:02:44,841 --> 03:02:47,878 NOW I UNDERSTAND WHICH VECTORS 3548 03:02:47,878 --> 03:02:49,146 UNDERSTAND THE VIRUS. 3549 03:02:49,146 --> 03:02:57,621 TO GIVE AN EXAMPLE, DBX21 THE 3550 03:02:57,621 --> 03:02:58,855 TRANSCRIPTION FACTOR IS A STRONG 3551 03:02:58,855 --> 03:02:59,789 PROTECTIVE SCREEN AGAINST THE 3552 03:02:59,789 --> 03:03:04,294 VIRUS BUT WE FOUND IT ACTUALLY 3553 03:03:04,294 --> 03:03:10,133 IS A HYPOTROPHIC VIRUS AND YOU 3554 03:03:10,133 --> 03:03:13,170 INCREASE THE LEVELS AND LOOKS 3555 03:03:13,170 --> 03:03:14,304 LIKE INFECTION OF THE VIRUS. 3556 03:03:14,304 --> 03:03:16,139 SO WE HAVE A LOT OF SITES FROM 3557 03:03:16,139 --> 03:03:17,274 THE DATA WE ARE GENERATING. 3558 03:03:17,274 --> 03:03:18,508 >> COOL. 3559 03:03:18,508 --> 03:03:20,710 AND DO YOU HAVE ANY EVIDENCE 3560 03:03:20,710 --> 03:03:28,818 PPID THE PROTEIN ENCODED IS 3561 03:03:28,818 --> 03:03:30,987 EXISTING INDEPENDENT OF 3562 03:03:30,987 --> 03:03:31,321 MITOCHONDRIA? 3563 03:03:31,321 --> 03:03:32,789 IT'S SUPPOSED TO BE IN THE 3564 03:03:32,789 --> 03:03:33,290 MITOCHONDRIAL MEMBRANE? 3565 03:03:33,290 --> 03:03:36,893 >> IT'S ALSO REPRESENTED IN THE 3566 03:03:36,893 --> 03:03:42,732 CYTOPLASM AND CAN HAVE 3567 03:03:42,732 --> 03:03:43,433 NUCLEARIZATION. 3568 03:03:43,433 --> 03:03:44,501 >> YOU WONDER IF IT HAS 3569 03:03:44,501 --> 03:03:46,002 SOMETHING TO DO WITH THE 3570 03:03:46,002 --> 03:03:46,369 MITOCHONDRIA. 3571 03:03:46,369 --> 03:03:50,140 >> WHAT WE'RE DOING RIGHT NOW IS 3572 03:03:50,140 --> 03:03:54,311 A SOMEWHAT SIMPLISTIC 3573 03:03:54,311 --> 03:03:54,611 EXPERIMENT. 3574 03:03:54,611 --> 03:03:56,313 WE LOOK AT THE STRONG POSITIVE 3575 03:03:56,313 --> 03:03:58,682 SELECTION AND TESTING PPID 3576 03:03:58,682 --> 03:03:59,349 PROTEINS FROM DIFFERENT SPECIES 3577 03:03:59,349 --> 03:04:04,154 TO FIGURE OUT IF ALL OF US ARE 3578 03:04:04,154 --> 03:04:07,991 MEDIATING THE FUNCTION. 3579 03:04:07,991 --> 03:04:11,995 A PART FROM THE PSYCYCLOPHILIN 3580 03:04:11,995 --> 03:04:13,697 ACTIVITY THERE'S SHEPHERDING 3581 03:04:13,697 --> 03:04:15,232 ACTIVITY AS WELL BUT IT'S AN 3582 03:04:15,232 --> 03:04:16,199 INTERESTING SITUATION. 3583 03:04:16,199 --> 03:04:18,134 >> THERE'S ALSO LITERATURE 3584 03:04:18,134 --> 03:04:23,273 FACTORS LIKE ARSENIC AND OTHER 3585 03:04:23,273 --> 03:04:25,342 DRUGS THAT AFFECT THE STATUS AND 3586 03:04:25,342 --> 03:04:28,178 THOSE AFFECT RESTRICTION FACTORS 3587 03:04:28,178 --> 03:04:30,313 IN WAYS WE DON'T UNDERSTAND. 3588 03:04:30,313 --> 03:04:32,282 >> VERY GOOD SESSION. 3589 03:04:32,282 --> 03:04:32,549 THANK YOU. 3590 03:04:32,549 --> 03:04:43,260 >> A QUESTION FOR NEF -- NEVIN 3591 03:04:43,260 --> 03:04:45,996 IF YOU DON'T MIND. 3592 03:04:45,996 --> 03:04:47,297 YOU PRESENTED THE INFORMATION 3593 03:04:47,297 --> 03:04:51,301 AND LOOKED LIKE IT IDENTIFIED 3594 03:04:51,301 --> 03:04:53,136 TWO DIFFERENT INTERFACES THAT 3595 03:04:53,136 --> 03:04:54,971 FORM THAT WILL AND YOU SAW THE 3596 03:04:54,971 --> 03:04:55,238 STRUCTURE. 3597 03:04:55,238 --> 03:04:56,239 HOW CLOSE WAS THE STRUCTURE TO 3598 03:04:56,239 --> 03:04:58,842 THE PREDICTED? 3599 03:04:58,842 --> 03:05:01,111 >> IT WAS -- I SHOULD HAVE SHOWN 3600 03:05:01,111 --> 03:05:03,747 THE PREDICTION WITH THE ACTUAL 3601 03:05:03,747 --> 03:05:04,014 STRUCTURE. 3602 03:05:04,014 --> 03:05:08,318 IT PREDICTED BOTH INTERFACES. 3603 03:05:08,318 --> 03:05:10,353 >> AND THEN THAT'S AWESOME. 3604 03:05:10,353 --> 03:05:11,755 WHEN IT WORKS IT WORKS. 3605 03:05:11,755 --> 03:05:15,158 AND WHAT ABOUT THE SECOND, THIRD 3606 03:05:15,158 --> 03:05:16,026 AND FOURTH COMPLEX? 3607 03:05:16,026 --> 03:05:17,994 >> WE'RE ON TWO, THREE AND FOUR 3608 03:05:17,994 --> 03:05:19,963 AND LOOKS LIKE NUMBER TWO IS 3609 03:05:19,963 --> 03:05:20,263 WORKING. 3610 03:05:20,263 --> 03:05:23,233 >> MAYBE 50/50. 3611 03:05:23,233 --> 03:05:23,433 GOOD. 3612 03:05:23,433 --> 03:05:25,235 >> WE CAN REPORT BACK SOON DAVID 3613 03:05:25,235 --> 03:05:30,006 ON THE SUCCESS RATE. 3614 03:05:30,006 --> 03:05:36,079 >> I HAVE A QUESTION ABOUT THE 3615 03:05:36,079 --> 03:05:36,379 TL2. 3616 03:05:36,379 --> 03:05:41,151 YOU FOUND WHEN YOU ADD IT YOU 3617 03:05:41,151 --> 03:05:42,485 FOUND POLYMERASE ALL OVER THE 3618 03:05:42,485 --> 03:05:43,420 PROVIRUS, YES? 3619 03:05:43,420 --> 03:05:47,290 SO DO YOU ANALYZE ABOUT THE 3620 03:05:47,290 --> 03:05:50,894 CELLULAR GENOME DOES THE SAME 3621 03:05:50,894 --> 03:05:51,594 PREDICT? 3622 03:05:51,594 --> 03:05:54,097 >> WE DEFINITELY SEE THE 3623 03:05:54,097 --> 03:05:57,334 CONSIDERATION OF IT IS A TOOL 3624 03:05:57,334 --> 03:06:00,003 COMPOUND AT HIGH MICROMOLAR 3625 03:06:00,003 --> 03:06:05,975 CONCENTRATIONS IT'S DEFINITELY 3626 03:06:05,975 --> 03:06:06,376 CYTOTOXIC. 3627 03:06:06,376 --> 03:06:08,411 ADDITIONALLY A LOT OF SEC 3628 03:06:08,411 --> 03:06:09,312 REGULATED GENES ARE ALONG THE 3629 03:06:09,312 --> 03:06:11,581 CELL CYCLE AND YOU SEE A REDUCED 3630 03:06:11,581 --> 03:06:22,125 CAPACITY TO PROLIVERATE BUT -- 3631 03:06:27,931 --> 03:06:32,302 PROLIFERATE BUT REDUCING THE 3632 03:06:32,302 --> 03:06:36,639 PROLIFERATORY CAPABILITY. 3633 03:06:36,639 --> 03:06:41,978 >> SO HAVE YOU TRIED TO LOOK AT 3634 03:06:41,978 --> 03:06:45,215 THE MONOCYTIC CELL LINE AND THE 3635 03:06:45,215 --> 03:06:45,949 ENTROPIC VIRUSES? 3636 03:06:45,949 --> 03:06:48,485 I'LL GIVE BACKGROUND ON THAT. 3637 03:06:48,485 --> 03:06:53,289 SO I HAD A HARC PILOT GRANT AND 3638 03:06:53,289 --> 03:06:58,828 WE JUST PUBLISHED A FEW MONTHS 3639 03:06:58,828 --> 03:06:59,295 BACK. 3640 03:06:59,295 --> 03:07:03,099 THIS PBMCs RESTRICT HIV AND 3641 03:07:03,099 --> 03:07:05,268 ESPECIALLY THE ACTIVATED ONE AND 3642 03:07:05,268 --> 03:07:06,870 HAVE RESTRICTION FACTORS 3643 03:07:06,870 --> 03:07:08,671 UPREGULATED. 3644 03:07:08,671 --> 03:07:11,107 NOW, IT THE REVIEWS FOR THE DATA 3645 03:07:11,107 --> 03:07:18,148 AND WHEN WE DID THAT, WE FOUND 3646 03:07:18,148 --> 03:07:27,390 THAT SICKLE CELL AND WONDER IF 3647 03:07:27,390 --> 03:07:32,695 THE T CELL CONTRIBUTE TO THE 3648 03:07:32,695 --> 03:07:34,697 RESTRICTION AND WONDERED IF YOU 3649 03:07:34,697 --> 03:07:35,865 LOOKED AT THAT PART? 3650 03:07:35,865 --> 03:07:37,567 >> WE DID GAIN OF FUNCTION 3651 03:07:37,567 --> 03:07:41,070 SCREENS WHICH MEANS YOU CAN 3652 03:07:41,070 --> 03:07:42,138 REVEAL PHENOTYPES NOT NATIVE TO 3653 03:07:42,138 --> 03:07:43,540 D CELLS. 3654 03:07:43,540 --> 03:07:48,711 WE FOUND THINGS LIKE CYCLIC 1 3655 03:07:48,711 --> 03:07:50,847 WHICH HAVE NO CONTEXT IN HIV 3656 03:07:50,847 --> 03:07:51,981 INFECTION AND WHEN YOU 3657 03:07:51,981 --> 03:07:54,150 OVEREXPRESS THOSE THEY SHOW A 3658 03:07:54,150 --> 03:07:56,152 SIMILAR PHENOTYPE ON DENDRITIC 3659 03:07:56,152 --> 03:07:57,287 CELLS WHEN YOU OVEREXPRESS AND 3660 03:07:57,287 --> 03:08:01,157 SEE THE SAME PHENOTYPE WE FOUND 3661 03:08:01,157 --> 03:08:04,661 WE'RE IDENTIFYING MACROPHAGES 3662 03:08:04,661 --> 03:08:05,995 AND MONOCYTES AND CAN DISTRIBUTE 3663 03:08:05,995 --> 03:08:07,096 THEM BY DOING SCREENS THROUGH 3664 03:08:07,096 --> 03:08:10,300 GAIN OF FUNCTION SCREENS. 3665 03:08:10,300 --> 03:08:12,001 >> THANK YOU. 3666 03:08:12,001 --> 03:08:14,137 >> HI. 3667 03:08:14,137 --> 03:08:18,107 YOU SAID PI16 IS INHIBITING THE 3668 03:08:18,107 --> 03:08:19,709 FUSION OF HIV. 3669 03:08:19,709 --> 03:08:25,315 DOES IT INHIBIT X4 AND R5? 3670 03:08:25,315 --> 03:08:26,115 >> SO NO. 3671 03:08:26,115 --> 03:08:29,486 SO IT SPECIFICALLY INFECTION FOR 3672 03:08:29,486 --> 03:08:31,387 TROPIC VIRUS. 3673 03:08:31,387 --> 03:08:32,856 I WAS INITIALLY SURPRISED TO SEE 3674 03:08:32,856 --> 03:08:38,761 THAT BUT WE FOUND OUT THROUGH 3675 03:08:38,761 --> 03:08:43,433 PROTEOMICS THE MOLECULES ARE 3676 03:08:43,433 --> 03:08:45,568 BELOW SIGNALLING AND DIFFERENT 3677 03:08:45,568 --> 03:08:46,002 HOW SFLALING WORKS. 3678 03:08:46,002 --> 03:08:50,740 -- SIGNALLING WORKS. 3679 03:08:50,740 --> 03:08:53,776 IT MAKES SENSE BUT DOESN'T 3680 03:08:53,776 --> 03:09:00,884 COMPARE TO PPID. 3681 03:09:00,884 --> 03:09:03,253 >> AND WE KNOW AT WHAT STAGE OF 3682 03:09:03,253 --> 03:09:05,421 HIV INFECTION IT INHIBITS THE 3683 03:09:05,421 --> 03:09:10,894 INFECTION. 3684 03:09:10,894 --> 03:09:12,762 PI16 NOT EXPRESSED IN MACK 3685 03:09:12,762 --> 03:09:19,769 REFATHERS BUT IN FIBROPLASTS AND 3686 03:09:19,769 --> 03:09:30,280 MISS EN -- MESSENCHIMAL CELLS. 3687 03:09:40,523 --> 03:09:41,824 >> DID YOU MEAN ESTABLISHMENT IN 3688 03:09:41,824 --> 03:09:45,562 INFECTION OR LOOKING AT THE 3689 03:09:45,562 --> 03:09:46,596 ENTIRE REPLICATION PROCESS? 3690 03:09:46,596 --> 03:09:47,864 IN OTHER WORDS, WERE VIRUS 3691 03:09:47,864 --> 03:09:48,965 PARTICLES BEING MADE IN THE 3692 03:09:48,965 --> 03:09:49,933 CELLS? 3693 03:09:49,933 --> 03:09:53,903 >> YES, SO THESE CELLS WE SAW -- 3694 03:09:53,903 --> 03:09:54,604 YEAH. 3695 03:09:54,604 --> 03:09:57,507 NO AFFECT OF THE KNOCKOUT OF THE 3696 03:09:57,507 --> 03:10:00,410 SUPER ELONGATION AND THE 3697 03:10:00,410 --> 03:10:02,879 ACTIVATED CELLS WERE INFECTED 3698 03:10:02,879 --> 03:10:04,781 WITH VIRUS AND NO CULTURE LIKE 3699 03:10:04,781 --> 03:10:09,519 NO ARC SO IT IT COULD HAVE BEEN 3700 03:10:09,519 --> 03:10:19,996 PRODUCING MORE VIREONES BUT 3701 03:10:21,331 --> 03:10:25,268 DIDN'T LOOK LIKE MORE WILL 3702 03:10:25,268 --> 03:10:27,737 REPLICATION FOR THE CULTURES. 3703 03:10:27,737 --> 03:10:37,914 [APPLAUSE] 3704 03:10:44,887 --> 03:10:45,388 >> ALL RIGHT. 3705 03:10:45,388 --> 03:10:49,292 SO THAT'S OUR MORNING SESSION. 3706 03:10:49,292 --> 03:10:51,728 PLEASE GO AHEAD AND GET YOUR 3707 03:10:51,728 --> 03:10:53,496 LUNCH AND MAKE SURE YOU MAKE IT 3708 03:10:53,496 --> 03:10:56,265 TO YOUR POSTERS. 3709 03:10:56,265 --> 03:10:57,900 WE HAVE POSTER PRESENTERS TO BE 3710 03:10:57,900 --> 03:10:59,402 AT THEIR POSTER AT 1:00. 3711 03:10:59,402 --> 03:11:01,170 NEXT SESSION SPEAKERS CAN EITHER 3712 03:11:01,170 --> 03:11:05,108 COME UP NOW, PREFERABLY NOW OR 3713 03:11:05,108 --> 03:11:10,146 BEFORE THE 2:30 RESTART TO GET 3714 03:11:10,146 --> 03:11:13,016 YOUR TALKS LOADED. 3715 03:11:13,016 --> 03:11:14,484 PLEASE TRY TO DO SO SO WE DON'T 3716 03:11:14,484 --> 03:11:18,634 HAVE TO TRACK YOU DOWN AT 2:30. 3717 03:11:24,487 --> 03:11:26,289 IS THAT GOOD? 3718 03:11:26,289 --> 03:11:26,523 OKAY. 3719 03:11:26,523 --> 03:11:27,924 JUST AS A BRIEF INTRODUCTION, 3720 03:11:27,924 --> 03:11:32,762 I'M SURE MANY OF YOU ARE AWARE 3721 03:11:32,762 --> 03:11:35,632 HIV BUDS FROM THE SURFACE OF THE 3722 03:11:35,632 --> 03:11:37,200 CELL, UNDERGOES A PROCESS CALLED 3723 03:11:37,200 --> 03:11:38,134 MATURATION. 3724 03:11:38,134 --> 03:11:40,637 ONCE IT'S DONE THIS, ONLY ONCE, 3725 03:11:40,637 --> 03:11:43,406 CAN IT FUSE WITH THE HOST CELL 3726 03:11:43,406 --> 03:11:43,673 MEMBRANE. 3727 03:11:43,673 --> 03:11:46,509 IN OTHER LAB WE THINK OF THIS ON 3728 03:11:46,509 --> 03:11:48,345 THREE LEVELS. 3729 03:11:48,345 --> 03:11:51,014 FIRST IS PROTEOLYTIC MATURE, SO 3730 03:11:51,014 --> 03:11:59,589 GAG IS CLEAVED INTO CONSTITUENT 3731 03:11:59,589 --> 03:12:01,291 DOMAIN, AND TWO PEPTIDES, 3732 03:12:01,291 --> 03:12:02,659 ACCOMPANIED BY STRUCTURAL 3733 03:12:02,659 --> 03:12:08,798 CHANGES AND THE BEST STUDIED IS 3734 03:12:08,798 --> 03:12:09,699 CAPSID. 3735 03:12:09,699 --> 03:12:11,101 THEN THERE'S ALSO ARCHITECTURAL 3736 03:12:11,101 --> 03:12:12,435 MATURATION, SO ARRANGEMENT OF 3737 03:12:12,435 --> 03:12:20,210 PROTEINS IN THE VIRUS ALSO 3738 03:12:20,210 --> 03:12:20,643 CHANGES. 3739 03:12:20,643 --> 03:12:25,582 SO, KERN IN OUR LAB DISCOVERED 3740 03:12:25,582 --> 03:12:30,253 LIKE CAPSID, MATRIX FORMS A 3741 03:12:30,253 --> 03:12:31,388 LATTICE IN VIRUSES. 3742 03:12:31,388 --> 03:12:32,589 OF PARTICULAR IMPORTANCE IS THE 3743 03:12:32,589 --> 03:12:34,758 FACT THAT THIS LATTICE ALSO 3744 03:12:34,758 --> 03:12:36,393 APPEARS TO UNDERGO STRUCTURAL 3745 03:12:36,393 --> 03:12:37,193 MATURATION. 3746 03:12:37,193 --> 03:12:41,531 SO IN THE IMMATURE VIRUS YOU GET 3747 03:12:41,531 --> 03:12:44,401 THE IMMATURE LATTICE, IN MATURE 3748 03:12:44,401 --> 03:12:49,205 WE GET MATURE LATTICE. 3749 03:12:49,205 --> 03:12:52,976 IT WAS OBSERVED A LIGAND WAS 3750 03:12:52,976 --> 03:12:55,345 BOUND TO MATRIX, ABSENT IN THE 3751 03:12:55,345 --> 03:12:55,745 IMMATURE LATTICE. 3752 03:12:55,745 --> 03:12:59,582 THIS IS A VIDEO THAT SUMMARIZES 3753 03:12:59,582 --> 03:12:59,783 THAT. 3754 03:12:59,783 --> 03:13:01,584 SO, I WILLING AND IS BOUND IN 3755 03:13:01,584 --> 03:13:02,786 THE MATURE FORM. 3756 03:13:02,786 --> 03:13:04,521 SO THERE'S SOME OPEN QUESTIONS 3757 03:13:04,521 --> 03:13:05,054 LEFT OVER. 3758 03:13:05,054 --> 03:13:08,057 FIRST IS WHAT IS THE 3759 03:13:08,057 --> 03:13:10,627 RELATIONSHIP BETWEEN CLEAVAGE OF 3760 03:13:10,627 --> 03:13:12,529 GAG AND MATURATION OF THE 3761 03:13:12,529 --> 03:13:12,896 LATTICE. 3762 03:13:12,896 --> 03:13:16,032 THE LAST THING HE LEFT US WAS 3763 03:13:16,032 --> 03:13:17,467 WITH OBSERVATION IT APPEARS 3764 03:13:17,467 --> 03:13:24,007 SIMPLE CLEAVAGE IS NOT THE 3765 03:13:24,007 --> 03:13:24,507 TRIGGER. 3766 03:13:24,507 --> 03:13:29,379 HE OBSERVED MAFP 1 IN LICK THE 3767 03:13:29,379 --> 03:13:30,613 NUCLEOCAPSID IS CLEAVED, LATTICE 3768 03:13:30,613 --> 03:13:34,384 WAS MATURE COMPLETE WITH BOUND 3769 03:13:34,384 --> 03:13:35,151 LIGAND. 3770 03:13:35,151 --> 03:13:38,321 NEXT HYPOTHESIS WAS ALLOSTERIC 3771 03:13:38,321 --> 03:13:44,594 NETWORK ACROSS SP 1 AND CAPSID, 3772 03:13:44,594 --> 03:13:48,164 FOUND NO EVIDENCE OF ALLOSTERIC 3773 03:13:48,164 --> 03:13:48,398 NETWORK. 3774 03:13:48,398 --> 03:13:50,800 MY JOB WAS TO INVESTIGATE THE 3775 03:13:50,800 --> 03:13:57,707 STATE OF MATRIX, FIRST USING 3776 03:13:57,707 --> 03:14:02,679 CO-ANALYSIS TO CLASSIFY GAG 3777 03:14:02,679 --> 03:14:03,713 LAYER AND DIRECTLY LOOK AT THE 3778 03:14:03,713 --> 03:14:06,783 STATE OF THE LATTICE. 3779 03:14:06,783 --> 03:14:09,986 SECOND WAY TO PERFORM FOURIER 3780 03:14:09,986 --> 03:14:14,891 ANALYSIS AS OBSERVED IN 2D 3781 03:14:14,891 --> 03:14:17,293 CLASSES, AS THE SPACINGS OF THE 3782 03:14:17,293 --> 03:14:18,862 TWO LATTICES ARE DISTINCT, 3783 03:14:18,862 --> 03:14:20,163 GIVING DISTINCT PROFILES. 3784 03:14:20,163 --> 03:14:26,503 IF WE START WITH IMMATURE VIRUS, 3785 03:14:26,503 --> 03:14:30,340 WE OBSERVE ELASTIC SPACE 3786 03:14:30,340 --> 03:14:33,309 CONSISTENT AND CAN GET A 3D. 3787 03:14:33,309 --> 03:14:35,912 IF WE CLEAVE THE BOTTOM DOMAIN 3788 03:14:35,912 --> 03:14:42,151 OF GAG P 6, WE DON'T HAVE 3D 3789 03:14:42,151 --> 03:14:43,353 CONSTRUCTION, BUT FOURIER 3790 03:14:43,353 --> 03:14:44,254 ANALYSIS LIKELY IMMATURE 3791 03:14:44,254 --> 03:14:44,521 ANALYSIS. 3792 03:14:44,521 --> 03:14:47,457 IF WE CLEAVE THE NEXT TWO, P6 3793 03:14:47,457 --> 03:14:51,494 AND SP2 WE SEE STRIKING CHANGE, 3794 03:14:51,494 --> 03:14:53,963 WE SEE A MATURE LATTICE. 3795 03:14:53,963 --> 03:14:56,633 IF WE KEEP GOING WE AGAIN 3796 03:14:56,633 --> 03:14:58,034 OBSERVE MATURE LATTICES, UP TO 3797 03:14:58,034 --> 03:15:02,505 THE MATURE VIRUS. 3798 03:15:02,505 --> 03:15:04,607 SO THIS START MAKING US THING 3799 03:15:04,607 --> 03:15:06,242 SP2 MIGHT BE IMPORTANT FOR 3800 03:15:06,242 --> 03:15:09,746 REGULATION OF MATURATION OF 3801 03:15:09,746 --> 03:15:10,213 MATRIX. 3802 03:15:10,213 --> 03:15:13,550 AND SHOULD WE GENERATE MUTE 3803 03:15:13,550 --> 03:15:15,752 RANTS WHERE SP2 WASN'T RELEASED, 3804 03:15:15,752 --> 03:15:17,587 HELD IN THE BUNDLE, THIS WAY WE 3805 03:15:17,587 --> 03:15:19,622 COULD APPEAR TO SUPPRESS THE 3806 03:15:19,622 --> 03:15:22,892 MATURATION OF THE MATRIX 3807 03:15:22,892 --> 03:15:23,426 LATTICE. 3808 03:15:23,426 --> 03:15:26,296 SO TO SUMMARIZE, WE BELIEVE 3809 03:15:26,296 --> 03:15:29,265 REALLY SP2 IS IMPORTANT FOR 3810 03:15:29,265 --> 03:15:30,867 MATRIX MATURATION. 3811 03:15:30,867 --> 03:15:34,304 INTERESTINGLY, SP2 IS BASICALLY 3812 03:15:34,304 --> 03:15:36,372 THE ONLY COMPONENT OF HIV WITH 3813 03:15:36,372 --> 03:15:38,141 NO ASSIGNED FUNCTION. 3814 03:15:38,141 --> 03:15:41,311 I ENCOURAGED THIS WHERE YOU CAN 3815 03:15:41,311 --> 03:15:45,515 SEE SP2 ANNOTATED WITH THE 3816 03:15:45,515 --> 03:15:46,716 FUNCTION, HIGHLY CONSERVED ALSO. 3817 03:15:46,716 --> 03:15:50,053 NEXT THING I DID WAS TRY TO 3818 03:15:50,053 --> 03:15:51,988 SOLVE THE HIGH RESOLUTION 3819 03:15:51,988 --> 03:15:53,389 STRUCTURE OF THESE LATTICES. 3820 03:15:53,389 --> 03:15:56,359 AND TO DO THIS USING SIMILAR 3821 03:15:56,359 --> 03:15:59,629 APPROACH MANY LABS ARE USING AT 3822 03:15:59,629 --> 03:16:04,467 THE MOMENT, I STARTED TO REALIZE 3823 03:16:04,467 --> 03:16:08,404 I COULD EXTRACT HIGH RESOLUTION 3824 03:16:08,404 --> 03:16:09,105 FEATURES USING THIS APPROACH, A 3825 03:16:09,105 --> 03:16:12,375 LOT OF HARD WORK BUT MANAGED TO 3826 03:16:12,375 --> 03:16:14,043 GET VERY HIGH QUALITY 3827 03:16:14,043 --> 03:16:18,581 RECONSTRUCTIONS OF THE MATRIX 3828 03:16:18,581 --> 03:16:19,282 LATTICE WITHIN VIRUSES. 3829 03:16:19,282 --> 03:16:22,085 AND THIS IS WHAT THEY LOOK LIKE. 3830 03:16:22,085 --> 03:16:25,254 THIS RECONSTRUCTION IS THREE 3831 03:16:25,254 --> 03:16:28,024 ANGSTROMS. 3832 03:16:28,024 --> 03:16:32,695 RESOLUTION WHERE WE CAN SOLVE 3833 03:16:32,695 --> 03:16:33,730 CHAINS. 3834 03:16:33,730 --> 03:16:36,432 FIRST THING WE DID WAS DESCRIBE 3835 03:16:36,432 --> 03:16:38,635 INTERFACES THAT HOLD THE MATURE 3836 03:16:38,635 --> 03:16:40,970 LATTICE TOGETHER, SEEMED TO BE 3837 03:16:40,970 --> 03:16:43,873 MADE OF VERY SPECIFIC 3838 03:16:43,873 --> 03:16:44,874 ELECTROSTATIC CONTACTS. 3839 03:16:44,874 --> 03:16:48,911 SO AS PREVIOUSLY MENTIONED 3840 03:16:48,911 --> 03:16:50,246 MATURE MAT RICKS LATTICE WAS 3841 03:16:50,246 --> 03:16:54,083 OBSERVED TO BIND A LIGAND AND WE 3842 03:16:54,083 --> 03:16:56,252 COULD SEE THIS LIGAND, COULD SEE 3843 03:16:56,252 --> 03:17:04,260 NEW FEATURES IN THE LIGAND AND 3844 03:17:04,260 --> 03:17:05,428 HAS VERY DEFINED SHAPE. 3845 03:17:05,428 --> 03:17:09,265 SO WHAT IS THIS LIGAND? 3846 03:17:09,265 --> 03:17:14,504 SO, BASED ON PREVIOUS STRUCTURAL 3847 03:17:14,504 --> 03:17:17,740 WORK THAT SHOWED MATRIX COMBINED 3848 03:17:17,740 --> 03:17:21,077 PIT 2 AND BASED ON SIZE OF THE 3849 03:17:21,077 --> 03:17:22,912 LIGAND IT WAS HYPOTHESIZED THAT 3850 03:17:22,912 --> 03:17:26,983 THE IDENTITY OF THE LIGAND WAS 3851 03:17:26,983 --> 03:17:27,984 PIT 2. 3852 03:17:27,984 --> 03:17:30,286 WHEN WE TRIED TO FIT THE 3853 03:17:30,286 --> 03:17:32,622 STRUCTURE IN THE NEW MAP WE 3854 03:17:32,622 --> 03:17:36,559 FOUND IT DIDN'T FIT, WE COULDN'T 3855 03:17:36,559 --> 03:17:41,230 GET IT TO BIND. 3856 03:17:41,230 --> 03:17:47,737 WE TRIED ALL THE OTHER LIPIDS, 3857 03:17:47,737 --> 03:17:49,772 INEFFICIENT QUANTITIES, WE 3858 03:17:49,772 --> 03:17:51,708 COULDN'T GET LIPIDS TO BIND NO 3859 03:17:51,708 --> 03:18:00,249 MATTER HOW HARD WE TRIED. 3860 03:18:00,249 --> 03:18:02,018 SO PREVIOUS OBSERVATION SP2 3861 03:18:02,018 --> 03:18:03,886 APPEARS TO REGULATE MATURATION 3862 03:18:03,886 --> 03:18:06,656 OF LATTICE, ATTEMPTED TO FIT SP2 3863 03:18:06,656 --> 03:18:08,324 INTO THIS DENSITY. 3864 03:18:08,324 --> 03:18:12,962 AND WE FOUND THAT THE C-TERMINAL 3865 03:18:12,962 --> 03:18:16,766 FIVE RESIDUES FIT INTO THIS 3866 03:18:16,766 --> 03:18:19,936 DENSITY QUITE WELL. 3867 03:18:19,936 --> 03:18:22,105 IN A CONFIRMATION THAT SUGGESTED 3868 03:18:22,105 --> 03:18:23,873 REMAINING TEN RESIDUES OF 3869 03:18:23,873 --> 03:18:26,709 PEPTIDE FIT ABOVE THE MATRIX 3870 03:18:26,709 --> 03:18:32,014 LAYER AND CONTACT THE MEMBRANE. 3871 03:18:32,014 --> 03:18:36,152 WHEN WE PERFORMED AUTOMATIC 3872 03:18:36,152 --> 03:18:39,322 MODEL BUILDING, WITHOUT INPUT 3873 03:18:39,322 --> 03:18:42,191 SEQUENCE WE FOUND THAT IT AGREED 3874 03:18:42,191 --> 03:18:44,260 WITH US IN OUR DETERMINATION, 3875 03:18:44,260 --> 03:18:46,863 AND QUITE CONSISTENTLY PREDICTED 3876 03:18:46,863 --> 03:18:51,234 THESE RESIDUES WERE SP2 3877 03:18:51,234 --> 03:18:52,335 RESIDUES. 3878 03:18:52,335 --> 03:18:58,441 DOMINICK IN OUR LAB WORKED ON 3879 03:18:58,441 --> 03:19:00,243 ASSEMBLING MATRIX LATTICES IN 3880 03:19:00,243 --> 03:19:03,079 VITRO, AND THEN ON LIPID 3881 03:19:03,079 --> 03:19:04,514 MONOLAYERS AND IMAGED ON GRIDS. 3882 03:19:04,514 --> 03:19:08,084 WHEN I INITIALLY TRIED HE FOUND 3883 03:19:08,084 --> 03:19:10,553 IT DIDN'T WORK VERY WELL, DID 3884 03:19:10,553 --> 03:19:13,489 GET SMALL AMOUNTS OF IMMATURE 3885 03:19:13,489 --> 03:19:15,758 AND MATURE LATTICE. 3886 03:19:15,758 --> 03:19:23,299 WHEN HE ADDED SP2 TO REACTION 3887 03:19:23,299 --> 03:19:23,966 MIXTURES SUDDENLY SAW 3888 03:19:23,966 --> 03:19:26,169 SIGNIFICANT AMOUNTS OF ONLY THE 3889 03:19:26,169 --> 03:19:27,003 MATURE LATTICE. 3890 03:19:27,003 --> 03:19:31,641 2D CLASSIFICATION OF 2D CRYSTALS 3891 03:19:31,641 --> 03:19:33,509 SHOWED BINDING OF SP2, IN THE 3892 03:19:33,509 --> 03:19:37,113 TOP ROW HOPEFULLY YOU CAN SEE WE 3893 03:19:37,113 --> 03:19:39,315 ACTUALLY DIRECTLY OBSERVE THE 3894 03:19:39,315 --> 03:19:44,053 BINDING OF A LIGAND, AND WHEN WE 3895 03:19:44,053 --> 03:19:46,122 STIMULATE OUR RESOLUTION 2D 3896 03:19:46,122 --> 03:19:54,363 CLASSES OF SP2 AND NOT SP 2 3897 03:19:54,363 --> 03:19:57,266 BOUND MATRIX IT MATCHES OUR DATA 3898 03:19:57,266 --> 03:19:58,267 WELL. 3899 03:19:58,267 --> 03:20:01,737 WE BELIEVE THE REMAINDER OF THE 3900 03:20:01,737 --> 03:20:04,307 PEPTIDE IS ABOVE MATRIX AND SEE 3901 03:20:04,307 --> 03:20:07,243 IN OUR RECONSTRUCTIONS AT LOW 3902 03:20:07,243 --> 03:20:08,277 THRESHOLD. 3903 03:20:08,277 --> 03:20:10,646 APPEARS TO BE CONTACTING THE 3904 03:20:10,646 --> 03:20:12,114 INTERFACE TO THE MEMBRANE. 3905 03:20:12,114 --> 03:20:14,750 WITH THAT WE COULD DESCRIBE THE 3906 03:20:14,750 --> 03:20:16,853 STRUCTURE OF SP2 BOUND MATRIX IN 3907 03:20:16,853 --> 03:20:18,120 THE VIRUS. 3908 03:20:18,120 --> 03:20:22,058 THIS IS THE MATURE LATTICE. 3909 03:20:22,058 --> 03:20:24,694 WE FOUND SP2 IS LIKELY 3910 03:20:24,694 --> 03:20:28,564 CRITICALLY INVOLVED IN THE 3911 03:20:28,564 --> 03:20:30,366 INTERTRIMERIC INTERFACE BETWEEN 3912 03:20:30,366 --> 03:20:32,001 MATRIX TRIMERS, SPECIFICALLY IN 3913 03:20:32,001 --> 03:20:38,574 THIS REGION HERE, WHERE THE 3914 03:20:38,574 --> 03:20:41,577 C-TERMINAL GROUP MUTUALIZES 3915 03:20:41,577 --> 03:20:43,079 ARGININE 22, ALLOWS THEM TO BE 3916 03:20:43,079 --> 03:20:43,846 IN CLOSE PROXIMITY TOGETHER. 3917 03:20:43,846 --> 03:20:45,748 THIS IS A SUMMARY OF WHAT WE 3918 03:20:45,748 --> 03:20:50,286 BELIEVE IS HAPPENING. 3919 03:20:50,286 --> 03:20:52,922 CLEAVAGE OF GAG, RELEASE OF SP2 3920 03:20:52,922 --> 03:20:55,591 WHICH BINDS MATRIX LAYER AND 3921 03:20:55,591 --> 03:20:57,360 INDUCES MATURATION OF MATRIX. 3922 03:20:57,360 --> 03:21:00,363 ALSO I'D LIKE TO POINT OUT WE 3923 03:21:00,363 --> 03:21:05,201 BELIEVE THIS MECHANISM IS QUITE 3924 03:21:05,201 --> 03:21:07,670 SIMILAR TO CAPSID, REGULATED BY 3925 03:21:07,670 --> 03:21:10,506 CLEAVAGE OF SP1, THE OTHER SPACE 3926 03:21:10,506 --> 03:21:17,013 PEPTIDE, IN THIS CAUSE IP2 IS 3927 03:21:17,013 --> 03:21:18,414 RELEASED, MATURE CAPSID LATTICE, 3928 03:21:18,414 --> 03:21:22,585 IN A SIMILAR WAY TO THE WAY SP2 3929 03:21:22,585 --> 03:21:27,390 TRIGGERS FORMATION OF MATURE 3930 03:21:27,390 --> 03:21:27,623 MATRIX. 3931 03:21:27,623 --> 03:21:32,328 I'D LIKE TO THANK MY LAB AND 3932 03:21:32,328 --> 03:21:34,964 Ph.D. SUPERVISOR JOHN BRICKS 3933 03:21:34,964 --> 03:21:36,465 AND COLLABORATOR DOMINICK AND 3934 03:21:36,465 --> 03:21:37,466 ALL THE COLLABORATORS THAT 3935 03:21:37,466 --> 03:21:39,302 HELPED US MAKE THE VIRUS. 3936 03:21:39,302 --> 03:21:40,102 THANKS VERY MUCH. 3937 03:21:40,102 --> 03:21:45,341 [APPLAUSE] 3938 03:21:45,341 --> 03:21:46,642 >> THANK YOU VERY MUCH, JAMES. 3939 03:21:46,642 --> 03:21:48,611 DO WE HAVE ANY QUESTIONS? 3940 03:21:48,611 --> 03:21:50,379 WE HAVE THREE TO FOUR MINUTES. 3941 03:21:50,379 --> 03:21:53,215 >> THAT WAS REALLY AN IMPRESSIVE 3942 03:21:53,215 --> 03:21:54,016 TALK. 3943 03:21:54,016 --> 03:21:54,517 CONGRATULATIONS. 3944 03:21:54,517 --> 03:21:55,418 HAVE YOU THOUGHT ABOUT WHETHER 3945 03:21:55,418 --> 03:21:56,953 OR NOT A PEPTIDE LIKE THAT MIGHT 3946 03:21:56,953 --> 03:22:00,656 BE PLAYING A ROLE IN OTHER 3947 03:22:00,656 --> 03:22:01,557 RETROVIRUSES? 3948 03:22:01,557 --> 03:22:05,061 LET'S SAY MURINE RETROVIRUSES? 3949 03:22:05,061 --> 03:22:07,797 >> YEAH, SO WE KNOW HIV-2 AT THE 3950 03:22:07,797 --> 03:22:13,035 VERY LEAST ALSO HAS THIS SP2 3951 03:22:13,035 --> 03:22:13,836 PEPTIDE, SLIGHTLY DIFFERENT 3952 03:22:13,836 --> 03:22:15,338 SEQUENCE, BUT I BELIEVE IT'S 3953 03:22:15,338 --> 03:22:16,906 PERFECTLY POSSIBLE BECAUSE IF 3954 03:22:16,906 --> 03:22:21,277 YOU LOOK AT THE GAG, GAG, THEY 3955 03:22:21,277 --> 03:22:22,244 HAVE SMALL DOMAINS, MANY OF 3956 03:22:22,244 --> 03:22:23,746 WHICH ARE NOT ASSIGNED TO ANY 3957 03:22:23,746 --> 03:22:26,882 PARTICULAR FUNCTION BUT WE DON'T 3958 03:22:26,882 --> 03:22:31,620 KNOW IF OTHER RETROVIRUSES 3959 03:22:31,620 --> 03:22:33,956 BASICALLY UNDERGO MATRIX 3960 03:22:33,956 --> 03:22:34,523 MATURATION, SOMETHING WE'RE 3961 03:22:34,523 --> 03:22:36,359 INTERESTED IN AND MIGHT TRY TO 3962 03:22:36,359 --> 03:22:37,860 EXPLORE, I DON'T KNOW. 3963 03:22:37,860 --> 03:22:38,961 >> VERY NICE TALK. 3964 03:22:38,961 --> 03:22:41,797 >> CAN YOU SHOW NAME TAG. 3965 03:22:41,797 --> 03:22:43,366 THANK YOU. 3966 03:22:43,366 --> 03:22:44,767 >> OH, YEAH, HERE. 3967 03:22:44,767 --> 03:22:52,141 DOES YOUR MODEL SUGGEST THAT SP2 3968 03:22:52,141 --> 03:22:53,743 REPLACES OR DISPLACES PIP 2? 3969 03:22:53,743 --> 03:22:56,679 >> I THINK THE ANSWER IS NO. 3970 03:22:56,679 --> 03:23:01,517 WE WOULD EXPECT TO OBSERVE PIP 2 3971 03:23:01,517 --> 03:23:03,786 WITHIN THE POCKET IN THE 3972 03:23:03,786 --> 03:23:04,987 IMMATURE MATRIX LATTICE WITH YOU 3973 03:23:04,987 --> 03:23:07,089 WE DON'T OBSERVE ANY LIGAND IN 3974 03:23:07,089 --> 03:23:11,527 THAT POCKET IN THE IMMATURE 3975 03:23:11,527 --> 03:23:11,761 LATTICE. 3976 03:23:11,761 --> 03:23:15,765 SO I DON'T THINK IT DISPLACES 3977 03:23:15,765 --> 03:23:18,901 IT, NO, BUT -- OR THE ROLE OF 3978 03:23:18,901 --> 03:23:26,575 PIP 2, I'M NOT SURE. 3979 03:23:26,575 --> 03:23:29,512 >> WOULD YOUR STRUCTURE ALLOW 3980 03:23:29,512 --> 03:23:32,615 YOU TO DESIGN A POINT MUTANT 3981 03:23:32,615 --> 03:23:34,984 THAT BREAK THIS, INDUCE 3982 03:23:34,984 --> 03:23:35,751 REPLICATION DEFECT THEN 3983 03:23:35,751 --> 03:23:38,154 COMPENSATED BY MUTATION IN 3984 03:23:38,154 --> 03:23:38,554 MATRIX? 3985 03:23:38,554 --> 03:23:40,890 >> SO THE PROBLEM WITH DESIGNING 3986 03:23:40,890 --> 03:23:44,226 MUTATIONS IN SP2 IS ACTUALLY THE 3987 03:23:44,226 --> 03:23:48,230 SEQUENCE OF SP2 OVERLAPS WITH 3988 03:23:48,230 --> 03:23:50,733 THE RIBOSOMAL SLIPPAGE SITE THAT 3989 03:23:50,733 --> 03:23:53,402 CONVERTS GAG TO GAG POLL. 3990 03:23:53,402 --> 03:23:58,707 WE'RE TRYING BUT IT'S TRICKY 3991 03:23:58,707 --> 03:24:04,814 BECAUSE WE RELY ON (INAUDIBLE). 3992 03:24:04,814 --> 03:24:07,149 >> ANY OTHER QUESTIONS? 3993 03:24:07,149 --> 03:24:11,253 IF NOT, NEXT SPEAKER IS 3994 03:24:11,253 --> 03:24:12,788 CHRISTIAN FRENIERE FROM YALE 3995 03:24:12,788 --> 03:24:15,124 UNIVERSITY. 3996 03:24:15,124 --> 03:24:15,791 ANOTHER APPLAUSE FOR JAMES. 3997 03:24:15,791 --> 03:24:22,698 [APPLAUSE] 3998 03:24:22,698 --> 03:24:25,334 >> ALL RIGHT. 3999 03:24:25,334 --> 03:24:25,634 AWESOME. 4000 03:24:25,634 --> 03:24:28,938 YEAH I'M CHRISTIAN FROM YALE, 4001 03:24:28,938 --> 03:24:32,408 FIFTH YEAR GRADUATE STUDENT IN 4002 03:24:32,408 --> 03:24:33,342 YONG JUNG'S LAB. 4003 03:24:33,342 --> 03:24:34,643 BEFORE I GET GOING I WANT TO 4004 03:24:34,643 --> 03:24:36,145 GIVE A QUICK SHOUT OUT TO THE 4005 03:24:36,145 --> 03:24:38,547 PEOPLE WHO MADE THIS WORK 4006 03:24:38,547 --> 03:24:40,883 POSSIBLE, SO MATT HAS BEEN MY 4007 03:24:40,883 --> 03:24:47,089 PARTNER IN CRIME, WE WORK VERY 4008 03:24:47,089 --> 03:24:48,591 INTIMATELY EVERY STOP ON THE 4009 03:24:48,591 --> 03:24:48,924 PROJECT. 4010 03:24:48,924 --> 03:24:52,495 CHARLIE TAUGHT ME EVERYTHING 4011 03:24:52,495 --> 03:24:54,463 ABOUT THE ELECTRON MICROSCOPE, 4012 03:24:54,463 --> 03:24:56,065 IMPORTANT. 4013 03:24:56,065 --> 03:24:57,733 FAITH IS A TALENTED 4014 03:24:57,733 --> 03:25:01,470 UNDERGRADUATE WORKING ON 4015 03:25:01,470 --> 03:25:02,104 ASSEMBLY AND CO-SEDIMENTATION 4016 03:25:02,104 --> 03:25:05,074 ASSAYS AND THE BOSS WHO MAKES 4017 03:25:05,074 --> 03:25:07,076 EVERYTHING POSSIBLE, ALSO REALLY 4018 03:25:07,076 --> 03:25:09,278 HELPS WITH DATA COLLECTION, DATA 4019 03:25:09,278 --> 03:25:10,980 PROCESSING, STUFF LIKE THAT. 4020 03:25:10,980 --> 03:25:12,548 ALSO VERY MUCH INVOLVED IN THE 4021 03:25:12,548 --> 03:25:12,948 WORK. 4022 03:25:12,948 --> 03:25:15,050 SO LET'S JUMP IN. 4023 03:25:15,050 --> 03:25:20,189 WE'LL DO HIV-2 FROM 50,000 FEET. 4024 03:25:20,189 --> 03:25:26,629 SO AROSE FROM DISTINCT SIMIAN 4025 03:25:26,629 --> 03:25:28,497 RETROVIRUS, CAN PROGRESS TO AIDS 4026 03:25:28,497 --> 03:25:31,534 OVER TIME, FEATURES SLOWER 4027 03:25:31,534 --> 03:25:32,334 PROGRESSION TO AIDS. 4028 03:25:32,334 --> 03:25:35,504 AND BURDEN OF THE DISEASE IS 4029 03:25:35,504 --> 03:25:38,240 SORT OF BORN ON SUB-SAHARAN WEST 4030 03:25:38,240 --> 03:25:40,876 AFRICA AND SORRY COUNTRIES HAD 4031 03:25:40,876 --> 03:25:41,744 COLONIAL EXCHANGE WITH THAT 4032 03:25:41,744 --> 03:25:44,380 REGION OF THE WORLD. 4033 03:25:44,380 --> 03:25:47,850 SO, YEAH, A LITTLE BIT ABOUT THE 4034 03:25:47,850 --> 03:25:51,487 CAPSID, TWO CAPSID PROTEINS, 80% 4035 03:25:51,487 --> 03:25:53,689 IDENTICAL, SO DESPITE THIS WE 4036 03:25:53,689 --> 03:25:55,758 KNOW THERE ARE DIFFERENT 4037 03:25:55,758 --> 03:26:02,464 PROVIRAL FACTORS THAT RECOGNIZE 4038 03:26:02,464 --> 03:26:04,233 THESE CAPSIDS DIFFERENTLY, 4039 03:26:04,233 --> 03:26:07,336 BINDING TO HIV-1 VERSUS HIV-2. 4040 03:26:07,336 --> 03:26:08,470 LIKEWISE RESTRICTION FACTORS 4041 03:26:08,470 --> 03:26:10,306 SUCH AS NONO HAVE DIFFERENT 4042 03:26:10,306 --> 03:26:12,908 BINDINGS, THERE'S REASON TO 4043 03:26:12,908 --> 03:26:14,143 BELIEVE DESPITE SIMILARITIES 4044 03:26:14,143 --> 03:26:15,878 THERE'S SOME DIFFERENCES, AND 4045 03:26:15,878 --> 03:26:17,146 ALTHOUGH THANKS TO MANY PEOPLE 4046 03:26:17,146 --> 03:26:19,181 IN THIS ROOM WE HAVE AWESOME 4047 03:26:19,181 --> 03:26:23,319 TOOLKIT FOR INVESTIGATION OF 4048 03:26:23,319 --> 03:26:24,687 HIV-1, FROM STRUCTURAL 4049 03:26:24,687 --> 03:26:25,187 STANDPOINT. 4050 03:26:25,187 --> 03:26:28,257 WE DON'T HAVE TOOLS FOR THE IN 4051 03:26:28,257 --> 03:26:29,425 VITRO ASSEMBLY AND STRUCTURAL 4052 03:26:29,425 --> 03:26:31,160 STUDY OF HIV CAPSID. 4053 03:26:31,160 --> 03:26:32,394 MOTIVATION OF THIS PROJECT IS 4054 03:26:32,394 --> 03:26:35,064 KIND OF TO TRY TO BEGIN BUILDING 4055 03:26:35,064 --> 03:26:40,836 A SIMILAR TOOLKIT FOR HIV-2. 4056 03:26:40,836 --> 03:26:42,071 ALL RIGHT. 4057 03:26:42,071 --> 03:26:45,140 SO, APPROACH WE TOOK IS INSPIRED 4058 03:26:45,140 --> 03:26:48,310 HEAVILY BY ROB DICK'S LAB, A BIG 4059 03:26:48,310 --> 03:26:49,645 SHOUT OUT TO ROB TO SAY THIS 4060 03:26:49,645 --> 03:26:52,348 METHOD IS EXTREMELY POWERFUL FOR 4061 03:26:52,348 --> 03:26:54,149 THE TEMPLATING OF DIFFERENT 4062 03:26:54,149 --> 03:26:55,217 CAPSID LATTICES. 4063 03:26:55,217 --> 03:26:59,688 MAKES USE OF LIPOSOMES WHICH 4064 03:26:59,688 --> 03:27:05,027 FEATURE A SMALL PERCENTAGE OF 4065 03:27:05,027 --> 03:27:12,935 NTA LIB IDENTITY THAT PROVIDES A 4066 03:27:12,935 --> 03:27:18,073 POINT, USING THE VARIANT OF 4067 03:27:18,073 --> 03:27:20,809 HIV-2 PROVIRUS, AND WE HIS TAG 4068 03:27:20,809 --> 03:27:24,847 ON THE C-TERMINAL TO ASSOCIATE 4069 03:27:24,847 --> 03:27:27,383 WITH THE NICKEL LIPOSOME, 4070 03:27:27,383 --> 03:27:28,884 ALLOWING FOR NUCLEATION, THE 4071 03:27:28,884 --> 03:27:31,987 EFFICIENT TEMPLATING OF LATTICE 4072 03:27:31,987 --> 03:27:35,924 AROUND THIS NUCLEATION POINT, AN 4073 03:27:35,924 --> 03:27:36,825 EXTREMELY POWERFUL TOOL. 4074 03:27:36,825 --> 03:27:38,994 SO WHAT DOES THIS LOOK LIKE WITH 4075 03:27:38,994 --> 03:27:39,762 HIV-2? 4076 03:27:39,762 --> 03:27:50,205 YOU CAN SEE CLEARLY WE GET 4077 03:27:51,974 --> 03:27:52,841 PRETTY DISTINCT CAPSULE-LIKE 4078 03:27:52,841 --> 03:27:55,678 PARTICLES, ON THE INSIDE OF THE 4079 03:27:55,678 --> 03:27:56,645 CAPSID-LIKE PARTICLE. 4080 03:27:56,645 --> 03:27:58,681 OH, THIS IS -- I'M GOING ALL 4081 03:27:58,681 --> 03:27:59,448 OVER THE PLACE. 4082 03:27:59,448 --> 03:28:02,518 ON THE INSIDE YOU CAN SEE 4083 03:28:02,518 --> 03:28:04,586 THERE'S LIPOSOME AND CLEARLY YOU 4084 03:28:04,586 --> 03:28:05,788 CAN SEE CAPSID TEMPLATED AROUND 4085 03:28:05,788 --> 03:28:09,892 IT INDICATING TO US THIS IS 4086 03:28:09,892 --> 03:28:10,225 WORKING. 4087 03:28:10,225 --> 03:28:11,627 WE CAN DO CLASS AVERAGING 4088 03:28:11,627 --> 03:28:13,429 SIMILAR TO WHAT OTHER PEOPLE 4089 03:28:13,429 --> 03:28:15,964 MENTIONED, TREATING THIS AS A 4090 03:28:15,964 --> 03:28:16,765 SINGLE PARTICLE. 4091 03:28:16,765 --> 03:28:19,201 WE SEE CLEAR TOP VIEWS OF THE 4092 03:28:19,201 --> 03:28:20,936 LATTICE, AND WE ALSO SEE AREAS 4093 03:28:20,936 --> 03:28:24,406 WHERE WE CAN SEE THE LIPOSOME 4094 03:28:24,406 --> 03:28:25,307 UNDERNEATH AS WELL. 4095 03:28:25,307 --> 03:28:29,144 SO THIS ALLOWS US TO GET HIGH 4096 03:28:29,144 --> 03:28:31,647 RESOLUTION RECONSTRUCTION OF CA 4097 03:28:31,647 --> 03:28:33,349 HEXAMER, ALLOWS FOR CONFIDENT 4098 03:28:33,349 --> 03:28:34,283 MODEL BUILDING. 4099 03:28:34,283 --> 03:28:36,685 SHOUT OUT TO MATT FOR THE MODEL 4100 03:28:36,685 --> 03:28:41,190 BUILDING BUT PERHAPS EVEN MORE 4101 03:28:41,190 --> 03:28:43,125 EXCITINGLY IN EARLY 4102 03:28:43,125 --> 03:28:44,360 HETEROGENEOUS ASSIGNMENTS 4103 03:28:44,360 --> 03:28:50,699 NOTICED PRESENCE OF PENTAMER 4104 03:28:50,699 --> 03:28:53,102 ADJACENT TO HEXAMER, SLIGHTLY 4105 03:28:53,102 --> 03:28:57,706 HIGHER RESOLUTION ALLOWS TO 4106 03:28:57,706 --> 03:29:02,444 BUILD VERY CONFIDENT MODELS. 4107 03:29:02,444 --> 03:29:04,847 AGAIN BIG SHOUT OUT TO MATT. 4108 03:29:04,847 --> 03:29:10,552 YOU MIGHT HAVE NOTICED THERE'S A 4109 03:29:10,552 --> 03:29:14,323 SMALL PERCENTAGE OF PARTICLES 4110 03:29:14,323 --> 03:29:21,997 WHICH DON'T LOOK SIMILAR. 4111 03:29:21,997 --> 03:29:27,369 NORMAL CAPSID-LIKE HAVING THE 4112 03:29:27,369 --> 03:29:28,370 MORPHOLOGY, REMINISCENT TO 4113 03:29:28,370 --> 03:29:38,013 PENTAMER WHICH WAS RECENTLY 4114 03:29:38,013 --> 03:29:38,514 PUBLISHED. 4115 03:29:38,514 --> 03:29:44,553 WE SAW TO SEE IF T EQUALS 1, WE 4116 03:29:44,553 --> 03:29:47,423 GET HIGH RESOLUTION, AGAIN WE'RE 4117 03:29:47,423 --> 03:29:49,057 ABLE TO BUILD MODELS. 4118 03:29:49,057 --> 03:29:51,360 I HAVE TO APOLOGIZE TO MATT FOR 4119 03:29:51,360 --> 03:29:57,699 THE PAIN OF HAVING SUCH HIGH 4120 03:29:57,699 --> 03:29:58,000 RESOLUTION. 4121 03:29:58,000 --> 03:29:59,468 YOU'RE ABLE TO SEE THE HOLES IN 4122 03:29:59,468 --> 03:30:02,538 THE AMINO ACID, AN EXCITING TIME 4123 03:30:02,538 --> 03:30:04,173 TO WORK ON CAPSID ASSEMBLY AND 4124 03:30:04,173 --> 03:30:05,374 USING THIS TECHNIQUE. 4125 03:30:05,374 --> 03:30:06,675 WE HAVE HIGH RESOLUTION 4126 03:30:06,675 --> 03:30:07,443 STRUCTURES OF LATTICE. 4127 03:30:07,443 --> 03:30:08,677 WHAT HAVE WE LEARNED? 4128 03:30:08,677 --> 03:30:10,612 ONE OF THE FIRST THINGS THAT WAS 4129 03:30:10,612 --> 03:30:15,751 IMMEDIATELY APPARENT TO US WAS 4130 03:30:15,751 --> 03:30:17,519 THIS LOOKS LIKE A HANDLE ON A 4131 03:30:17,519 --> 03:30:19,688 BUCKET YOU CAN PICK UP BUT IF I 4132 03:30:19,688 --> 03:30:23,292 SHOW YOU A SIMILARLY SHARPENED 4133 03:30:23,292 --> 03:30:27,429 MAP FROM HIV-1, SUV TEMPLATED 4134 03:30:27,429 --> 03:30:29,865 ASSEMBLY WE DON'T SEE THE SAME 4135 03:30:29,865 --> 03:30:33,268 AMOUNT OF CYP 1, SUGGESTING 4136 03:30:33,268 --> 03:30:38,707 BINDING OF HOSE FACTORS TO THE 4137 03:30:38,707 --> 03:30:39,875 CYP-A COULD BE DIFFERENT. 4138 03:30:39,875 --> 03:30:46,048 ANOTHER INTERESTING POINT OF 4139 03:30:46,048 --> 03:30:49,418 DIVERGENCE AND CONFIRMATION OF 4140 03:30:49,418 --> 03:30:52,721 WORK IS THAT THIS N-TERMINAL 4141 03:30:52,721 --> 03:30:54,790 BETA HAIRPIN, POSE TO PH SENSOR, 4142 03:30:54,790 --> 03:30:57,259 MAY HAVE ROLE A NUCLEOTIDE 4143 03:30:57,259 --> 03:30:57,793 IMPORT. 4144 03:30:57,793 --> 03:31:00,796 IN HIV-2 WE ALWAYS OBSERVE IT IN 4145 03:31:00,796 --> 03:31:03,999 THIS OPEN STATE, WHEREAS AT THIS 4146 03:31:03,999 --> 03:31:05,133 PH IN HIV-1 WOULD HAVE OBSERVED 4147 03:31:05,133 --> 03:31:06,735 IN THE CLOSED STATE. 4148 03:31:06,735 --> 03:31:09,671 THIS MAY HAVE SOME IMPLICATION 4149 03:31:09,671 --> 03:31:11,740 ON NUCLEOTIDE IMPORT. 4150 03:31:11,740 --> 03:31:13,775 ALL RIGHT. 4151 03:31:13,775 --> 03:31:17,613 SO, ONE OF THE OTHER SORT OF 4152 03:31:17,613 --> 03:31:20,249 DIVERGENCES WE SAW WITH HIV-2 IS 4153 03:31:20,249 --> 03:31:23,285 IN THE STRUCTURAL SWITCHING. 4154 03:31:23,285 --> 03:31:27,422 SO THE TVDG LOOK WHICH NOW IS 4155 03:31:27,422 --> 03:31:29,291 BECOMING FAMOUS FOR HIV-2 4156 03:31:29,291 --> 03:31:32,127 STRUCTURAL SWITCHING, ACTUALLY 4157 03:31:32,127 --> 03:31:34,463 IS A CVGD LOOP IN HIV-1, AND WE 4158 03:31:34,463 --> 03:31:37,366 SEE SOMETHING DIFFERENT AS WELL. 4159 03:31:37,366 --> 03:31:40,869 IN HIV-1 AGAIN THIS IS WORK BY 4160 03:31:40,869 --> 03:31:46,275 OWEN, JOHN, AND ROB'S GROUP THAT 4161 03:31:46,275 --> 03:31:47,576 ELEGANTLY ELIMINATED THE 4162 03:31:47,576 --> 03:31:50,546 SWITCHING IN THE TVGG LOOP, 4163 03:31:50,546 --> 03:31:52,614 PRONOUNCED DIFFERENCE BETWEEN 4164 03:31:52,614 --> 03:31:55,350 THE HEXAMER AND PENTAMERIC 4165 03:31:55,350 --> 03:31:56,118 FORMS. 4166 03:31:56,118 --> 03:31:58,854 IN HIV-2 WE DON'T HAVE AS 4167 03:31:58,854 --> 03:32:02,257 DRAMATIC A REARRANGEMENT. 4168 03:32:02,257 --> 03:32:04,192 HIV-2 HEXAMER LOOP ALREADY LOOKS 4169 03:32:04,192 --> 03:32:07,396 LIKE THE PENTAMER LOOP FOR 4170 03:32:07,396 --> 03:32:08,931 HIV-1, AND PENTAMER LOOP IS 4171 03:32:08,931 --> 03:32:09,898 SIMILAR STORY, RIGHT? 4172 03:32:09,898 --> 03:32:12,067 LESS OF A CHANGE THERE. 4173 03:32:12,067 --> 03:32:14,169 SO THIS IS LEADING US TO 4174 03:32:14,169 --> 03:32:15,270 THINKING THAT IN HIV-2 THIS IS 4175 03:32:15,270 --> 03:32:17,673 LESS OF A SWITCH AND MORE OF A 4176 03:32:17,673 --> 03:32:19,675 SLIDER IN THIS SORT OF AREA. 4177 03:32:19,675 --> 03:32:21,743 SO MORE SUBTLE CHANGE. 4178 03:32:21,743 --> 03:32:26,648 WHAT WE DO SEE IN TERMS OF NEW 4179 03:32:26,648 --> 03:32:30,152 CONTEXT BETWEEN THE HEXAMER AND 4180 03:32:30,152 --> 03:32:35,524 PENTAMER IS IS ACTUALLY IN THE 4181 03:32:35,524 --> 03:32:37,292 HELIX 2-3 AREA, AND THE 4182 03:32:37,292 --> 03:32:37,759 JUNCTION. 4183 03:32:37,759 --> 03:32:40,462 WE HAVE APPEARANCE OF -- OR 4184 03:32:40,462 --> 03:32:43,165 SLIDING OF DIFFERENT POLAR 4185 03:32:43,165 --> 03:32:47,035 CONTEXT BETWEEN HELIX 2 AND 3 4186 03:32:47,035 --> 03:32:47,669 BETWEEN HEXAMERRIC AND 4187 03:32:47,669 --> 03:32:48,770 PENTAMERIC FORM. 4188 03:32:48,770 --> 03:32:51,406 I'LL SHOW YOU A MOVIE. 4189 03:32:51,406 --> 03:32:59,181 WE HAVE THIS INTERCHANGE OF 4190 03:32:59,181 --> 03:33:01,550 POLAR CONTACT IN THE NTD. 4191 03:33:01,550 --> 03:33:02,551 SIMILARLY WE HAVE A SIMILAR 4192 03:33:02,551 --> 03:33:06,989 STORY GOING ON IN THE NTD-CTD 4193 03:33:06,989 --> 03:33:09,291 JUNG WHERE POLAR CONTACTS ARE 4194 03:33:09,291 --> 03:33:11,560 SLIGHTLY DIFFERENT BETWEEN THE 4195 03:33:11,560 --> 03:33:13,228 HEXAMER AND PENTAMER, WE BELIEVE 4196 03:33:13,228 --> 03:33:19,234 THERE'S A SLIDING OF THE POLAR 4197 03:33:19,234 --> 03:33:21,403 CONTACTS ACHIEVED IN THESE 4198 03:33:21,403 --> 03:33:21,837 FORMS. 4199 03:33:21,837 --> 03:33:24,206 SO, ONE POINT OF SIMILARITY I 4200 03:33:24,206 --> 03:33:26,341 FOCUSED MOSTLY ON DIFFERENCES 4201 03:33:26,341 --> 03:33:28,877 HERE IS BINDING OF FG PEPTIDES, 4202 03:33:28,877 --> 03:33:34,449 WERE ABLE TO RESOLVE STRUCTURES 4203 03:33:34,449 --> 03:33:36,418 WITH BOTH, THEY ALIGNED CLOSELY 4204 03:33:36,418 --> 03:33:39,588 WITH THE HIV 1 STRUCTURES, AND 4205 03:33:39,588 --> 03:33:41,556 SIMILARLY WE ONLY OBSERVED 4206 03:33:41,556 --> 03:33:43,025 DENSITY FOR THESE FG PEPTIDES IN 4207 03:33:43,025 --> 03:33:43,692 THE HEXAMER. 4208 03:33:43,692 --> 03:33:46,395 WE DON'T SEE IT IN THE PENTAMER. 4209 03:33:46,395 --> 03:33:48,363 THIS IS SLIGHTLY INTRIGUING TO 4210 03:33:48,363 --> 03:33:50,699 US BECAUSE IN THE HEXAMER THERE 4211 03:33:50,699 --> 03:33:54,803 IS NO LONGER -- SORRY, IN THE 4212 03:33:54,803 --> 03:33:58,306 PENTAMER THERE'S NO LONGER 4213 03:33:58,306 --> 03:34:00,809 STERIC OCCLUSION, SOME OTHER 4214 03:34:00,809 --> 03:34:01,943 MECHANISM IS PREVENTING THE FG 4215 03:34:01,943 --> 03:34:05,414 PEPTIDES FROM BINDING TO THE 4216 03:34:05,414 --> 03:34:06,715 PENTAMER. 4217 03:34:06,715 --> 03:34:06,915 OKAY. 4218 03:34:06,915 --> 03:34:08,383 SO, FINAL TAKEHOME POINTS, WHAT 4219 03:34:08,383 --> 03:34:09,685 HAVE WE LEARNED IN COMMON 4220 03:34:09,685 --> 03:34:12,554 FEATURES, SOME I DIDN'T TALK 4221 03:34:12,554 --> 03:34:20,095 ABOUT, YOU CAN COME TO MY POSTER 4222 03:34:20,095 --> 03:34:20,529 TOMORROW. 4223 03:34:20,529 --> 03:34:22,397 THE OVERALL TERTIARY IS SIMILAR, 4224 03:34:22,397 --> 03:34:24,966 IP6 I DIDN'T TOUCH ON TODAY, 4225 03:34:24,966 --> 03:34:27,669 IT'S ALSO VERY SIMILAR. 4226 03:34:27,669 --> 03:34:33,141 FG POCKET BINDING SEEMS TO BE 4227 03:34:33,141 --> 03:34:36,845 NEARLY IDENTICAL. 4228 03:34:36,845 --> 03:34:40,682 SO, CYP- A, THIS COULD HAVE 4229 03:34:40,682 --> 03:34:42,350 IMPLICATIONS FOR HOST FACTORS, 4230 03:34:42,350 --> 03:34:43,618 POTENTIALLY NUCLEAR IMPORT. 4231 03:34:43,618 --> 03:34:46,488 WE DO SEE SUBTLE DIFFERENCE IN 4232 03:34:46,488 --> 03:34:49,758 THE GATING OF THAT PH SENSITIVE 4233 03:34:49,758 --> 03:34:52,194 LOOP STUCK IN OPEN CONFIRMATION, 4234 03:34:52,194 --> 03:34:56,465 AND OUR MECHANISM OF ACHIEVING 4235 03:34:56,465 --> 03:34:57,566 THE PENTAMER-HEXAMER TRANSITION 4236 03:34:57,566 --> 03:34:58,567 IS SUBTLY DIFFERENT. 4237 03:34:58,567 --> 03:35:01,470 WE HAVE JUST THE SLIDING OF 4238 03:35:01,470 --> 03:35:03,038 POLAR CONTACTSES RATHER THAN 4239 03:35:03,038 --> 03:35:06,641 REARRANGEMENT OF THIS LOOP 4240 03:35:06,641 --> 03:35:07,242 REGION. 4241 03:35:07,242 --> 03:35:08,376 SO, YEAH, AGAIN, I WILL JUST 4242 03:35:08,376 --> 03:35:10,078 HIGHLIGHT THE PEOPLE THAT DID 4243 03:35:10,078 --> 03:35:12,647 THE WORK AND I'VE GOT A MINUTE 4244 03:35:12,647 --> 03:35:13,615 OR SO FOR QUESTIONS. 4245 03:35:13,615 --> 03:35:15,617 [APPLAUSE] 4246 03:35:15,617 --> 03:35:20,789 4247 03:35:20,789 --> 03:35:22,724 >> THANK YOU VERY MUCH, 4248 03:35:22,724 --> 03:35:23,158 CHRISTIAN. 4249 03:35:23,158 --> 03:35:30,632 I REALLY DO HAVE A QUESTION 4250 03:35:30,632 --> 03:35:31,399 MYSELF. 4251 03:35:31,399 --> 03:35:36,571 A COMPOUND, IT'S A CYCLOSPORIN 4252 03:35:36,571 --> 03:35:38,840 ANALOG, IT ONLY WORKS FOR HIV-1. 4253 03:35:38,840 --> 03:35:40,242 DOESN'T WORK FOR HIV-2. 4254 03:35:40,242 --> 03:35:44,045 MAYBE THAT HAS SOMETHING TO DO 4255 03:35:44,045 --> 03:35:46,615 WITH THE CA LOOP RIGIDITY. 4256 03:35:46,615 --> 03:35:49,518 CAN ANYONE COMMENT ON THAT? 4257 03:35:49,518 --> 03:35:55,357 THIS MECHANISM OF CYCLOCHLORINE 4258 03:35:55,357 --> 03:35:57,425 BINDING ONLY WORKS FOR HIV-1, IS 4259 03:35:57,425 --> 03:35:58,927 THAT CORRECT? 4260 03:35:58,927 --> 03:36:01,663 DOESN'T WORK FOR HIV-2, THAT'S 4261 03:36:01,663 --> 03:36:02,564 MY UNDERSTANDING. 4262 03:36:02,564 --> 03:36:03,398 >> PARTICULARLY INTERESTING, 4263 03:36:03,398 --> 03:36:08,670 MANY PEOPLE IN THE FIELD SUGGEST 4264 03:36:08,670 --> 03:36:11,439 HANDOFF BETWEEN CYP A MIGHT BE 4265 03:36:11,439 --> 03:36:12,641 IMPORTANT, IF ENCOURAGING MORE 4266 03:36:12,641 --> 03:36:14,242 BINDING THE INTERACTION OF HOST 4267 03:36:14,242 --> 03:36:17,345 FACTORS AND TIMING OF THE 4268 03:36:17,345 --> 03:36:18,947 DIFFERENT STEPS COULD BE, YEAH, 4269 03:36:18,947 --> 03:36:24,853 DIFFERENT JUST BECAUSE OF THE 4270 03:36:24,853 --> 03:36:26,955 CYP A BINDING. 4271 03:36:26,955 --> 03:36:29,024 >> IT'S HIV-1 SPECIFIC SO 4272 03:36:29,024 --> 03:36:29,791 THERE'S DIFFERENCES BETWEEN 4273 03:36:29,791 --> 03:36:31,226 HIV-1 AND 2 WHICH MAKES IT 4274 03:36:31,226 --> 03:36:33,628 HARDER TO TEST THOSE COMPOUNDS 4275 03:36:33,628 --> 03:36:38,867 IN AN ANIMAL MODEL, FOR EXAMPLE. 4276 03:36:38,867 --> 03:36:49,444 >> SO THE ROLE OF CYCLOPHILIN IS 4277 03:36:50,679 --> 03:36:51,880 MORE COMPLICATED. 4278 03:36:51,880 --> 03:37:01,523 THE READOUT FOR THE DUG EFFECT, 4279 03:37:01,523 --> 03:37:04,092 DIFFERENT SIVs RESPOND 4280 03:37:04,092 --> 03:37:06,995 DIFFERENTLY IN TERMS OF BINDING. 4281 03:37:06,995 --> 03:37:09,598 >> MAJOR RESTRICTION IN MICE, IS 4282 03:37:09,598 --> 03:37:09,898 THAT RIGHT? 4283 03:37:09,898 --> 03:37:12,467 I MEAN, ONE OF THE REASONS HIV 4284 03:37:12,467 --> 03:37:20,208 DOESN'T INFECT MICE, I MEAN, 4285 03:37:20,208 --> 03:37:25,447 CYCLOPHILIN, CAPSID ALTERS 4286 03:37:25,447 --> 03:37:27,048 BINDING, SPECIFIC IN ACTIVITIES, 4287 03:37:27,048 --> 03:37:34,356 SO EVEN HIV 1 IN SOME NON-HUMAN 4288 03:37:34,356 --> 03:37:35,624 PRIMATE SPECIES, CYCLOPHILIN 4289 03:37:35,624 --> 03:37:38,693 PROMOTES BINDING SO IT DEPENDS. 4290 03:37:38,693 --> 03:37:40,328 IT'S VERY DYNAMIC. 4291 03:37:40,328 --> 03:37:40,729 >> COMPLICATED. 4292 03:37:40,729 --> 03:37:42,530 >> THANK YOU. 4293 03:37:42,530 --> 03:37:43,999 NEXT QUESTION? 4294 03:37:43,999 --> 03:37:45,300 >> GREAT TALK. 4295 03:37:45,300 --> 03:37:47,769 DID YOU LOOK AT THE LOOP WITH 4296 03:37:47,769 --> 03:37:49,304 DIFFERENT PHs? 4297 03:37:49,304 --> 03:37:51,373 >> THE ASSEMBLIES AT DIFFERENT 4298 03:37:51,373 --> 03:37:52,340 PHs? 4299 03:37:52,340 --> 03:37:52,807 >> UH-HUH. 4300 03:37:52,807 --> 03:37:55,577 >> YES, ASSEMBLED EFFICIENTLY AT 4301 03:37:55,577 --> 03:38:00,382 ALL PHs, SO WE GO LOW LIKE PH 4302 03:38:00,382 --> 03:38:03,718 6 SOME MESSINESS BUT WE CAN GO 4303 03:38:03,718 --> 03:38:08,657 AS LOW AS PHYSIOLOGIC 7- 2 AREA. 4304 03:38:08,657 --> 03:38:09,958 >> AND YOUR T1s DO YOU SEE 4305 03:38:09,958 --> 03:38:12,727 INDICATION OF A ROLE FOR HIS TAG 4306 03:38:12,727 --> 03:38:14,062 IN FORMATION OF THESE? 4307 03:38:14,062 --> 03:38:18,066 >> SO, THAT'S AN INTERESTING 4308 03:38:18,066 --> 03:38:18,433 QUESTION. 4309 03:38:18,433 --> 03:38:20,568 SO THERE IS -- I SURE SAY WE 4310 03:38:20,568 --> 03:38:23,872 DO -- I BELIEVE THE T EQUALS 1 4311 03:38:23,872 --> 03:38:31,212 FORM EVEN WITHOUT THE MICELLE 4312 03:38:31,212 --> 03:38:32,814 TEMPLATING, THE PENT MORE IS 4313 03:38:32,814 --> 03:38:36,017 MORE EASILY FORMED BUT THERE'S 4314 03:38:36,017 --> 03:38:37,319 SOME INDICATION, VERY C-TERMINAL 4315 03:38:37,319 --> 03:38:40,689 WE HAD RESIDUES THAT APPEAR TO 4316 03:38:40,689 --> 03:38:43,525 BE IN AN ODD CONFIRMATION, 4317 03:38:43,525 --> 03:38:48,363 PROBABLY SEEING THEM PINNED DOWN 4318 03:38:48,363 --> 03:38:48,964 BY THE HISTAG. 4319 03:38:48,964 --> 03:38:51,433 I DON'T KNOW IF THAT FULLY 4320 03:38:51,433 --> 03:38:52,634 ANSWERS YOUR QUESTION. 4321 03:38:52,634 --> 03:38:54,369 >> LAST QUESTION IN 4322 03:38:54,369 --> 03:38:55,570 HETEROGENEITY REFINEMENTS DO YOU 4323 03:38:55,570 --> 03:39:00,308 SEE INDICATIONS OF PENTAMERS 4324 03:39:00,308 --> 03:39:01,176 NEXT TO PENTAMERS? 4325 03:39:01,176 --> 03:39:04,212 >> I'VE NEVER SEEN THAT BEFORE. 4326 03:39:04,212 --> 03:39:05,113 >> THANK YOU. 4327 03:39:05,113 --> 03:39:13,088 >> IF NO OTHER QUESTIONS ANOTHER 4328 03:39:13,088 --> 03:39:15,490 APPLAUSE FOR CHRISTIAN. 4329 03:39:15,490 --> 03:39:17,892 [APPLAUSE] 4330 03:39:17,892 --> 03:39:18,259 4331 03:39:18,259 --> 03:39:21,930 >> NEXT IS WILLIAM ARNDT FROM 4332 03:39:21,930 --> 03:39:32,374 UNIVERSITY OF MINNESOTA. 4333 03:40:07,542 --> 03:40:17,519 4334 03:42:08,830 --> 03:42:11,733 >> WEIR 4335 03:42:11,733 --> 03:42:15,703 >> WE'LL MOVE TO THE NEXT 4336 03:42:15,703 --> 03:42:19,941 SPEAKER AND PUT WILLIAM LAST. 4337 03:42:19,941 --> 03:42:21,376 NEXT SPEAKER IS JOSHUA HOPE, 4338 03:42:21,376 --> 03:42:25,313 UNIVERSITY OF OXFORD. 4339 03:42:25,313 --> 03:42:29,918 GO AHEAD, JOSHUA. 4340 03:42:29,918 --> 03:42:40,128 >> OKAY. 4341 03:42:50,171 --> 03:42:53,808 TODAY I'M GOING TO TALK TO YOU 4342 03:42:53,808 --> 03:42:57,612 ABOUT OUR PROJECT TO 4343 03:42:57,612 --> 03:43:00,081 CHARACTERIZE -- SORRY, TO 4344 03:43:00,081 --> 03:43:05,286 CHARACTERIZE THE INTERACTION OF 4345 03:43:05,286 --> 03:43:13,595 LENT VIRAL INTEGRATION MACHINERY 4346 03:43:13,595 --> 03:43:14,162 AND CHROMATIN. 4347 03:43:14,162 --> 03:43:17,532 MULTIMERIC, UPON HOST DNA 4348 03:43:17,532 --> 03:43:18,399 DOCKING CAUSE COVALENT INSERTION 4349 03:43:18,399 --> 03:43:21,369 OF THE VIRAL DNA INTO THE HOST 4350 03:43:21,369 --> 03:43:25,006 DNA TO FORM A COMPLEX STRAND 4351 03:43:25,006 --> 03:43:25,406 TRANSFER. 4352 03:43:25,406 --> 03:43:28,710 AFTER WHICH TIME THE HOST 4353 03:43:28,710 --> 03:43:29,711 MACHINERY TAKES OVER COMPLETELY 4354 03:43:29,711 --> 03:43:34,249 TO REPAIR THE SCARS OF 4355 03:43:34,249 --> 03:43:36,584 INTEGRATION AND STABLE 4356 03:43:36,584 --> 03:43:38,119 TRANSCRIPTION OF THIS PROVIRUS 4357 03:43:38,119 --> 03:43:40,855 CAN TAKE PLACE. 4358 03:43:40,855 --> 03:43:51,399 WE KNOW A LOT STRUCTURALLY ABOUT 4359 03:43:54,502 --> 03:43:57,605 INTASOMES, NVV FORMS STABLE 4360 03:43:57,605 --> 03:43:57,939 NUGGETS. 4361 03:43:57,939 --> 03:44:01,876 NOW, THE VAST MAJORITY OF 4362 03:44:01,876 --> 03:44:03,645 RESEARCH IS FOCUSED ON 4363 03:44:03,645 --> 03:44:05,380 INTEGRATION INTO NAKED DNA 4364 03:44:05,380 --> 03:44:05,847 SUBSTRATES. 4365 03:44:05,847 --> 03:44:09,984 THE TRUE SUBSTRATE FOR 4366 03:44:09,984 --> 03:44:12,086 INTEGRATION IS IN FACT 4367 03:44:12,086 --> 03:44:16,858 CHROMATIN, 80% OF MAMMALIAN 4368 03:44:16,858 --> 03:44:20,695 GENOME IS FOOTPRINTED BY 4369 03:44:20,695 --> 03:44:21,596 NUCLEOSOMES. 4370 03:44:21,596 --> 03:44:23,431 MANY RETROVIRUSES HAVE EVOLVED 4371 03:44:23,431 --> 03:44:28,603 MECHANISMS TO INTERACT WITH THE 4372 03:44:28,603 --> 03:44:35,109 CHROMATIN, STRIKINGLY NOT 4373 03:44:35,109 --> 03:44:37,078 CONSERVED. 4374 03:44:37,078 --> 03:44:38,146 PFV INTERACTS WITH HISTONES, 4375 03:44:38,146 --> 03:44:40,315 OTHERS SUV AS HIV AND MLV SEEM 4376 03:44:40,315 --> 03:44:42,717 TO USE THESE TETHERING FACTORS 4377 03:44:42,717 --> 03:44:44,285 TO TETHER THE PRE-INTEGRATION 4378 03:44:44,285 --> 03:44:46,587 COMPLEX TO THE CHROMATIN. 4379 03:44:46,587 --> 03:44:49,424 THIS HAS IMPLICATIONS FOR 4380 03:44:49,424 --> 03:44:52,694 INTEGRATION SITE TARGETING AND 4381 03:44:52,694 --> 03:44:53,895 ALSO POTENTIALLY 4382 03:44:53,895 --> 03:44:55,663 POST-INTEGRATION SITE REPAIR AND 4383 03:44:55,663 --> 03:44:57,598 SUBSEQUENT TRANSCRIPTION. 4384 03:44:57,598 --> 03:45:02,036 WORK IN OUR LAB IN 2015 FOUND 4385 03:45:02,036 --> 03:45:03,071 THAT INTEGRATION TAKES PLACE 4386 03:45:03,071 --> 03:45:06,441 REALLY IN THE CORE OF THE 4387 03:45:06,441 --> 03:45:09,744 NUCLEOSOME BY THE PROTEASOMIC 4388 03:45:09,744 --> 03:45:11,979 VIRUS INTERACTING STRONGLY WITH 4389 03:45:11,979 --> 03:45:18,820 H2A AND H2V IN THE CONTEXT OF 4390 03:45:18,820 --> 03:45:20,555 THE NUCLEOSOME. 4391 03:45:20,555 --> 03:45:23,291 IT'S LESS CLEAR WITH 4392 03:45:23,291 --> 03:45:26,394 LENTIVIRUSES BUT ONE KEY 4393 03:45:26,394 --> 03:45:31,332 CANDIDATE IS THE HOST FACTOR 4394 03:45:31,332 --> 03:45:35,036 LEDGER, TWO DOMAIN PROTEIN WITH 4395 03:45:35,036 --> 03:45:38,539 INTEGRATED BINDING DOMAIN AND 4396 03:45:38,539 --> 03:45:42,810 PWWP DEMAIN, IT BINDS TO 4397 03:45:42,810 --> 03:45:48,282 INTEGRASE, PWWP DOMAIN BINDS 4398 03:45:48,282 --> 03:45:50,585 STRONGLY TO LYSINE, THIS DIRECTS 4399 03:45:50,585 --> 03:45:52,553 INTO THESE ACTIVELY TRANSCRIBED 4400 03:45:52,553 --> 03:45:55,723 REGIONS THAT BEAR THIS 4401 03:45:55,723 --> 03:45:57,358 POST-TRANSLATIONAL MODIFICATION. 4402 03:45:57,358 --> 03:45:59,660 SO WE WANTED TO STUDY THIS. 4403 03:45:59,660 --> 03:46:05,133 AND WHILE THERE'S A WEALTH OF 4404 03:46:05,133 --> 03:46:07,969 DATA IN CELLS OF LEDGF, THE 4405 03:46:07,969 --> 03:46:09,771 PICTURE IS NOT WELL UNDERSTOOD. 4406 03:46:09,771 --> 03:46:12,573 SO TO TAKE A STEP BACK IN VITRO, 4407 03:46:12,573 --> 03:46:16,210 WE WANTED MODELS OF CHROMATIN. 4408 03:46:16,210 --> 03:46:19,514 WE TURNED TO POLY NUCLEOSOMAL 4409 03:46:19,514 --> 03:46:24,352 ARRAYS WHICH WE DEPOSITED 4410 03:46:24,352 --> 03:46:27,422 HISTONES ONTO 12 REPEATS, YOU 4411 03:46:27,422 --> 03:46:29,157 CAN SEE THIS BEAUTIFUL BEADS ON 4412 03:46:29,157 --> 03:46:32,927 A STRING OF THE NUCLEOSOMES. 4413 03:46:32,927 --> 03:46:36,631 WE HAVE HAVE THEM UNMODIFIED OR 4414 03:46:36,631 --> 03:46:39,834 TRIMETHYLATED, BECOMING THE TRUE 4415 03:46:39,834 --> 03:46:42,336 SUBSTRATE OF LEDGF. 4416 03:46:42,336 --> 03:46:44,806 SO, IF I EXTRACT THESE 4417 03:46:44,806 --> 03:46:46,674 PRE-INTEGRATION COMPLEXES FROM 4418 03:46:46,674 --> 03:46:49,410 ACUTELY INFECTED CELLS I GET AN 4419 03:46:49,410 --> 03:46:52,280 INCREASED PREFERENCE FOR THIS 4420 03:46:52,280 --> 03:46:53,681 TRIMETHYLATED ARRAYS, IN 4421 03:46:53,681 --> 03:46:55,450 AGREEMENT WITH WORK PUBLISHED BY 4422 03:46:55,450 --> 03:46:57,185 THE DASH LAB. 4423 03:46:57,185 --> 03:47:02,957 AND IF I EXTRACT THESE PICs, 4424 03:47:02,957 --> 03:47:04,959 THAT PREFERENCE IS JUST 4425 03:47:04,959 --> 03:47:05,460 NON-EXISTENT. 4426 03:47:05,460 --> 03:47:09,063 ADDING BACK RECOMBINANT LEDGF WE 4427 03:47:09,063 --> 03:47:14,469 CAN RESCUE THIS PREFERENCE FOR 4428 03:47:14,469 --> 03:47:15,002 TRIMETHYLATED ARRAYS. 4429 03:47:15,002 --> 03:47:22,243 WE WANTED TO DO THIS WITH 4430 03:47:22,243 --> 03:47:23,277 INTOSOMES. 4431 03:47:23,277 --> 03:47:25,079 BY USING INTEGRASE, LEDGF AND 4432 03:47:25,079 --> 03:47:33,754 SHORT VIRAL DNA MIMICS WE CAN 4433 03:47:33,754 --> 03:47:37,158 PURCHASE PHI AND ASSEMBLE. 4434 03:47:37,158 --> 03:47:39,227 WE GET INCREASED PREFERENCE, IF 4435 03:47:39,227 --> 03:47:43,364 YOU CHOP OFF THE PWWP DOMAIN. 4436 03:47:43,364 --> 03:47:50,071 AND STRIKINGLY WHEN YOU ADD 4437 03:47:50,071 --> 03:47:51,672 LEDGF TO THE REACTION, 4438 03:47:51,672 --> 03:47:58,813 DOSE-RESPONSE, WE SEEM TO GET 4439 03:47:58,813 --> 03:48:01,415 SELECTIVE INHIBITION INTO 4440 03:48:01,415 --> 03:48:03,317 UNMODIFIED CHROMATIN, 4441 03:48:03,317 --> 03:48:04,352 TRIMETHYLATED STAGE REASONABLY 4442 03:48:04,352 --> 03:48:05,119 CONSTANT. 4443 03:48:05,119 --> 03:48:07,321 WITH THAT WE TURNED TO 4444 03:48:07,321 --> 03:48:12,560 INTEGRATION SITE SEQUENCING TO 4445 03:48:12,560 --> 03:48:14,795 SEE HOW EXACTLY THIS INTOSOMES 4446 03:48:14,795 --> 03:48:17,331 IS REACTION. 4447 03:48:17,331 --> 03:48:19,767 WE USE PCR 3 APPROACH WITH 4448 03:48:19,767 --> 03:48:21,602 NANOPORE SEQUENCING TO GET LONG 4449 03:48:21,602 --> 03:48:25,573 READS OF INTEGRATION ACROSS THE 4450 03:48:25,573 --> 03:48:26,574 ENTIRE ARRAY. 4451 03:48:26,574 --> 03:48:29,410 COMPRESSING OUR DATA OF 4452 03:48:29,410 --> 03:48:31,846 INTEGRATION SITES ONTO A SINGLE 4453 03:48:31,846 --> 03:48:34,849 NUCLEOSOME HERE, WE HAVE THE 4454 03:48:34,849 --> 03:48:37,084 NUCLEOSOME FOOTPRINT IN THE GRAY 4455 03:48:37,084 --> 03:48:39,020 BOX, THEN THE FLANKING WHITE 4456 03:48:39,020 --> 03:48:46,961 SPACE WILL BE THE LINKAGE THE 4457 03:48:46,961 --> 03:48:47,495 NUCLEOSOME. 4458 03:48:47,495 --> 03:48:50,364 WHAT WE SEE IS -- WHAT WE EXPECT 4459 03:48:50,364 --> 03:48:54,201 FOR NAKED DNA SUBSTRATE HIGHLY 4460 03:48:54,201 --> 03:48:55,069 PROMISCUOUS INTEGRATION ACROSS 4461 03:48:55,069 --> 03:48:57,772 THE BODY OF THE NAKED DNA. 4462 03:48:57,772 --> 03:49:00,408 BUT WITH STILL SOME SEQUENCE 4463 03:49:00,408 --> 03:49:02,443 SPECIFICITY EXPECTED. 4464 03:49:02,443 --> 03:49:03,945 WHEN WE WRAP THESE IN 4465 03:49:03,945 --> 03:49:06,347 NUCLEOSOMES WE SEE PUSHING OUT 4466 03:49:06,347 --> 03:49:08,416 OF INTEGRATION FROM THE CORE OF 4467 03:49:08,416 --> 03:49:11,852 THE NUCLEOSOME TO LINKERS 4468 03:49:11,852 --> 03:49:14,488 BETWEEN THE NUCLEOSOMES. 4469 03:49:14,488 --> 03:49:16,190 IF WE AGGREGATE DATA TO LOOK AT 4470 03:49:16,190 --> 03:49:18,926 INTEGRATION JUST INTO THE LINKER 4471 03:49:18,926 --> 03:49:23,431 OF THE TOTAL INTEGRATION, WHAT 4472 03:49:23,431 --> 03:49:27,301 WE SEE IS INCREASED INTEGRATION 4473 03:49:27,301 --> 03:49:32,607 INTO THE LINK 2007 TRISOMY 4474 03:49:32,607 --> 03:49:34,675 MARKER IS PRESENT, GREATLY 4475 03:49:34,675 --> 03:49:37,111 RESISTED WHEN YOU RAMP UP TO 4476 03:49:37,111 --> 03:49:37,378 LEDGF. 4477 03:49:37,378 --> 03:49:39,714 AND THIS KIND OF SUMPED US. 4478 03:49:39,714 --> 03:49:42,550 WHAT IS GOING ON? 4479 03:49:42,550 --> 03:49:44,318 HOW IS INTEGRATION MOVING INTO 4480 03:49:44,318 --> 03:49:48,356 THE CORE OF THE NUCLEOSOME WHEN 4481 03:49:48,356 --> 03:49:49,457 THIS MARKER IS NOT PRESENT? 4482 03:49:49,457 --> 03:49:50,791 THERE'S SOME SITES WHICH I'LL 4483 03:49:50,791 --> 03:49:52,560 TRY AND POINT OUT. 4484 03:49:52,560 --> 03:49:55,029 HERE WHICH WOULD BE COMPLETELY 4485 03:49:55,029 --> 03:49:56,497 REFRACTORY TO INTEGRATION IN THE 4486 03:49:56,497 --> 03:49:59,000 CONTEXT OF THE NUCLEOSOME 4487 03:49:59,000 --> 03:50:02,803 THERE'S NO WAY THAT INTASOME CAN 4488 03:50:02,803 --> 03:50:04,538 GET IN THERE. 4489 03:50:04,538 --> 03:50:05,573 NO REACTION IS 100%. 4490 03:50:05,573 --> 03:50:07,174 MAYBE THERE'S A SMALL AMOUNT OF 4491 03:50:07,174 --> 03:50:10,745 NAKED DNA IN OUR SAMPLE, AND 4492 03:50:10,745 --> 03:50:13,214 ACTUALLY INTEGRATION IS GOING 4493 03:50:13,214 --> 03:50:13,748 THERE. 4494 03:50:13,748 --> 03:50:15,983 WE BUILT EXPERIMENTS WITH NAKED 4495 03:50:15,983 --> 03:50:17,718 DNA, DIFFERENT SEQUENCE, RUN 4496 03:50:17,718 --> 03:50:19,620 ALIGNMENT OF WHERE INTEGRATION 4497 03:50:19,620 --> 03:50:20,154 GOES. 4498 03:50:20,154 --> 03:50:22,857 WHEN THE CHROMATIN IS 4499 03:50:22,857 --> 03:50:24,425 UNMETHYLATED WE GET SPARING 4500 03:50:24,425 --> 03:50:26,360 INTEGRATION INTO THE UNMODIFIED 4501 03:50:26,360 --> 03:50:28,696 CHROMATIN BUT WHEN IT'S 4502 03:50:28,696 --> 03:50:30,197 TRIMETHYLATED IT REALLY GOES 4503 03:50:30,197 --> 03:50:33,267 INTO THAT CHROMATIN BUT STILL IN 4504 03:50:33,267 --> 03:50:35,002 THAT LINKER BETWEEN THE 4505 03:50:35,002 --> 03:50:35,670 NUCLEOSOMES. 4506 03:50:35,670 --> 03:50:38,205 WE EXPAND THIS DATASET ACROSS 4507 03:50:38,205 --> 03:50:39,840 INTEGRATION ACROSS THE WHOLE 4508 03:50:39,840 --> 03:50:46,180 ARRAY AND SEE SOMETHING REALLY 4509 03:50:46,180 --> 03:50:47,481 COOL. 4510 03:50:47,481 --> 03:50:49,116 SO, WHEN THERE'S NO MARK PRESENT 4511 03:50:49,116 --> 03:50:51,085 THERE'S NO PREFERENCE FOR WHICH 4512 03:50:51,085 --> 03:50:52,720 NUCLEOSOME IT GOES INTO ACROSS 4513 03:50:52,720 --> 03:50:55,356 THE ARRAY, STILL TARGETING THAT 4514 03:50:55,356 --> 03:50:57,358 NAKED LINKER BUT DOESN'T CARE 4515 03:50:57,358 --> 03:50:58,325 WHICH NUCLEOSOME IT IS. 4516 03:50:58,325 --> 03:51:01,862 WHEN WE HAVE THE LEDGF AND 4517 03:51:01,862 --> 03:51:04,165 MARKER PRESENT THERE'S A PUSHING 4518 03:51:04,165 --> 03:51:06,100 OF INTEGRATION INTO THE BODY OF 4519 03:51:06,100 --> 03:51:08,369 THE NUCLEOSOME WHICH WE THINK IS 4520 03:51:08,369 --> 03:51:10,137 PROBABLY EXPLAINED BY THE FACT 4521 03:51:10,137 --> 03:51:12,306 THAT THIS HIGHLY DISORDERED 4522 03:51:12,306 --> 03:51:18,679 LEDGF PROTEIN CAN MAKE SEVERAL 4523 03:51:18,679 --> 03:51:20,448 CONTACTS ACROSS SEVERAL 4524 03:51:20,448 --> 03:51:21,716 NUCLEOSOMES, TOWARDS THE CENTER 4525 03:51:21,716 --> 03:51:25,019 YOU'RE LIKELY TO GET MORE THAN 4526 03:51:25,019 --> 03:51:26,454 AT THE ENDS. 4527 03:51:26,454 --> 03:51:29,924 I'M GOING TO SUMMARIZE WITH 4528 03:51:29,924 --> 03:51:31,892 REALLY HOT OFF THE PRESS 4529 03:51:31,892 --> 03:51:33,427 STRUCTURES THAT A POSTDOC IN OUR 4530 03:51:33,427 --> 03:51:35,963 LAB HAS MANAGED TO SOLVE. 4531 03:51:35,963 --> 03:51:44,205 THIS IS THE CHROMATIN BINDING 4532 03:51:44,205 --> 03:51:47,074 PLATFORM OF THE INTOSOME. 4533 03:51:47,074 --> 03:51:49,977 WE USE LEDGF BUT CAN ONLY SEE 4534 03:51:49,977 --> 03:51:52,947 RESOLUTION FOR LEDGF INTEGRASE 4535 03:51:52,947 --> 03:51:54,515 BINDING DOMAINS WHEN SATURATED 4536 03:51:54,515 --> 03:51:55,816 WITH LEDGF. 4537 03:51:55,816 --> 03:51:59,587 WE SEE A MORE LEDGF BINDING 4538 03:51:59,587 --> 03:52:03,891 MOLECULES ON THIS, THE TARGET 4539 03:52:03,891 --> 03:52:06,393 DNA FACING SIDE, AND THAT HELPS 4540 03:52:06,393 --> 03:52:08,362 TO EXPLAIN THESE MULTIVALENT 4541 03:52:08,362 --> 03:52:16,937 INTERACTIONS THAT CAN OCCUR 4542 03:52:16,937 --> 03:52:22,243 BECAUSE THE INTOSOME CAN BIND. 4543 03:52:22,243 --> 03:52:25,212 WHEN THIS TARGET DNA BINDING 4544 03:52:25,212 --> 03:52:29,116 GROUP, SO THESE LEDGF MOLECULES 4545 03:52:29,116 --> 03:52:34,522 FRAME THE SITE WHERE THIS TARGET 4546 03:52:34,522 --> 03:52:35,422 DNA COMBINED, AND REALLY 4547 03:52:35,422 --> 03:52:39,894 RESTRICT THAT THE DNA CAN BE IN. 4548 03:52:39,894 --> 03:52:43,531 WHEN WE'VE LOOKED AT THIS, AND 4549 03:52:43,531 --> 03:52:45,466 TRY DOCK WHERE A NUCLEOSOME 4550 03:52:45,466 --> 03:52:46,934 WOULD FIT INTO THIS STRUCTURE 4551 03:52:46,934 --> 03:52:51,005 WHEN IT'S STAT RATED WITH LEDGF 4552 03:52:51,005 --> 03:52:52,506 YOU JUST CAN'T FIT IT IN. 4553 03:52:52,506 --> 03:52:58,546 WHEN WE HAVE THE PWWP PRESENT, 4554 03:52:58,546 --> 03:53:00,347 YES IT'S TARGETING CHROMATIN, 4555 03:53:00,347 --> 03:53:02,583 BUT WHEN THE LEDGF IS PART OF 4556 03:53:02,583 --> 03:53:11,292 THIS KIND OF CHROMATIN BINDING 4557 03:53:11,292 --> 03:53:14,728 PLATFORM IBDs ARE RESTRICTING, 4558 03:53:14,728 --> 03:53:19,366 PREVENTS DIRECT INTERACTION WITH 4559 03:53:19,366 --> 03:53:21,235 NUCLEOSOME, TARGETS NAKED 4560 03:53:21,235 --> 03:53:22,002 SUBSTRATES. 4561 03:53:22,002 --> 03:53:28,375 WITH THAT I'D LIKE TO THANK MY 4562 03:53:28,375 --> 03:53:30,744 SUPERVISOR, MENTOR IN PETER'S 4563 03:53:30,744 --> 03:53:37,084 LAB, AND A LOT OF THE STRUCTURAL 4564 03:53:37,084 --> 03:53:45,659 WORK FOR THIS PROJECT, AND 4565 03:53:45,659 --> 03:53:48,295 NICOLA COLLECTED DATA. 4566 03:53:48,295 --> 03:53:49,063 THANK YOU. 4567 03:53:49,063 --> 03:53:52,733 [APPLAUSE] 4568 03:53:52,733 --> 03:53:54,602 >> ANY QUESTIONS FOR JOSHUA? 4569 03:53:54,602 --> 03:53:56,570 >> HI, JOSHUA. 4570 03:53:56,570 --> 03:53:56,937 BEAUTIFUL WORK. 4571 03:53:56,937 --> 03:53:57,605 ESPECIALLY THE STRUCTURES AT THE 4572 03:53:57,605 --> 03:53:57,771 END. 4573 03:53:57,771 --> 03:54:00,074 I WANTED TO ASK ABOUT THE 4574 03:54:00,074 --> 03:54:01,075 INITIAL SEQUENCING DATA. 4575 03:54:01,075 --> 03:54:05,346 SO YOU SEEM TO HAVE A PREFERENCE 4576 03:54:05,346 --> 03:54:07,781 FOR THE EXIT SITE OF THE 4577 03:54:07,781 --> 03:54:09,383 NUCLEOSOME BUT NOT THE ENTRY 4578 03:54:09,383 --> 03:54:10,150 SITE INTEGRATION. 4579 03:54:10,150 --> 03:54:13,120 DO YOU HAVE ANY EXPLANATION FOR 4580 03:54:13,120 --> 03:54:13,854 THAT? 4581 03:54:13,854 --> 03:54:17,258 >> SO, WE THINK THAT THE 4582 03:54:17,258 --> 03:54:19,326 SEQUENCE SPECIFICITY, YOU KIND 4583 03:54:19,326 --> 03:54:22,496 OF HAVE MORE SPECIFICITY FOR 4584 03:54:22,496 --> 03:54:24,198 THAT EXIT SITE BECAUSE IT'S 4585 03:54:24,198 --> 03:54:25,232 SYMMETRIC, RIGHT? 4586 03:54:25,232 --> 03:54:28,202 THERE SHOULDN'T BE A DIFFERENCE. 4587 03:54:28,202 --> 03:54:30,604 WE'RE WORKING ON GETTING 4588 03:54:30,604 --> 03:54:33,674 INTEGRATION SITE DATA FOR -- 4589 03:54:33,674 --> 03:54:35,376 WITHOUT ANY SEQUENCE 4590 03:54:35,376 --> 03:54:36,076 SPECIFICITY, JUST RANDOM 4591 03:54:36,076 --> 03:54:38,712 SEQUENCES TO AVERAGE THAT OUT. 4592 03:54:38,712 --> 03:54:42,283 >> DO YOU THINK IT MIGHT HAVE TO 4593 03:54:42,283 --> 03:54:44,852 DO WITH NORMAL MALLEABILITY OF 4594 03:54:44,852 --> 03:54:49,390 THE DNA, HOW THE SEQUENCE -- IS 4595 03:54:49,390 --> 03:54:51,292 THIS W 6 O 1? 4596 03:54:51,292 --> 03:54:52,626 >> IT IS. 4597 03:54:52,626 --> 03:54:54,028 MAYBE IT'S FLOPPIER AT EXIT 4598 03:54:54,028 --> 03:54:56,330 SITE, YEAH, THAT COULD BE THE 4599 03:54:56,330 --> 03:54:56,530 CASE. 4600 03:54:56,530 --> 03:54:59,733 >> THANK YOU. 4601 03:54:59,733 --> 03:55:05,406 >> DO YOU HAVE ANY ESTIMATE HOW 4602 03:55:05,406 --> 03:55:07,675 MANY LEDGFs ARE IN YOUR 4603 03:55:07,675 --> 03:55:08,142 ISOLATED PICKS? 4604 03:55:08,142 --> 03:55:13,247 >> OH, WE DON'T HAVE ANY 4605 03:55:13,247 --> 03:55:13,514 ESTIMATE. 4606 03:55:13,514 --> 03:55:15,249 BUT THEY ARE NUCLEAR EXTRACTED 4607 03:55:15,249 --> 03:55:18,419 PITS, SO THERE'S A LOT OF IT. 4608 03:55:18,419 --> 03:55:21,722 AND WHEN WE GET IT FROM THESE 4609 03:55:21,722 --> 03:55:23,557 KNOCKOUT PICKS, LEDGF KNOCKOUT 4610 03:55:23,557 --> 03:55:27,828 PICKS, WE START AT ONE NANOMOLAR 4611 03:55:27,828 --> 03:55:33,534 TO KICK THAT PREFERENCE, YOU 4612 03:55:33,534 --> 03:55:39,740 STILL GET REALLY HIGH TARGETING 4613 03:55:39,740 --> 03:55:41,175 TO TRIOMY REGIONS SO I DON'T 4614 03:55:41,175 --> 03:55:43,510 THINK YOU NEED A LOT BASICALLY 4615 03:55:43,510 --> 03:55:45,346 TO TARGET THIS. 4616 03:55:45,346 --> 03:55:45,913 >> OKAY. 4617 03:55:45,913 --> 03:55:49,750 NO MORE QUESTIONS, APPLAUSE 4618 03:55:49,750 --> 03:55:51,719 AGAIN FOR JOSHUA. 4619 03:55:51,719 --> 03:55:52,219 [APPLAUSE] 4620 03:55:52,219 --> 03:55:53,988 SO SLIGHT CHANGE IN SCHEDULE, 4621 03:55:53,988 --> 03:55:56,256 ONE MORE SPEAKER AND THEN TAKE 4622 03:55:56,256 --> 03:55:59,660 THE BREAK EARLY AND GO BACK TO 4623 03:55:59,660 --> 03:56:01,228 WILLIAM TO MAKE SURE HIS SLIDES 4624 03:56:01,228 --> 03:56:04,264 ARE WORKING. 4625 03:56:04,264 --> 03:56:09,069 NEXT SLEEKER IS AVIK BISWAS, 4626 03:56:09,069 --> 03:56:11,605 SALK INSTITUTE FOR BIOLOGICAL 4627 03:56:11,605 --> 03:56:21,682 STUDIES. 4628 03:56:21,682 --> 03:56:27,988 TWO MORE SPEAKERS AND THEN 4629 03:56:27,988 --> 03:56:28,655 BREAK. 4630 03:56:28,655 --> 03:56:29,890 ONE, TWO, THREE. 4631 03:56:29,890 --> 03:56:31,158 >> YES, THANK YOU FOR THE 4632 03:56:31,158 --> 03:56:31,959 INVITATION TO SPEAK. 4633 03:56:31,959 --> 03:56:33,694 SORRY ABOUT THE SLIDE ISSUE. 4634 03:56:33,694 --> 03:56:37,731 I'LL TALK ABOUT OUR STRUCTURAL 4635 03:56:37,731 --> 03:56:40,567 ANALYSIS OF THE HW 1 IMMATURE 4636 03:56:40,567 --> 03:56:45,739 CAPSID LATTICE, I'M A GRADUATE 4637 03:56:45,739 --> 03:56:47,775 STUDENT, CO-MENTORED, UNIVERSITY 4638 03:56:47,775 --> 03:56:57,718 OF MINNESOTA. 4639 03:56:57,718 --> 03:56:59,186 4640 03:56:59,186 --> 03:57:01,755 SO HUMAN T CELL TYPE 1 INFECTS 4641 03:57:01,755 --> 03:57:04,324 10 TO 15 MILLION PEOPLE 4642 03:57:04,324 --> 03:57:04,591 WORLDWIDE. 4643 03:57:04,591 --> 03:57:06,527 AND IS THE CAUSATIVE AGENT OF 4644 03:57:06,527 --> 03:57:13,734 ADULT T CELL LEUKEMIA LYMPHOMA 4645 03:57:13,734 --> 03:57:18,672 AND MYELOPATHY AND A 4646 03:57:18,672 --> 03:57:20,307 NEUROLOGICAL DISORDER. 4647 03:57:20,307 --> 03:57:25,679 DIFFERENT RETROVIRUSES HAVE 4648 03:57:25,679 --> 03:57:26,647 DISTINCT MORPHOLOGIES, IT OFTEN 4649 03:57:26,647 --> 03:57:28,715 HAS FLAT REGIONS OF THE LATTICE 4650 03:57:28,715 --> 03:57:29,616 AND REGIONS THAT DON'T ALWAYS 4651 03:57:29,616 --> 03:57:37,925 FOLLOW THE CURVE CURVATURE. 4652 03:57:37,925 --> 03:57:39,226 THESE ARE 2D CRYOIMAGES, HOW 4653 03:57:39,226 --> 03:57:44,465 DOES THIS LOOK IN THREE 4654 03:57:44,465 --> 03:57:45,065 DIMENSIONS? 4655 03:57:45,065 --> 03:57:50,737 COLLECTED TOMOGRAPHY DATASET, 4656 03:57:50,737 --> 03:57:52,506 YOU CAN SEE GAPS IN THE LATTICE 4657 03:57:52,506 --> 03:57:54,908 AS WELL AND FLAT REGIONS. 4658 03:57:54,908 --> 03:58:01,615 WE USED SUBTOMOGRAM AVERAGING, 4659 03:58:01,615 --> 03:58:03,283 GAVE IT TO A SCIENTIST AT THE 4660 03:58:03,283 --> 03:58:04,718 UNIVERSITY WHO WROTE A PROGRAM 4661 03:58:04,718 --> 03:58:08,422 WHERE HE CAN CALCULATE WITH 4662 03:58:08,422 --> 03:58:09,890 LOCAL CURVATURE AT PER-HEXAMER 4663 03:58:09,890 --> 03:58:10,257 LEVEL. 4664 03:58:10,257 --> 03:58:14,561 THOSE IN BLUE TEND TO HAVE MORE 4665 03:58:14,561 --> 03:58:16,763 FLAT LATTICE, LARGER RADIUS, UP 4666 03:58:16,763 --> 03:58:19,466 TO 75 NANOMETERS. 4667 03:58:19,466 --> 03:58:21,869 HEXAMERS IN RED HAVE HIGHER 4668 03:58:21,869 --> 03:58:24,838 CURVATURE OR MORE CURVED AND 4669 03:58:24,838 --> 03:58:27,174 SMALLER RADIUS DOWN TO 28 4670 03:58:27,174 --> 03:58:27,708 NANOMETERS. 4671 03:58:27,708 --> 03:58:30,844 EVEN WITH THE THE SAME PARTICLE 4672 03:58:30,844 --> 03:58:32,279 YOU HAVE DRASTIC DIFFERENCES, 4673 03:58:32,279 --> 03:58:33,614 NOT JUST FOLLOWING SIMPLE 4674 03:58:33,614 --> 03:58:34,815 SPHERE. 4675 03:58:34,815 --> 03:58:36,183 ANOTHER THING WE'VE NOTICED IS 4676 03:58:36,183 --> 03:58:37,851 THAT EVEN WITHIN THE SAME 4677 03:58:37,851 --> 03:58:39,153 PARTICLE WE'RE ON THE RIGHT 4678 03:58:39,153 --> 03:58:41,121 COLORING THEM BASED OFF DISTANCE 4679 03:58:41,121 --> 03:58:44,091 FROM CAPSID TO MEMBRANE, YOU CAN 4680 03:58:44,091 --> 03:58:45,659 HAVE DIFFERENT DISTANCES EVEN 4681 03:58:45,659 --> 03:58:46,827 WITHIN THE SAME HEXAMER. 4682 03:58:46,827 --> 03:58:49,630 WITHIN THE SAME PARTICLE. 4683 03:58:49,630 --> 03:58:55,269 SO OVERALL THIS IS SHOWING 4684 03:58:55,269 --> 03:58:57,237 HTLV-1 PARTICLES ARE 4685 03:58:57,237 --> 03:58:58,405 HETEROGENEOUS, RECENT 4686 03:58:58,405 --> 03:58:59,740 bioRxiv PUBLICATION DID A 4687 03:58:59,740 --> 03:59:04,711 NICE JOB SHOWING ARC SEC TIRE OF 4688 03:59:04,711 --> 03:59:06,446 IMMATURE LATTICE AND GOT HIGH 4689 03:59:06,446 --> 03:59:11,351 RESOLUTION USING HELICAL TUBES. 4690 03:59:11,351 --> 03:59:13,120 THE CTD IN RECONSTRUCTION WASN'T 4691 03:59:13,120 --> 03:59:14,721 AS WELL RESOLVED DUE TO 4692 03:59:14,721 --> 03:59:15,222 FLEXIBILITY. 4693 03:59:15,222 --> 03:59:18,258 WE WANTED TO USE SINGLE PARTICLE 4694 03:59:18,258 --> 03:59:19,660 ANALYSIS ON IMMATURE PARTICLES 4695 03:59:19,660 --> 03:59:22,596 THAT WERE PRODUCED BY 4696 03:59:22,596 --> 03:59:24,264 TRANSFECTING GAG INTO CELLS AND 4697 03:59:24,264 --> 03:59:24,631 PURIFYING THEM. 4698 03:59:24,631 --> 03:59:26,900 SO ON THE LEFT IS JUST SCHEMATIC 4699 03:59:26,900 --> 03:59:29,102 OF WHAT THE OVERALL 4700 03:59:29,102 --> 03:59:29,870 PARTICLE-PICKING SCHEME WOULD 4701 03:59:29,870 --> 03:59:30,737 BE, ESSENTIALLY PICKING ALL 4702 03:59:30,737 --> 03:59:36,343 PARTS OF THE LATTICE UNDER THE 4703 03:59:36,343 --> 03:59:37,077 VIRAL MEMBRANE. 4704 03:59:37,077 --> 03:59:40,013 WE CAN GET SIDE VIEWS AND MOST 4705 03:59:40,013 --> 03:59:41,715 IMPORTANTLY RECOVER TOP VIEWS OF 4706 03:59:41,715 --> 03:59:42,883 THE LATTICE AS WELL. 4707 03:59:42,883 --> 03:59:47,454 AND SO WITH THAT WE'RE ABLE TO 4708 03:59:47,454 --> 03:59:52,726 ACHIEVE A 3.4-ANGSTROM 4709 03:59:52,726 --> 03:59:55,195 RECONSTRUCTION, TOP VIEW, 4710 03:59:55,195 --> 03:59:56,463 CENTRAL HEXAMER, SURROUNDING 4711 03:59:56,463 --> 03:59:56,697 HEXAMER. 4712 03:59:56,697 --> 04:00:02,169 ON ITS SLIDE IN BLUE IS 4713 04:00:02,169 --> 04:00:07,074 N-TERMINAL DOMAIN, GOLD IS 4714 04:00:07,074 --> 04:00:09,376 C-TERMINAL DOMAIN. 4715 04:00:09,376 --> 04:00:12,346 NO SPACER PEPTIDE REGION, 4716 04:00:12,346 --> 04:00:13,981 STRAIGHT TO NUCLEOCAPSID. 4717 04:00:13,981 --> 04:00:15,515 THERE'S DENSITY BUT THAT'S 4718 04:00:15,515 --> 04:00:22,289 MOSTLY ATTRIBUTED TO NUCLEOCAP 4719 04:00:22,289 --> 04:00:22,522 C 4720 04:00:22,522 --> 04:00:23,290 APSID. 4721 04:00:23,290 --> 04:00:25,225 TOP RIGHT IS ESTIMATE, IN THE 4722 04:00:25,225 --> 04:00:27,961 N-TERMINAL DOMAIN ABLE TO 4723 04:00:27,961 --> 04:00:29,029 ACHIEVE HIGHER RESOLUTION THAN 4724 04:00:29,029 --> 04:00:30,230 C-TERMINAL DOMAIN, APPEARS TO BE 4725 04:00:30,230 --> 04:00:31,698 MORE FLEXIBLE. 4726 04:00:31,698 --> 04:00:33,567 THIS MOVIE PLAYING IN THE BOTTOM 4727 04:00:33,567 --> 04:00:38,505 RIGHT IS A GREAT DEPICTION OF 4728 04:00:38,505 --> 04:00:41,041 WHY, PICKING UP ON 4729 04:00:41,041 --> 04:00:41,375 HETEROGENEITY. 4730 04:00:41,375 --> 04:00:42,709 THE MAIN HETEROGENEITY IT'S 4731 04:00:42,709 --> 04:00:44,778 PICKING UP IS DIFFERENT 4732 04:00:44,778 --> 04:00:46,613 CURVATURE STATES OF THE LATTICE. 4733 04:00:46,613 --> 04:00:51,018 THE TOP OF THE NTD IS ACTING AS 4734 04:00:51,018 --> 04:00:53,287 HINGE POINT ALLOWING FOR THE CTD 4735 04:00:53,287 --> 04:00:58,025 TO MOVE OUT AND GET VARYING 4736 04:00:58,025 --> 04:00:58,959 CURVATURE. 4737 04:00:58,959 --> 04:01:01,695 LIKELY WHY WE'RE SEEING WORSENED 4738 04:01:01,695 --> 04:01:02,129 RESOLUTION. 4739 04:01:02,129 --> 04:01:04,264 WITHOUT ABSENCE OF SIX HELIX 4740 04:01:04,264 --> 04:01:05,299 BUNDLE IT'S MORE FLEXIBLE TO 4741 04:01:05,299 --> 04:01:05,799 MOVE. 4742 04:01:05,799 --> 04:01:09,403 HERE IS A MODEL OF WHAT OUR 4743 04:01:09,403 --> 04:01:10,904 LATTICS LOOK LIKE. 4744 04:01:10,904 --> 04:01:13,640 COLOR RECONSTRUCTION BASED ON 4745 04:01:13,640 --> 04:01:15,275 HEXAMER, SIDE VIEW, BACK TO THE 4746 04:01:15,275 --> 04:01:18,912 TOP, FIT INTO A MODEL. 4747 04:01:18,912 --> 04:01:19,513 AND CROSS-SECTION THROUGH THE 4748 04:01:19,513 --> 04:01:25,686 MODEL TO LOOK AT TRIMERIC NTD 4749 04:01:25,686 --> 04:01:27,421 INTERFACE, CTD WITH DIAPER 4750 04:01:27,421 --> 04:01:29,089 INTERFACE, ROTATE ON ITS SIDE 4751 04:01:29,089 --> 04:01:32,893 AND DO A CROSS-SECTION, AGAIN TO 4752 04:01:32,893 --> 04:01:33,994 SHOW NTD AND CTD. 4753 04:01:33,994 --> 04:01:35,862 HERE SHOWING THE MODEL IN THE 4754 04:01:35,862 --> 04:01:45,372 CENTRAL HEXAMER, BLUE IS NTD, 4755 04:01:45,372 --> 04:01:45,939 GOLD CTD. 4756 04:01:45,939 --> 04:01:50,544 I WON'T GO INTO DETAIL BECAUSE 4757 04:01:50,544 --> 04:01:53,080 THAT'S THE MAIN FOCUS OF DR. 4758 04:01:53,080 --> 04:01:54,147 FULLER'S PUBLICATION. 4759 04:01:54,147 --> 04:01:56,350 WE'RE IN GOOD AGREEMENT WITH THE 4760 04:01:56,350 --> 04:01:57,651 TUBE ASSEMBLY WITH VIRAL-LIKE 4761 04:01:57,651 --> 04:02:02,022 PARTICLES WHERE WE'RE SEEING 4762 04:02:02,022 --> 04:02:04,224 TRIMERIC NTD INTERFACE 4763 04:02:04,224 --> 04:02:07,060 STABILIZED BY HELIX 4 4764 04:02:07,060 --> 04:02:07,394 INTERACTIONS. 4765 04:02:07,394 --> 04:02:09,363 LOOKING AT CTD IT FORMS A DIMER 4766 04:02:09,363 --> 04:02:10,130 INTERFACE. 4767 04:02:10,130 --> 04:02:13,100 WE DON'T SEE INTERACTION BETWEEN 4768 04:02:13,100 --> 04:02:15,035 CTD MONOMERS WITHIN THE SAME 4769 04:02:15,035 --> 04:02:16,336 HEXAMER. 4770 04:02:16,336 --> 04:02:17,270 IT'S ONLY FORMING INTERACTIONS 4771 04:02:17,270 --> 04:02:20,006 WITH MONOMER ON THE ADJACENT 4772 04:02:20,006 --> 04:02:21,675 HEXAMER THROUGH THE DIMER 4773 04:02:21,675 --> 04:02:24,678 INTERFACE, MOSTLY COMPOSED OF 4774 04:02:24,678 --> 04:02:32,986 HELIX 8 AND HELIX 9, HELIX 9 4775 04:02:32,986 --> 04:02:33,453 INTERACTIONS. 4776 04:02:33,453 --> 04:02:40,660 IT HAS HYDROPHOBIC POCKET, AND 4777 04:02:40,660 --> 04:02:42,829 THEN ALSO TRYPTOPHAN 133, A 4778 04:02:42,829 --> 04:02:48,435 MUTATION OF RESIDUES TO ALANINE 4779 04:02:48,435 --> 04:02:50,270 DECREASES IMMATURE PARTICLE 4780 04:02:50,270 --> 04:02:50,804 PRODUCTION. 4781 04:02:50,804 --> 04:02:54,141 SIMILARLY AT THE BOTTOM OF HELIX 4782 04:02:54,141 --> 04:03:02,315 8 AND 9 HYDROGEN BONDING, 4783 04:03:02,315 --> 04:03:03,450 DIMINISHED PARTICLE PRODUCTION. 4784 04:03:03,450 --> 04:03:06,620 THE INTERFACE IS ACTUALLY QUITE 4785 04:03:06,620 --> 04:03:10,023 CRITICAL FOR PARTICLE ASSEMBLY, 4786 04:03:10,023 --> 04:03:11,024 MOSTLY UNDERAPPRECIATED, MOSTLY 4787 04:03:11,024 --> 04:03:15,896 NTD THAT IS IMPORTANT FOR 4788 04:03:15,896 --> 04:03:17,230 PARTICLE ASSEMBLY. 4789 04:03:17,230 --> 04:03:19,332 ANOTHER THING WE NOTICED WAS IN 4790 04:03:19,332 --> 04:03:21,935 THE N-TERMINAL DOMAIN WE HAVE 4791 04:03:21,935 --> 04:03:24,871 THIS SMALL EXTRA DENSITY, 4792 04:03:24,871 --> 04:03:25,472 CENTRAL HEXAMER, ADJACENT 4793 04:03:25,472 --> 04:03:26,440 HEXAMERS AS WELL. 4794 04:03:26,440 --> 04:03:29,142 I'LL COLOR IT SO YOU CAN SEE IT 4795 04:03:29,142 --> 04:03:29,676 BETTER. 4796 04:03:29,676 --> 04:03:31,311 IF YOU FOCUS ON WHAT THAT 4797 04:03:31,311 --> 04:03:37,951 DENSITY IS WE HAVE A RING OF 4798 04:03:37,951 --> 04:03:41,688 LYSINE 18, ANALOGOUS TO WHERE 4799 04:03:41,688 --> 04:03:46,293 YOU EXPECT TO SEE IP6 IN THE 4800 04:03:46,293 --> 04:03:47,394 MATURE STRUCTURE. 4801 04:03:47,394 --> 04:03:50,530 WE USE THESE KNOCKOUT CELL 4802 04:03:50,530 --> 04:03:54,935 LINES, IPPK IS KINASE THAT 4803 04:03:54,935 --> 04:03:58,305 CONVERTS TO IPK, AND WE ALSO 4804 04:03:58,305 --> 04:04:01,808 TRANSFECT IN CP 1, A PHOSPHATASE 4805 04:04:01,808 --> 04:04:06,546 THAT CONVERTS IP6 BACK TO IP 5. 4806 04:04:06,546 --> 04:04:10,484 WHEN WE TRANSFECT, IT DOES NOT 4807 04:04:10,484 --> 04:04:11,551 AFFECT IMMATURE PARTICLE 4808 04:04:11,551 --> 04:04:13,553 PRODUCTION, WE'RE USING THE 4809 04:04:13,553 --> 04:04:16,590 CONTROL, AS YOU'D EXPECT WE GET 4810 04:04:16,590 --> 04:04:18,024 DEPRESSED PARTICLE PRODUCTION. 4811 04:04:18,024 --> 04:04:21,962 OVERALL IT SEEMS LIKE IP6 IS NOT 4812 04:04:21,962 --> 04:04:23,597 REQUIRED FOR IMMATURE PARTICLE 4813 04:04:23,597 --> 04:04:23,897 PRODUCTION. 4814 04:04:23,897 --> 04:04:27,000 LAST THING WAS THE LATTICE WAS 4815 04:04:27,000 --> 04:04:28,134 INTACT. 4816 04:04:28,134 --> 04:04:30,504 SO WE TOOK CRYOIMAGES OF 4817 04:04:30,504 --> 04:04:37,077 PARTICLES FROM THE KNOCKOUT 4818 04:04:37,077 --> 04:04:40,881 CELLS, OVEREXPRESSING 4819 04:04:40,881 --> 04:04:42,649 PHOSPHATASE, IT'S NOT AFFECTING 4820 04:04:42,649 --> 04:04:45,051 PARTICLE MORPHOLOGY EITHER. 4821 04:04:45,051 --> 04:04:52,492 SO, OVERALL CONCLUSIONS THAT 4822 04:04:52,492 --> 04:04:55,896 IMMATURE HTLH-1 PARTICLES, THE 4823 04:04:55,896 --> 04:04:59,266 LATTICE IS STABILIZED BY 4824 04:04:59,266 --> 04:05:01,568 INTERFACE AND DISRUPTION CAN 4825 04:05:01,568 --> 04:05:02,602 DIMINISH PARTICLES PRODUCTION. 4826 04:05:02,602 --> 04:05:05,105 IP6 IS NOT REQUIRED FOR IMMATURE 4827 04:05:05,105 --> 04:05:09,042 PARTICLE ASSEMBLY BUT LIKELY 4828 04:05:09,042 --> 04:05:14,180 STILL REQUIRES DIFFERENT POLY 4829 04:05:14,180 --> 04:05:18,351 ANINE TO LYSINE RING. 4830 04:05:18,351 --> 04:05:20,320 I'D LIKE TO THANK ALL MEMBERS OF 4831 04:05:20,320 --> 04:05:28,328 THE LAB AND OUR COLLABORATORS, 4832 04:05:28,328 --> 04:05:31,665 ESPECIALLY MY CO-MENTOR. 4833 04:05:31,665 --> 04:05:33,667 I WISH I KNEW WHO WROTE THE 4834 04:05:33,667 --> 04:05:34,568 PROGRAM. 4835 04:05:34,568 --> 04:05:40,240 AND OUR LAB RATERS AND THOSE WHO 4836 04:05:40,240 --> 04:05:41,274 PROVIDED REAGENTS FOR US. 4837 04:05:41,274 --> 04:05:43,410 I'D BE HAPPY TO TAKE QUESTIONS. 4838 04:05:43,410 --> 04:05:44,744 THANK YOU. 4839 04:05:44,744 --> 04:05:45,211 [APPLAUSE] 4840 04:05:45,211 --> 04:05:46,613 >> WE'RE DOING EXCELLENT ON 4841 04:05:46,613 --> 04:05:47,581 TIME. 4842 04:05:47,581 --> 04:05:50,283 ANY QUESTIONS FOR WILLIAM? 4843 04:05:50,283 --> 04:05:51,017 >> GREAT TALK. 4844 04:05:51,017 --> 04:05:53,186 HAVE YOU LOOKED AND IF SO DO YOU 4845 04:05:53,186 --> 04:05:55,255 SEE ANY INDICATION OF REGULARITY 4846 04:05:55,255 --> 04:05:57,657 IN MATRIX LAYER IN YOUR 4847 04:05:57,657 --> 04:05:58,091 PARTICLES? 4848 04:05:58,091 --> 04:06:01,728 >> YEAH, I MEAN, I CAN SEE 4849 04:06:01,728 --> 04:06:03,396 MATRIX IN MICROGRAPHS AND I 4850 04:06:03,396 --> 04:06:04,898 TYPICALLY DON'T BOX LARGE ENOUGH 4851 04:06:04,898 --> 04:06:07,734 TO SEE MATRIX BUT HAVE FOCUSED 4852 04:06:07,734 --> 04:06:09,636 IN, YOU CAN SEE ORDERED LAYER OF 4853 04:06:09,636 --> 04:06:11,538 MATRIX IN THESE PARTICLES, MAYBE 4854 04:06:11,538 --> 04:06:13,573 NOT COMPLETE THROUGHOUT THE 4855 04:06:13,573 --> 04:06:15,075 ENTIRE PARTICLE AS YOU'D EXPECT 4856 04:06:15,075 --> 04:06:16,476 BASED OFF HETEROGENEITY IN THE 4857 04:06:16,476 --> 04:06:18,445 LATTICE BUT I THINK IT IS 4858 04:06:18,445 --> 04:06:19,546 DEFINITELY THERE. 4859 04:06:19,546 --> 04:06:20,547 >> COOL. 4860 04:06:20,547 --> 04:06:21,848 THANK YOU. 4861 04:06:21,848 --> 04:06:23,516 >> YEAH, THANK YOU. 4862 04:06:23,516 --> 04:06:26,553 >> ANY OTHER QUESTIONS OF 4863 04:06:26,553 --> 04:06:26,853 WILLIAM? 4864 04:06:26,853 --> 04:06:32,125 IF NOT THEN LET'S GIVE HIM 4865 04:06:32,125 --> 04:06:32,559 ANOTHER APPLAUSE. 4866 04:06:32,559 --> 04:06:38,732 [APPLAUSE] 4867 04:06:38,732 --> 04:06:46,239 AND NOW AVIK BISWAS FROM SALK 4868 04:06:46,239 --> 04:06:48,241 INSTITUTE FOR BIOLOGICAL 4869 04:06:48,241 --> 04:06:48,475 STUDIES. 4870 04:06:48,475 --> 04:06:56,783 GO AHEAD, AVIK. 4871 04:06:56,783 --> 04:07:06,826 4872 04:07:11,665 --> 04:07:13,066 >> ALL RIGHT. 4873 04:07:13,066 --> 04:07:14,234 GOOD AFTERNOON, EVERYONE. 4874 04:07:14,234 --> 04:07:17,470 I'M AVIK, POSTDOC AT THE SALK 4875 04:07:17,470 --> 04:07:18,304 INSTITUTE FOR BIOLOGICAL STUDIES 4876 04:07:18,304 --> 04:07:21,074 IN TODAY I'M GOING TO BE TALKING 4877 04:07:21,074 --> 04:07:25,311 ABOUT SOME OF OUR RECENT EFFORTS 4878 04:07:25,311 --> 04:07:26,746 LOOKING AT PATHWAYS AND 4879 04:07:26,746 --> 04:07:29,249 MECHANISMS OF HOW DRUG 4880 04:07:29,249 --> 04:07:31,451 RESISTANCE EVOLVES IN HIV, IN 4881 04:07:31,451 --> 04:07:35,388 COLLABORATION WITH RON LEVI'S 4882 04:07:35,388 --> 04:07:37,490 GROUP AT TEMPLE, MY Ph.D. 4883 04:07:37,490 --> 04:07:37,724 ADVISOR. 4884 04:07:37,724 --> 04:07:39,859 THIS WORK BEGAN IN RON'S GROUP. 4885 04:07:39,859 --> 04:07:41,428 THE KEY QUESTION WE ASKED HERE 4886 04:07:41,428 --> 04:07:44,798 IS WHY DO SOME DRUG RESISTANCE 4887 04:07:44,798 --> 04:07:45,899 MUTATIONS ARISE FASTER THAN 4888 04:07:45,899 --> 04:07:46,299 OTHERS? 4889 04:07:46,299 --> 04:07:51,404 SO IF YOU TAKE THE EXAMPLE FROM 4890 04:07:51,404 --> 04:07:54,340 HIV INTEGRASE WE SEE THE 4891 04:07:54,340 --> 04:07:56,910 MUTATION AND Q1 48 MUTATION ARE 4892 04:07:56,910 --> 04:07:59,446 PRESENT IN DRUG EXPERIENCE 4893 04:07:59,446 --> 04:08:00,513 PATIENT SAMPLES IN STANFORD 4894 04:08:00,513 --> 04:08:04,284 DATABASE FOR EXAMPLE WITH 4895 04:08:04,284 --> 04:08:06,453 ROUGHLY EQUAL FREQUENCES YET N 4896 04:08:06,453 --> 04:08:09,989 155H IS REPORTED TO BE FASTER 4897 04:08:09,989 --> 04:08:11,624 THAN THE Q148H MUTATION, WE WANT 4898 04:08:11,624 --> 04:08:13,593 TO KNOW HOW AND WHY AND MORE 4899 04:08:13,593 --> 04:08:14,994 BROADLY WHAT ARE THE KINETIC 4900 04:08:14,994 --> 04:08:19,265 PATHWAYS THAT LEAD TO DRUG 4901 04:08:19,265 --> 04:08:20,600 RESISTANCE STARTING FROM DRUG 4902 04:08:20,600 --> 04:08:21,768 NAIVE PATIENT POPULATION, IF WE 4903 04:08:21,768 --> 04:08:24,204 CAN FINDS A BASIS FOR THIS SLOW 4904 04:08:24,204 --> 04:08:26,372 OR FAST ACQUISITION OF 4905 04:08:26,372 --> 04:08:26,773 RESISTANCE. 4906 04:08:26,773 --> 04:08:28,675 SO WITH THIS IN MIND WHAT WE 4907 04:08:28,675 --> 04:08:31,745 WANTED TO DO IS START FROM DRUG 4908 04:08:31,745 --> 04:08:33,847 NAIVE PATIENT SAMPLES, IN THE 4909 04:08:33,847 --> 04:08:35,014 STANFORD DATABASE, EVOLVE THEM 4910 04:08:35,014 --> 04:08:37,550 SUCH THAT THEY THEN BECOME DRUG 4911 04:08:37,550 --> 04:08:38,752 EXPERIENCE PATIENT SAMPLES IN 4912 04:08:38,752 --> 04:08:39,619 STANFORD DATABASE. 4913 04:08:39,619 --> 04:08:43,323 SO WE CAME UP WITH A KINETIC 4914 04:08:43,323 --> 04:08:46,192 KIEM TO DO THIS, FIRST BUILD A 4915 04:08:46,192 --> 04:08:50,330 MODEL OF THE PROTEIN FITNESS 4916 04:08:50,330 --> 04:08:53,533 LANDSCAPE BASED ON DRUG 4917 04:08:53,533 --> 04:08:54,567 EXPERIENCE IN STANFORD COUPLED 4918 04:08:54,567 --> 04:08:56,369 TO THE PROCESS WHERE WE START 4919 04:08:56,369 --> 04:09:00,540 FROM THE DRUG NAIVE PATIENT 4920 04:09:00,540 --> 04:09:11,084 SAMPLES, SELECT SEQUENCE AND TRY 4921 04:09:13,186 --> 04:09:17,090 MUTATE RANDOM, WE CAN ITERATE 4922 04:09:17,090 --> 04:09:21,628 AND THAT MAKES DRUG NAIVE 4923 04:09:21,628 --> 04:09:23,096 SEQUENCE BECOME THOSE IN THE 4924 04:09:23,096 --> 04:09:23,963 STANFORD DATABASE. 4925 04:09:23,963 --> 04:09:27,700 WE CAN TRACK HOW THESE MUTATIONS 4926 04:09:27,700 --> 04:09:28,902 ARE EVOLVING STARTING WITH DRUG 4927 04:09:28,902 --> 04:09:30,703 NAIVE STATE. 4928 04:09:30,703 --> 04:09:32,105 WHAT DO WE GET? 4929 04:09:32,105 --> 04:09:34,073 FROM THIS PROCESS I'M SHOWING 4930 04:09:34,073 --> 04:09:36,576 TWO EXAMPLES, ON THE TOP RIGHT 4931 04:09:36,576 --> 04:09:40,513 IS A FAST RISING DRUG RESISTANCE 4932 04:09:40,513 --> 04:09:42,515 MUTATION, BOTTOM RIGHT IS SLOW 4933 04:09:42,515 --> 04:09:43,483 ARISING DRUG RESISTANCE 4934 04:09:43,483 --> 04:09:43,817 MUTATION. 4935 04:09:43,817 --> 04:09:46,252 IN EITHER CASE WE SEE THE 4936 04:09:46,252 --> 04:09:48,254 MUTATION STARTS FROM CLOSE TO 4937 04:09:48,254 --> 04:09:52,125 ZERO FREQUENCY IN THE DRUG-NAIVE 4938 04:09:52,125 --> 04:09:53,993 PATIENT SAMPLES, GRADUALLY 4939 04:09:53,993 --> 04:09:56,162 ARISES TO EQUAL DRUG EXPERIENCE 4940 04:09:56,162 --> 04:09:58,164 FREQUENCY IN THE DRUG 4941 04:09:58,164 --> 04:09:59,065 EXPERIENCED PATIENT. 4942 04:09:59,065 --> 04:10:02,902 HOWEVER, IT'S ALSO TRUE IN OUR 4943 04:10:02,902 --> 04:10:05,138 SIMULATION THE SLOWER MUTATION 4944 04:10:05,138 --> 04:10:07,974 TAKES MUCH LONGER THAT EQUAL ITS 4945 04:10:07,974 --> 04:10:08,975 DRUG EXPERIENCE FREQUENCIES. 4946 04:10:08,975 --> 04:10:13,479 CAN WE USE THIS PROCESS TO 4947 04:10:13,479 --> 04:10:15,081 PREDICT THE TIMELINE FOR DRUG 4948 04:10:15,081 --> 04:10:16,649 RESISTANCE MUTATIONS? 4949 04:10:16,649 --> 04:10:19,385 TAKING THE EXAMPLE FROM HIV 4950 04:10:19,385 --> 04:10:21,254 INTEGRASE, HERE I'M SHOWING YOU 4951 04:10:21,254 --> 04:10:23,723 THE TIME CONSTANT WHICH COMES 4952 04:10:23,723 --> 04:10:25,225 FROM OUR SIMULATIONS, AND THAT 4953 04:10:25,225 --> 04:10:26,593 GIVES THE HEIGHT OF THESE BAR 4954 04:10:26,593 --> 04:10:28,695 GRAPHS HERE AND THE COLOR OF THE 4955 04:10:28,695 --> 04:10:29,996 BAR GRAPHS ARE ACTUALLY THE TIME 4956 04:10:29,996 --> 04:10:32,398 THAT WE FIND IN THE CLINICAL 4957 04:10:32,398 --> 04:10:33,366 LITERATURE FOR THESE MUTATIONS 4958 04:10:33,366 --> 04:10:35,235 TO ARISE IN PATIENTS. 4959 04:10:35,235 --> 04:10:37,871 AND FROM THE GRADIENT WE CAN SEE 4960 04:10:37,871 --> 04:10:39,939 THAT THERE'S A VERY GOOD 4961 04:10:39,939 --> 04:10:41,274 CORRELATION WITH THE TIME 4962 04:10:41,274 --> 04:10:42,909 CONSTANTS THAT PREDICTED FROM 4963 04:10:42,909 --> 04:10:44,878 OUR SIMULATIONS WITH WHAT'S SEEN 4964 04:10:44,878 --> 04:10:47,780 IN THE CLINICAL LITERATURE. 4965 04:10:47,780 --> 04:10:49,082 THE CORRELATION COEFFICIENT IS 4966 04:10:49,082 --> 04:10:49,716 .9. 4967 04:10:49,716 --> 04:10:52,685 THEN WE ASKED THE QUESTION THAT 4968 04:10:52,685 --> 04:10:53,620 WHAT ABOUT OTHER FACTORS THAT 4969 04:10:53,620 --> 04:10:54,954 CAN PLAY A ROLE? 4970 04:10:54,954 --> 04:10:56,522 IS THIS RELATED, FOR EXAMPLE, TO 4971 04:10:56,522 --> 04:10:59,325 THE PREVALENCE OF THE MUTATIONS 4972 04:10:59,325 --> 04:11:00,560 OR ITS DRUG EXPERIENCE 4973 04:11:00,560 --> 04:11:01,861 FREQUENCIES WHICH WE CAN THINK 4974 04:11:01,861 --> 04:11:04,197 OF AS A PROXY FOR THE FITNESS OF 4975 04:11:04,197 --> 04:11:06,199 THE MUTATION, OR HOW DOES THE 4976 04:11:06,199 --> 04:11:09,602 GENETIC CODE SUCH AS TRANSITION 4977 04:11:09,602 --> 04:11:13,806 VERSUS TRANSVERSION PLAY, AND 4978 04:11:13,806 --> 04:11:16,643 USUALLY AALONE DO NOT CORRELATE 4979 04:11:16,643 --> 04:11:18,311 WELL OR WITH PREDICTED TIME 4980 04:11:18,311 --> 04:11:20,580 CONSTANTS FROM SIMULATIONS. 4981 04:11:20,580 --> 04:11:23,750 SO WHAT IS IT THAT DETERMINES 4982 04:11:23,750 --> 04:11:25,752 KINETICS, WHY IS IT SOME 4983 04:11:25,752 --> 04:11:27,587 MUTATIONS ARE SLOW, SOME ARE 4984 04:11:27,587 --> 04:11:27,787 FAST? 4985 04:11:27,787 --> 04:11:30,323 WE FIND THAT THERE ARE GENERALLY 4986 04:11:30,323 --> 04:11:32,525 TWO BROAD PROCESSES WHICH CAN 4987 04:11:32,525 --> 04:11:34,460 EXPLAIN WHY SOME MUTATIONS ARE 4988 04:11:34,460 --> 04:11:35,561 SLOW, SOME MUTATIONS ARE FAST. 4989 04:11:35,561 --> 04:11:38,564 SO IN CASE OF THE FAST MUTATION, 4990 04:11:38,564 --> 04:11:39,666 OR FAST ARISING MUTATIONS, WHAT 4991 04:11:39,666 --> 04:11:42,535 WE SEE IS THERE'S A PREEXISTING 4992 04:11:42,535 --> 04:11:44,904 BIAS THAT'S ALREADY PRESENT IN 4993 04:11:44,904 --> 04:11:46,973 THE DRUG NAIVE PATIENT 4994 04:11:46,973 --> 04:11:49,142 SEQUENCES, TO GET THIS MUTATION. 4995 04:11:49,142 --> 04:11:50,476 THAT BECOMES APPARENT WHEN DRUG 4996 04:11:50,476 --> 04:11:52,779 PRESSURE IS TURNED ON IN OUR 4997 04:11:52,779 --> 04:11:56,015 SIMULATIONS SO IN THIS CASE, FOR 4998 04:11:56,015 --> 04:11:59,786 EXAMPLE, AGAIN THIS IS HIV 4999 04:11:59,786 --> 04:12:02,555 INTEGRASE, THE SLOW ONE IS Q1 5000 04:12:02,555 --> 04:12:05,758 48H, WE SEE BOTH OF THEM START 5001 04:12:05,758 --> 04:12:07,360 OFF AT ZERO FREQUENCIES, THAT'S 5002 04:12:07,360 --> 04:12:09,996 THE BLACK AND YELLOW LINES OVER 5003 04:12:09,996 --> 04:12:10,196 HERE. 5004 04:12:10,196 --> 04:12:12,065 HOWEVER, THE FAST MUTATION WHICH 5005 04:12:12,065 --> 04:12:15,601 IS IN THE -- HAS MUCH HIGHER 5006 04:12:15,601 --> 04:12:18,371 PROPENSITY OR BIAS IN DRUG NAIVE 5007 04:12:18,371 --> 04:12:19,973 SAMPLE CLOSE TO FINAL FREQUENCY, 5008 04:12:19,973 --> 04:12:24,110 THE GREEN DASHED LINE HERE FOR 5009 04:12:24,110 --> 04:12:29,282 N155H MUTATION. 5010 04:12:29,282 --> 04:12:32,986 ALSO CONTRIBUTION FROM ACCESSORY 5011 04:12:32,986 --> 04:12:33,886 MUTATION, SPECIFIC ACCESSORY 5012 04:12:33,886 --> 04:12:42,095 MUTATION THAT IT'S COUPLED TOO. 5013 04:12:42,095 --> 04:12:44,697 IT BECOMES CONTINGENT ON THE 5014 04:12:44,697 --> 04:12:46,032 OTHER MUTATION BEING PRESENT 5015 04:12:46,032 --> 04:12:47,800 BEFORE IT OR AT THE TIME WHEN 5016 04:12:47,800 --> 04:12:49,202 IT'S OCCURRING IN THE SEQUENCE 5017 04:12:49,202 --> 04:12:51,838 THAT IT'S OCCURRING IN. 5018 04:12:51,838 --> 04:12:55,008 SO THESE TWO PROCESSES GENERALLY 5019 04:12:55,008 --> 04:12:57,610 EXPLAIN THE KINETICS OF OUR SLOW 5020 04:12:57,610 --> 04:12:58,111 VERSUS FAST MUTATIONS. 5021 04:12:58,111 --> 04:12:59,479 HOWEVER I'D LIKE TO POINT OUT 5022 04:12:59,479 --> 04:13:01,781 IT'S NOT THAT THESE TWO ALONE 5023 04:13:01,781 --> 04:13:03,916 CORRELATE WELL BUT IT'S ACTUALLY 5024 04:13:03,916 --> 04:13:07,487 THE RELATIVE STRENGTH OF THESE 5025 04:13:07,487 --> 04:13:08,988 THAT CORRELATE WITH THE 5026 04:13:08,988 --> 04:13:09,789 TIMELINES WE SEE. 5027 04:13:09,789 --> 04:13:11,958 SO WITH THAT WE THEN WANTED TO 5028 04:13:11,958 --> 04:13:14,160 ASK THE QUESTION WHAT IF WE TAKE 5029 04:13:14,160 --> 04:13:16,896 ONE OF THE SLOWEST ARISING 5030 04:13:16,896 --> 04:13:19,098 MUTATIONS, ALONG WITH ASSOCIATED 5031 04:13:19,098 --> 04:13:23,703 PATTERN OF MUTATIONS, AND CAN WE 5032 04:13:23,703 --> 04:13:26,005 PREDICT THE SEQUENCE THAT LET TO 5033 04:13:26,005 --> 04:13:28,708 THE PATTERN OF MUTATIONS? 5034 04:13:28,708 --> 04:13:31,144 I'M SHOWING TWO EXAMPLES, 5035 04:13:31,144 --> 04:13:34,080 STARTING FROM NL SEQUENCE, USING 5036 04:13:34,080 --> 04:13:36,315 FITNESS MODEL TO GIVE US A 5037 04:13:36,315 --> 04:13:38,351 LIKELIHOOD OF EACH OF THE 5038 04:13:38,351 --> 04:13:39,452 MUTATIONS STARTING FROM THIS 5039 04:13:39,452 --> 04:13:40,653 PARTICULAR SEQUENCE. 5040 04:13:40,653 --> 04:13:42,422 AND THEN ONCE THAT MUTATION HAS 5041 04:13:42,422 --> 04:13:43,890 EVOLVED IN THE SEQUENCE WE CAN 5042 04:13:43,890 --> 04:13:46,993 ASK WHAT'S THE NEXT MOST LIKELY 5043 04:13:46,993 --> 04:13:48,895 MUTATIONAL EVENT BASED ON THIS 5044 04:13:48,895 --> 04:13:50,963 EVOLVED SEQUENCE BACKGROUND, AND 5045 04:13:50,963 --> 04:13:54,534 THIS WAY WE CAN DERIVE 5046 04:13:54,534 --> 04:13:56,202 PROBABILISTIC PATHWAY THAT LEADS 5047 04:13:56,202 --> 04:14:06,279 TO THE PATTERN OF MUTATIONS. 5048 04:14:06,279 --> 04:14:08,581 E138K, G140S AND Q148H, AS WELL 5049 04:14:08,581 --> 04:14:11,951 AS THESE, IN INTEGRASE, IN THESE 5050 04:14:11,951 --> 04:14:13,820 TWO CASES THE RESIDUE SITES ARE 5051 04:14:13,820 --> 04:14:18,391 THE SAME EXCEPT THAT THE 5052 04:14:18,391 --> 04:14:21,194 MUTATIONS HAPPENING AT 140 ARE 5053 04:14:21,194 --> 04:14:22,395 DIFFERENT AND THAT SWITCHES 5054 04:14:22,395 --> 04:14:24,730 WHICH WOULD BE THE MOST LIKELY 5055 04:14:24,730 --> 04:14:26,966 PATHWAY LEADING TO THIS TRIPLE 5056 04:14:26,966 --> 04:14:27,733 MUTANT. 5057 04:14:27,733 --> 04:14:29,535 SO THEN WE ASKED THE QUESTION 5058 04:14:29,535 --> 04:14:31,938 CAN WE THEN RATIONALIZE THESE 5059 04:14:31,938 --> 04:14:35,374 PREDICTED TEMPORAL ORDERS OR 5060 04:14:35,374 --> 04:14:38,344 PREFERRED PATHWAYS WITH 5061 04:14:38,344 --> 04:14:41,080 STRUCTURAL BIOLOGY USING HIGH 5062 04:14:41,080 --> 04:14:41,981 RESOLUTION CryoEM STRUCTURES 5063 04:14:41,981 --> 04:14:43,149 ALONG THE SPECIFIC PATHWAY, 5064 04:14:43,149 --> 04:14:47,320 STARTING FROM WILD TYPE HERE, 5065 04:14:47,320 --> 04:14:52,792 NL43 AND I'VE HIGHLIGHTED KEY 5066 04:14:52,792 --> 04:14:53,659 RESIDUES WITH THE HISTIDINE, 5067 04:14:53,659 --> 04:14:54,961 THAT BECOMES IMPORTANT LATER ON 5068 04:14:54,961 --> 04:14:57,930 IN THE STORY. 5069 04:14:57,930 --> 04:14:59,665 SO WHEN THE E138K HAPPENS FIRST, 5070 04:14:59,665 --> 04:15:01,434 THIS THEN LEADS TO A SLIGHT 5071 04:15:01,434 --> 04:15:03,503 SHIFT IN THE DNA OVER HERE. 5072 04:15:03,503 --> 04:15:06,572 THAT THEN ALLOWS ROOM HERE AT 5073 04:15:06,572 --> 04:15:10,510 148 TO MUTATE TO A LYSINE. 5074 04:15:10,510 --> 04:15:12,812 LAST THIS 140 MUTATES TO ALANINE 5075 04:15:12,812 --> 04:15:17,617 WHICH CAUSES SHIFT IN 5076 04:15:17,617 --> 04:15:21,888 CONFIRMATION OF 148 LYSINE 5077 04:15:21,888 --> 04:15:23,656 TOWARDS THE DRUG, LEADS TO DRUG 5078 04:15:23,656 --> 04:15:26,726 EXCISION, THIS AGREES WITH THE 5079 04:15:26,726 --> 04:15:29,395 PATHWAY WE PREDICTED USING THE 5080 04:15:29,395 --> 04:15:34,534 FITNESS MODEL. 5081 04:15:34,534 --> 04:15:35,935 HOW IF Q 148K WOULD HAVE BEEN 5082 04:15:35,935 --> 04:15:39,305 THE FIRST? 5083 04:15:39,305 --> 04:15:41,174 WE SEE IF Q 148K WAS FIRST THIS 5084 04:15:41,174 --> 04:15:44,243 COULD HAVE CAUSED SHIFT IN THE 5085 04:15:44,243 --> 04:15:45,912 BACKBONE OF THE RESIDUES 139 TO 5086 04:15:45,912 --> 04:15:48,181 141 WHICH WOULD HAVE BEEN 5087 04:15:48,181 --> 04:15:52,018 UNFAVORABLE. 5088 04:15:52,018 --> 04:15:54,120 SIMILARLY THIS HISTIDINE 114 IN 5089 04:15:54,120 --> 04:15:55,121 THE SAME CONFIRMATION BEFORE IS 5090 04:15:55,121 --> 04:16:00,193 NOW IN A VERY UNFAVORABLE 5091 04:16:00,193 --> 04:16:00,526 CONFIRMATION. 5092 04:16:00,526 --> 04:16:07,967 SIMILARLY G 140 LEADS TO 5093 04:16:07,967 --> 04:16:09,635 UNFAVORABLE CONFIRMATION AT 148, 5094 04:16:09,635 --> 04:16:11,103 RATIONALIZING THE PATHWAY USING 5095 04:16:11,103 --> 04:16:12,171 THE FITNESS MODEL. 5096 04:16:12,171 --> 04:16:13,940 THIS IS STILL A WORK IN PROGRESS 5097 04:16:13,940 --> 04:16:18,644 AND WE'RE NOW LOOKING AT OTHER 5098 04:16:18,644 --> 04:16:19,845 POSSIBLE PATHWAYS, INCLUDING 5099 04:16:19,845 --> 04:16:21,714 THOSE TO OTHER PATTERNS OF 5100 04:16:21,714 --> 04:16:22,815 RESISTANCE MUTATIONS INCLUDING 5101 04:16:22,815 --> 04:16:24,383 MORE COMPLEX PATTERNS THAT 5102 04:16:24,383 --> 04:16:27,220 INVOLVE FIVE OR MORE MUTATIONS. 5103 04:16:27,220 --> 04:16:29,388 SO WITH THAT I'D SUMMARIZE BY 5104 04:16:29,388 --> 04:16:31,891 SAYING HERE WE'VE USED A MODEL 5105 04:16:31,891 --> 04:16:34,527 OF HIV FITNESS LANDSCAPE COUPLED 5106 04:16:34,527 --> 04:16:35,928 TO KINETIC MONTE CARLO 5107 04:16:35,928 --> 04:16:38,364 SIMULATION WHICH WE SEE CAN GIVE 5108 04:16:38,364 --> 04:16:41,400 US TEMPORAL BEHAVIOR OF THESE 5109 04:16:41,400 --> 04:16:43,469 RESISTANCE MUTATIONS IN HIV. 5110 04:16:43,469 --> 04:16:46,439 AND TO EXPLAIN THE KINETICS OF 5111 04:16:46,439 --> 04:16:48,941 THESE FAST VERSUS SLOW MUTATIONS 5112 04:16:48,941 --> 04:16:50,943 THERE ARE GENERALLY TWO BROAD 5113 04:16:50,943 --> 04:16:53,446 PROCESSES THAT CONTRIBUTE. 5114 04:16:53,446 --> 04:16:54,614 ONCE A PREEXISTING BIAS ALREADY 5115 04:16:54,614 --> 04:16:56,515 IN THE DRUG NAIVE PATIENT 5116 04:16:56,515 --> 04:16:57,617 POPULATION THAT BECOMES APPARENT 5117 04:16:57,617 --> 04:17:00,486 WHEN DRUG PRESSURE IS TURNED ON, 5118 04:17:00,486 --> 04:17:03,089 WHICH IS ESPECIALLY HIGH FOR THE 5119 04:17:03,089 --> 04:17:05,491 FAST ARISING MUTATIONS, AND THE 5120 04:17:05,491 --> 04:17:08,194 OTHER IS THE CONTRIBUTION FROM 5121 04:17:08,194 --> 04:17:09,829 ACCESSORY MUTATIONS, IN 5122 04:17:09,829 --> 04:17:12,164 PARTICULAR VERY STRONGLY 5123 04:17:12,164 --> 04:17:13,266 AFFECTING SLOWLY ARISING 5124 04:17:13,266 --> 04:17:15,434 RESISTANCE MUTATIONS COUPLED TO 5125 04:17:15,434 --> 04:17:17,870 SPECIFIC OTHER MUTATIONS AND 5126 04:17:17,870 --> 04:17:19,071 BECOME CONTINGENT ON MUTATIONS 5127 04:17:19,071 --> 04:17:20,406 BEING PRESENT WITH THEM. 5128 04:17:20,406 --> 04:17:23,242 WE CAN ALSO USE THIS FITNESS 5129 04:17:23,242 --> 04:17:24,577 MODEL TO PREDICT SEQUENTIAL 5130 04:17:24,577 --> 04:17:27,113 ORDER OF EVENTS THAT LEAD TO 5131 04:17:27,113 --> 04:17:28,180 SPECIFIC PATTERNS OF RESISTANCE 5132 04:17:28,180 --> 04:17:30,283 MUTATIONS AND THAT THESE 5133 04:17:30,283 --> 04:17:32,451 PATTERNS CAN THEN BE 5134 04:17:32,451 --> 04:17:33,686 RATIONALIZED USING STRUCTURAL 5135 04:17:33,686 --> 04:17:33,919 BIOLOGY. 5136 04:17:33,919 --> 04:17:35,921 WITH THAT I'D LIKE TO THANK 5137 04:17:35,921 --> 04:17:40,760 EVERYONE INVOLVEDIN THIS WORK, 5138 04:17:40,760 --> 04:17:42,928 ESPECIALLY DMITRY AND RON, ALSO 5139 04:17:42,928 --> 04:17:43,663 COLLABORATIONS AND FUNDING. 5140 04:17:43,663 --> 04:17:47,099 THANK YOU FOR YOUR TIME. 5141 04:17:47,099 --> 04:17:55,641 [APPLAUSE] 5142 04:17:55,641 --> 04:18:05,318 >> NICE WORK. 5143 04:18:05,318 --> 04:18:07,320 YOU MUST TAKE INTO ACCOUNT, I 5144 04:18:07,320 --> 04:18:09,622 TRY TO GO THROUGH QUICKLY, YOU 5145 04:18:09,622 --> 04:18:12,024 PRESENT EVERYTHING ON THE 5146 04:18:12,024 --> 04:18:13,592 PROTEIN SIDE BUT CHANGES TAKE 5147 04:18:13,592 --> 04:18:15,995 PLACE AT THE NUCLEIC ACID. 5148 04:18:15,995 --> 04:18:18,931 I THINK EVERYTHING YOU THREW AT 5149 04:18:18,931 --> 04:18:20,166 US TODAY IS A SINGLE NUCLEOTIDE 5150 04:18:20,166 --> 04:18:23,235 CHANGE, YOU MUST THINK ABOUT 5151 04:18:23,235 --> 04:18:23,969 THAT. 5152 04:18:23,969 --> 04:18:25,838 >> MOST OF THESE RESISTANCE 5153 04:18:25,838 --> 04:18:28,374 MUTATIONS, VAST MAJORITY OF 5154 04:18:28,374 --> 04:18:30,209 THEM, SINGLE NUCLEOTIDE CHANGES. 5155 04:18:30,209 --> 04:18:32,745 THERE ARE A COUPLE DOUBLE, THAT 5156 04:18:32,745 --> 04:18:34,046 DOESN'T CORRELATE WELL WITH THE 5157 04:18:34,046 --> 04:18:34,313 TIMELINES. 5158 04:18:34,313 --> 04:18:35,348 >> WHY DO YOU THINK THAT? 5159 04:18:35,348 --> 04:18:36,782 YOU WOULD THINK IF YOU NEED 5160 04:18:36,782 --> 04:18:39,952 TWICE AS MANY CHANGES IN THE 5161 04:18:39,952 --> 04:18:42,054 TEMPLATE THAT WOULD AFFECT 5162 04:18:42,054 --> 04:18:42,688 KINETICS. 5163 04:18:42,688 --> 04:18:43,489 >> RIGHT. 5164 04:18:43,489 --> 04:18:45,157 I THINK IN TERMS OF THE 5165 04:18:45,157 --> 04:18:47,626 CORRELATION THERE'S SO FEW OF 5166 04:18:47,626 --> 04:18:48,828 THE DOUBLE NUCLEOTIDE CHANGES IN 5167 04:18:48,828 --> 04:18:50,696 TERMS OF THE PRIMARY RESISTANCE 5168 04:18:50,696 --> 04:18:52,898 MUTATION, SO WE CAN FIND 5169 04:18:52,898 --> 04:18:56,702 EXAMPLES OF A DOUBLE NUCLEOTIDE 5170 04:18:56,702 --> 04:19:00,272 CHAIN THAT'S SLOWERTHAN SINGLE 5171 04:19:00,272 --> 04:19:01,974 NUCLEOTIDE CHANGE. 5172 04:19:01,974 --> 04:19:03,809 >> ANY OTHER QUESTIONS FOR AVIK? 5173 04:19:03,809 --> 04:19:05,111 I HAVE A QUESTION. 5174 04:19:05,111 --> 04:19:11,817 HOW DO YOU TAKE INTO ACCOUNT 5175 04:19:11,817 --> 04:19:13,552 BIOFITNESS PRIMARY ISOLATE, THEY 5176 04:19:13,552 --> 04:19:15,521 REPLICATE FASTER THAN OTHER 5177 04:19:15,521 --> 04:19:18,391 VIRUSES, CONSTANTLY CHANGES, 5178 04:19:18,391 --> 04:19:20,893 PRIMARY ISOLATE, HARD TO PREDICT 5179 04:19:20,893 --> 04:19:22,528 IN THE ENVIRONMENT. 5180 04:19:22,528 --> 04:19:24,163 >> WHAT WE'RE DOING HERE -- 5181 04:19:24,163 --> 04:19:27,333 >> MICROPHONE PLEASE. 5182 04:19:27,333 --> 04:19:27,867 >> SORRY. 5183 04:19:27,867 --> 04:19:29,769 WE'RE ACTUALLY TAKING PATIENT 5184 04:19:29,769 --> 04:19:30,936 SAMPLES FROM DATABASES, BECAUSE 5185 04:19:30,936 --> 04:19:35,775 THERE'S A DIVERSITY OF PATIENTS, 5186 04:19:35,775 --> 04:19:37,076 WE'RE ESSENTIALLY ASSUMING WE'RE 5187 04:19:37,076 --> 04:19:40,312 ARRANGING OVER -- SORRY, 5188 04:19:40,312 --> 04:19:41,814 IMMUNOLOGIC ASPECTS OF HOW OF 5189 04:19:41,814 --> 04:19:45,518 VIRUS IS RESPONDING TO DIFFERENT 5190 04:19:45,518 --> 04:19:45,785 PATIENTS. 5191 04:19:45,785 --> 04:19:48,053 >> OKAY. 5192 04:19:48,053 --> 04:19:53,225 THANK YOU. 5193 04:19:53,225 --> 04:19:54,860 >> HI. 5194 04:19:54,860 --> 04:19:56,162 A FOLLOW-UP, MOST ARE SINGLE 5195 04:19:56,162 --> 04:19:59,799 NUCLEOTIDE CHANGE BUT DIFFERENT 5196 04:19:59,799 --> 04:20:01,400 MUTATIONS HAVE DIFFERENT RATES, 5197 04:20:01,400 --> 04:20:06,739 MUCH MORE RARE THAN TRANSITIONS 5198 04:20:06,739 --> 04:20:08,641 SO HAVE YOU COMPARED RATES OF 5199 04:20:08,641 --> 04:20:11,143 DIFFERENT CHANGES THAT MIGHT BE 5200 04:20:11,143 --> 04:20:11,944 REQUIRED? 5201 04:20:11,944 --> 04:20:17,249 >> YEAH, LOOKED AT TRANSITION 5202 04:20:17,249 --> 04:20:18,918 VERSUS TRANSVERSION, DOES THAT 5203 04:20:18,918 --> 04:20:20,419 EXPLAIN THE TIMELINES WE 5204 04:20:20,419 --> 04:20:23,489 OBSERVED BUT THAT DOESN'T. 5205 04:20:23,489 --> 04:20:24,056 >> OKAY. 5206 04:20:24,056 --> 04:20:24,824 THANK YOU. 5207 04:20:24,824 --> 04:20:25,157 >> OKAY. 5208 04:20:25,157 --> 04:20:28,260 IF THERE ARE NO MORE QUESTIONS 5209 04:20:28,260 --> 04:20:29,995 FOR AVIK LET'S GIVE HIM ANOTHER 5210 04:20:29,995 --> 04:20:30,563 APPLAUSE. 5211 04:20:30,563 --> 04:20:33,098 [APPLAUSE] 5212 04:20:33,098 --> 04:20:35,334 5213 04:20:35,334 --> 04:20:39,705 AND WE HAVE A VERY NICE LAPTOP. 5214 04:20:39,705 --> 04:20:41,373 IS IT YOURS? 5215 04:20:41,373 --> 04:20:45,544 I WAS JUST ABOUT TO AUCTION IT 5216 04:20:45,544 --> 04:20:46,512 OFF. 5217 04:20:46,512 --> 04:20:46,779 [LAUGHTER] 5218 04:20:46,779 --> 04:20:48,747 IN ANY CASE, THANKS TO ALL THE 5219 04:20:48,747 --> 04:20:49,582 PREVIOUS SPEAKERS. 5220 04:20:49,582 --> 04:20:51,116 IT WAS A GREAT SESSION. 5221 04:20:51,116 --> 04:20:53,853 AND WE'LL TAKE A BREAK NOW UNTIL 5222 04:20:53,853 --> 04:21:00,726 4:00 AND IF THE NEXT FOUR 5223 04:21:00,726 --> 04:21:01,927 SPEAKERS COULD JUST QUICKLY 5224 04:21:01,927 --> 04:21:03,762 CHECK IN WITH ME. 5225 04:21:03,762 --> 04:21:13,255 SEE YOU AT 4:00. 5226 04:21:13,255 --> 04:21:15,957 >> THIS IS THE SECOND PART OF 5227 04:21:15,957 --> 04:21:17,959 THE ABSTRACT SESSION TODAY. 5228 04:21:17,959 --> 04:21:25,267 FIRST SPEAKER IS HUNG DO FROM 5229 04:21:25,267 --> 04:21:29,104 LOS ALAMOS NATIONAL LABORATORY. 5230 04:21:29,104 --> 04:21:33,408 >> I'M AT LOS ALAMOS LABORATORY, 5231 04:21:33,408 --> 04:21:40,048 TALKING ABOUT DEVELOPMENT OF THE 5232 04:21:40,048 --> 04:21:42,984 WORKFLOW TO ACCELERATE 5233 04:21:42,984 --> 04:21:46,588 CONFIRMATION OF SAMPLING OF 5234 04:21:46,588 --> 04:21:47,689 BIOMOLECULAR MECHANICS, I'D LIKE 5235 04:21:47,689 --> 04:21:50,625 TO RECOGNIZE THE ORIGINAL PERSON 5236 04:21:50,625 --> 04:21:58,600 WHO DEVELOPED THE IDEA FOR THE 5237 04:21:58,600 --> 04:22:05,940 WORKFLOW, BEFORE I CAME. 5238 04:22:05,940 --> 04:22:08,643 FIRST HIV RELEVANT PROCESS 5239 04:22:08,643 --> 04:22:11,646 INVOLVED CONFIRMATIONAL CHANGE 5240 04:22:11,646 --> 04:22:14,082 OCCUR OVER MICROSECOND, HIV 5241 04:22:14,082 --> 04:22:16,951 VIRAL MEMBRANE HIGHLY COMPLEX, 5242 04:22:16,951 --> 04:22:18,987 CONSIST OF SEVERAL LIPID TYPE 5243 04:22:18,987 --> 04:22:24,492 WHICH WILL BE DISCUSSED IN 5244 04:22:24,492 --> 04:22:29,864 POSTER, 78 AND 89, COLORFUL 5245 04:22:29,864 --> 04:22:30,332 MEMBRANE. 5246 04:22:30,332 --> 04:22:34,469 WE SHOW THE DIFFERENT STATE 5247 04:22:34,469 --> 04:22:37,539 IMMUNOGEN IMLID COMPOSITION AND 5248 04:22:37,539 --> 04:22:43,978 PROSECUTES ITTY DISCUSSED IN 5249 04:22:43,978 --> 04:22:45,113 ABSTRACTS. 5250 04:22:45,113 --> 04:22:50,218 CAME UP WITH THE IDEA TO COMBINE 5251 04:22:50,218 --> 04:22:52,654 CO-STIMULATION, WHICH ARE FAST, 5252 04:22:52,654 --> 04:22:53,755 MANY CONFIRMATIONAL TRANSITION 5253 04:22:53,755 --> 04:22:56,624 DUE TO FLAT UNDER SURFACE, 5254 04:22:56,624 --> 04:23:05,934 THERE'S NO BARRIER RIGHT HERE SO 5255 04:23:05,934 --> 04:23:08,136 CAN SAMPLE ACROSS LANDSCAPE, LOW 5256 04:23:08,136 --> 04:23:11,873 BUT PROVIDES DETAIL DUE TO 5257 04:23:11,873 --> 04:23:12,741 COMPLEX SURFACE, MANY LOW 5258 04:23:12,741 --> 04:23:16,111 ENERGY, MANY HIGH ENERGY 5259 04:23:16,111 --> 04:23:17,679 BARRIER, TRANSITION FROM ONE 5260 04:23:17,679 --> 04:23:21,483 STATE TO ANOTHER IS VERY SLOW. 5261 04:23:21,483 --> 04:23:25,653 THAT'S WHY THE ITERATED 5262 04:23:25,653 --> 04:23:31,226 MULTI-SCALE WORKFLOW IS 5263 04:23:31,226 --> 04:23:35,063 POWERFUL, IN PARTICULAR FOR 5264 04:23:35,063 --> 04:23:39,801 MODELING MEMBRANE PROTEIN CAN 5265 04:23:39,801 --> 04:23:45,140 USE CLOSE RANGE, TO GET INTO THE 5266 04:23:45,140 --> 04:23:50,945 CORRECT POSITION, AROUND 5267 04:23:50,945 --> 04:23:52,580 MEMBRANE PROTEIN. 5268 04:23:52,580 --> 04:23:57,719 ONE BIG CHALLENGE REMAINED, 5269 04:23:57,719 --> 04:23:59,020 MULTI-SCALE CONSISTENCY, NO 5270 04:23:59,020 --> 04:24:01,423 RELATIONSHIP BETWEEN THE 5271 04:24:01,423 --> 04:24:04,225 FUNCTIONS HOW HOW CAN WE COMBINE 5272 04:24:04,225 --> 04:24:05,660 THE TWO ACCURATELY, ONE QUESTION 5273 04:24:05,660 --> 04:24:08,163 THAT REMAINS TO THINK ABOUT. 5274 04:24:08,163 --> 04:24:11,433 I WILL TALK ABOUT OUR CURRENT 5275 04:24:11,433 --> 04:24:12,767 IMPLEMENTATION OF THE 5276 04:24:12,767 --> 04:24:15,270 MULTI-SCALE WORKFLOW IN THE 5277 04:24:15,270 --> 04:24:17,439 PACKAGE, ALONG WITH SOME 5278 04:24:17,439 --> 04:24:23,244 INTEREST AND CHALLENGE FROM 5279 04:24:23,244 --> 04:24:30,051 APPLYING TO SIMILAR. 5280 04:24:30,051 --> 04:24:31,252 FOR THE SIMULATION, CYCLOPHILIN, 5281 04:24:31,252 --> 04:24:34,556 IF YOU'RE INTERESTED COME TO OUR 5282 04:24:34,556 --> 04:24:39,627 POSTER 77 AND WE CAN DISCUSS 5283 04:24:39,627 --> 04:24:49,904 MORE TOMORROW. 5284 04:24:53,374 --> 04:24:54,709 LET'S TALK ABOUT METHODOLOGY, 5285 04:24:54,709 --> 04:24:58,613 YOU DON'T NEED TO SET UP THE 5286 04:24:58,613 --> 04:24:59,614 SIMULATION SYSTEM BEFOREHAND. 5287 04:24:59,614 --> 04:25:01,115 WORKFLOW SET UP THE SIMULATION 5288 04:25:01,115 --> 04:25:11,659 SYSTEM FOR YOU AND RUN THE FIRST 5289 04:25:12,594 --> 04:25:18,233 ATOM STIMULATION, CONVERT USING 5290 04:25:18,233 --> 04:25:23,104 MARTINIZE, WE CAN RUN THE FINAL 5291 04:25:23,104 --> 04:25:29,310 CONFIRMATION OF THE 5292 04:25:29,310 --> 04:25:32,413 COARSE-GRAINED, CONVERT BACK 5293 04:25:32,413 --> 04:25:36,050 USING M TOOL. 5294 04:25:36,050 --> 04:25:42,323 THEN BEFORE AND ATOM CYCLE TO 5295 04:25:42,323 --> 04:25:44,792 CONTINUE MULTI-SCALE SIMULATION 5296 04:25:44,792 --> 04:25:49,797 WORKFLOW. 5297 04:25:49,797 --> 04:25:50,498 WE DEMONSTRATED THE MULTI-SCALE 5298 04:25:50,498 --> 04:26:00,375 APPROACH BY SHOWING THAT IT CAN 5299 04:26:00,375 --> 04:26:07,682 CAPTURE, TWO MICROSECOND, AND 5300 04:26:07,682 --> 04:26:11,653 ONE WITH 15 NANOSECOND COMPARED 5301 04:26:11,653 --> 04:26:14,656 TO THE REQUIRED 14 MICROSECOND 5302 04:26:14,656 --> 04:26:16,324 TIME WITH PURE CONVENTIONAL. 5303 04:26:16,324 --> 04:26:26,801 YOU CAN SEE WE STARTED FROM 5304 04:26:27,769 --> 04:26:30,638 FOLDED STATE, TWO ITER RATION 5305 04:26:30,638 --> 04:26:32,740 WITH OBTAIN, OBSERVED FROM THE 5306 04:26:32,740 --> 04:26:33,374 PLOT RIGHT HERE. 5307 04:26:33,374 --> 04:26:38,846 AT THE END OF THE FIRST 5308 04:26:38,846 --> 04:26:40,515 SIMULATION PROTEIN IS STUCK, 5309 04:26:40,515 --> 04:26:43,351 RIGHT HERE, WITH SOME HELICAL 5310 04:26:43,351 --> 04:26:45,119 SECONDARY STRUCTURE ALREADY 5311 04:26:45,119 --> 04:26:50,892 FORM, STUCK RIGHT HERE, NO WAY 5312 04:26:50,892 --> 04:27:00,201 TO TRANSITION, THAT'S WHY WE GO 5313 04:27:00,201 --> 04:27:05,707 BACK, BACK PROTEIN CONFIRMATION, 5314 04:27:05,707 --> 04:27:08,743 TO FOLDED STATE, VERY STABLE IN 5315 04:27:08,743 --> 04:27:11,713 THE SECOND ITERATION. 5316 04:27:11,713 --> 04:27:14,148 WE ALSO OBSERVED MOLECULAR 5317 04:27:14,148 --> 04:27:17,952 DIFFUSE THREE TO FIVE TIME 5318 04:27:17,952 --> 04:27:28,363 FASTER COMPARED FROM THE 5319 04:27:28,696 --> 04:27:31,599 STIMULATION, BASED ON THE 5320 04:27:31,599 --> 04:27:33,568 COEFFICIENT, FROM THE 5321 04:27:33,568 --> 04:27:35,837 SIMULATION, THE BAR FOR THE 5322 04:27:35,837 --> 04:27:38,640 DIFFUSION CO-F OF THE MOLECULAR 5323 04:27:38,640 --> 04:27:41,409 TWO TO THREE TIME HIGHER THAN 5324 04:27:41,409 --> 04:27:46,414 THE BAR FOR THE SIMULATION SO 5325 04:27:46,414 --> 04:27:48,349 SUBMIT THE ORIGINAL DEVELOPMENT 5326 04:27:48,349 --> 04:27:48,883 PURPOSE. 5327 04:27:48,883 --> 04:27:51,853 BUT ONE PROBLEM, ONE CHALLENGE 5328 04:27:51,853 --> 04:27:52,420 REMAIN. 5329 04:27:52,420 --> 04:27:59,127 WE OBSERVED SECONDARY STRUCTURE 5330 04:27:59,127 --> 04:28:05,233 OF SOLUBLE REGION OF PROTEIN, 5331 04:28:05,233 --> 04:28:08,636 WHEN WE PERFORMED SIMULATION, 5332 04:28:08,636 --> 04:28:11,572 RIGHT HERE HAD SIP LAYINGS OF 5333 04:28:11,572 --> 04:28:22,050 RECEPTOR, WHEN WE START HAVE 5334 04:28:23,618 --> 04:28:24,085 CLEAR SECONDARY STRUCTURE, 5335 04:28:24,085 --> 04:28:32,593 SECONDARY STRUCTURE OF THE 5336 04:28:32,593 --> 04:28:34,562 REGION BECOME COMPLETELY 5337 04:28:34,562 --> 04:28:36,097 DISTORTED. 5338 04:28:36,097 --> 04:28:38,700 WHEN WE PERFORM SIMULATION AND 5339 04:28:38,700 --> 04:28:41,235 RESTRAINED THE POSITION, THE 5340 04:28:41,235 --> 04:28:51,713 PROTEIN SOLUBLE DOMAIN STILL 5341 04:28:52,113 --> 04:28:57,385 BECAME DISTOWARDED. 5342 04:28:57,385 --> 04:28:59,487 RIGHT HERE 400 NANOSECOND, STILL 5343 04:28:59,487 --> 04:29:00,822 RETAIN STRUCTURE IN THE 5344 04:29:00,822 --> 04:29:01,355 SOLUTION. 5345 04:29:01,355 --> 04:29:11,833 WHAT COULD BE THE CHALLENGE 5346 04:29:17,905 --> 04:29:19,006 HERE? 5347 04:29:19,006 --> 04:29:22,744 THE THIS INVOLVED MINIMIZATION, 5348 04:29:22,744 --> 04:29:24,479 HE SOLVED THIS CHALLENGE. 5349 04:29:24,479 --> 04:29:26,247 BECAUSE THE BIG PROBLEM WE 5350 04:29:26,247 --> 04:29:30,184 ALREADY MENTIONED IN THE FIRST 5351 04:29:30,184 --> 04:29:32,019 SLIDE BASICALLY THE FUNCTION OF 5352 04:29:32,019 --> 04:29:35,790 THE CLOSE RANGE REPRESENTATION 5353 04:29:35,790 --> 04:29:39,393 HAVE NO RELATIONSHIP WHATSOEVER, 5354 04:29:39,393 --> 04:29:43,865 THE OPTIMAL FOR THE PROTEIN ATOM 5355 04:29:43,865 --> 04:29:46,601 ARE DIFFERENT FROM OPTIMAL FOR 5356 04:29:46,601 --> 04:29:50,772 THOSE AT CLOSE RANGE 5357 04:29:50,772 --> 04:30:01,249 STIMULATION, VICE VERSA, THE 5358 04:30:03,384 --> 04:30:06,154 CURRENT PROTOCOLS BACK MAP, THE 5359 04:30:06,154 --> 04:30:07,155 DISTORTION BECOME MINOR BUT IF 5360 04:30:07,155 --> 04:30:10,224 YOU RUN THE CYCLE OVER MULTIPLE 5361 04:30:10,224 --> 04:30:11,993 TIME THEN THE DISTORTION BECOME 5362 04:30:11,993 --> 04:30:14,061 ACCUMULATED AND EVENTUALLY IT 5363 04:30:14,061 --> 04:30:15,163 BECOME VERY OBVIOUS. 5364 04:30:15,163 --> 04:30:16,497 SO AS YOU CAN SEE FROM THIS 5365 04:30:16,497 --> 04:30:21,369 FIGURE RIGHT HERE FOR SIMULATION 5366 04:30:21,369 --> 04:30:27,108 OF THE FINAL CONFIRMATION OF THE 5367 04:30:27,108 --> 04:30:29,477 FIRST CLOSE RANGE SIMULATION 5368 04:30:29,477 --> 04:30:35,383 LOOKS HIGHLY SIMILAR BUT WHEN WE 5369 04:30:35,383 --> 04:30:41,088 BACK MAP, WE GO BACK FROM FINAL 5370 04:30:41,088 --> 04:30:45,893 CG1 TO AA2 OBSERVE DISPORTION IN 5371 04:30:45,893 --> 04:30:49,297 THE BACKBONE, CIRCLE IN RED 5372 04:30:49,297 --> 04:30:50,832 RIGHT HERE. 5373 04:30:50,832 --> 04:30:56,204 RIGHT NOW WE DON'T HAVE A BACK 5374 04:30:56,204 --> 04:31:01,609 MAPPING PROTOCOL, SO ONE 5375 04:31:01,609 --> 04:31:04,979 PROPOSED SOLUTION WE BACK MAP 5376 04:31:04,979 --> 04:31:09,817 FROM CLOSE RANGE STIMULATION TO 5377 04:31:09,817 --> 04:31:11,319 AND COMBINE WITH ORIGINAL 5378 04:31:11,319 --> 04:31:13,387 CONFIRMATION BEFORE CONVERSION 5379 04:31:13,387 --> 04:31:18,759 TO CLOSE RANGE AND PROCEED TO 5380 04:31:18,759 --> 04:31:19,861 ATOMISTIC ITERATION, RETAIN 5381 04:31:19,861 --> 04:31:22,363 SECONDARY STRUCTURE OF THE 5382 04:31:22,363 --> 04:31:22,630 PROTEIN. 5383 04:31:22,630 --> 04:31:25,333 SO WITH THAT, THE CURRENT 5384 04:31:25,333 --> 04:31:27,168 MULTI-SCALE IMPLEMENTATION FOR 5385 04:31:27,168 --> 04:31:28,636 THE MEMBRANE PROTEIN SIMULATION 5386 04:31:28,636 --> 04:31:32,440 WE ONLY USE THE ATOMISTIC TO 5387 04:31:32,440 --> 04:31:34,308 SAMPLE PROTEIN INTERACTION AND 5388 04:31:34,308 --> 04:31:37,044 USE CLOSE CHANGE TO FACILITATE 5389 04:31:37,044 --> 04:31:39,247 LIPID MIXING. 5390 04:31:39,247 --> 04:31:44,585 SO WITH THAT I WANT TO -- WE TO 5391 04:31:44,585 --> 04:31:46,888 DEVELOP ITERATED MULTI-SCALE 5392 04:31:46,888 --> 04:31:52,159 WORK FLOW COMBINING CLOSE RANGE 5393 04:31:52,159 --> 04:32:01,802 BUT SIMULATION MUCH FASTER, AND 5394 04:32:01,802 --> 04:32:05,139 WE HAD DEMONSTRATED ENHANCE THE 5395 04:32:05,139 --> 04:32:09,010 SAMPLING CAPABILITY OF WORKFLOW 5396 04:32:09,010 --> 04:32:19,553 ON FACILITATING LIPID DIFFUSION, 5397 04:32:19,787 --> 04:32:23,991 METHODOLOGY USED IN UNCOVERING 5398 04:32:23,991 --> 04:32:25,760 ANTIGEN-DEPENDENT ACTIVATION 5399 04:32:25,760 --> 04:32:27,762 MECHANISM, IN GNANA'S TALK 5400 04:32:27,762 --> 04:32:28,229 TOMORROW. 5401 04:32:28,229 --> 04:32:29,897 FIRST QUESTION IS MULTI-KALE 5402 04:32:29,897 --> 04:32:34,936 CONSISTENCY, HOW CAN WE COMBINE 5403 04:32:34,936 --> 04:32:40,641 THESE TWO VERY DIFFERENT 5404 04:32:40,641 --> 04:32:41,575 RESOLUTION, WITH NO 5405 04:32:41,575 --> 04:32:43,077 RELATIONSHIP, AND BECAUSE OF THE 5406 04:32:43,077 --> 04:32:44,912 FIRST CHALLENGE WE HAD SECOND 5407 04:32:44,912 --> 04:32:48,382 CHALLENGE BECAUSE OF THE CURRENT 5408 04:32:48,382 --> 04:32:50,151 BACK MAPPING PROTOCOL USED SOME 5409 04:32:50,151 --> 04:32:54,388 FIRM SO HOW CAN WE BACKMAP 5410 04:32:54,388 --> 04:32:56,824 ACCURATELY, MAYBE IN THE FUTURE 5411 04:32:56,824 --> 04:33:00,461 WE'LL NEED A BACKMAPPING 5412 04:33:00,461 --> 04:33:02,196 PROTOCOL THAT DON'T USE 5413 04:33:02,196 --> 04:33:03,664 MINIMIZATION, WITH THAT I WANT 5414 04:33:03,664 --> 04:33:05,800 TO THANK OUR FUNDING SOURCE, SO 5415 04:33:05,800 --> 04:33:10,104 DUKE CENTER FOR HIV STRUCTURAL 5416 04:33:10,104 --> 04:33:14,575 BIOLOGY, NIH, LOS ALAMOS 5417 04:33:14,575 --> 04:33:15,476 NATIONAL LABORATORY, DR. KRISHNU 5418 04:33:15,476 --> 04:33:24,452 ANDER WON -- AND EVERYONE IN OR 5419 04:33:24,452 --> 04:33:26,520 GROUP -- DR. CHRIS NEALE AND 5420 04:33:26,520 --> 04:33:27,288 EVERYONE IN OUR GROUP. 5421 04:33:27,288 --> 04:33:27,722 [APPLAUSE] 5422 04:33:27,722 --> 04:33:29,590 >> THANK YOU. 5423 04:33:29,590 --> 04:33:34,495 ANY QUESTIONS FOR HUNG? 5424 04:33:34,495 --> 04:33:35,229 DON'T BE SHY. 5425 04:33:35,229 --> 04:33:37,031 THEN WE'LL MOVE TO THE NEXT 5426 04:33:37,031 --> 04:33:37,565 SPEAKER. 5427 04:33:37,565 --> 04:33:39,100 THANK YOU VERY MUCH, HUNG. 5428 04:33:39,100 --> 04:33:46,307 LET'S GIVE HIM ANOTHER ROUND OF 5429 04:33:46,307 --> 04:33:46,707 APPLAUSE. 5430 04:33:46,707 --> 04:33:47,074 [APPLAUSE] 5431 04:33:47,074 --> 04:33:51,345 WE'LL MOVE TO THE NEXT SPEAKER, 5432 04:33:51,345 --> 04:33:53,347 SHI-JIE CHEN, UNIVERSITY OF 5433 04:33:53,347 --> 04:34:01,188 MISSOURI. 5434 04:34:01,188 --> 04:34:11,232 5435 04:34:30,818 --> 04:34:31,652 >> YOU'LL GET MORE MINUTES IF 5436 04:34:31,652 --> 04:34:38,492 YOU SPEAK IN THE MICROPHONE. 5437 04:34:38,492 --> 04:34:39,260 [LAUGHTER] 5438 04:34:39,260 --> 04:34:42,563 >> I'M GOING TO TALK ABOUT OUR 5439 04:34:42,563 --> 04:34:43,631 RECENT WORK IN COLLABORATION 5440 04:34:43,631 --> 04:34:48,569 WITH THE LAB ON THE KINETIC 5441 04:34:48,569 --> 04:34:55,242 PATHWAYS OF HIV TAR RNA. 5442 04:34:55,242 --> 04:34:57,178 I'M FROM UNIVERSITY OF MISSOURI. 5443 04:34:57,178 --> 04:34:59,313 THIS SLIDE SHOWS TWO DIMENSIONAL 5444 04:34:59,313 --> 04:35:01,248 STRUCTURE OF THE FIVE PRIME UTR, 5445 04:35:01,248 --> 04:35:03,317 I DON'T THINK I HAVE TO EXPLAIN 5446 04:35:03,317 --> 04:35:03,984 WHAT THIS IS. 5447 04:35:03,984 --> 04:35:06,954 I FEEL LIKE IF I TALK TOO MUCH 5448 04:35:06,954 --> 04:35:10,157 ABOUT THIS I'M LIKE PREACHING OF 5449 04:35:10,157 --> 04:35:14,628 GOD, TODAY I'M GOING TO FOCUS ON 5450 04:35:14,628 --> 04:35:23,704 PAR, THIS SHOWS NATIVE 5451 04:35:23,704 --> 04:35:28,509 STRUCTURE, RNA FOLDING IS 5452 04:35:28,509 --> 04:35:29,310 CO-TRANSCRIPTIONAL. 5453 04:35:29,310 --> 04:35:33,147 LET ME START WITH EXPERIMENTAL 5454 04:35:33,147 --> 04:35:33,547 RESULTS. 5455 04:35:33,547 --> 04:35:39,053 THIS SLIDE, THIS FIGURE SHOWS 5456 04:35:39,053 --> 04:35:41,355 THE CLASSICAL EXPERIMENTAL DATA 5457 04:35:41,355 --> 04:35:49,029 FROM ROB'S LAB, AND FROM THIS WE 5458 04:35:49,029 --> 04:35:59,507 CAN SEE ELONGase, THERE'S A 5459 04:35:59,807 --> 04:36:08,182 TRANSCRIPTION PATH HERE, THE 5460 04:36:08,182 --> 04:36:09,383 PATH OCCURS. 5461 04:36:09,383 --> 04:36:13,854 IN THE POLYMERASE THESE 18 5462 04:36:13,854 --> 04:36:15,256 NUCLEOSIDES THEY DON'T 5463 04:36:15,256 --> 04:36:18,759 PARTICIPATE IN FOLDING. 5464 04:36:18,759 --> 04:36:23,297 WITH THIS 44 NUCLEOTIDES WE HAVE 5465 04:36:23,297 --> 04:36:25,966 NATIVE STRUCTURE, THESE APPEARS 5466 04:36:25,966 --> 04:36:34,341 IN THE NATIVE FINAL FOUR TAR 5467 04:36:34,341 --> 04:36:35,409 STRUCTURE. 5468 04:36:35,409 --> 04:36:35,943 LANDIC'S EXPERIMENT, THREE 5469 04:36:35,943 --> 04:36:36,310 CONCLUSIONS. 5470 04:36:36,310 --> 04:36:37,811 FIRST, YOU HAVE THE PATH HERE, 5471 04:36:37,811 --> 04:36:42,116 WE SEE THAT. 5472 04:36:42,116 --> 04:36:45,486 SECOND, THE PATH IS INDUCED WE 5473 04:36:45,486 --> 04:36:49,223 THE OTHER STRUCTURE, P4 5474 04:36:49,223 --> 04:36:50,357 STRUCTURE. 5475 04:36:50,357 --> 04:36:51,759 IT'S OUR NOTATIONS, P4 5476 04:36:51,759 --> 04:36:52,026 STRUCTURE. 5477 04:36:52,026 --> 04:36:55,696 THIS STRUCTURE IS A MISFOLDED 5478 04:36:55,696 --> 04:36:58,299 STRUCTURE, MEANING BASE PAIRS 5479 04:36:58,299 --> 04:37:00,167 ARE COMPLETELY DIFFERENT FROM 5480 04:37:00,167 --> 04:37:02,336 NATIVE PATH. 5481 04:37:02,336 --> 04:37:06,707 IT'S MISFOLDED. 5482 04:37:06,707 --> 04:37:14,181 ONCE THIS IS FORMED, THEY OCCUR. 5483 04:37:14,181 --> 04:37:16,517 70% GETS PASSED, 30% THROUGH. 5484 04:37:16,517 --> 04:37:21,288 THAT'S 70 VERSUS 30 THIS RATIO. 5485 04:37:21,288 --> 04:37:23,057 ANOTHER NUMBER IS 22 SECONDS, 5486 04:37:23,057 --> 04:37:27,962 PAUSE FOR 22 SECONDS. 5487 04:37:27,962 --> 04:37:32,600 WE WANT TO UNDERSTAND WHY 70/30, 5488 04:37:32,600 --> 04:37:35,402 WHY 22 SECONDS, AND WHAT IS 5489 04:37:35,402 --> 04:37:39,039 EXACTLY HAPPENED WHEN THE TAR IS 5490 04:37:39,039 --> 04:37:43,444 PASSED FROM STRUCTURE POINT OF 5491 04:37:43,444 --> 04:37:43,811 VIEW. 5492 04:37:43,811 --> 04:37:44,845 CO-TRANSCRIPTION FOLDING IS 5493 04:37:44,845 --> 04:37:48,916 DIFFERENT FROM REFOLDING. 5494 04:37:48,916 --> 04:37:53,621 WE HOLDING I MEAN YOU GET A VERY 5495 04:37:53,621 --> 04:37:57,491 DIFFERENT FOLDING BECAUSE IT'S A 5496 04:37:57,491 --> 04:37:59,426 PROCESS, COMPETITION BETWEEN 5497 04:37:59,426 --> 04:38:02,796 THREE RATE PROCESSES, ONE IS 5498 04:38:02,796 --> 04:38:05,332 TRANSCRIPTION RATE, ELONGATION 5499 04:38:05,332 --> 04:38:05,532 RATE. 5500 04:38:05,532 --> 04:38:07,434 SECOND PROCESS IS FOLDING, THIRD 5501 04:38:07,434 --> 04:38:10,404 PROCESS IS UNFOLDING, IS IT 5502 04:38:10,404 --> 04:38:13,374 COMPETITION, SO LET ME QUICKLY 5503 04:38:13,374 --> 04:38:18,445 GO THROUGH THIS SLIDE, 5504 04:38:18,445 --> 04:38:25,419 SUPPOSEDLY YOU HAVE TWO HAIRPIN 5505 04:38:25,419 --> 04:38:35,596 STRUCTURE, THE HELICES, LOOMS. 5506 04:38:35,596 --> 04:38:39,533 THE RNA WILL QUICKLY FORM THIS 5507 04:38:39,533 --> 04:38:49,143 LOCAL HAIRPIN STRUCTURE, IT'S 5508 04:38:49,143 --> 04:38:50,811 FAST, DOWN HERE. 5509 04:38:50,811 --> 04:38:52,680 THIS IS FORM BY BASE PAIRING OF 5510 04:38:52,680 --> 04:38:53,347 GREEN STRANDS. 5511 04:38:53,347 --> 04:38:57,351 IN ORDER TO FORM THIS STRUCTURE 5512 04:38:57,351 --> 04:39:03,390 YOU HAVE TO BREAK DISRUPT, 5513 04:39:03,390 --> 04:39:04,191 THAT'S SLOW. 5514 04:39:04,191 --> 04:39:05,359 ELONGATION KEEPS GOING. 5515 04:39:05,359 --> 04:39:09,196 VERY LIKELY YOU FORM THIS 5516 04:39:09,196 --> 04:39:13,233 STRUCTURE INSTEAD OF 5517 04:39:13,233 --> 04:39:14,234 THERMODYNAMIC STABLE STRUCTURE, 5518 04:39:14,234 --> 04:39:16,537 DIFFERENCE BETWEEN 5519 04:39:16,537 --> 04:39:20,274 CO-TRANSCRIPTION OF FOLDING AND 5520 04:39:20,274 --> 04:39:21,008 REFOLDING. 5521 04:39:21,008 --> 04:39:23,410 IN OUR MODEL, I'LL QUICKLY GO 5522 04:39:23,410 --> 04:39:24,945 THROUGH THE KEY POINT OF THE 5523 04:39:24,945 --> 04:39:25,412 MODEL. 5524 04:39:25,412 --> 04:39:27,348 I WON'T GO THROUGH DETAILS. 5525 04:39:27,348 --> 04:39:30,317 IN OUR MODEL WE WANT TO USE 5526 04:39:30,317 --> 04:39:31,618 COMPUTATIONAL MODEL TO 5527 04:39:31,618 --> 04:39:33,354 UNDERSTAND WHAT'S GOING ON FROM 5528 04:39:33,354 --> 04:39:35,155 STRUCTURE POINT OF VIEW, WANT TO 5529 04:39:35,155 --> 04:39:36,957 UNDERSTAND THE PATHWAYS. 5530 04:39:36,957 --> 04:39:42,129 SO IN OUR MODEL WE MODEL THE 5531 04:39:42,129 --> 04:39:47,368 TRANSCRIPTION LIKE MOVIE FRAME, 5532 04:39:47,368 --> 04:39:48,469 EACH FRAME RNA MOLECULE 5533 04:39:48,469 --> 04:39:51,538 STRUCTURE EVOLVE, KINETIC FROM 5534 04:39:51,538 --> 04:39:56,677 INITIAL TO FINAL STRUCTURE, 5535 04:39:56,677 --> 04:39:59,513 ELONGATE BY ONE UNIT, TIME 5536 04:39:59,513 --> 04:40:00,714 DURATION DETERMINED BY THE 5537 04:40:00,714 --> 04:40:02,683 TRANSCRIPTION SPEED. 5538 04:40:02,683 --> 04:40:04,685 OUR MODEL CAN MODEL THE 5539 04:40:04,685 --> 04:40:08,822 VARIATION OF THE TRANSCRIPTION 5540 04:40:08,822 --> 04:40:11,125 SPEED, AND ANOTHER IMPORTANT 5541 04:40:11,125 --> 04:40:16,263 POINT CO-TRANSCRIPTION FOLDING 5542 04:40:16,263 --> 04:40:17,431 IS DIFFERENT FROM REFOLDING, 5543 04:40:17,431 --> 04:40:19,867 SOMETIMES PEOPLE LIKE TO DO 5544 04:40:19,867 --> 04:40:22,269 FOLDING AT DIFFERENT THAN MAKE 5545 04:40:22,269 --> 04:40:27,841 CONNECTION TOGETHER, YOU THINK 5546 04:40:27,841 --> 04:40:29,276 THIS IS PATHWAY BUT THAT'S NOT 5547 04:40:29,276 --> 04:40:30,244 ALWAYS THE CASE. 5548 04:40:30,244 --> 04:40:32,212 WE HAVE TO BE CAREFUL. 5549 04:40:32,212 --> 04:40:35,449 ANOTHER COMPONENT IS FOLDING 5550 04:40:35,449 --> 04:40:36,383 MODEL, STRUCTURE PREDICTION 5551 04:40:36,383 --> 04:40:36,817 MODEL. 5552 04:40:36,817 --> 04:40:45,459 SO IN OUR MOLDING MODEL WE 5553 04:40:45,459 --> 04:40:52,266 DEVELOPED A SIMULATION, AND IN 5554 04:40:52,266 --> 04:40:56,570 OUR MODEL WE USE COARSE-GRAINED, 5555 04:40:56,570 --> 04:40:59,306 TWO DIMENSIONAL COMPARING THE 5556 04:40:59,306 --> 04:41:03,710 MODEL SIMULATE MOLECULE, THIS 5557 04:41:03,710 --> 04:41:05,546 ONE IS 412 NUCLEOTIDES, CAN GET 5558 04:41:05,546 --> 04:41:10,484 THE FINAL STRUCTURE, IT'S NOT 5559 04:41:10,484 --> 04:41:12,853 PERFECT, IT'S 17-ANGSTROM BUT I 5560 04:41:12,853 --> 04:41:16,824 JUST WANT TO SHOW THE 5561 04:41:16,824 --> 04:41:21,061 SIMULATION, WE CAN GET DOWN TO 5562 04:41:21,061 --> 04:41:31,572 THE 10-ANGSTROM SO IT WORK. 5563 04:41:32,873 --> 04:41:35,843 HERE IS MY LAB, FIRST ONE IS 5564 04:41:35,843 --> 04:41:37,044 FROM COMMERCIAL COMPANY, 5565 04:41:37,044 --> 04:41:38,579 INTERNATIONAL COMPANY. 5566 04:41:38,579 --> 04:41:42,316 SO LET'S COME BACK TO OUR REAL 5567 04:41:42,316 --> 04:41:45,419 TOPIC TODAY, THAT'S THE TAR 5568 04:41:45,419 --> 04:41:45,886 FOLDING. 5569 04:41:45,886 --> 04:41:48,222 SO WE FOUND WITH OUR MODEL WE 5570 04:41:48,222 --> 04:41:51,558 FOUND THAT WE HAVE THE TAR FOLDS 5571 04:41:51,558 --> 04:41:54,895 AROUND TWO PARALLEL PATHWAYS BUT 5572 04:41:54,895 --> 04:41:56,864 THIS SLIDE SHOWS IT'S TOO BUSY, 5573 04:41:56,864 --> 04:42:01,101 SO TO HELP THE VISUAL INSPECTION 5574 04:42:01,101 --> 04:42:04,905 I DRAW TWO LINES, RED AND BLUE, 5575 04:42:04,905 --> 04:42:06,306 TWO PATHWAYS, FIRST PATHWAY IS 5576 04:42:06,306 --> 04:42:10,010 BLUE ONE. 5577 04:42:10,010 --> 04:42:14,882 BASE PAIRS FOR THE NATIVE BASE 5578 04:42:14,882 --> 04:42:17,784 PAIRS, NATIVE STRUCTURES, WE CAN 5579 04:42:17,784 --> 04:42:19,653 ALSO GET THREE DIMENSIONAL 5580 04:42:19,653 --> 04:42:25,259 STRUCTURES AND THIS MOVIE SHOWS 5581 04:42:25,259 --> 04:42:28,762 HOW THE STRUCTURE EVOLVED, AS 5582 04:42:28,762 --> 04:42:29,930 TRANSCRIPTION PROCEEDS. 5583 04:42:29,930 --> 04:42:31,899 AND LET ME SKIP THIS SLIDE. 5584 04:42:31,899 --> 04:42:34,668 I DON'T HAVE TIME TO FINISH ALL 5585 04:42:34,668 --> 04:42:36,169 THESE MOVIES. 5586 04:42:36,169 --> 04:42:38,672 AND THE OTHER PATHWAY, THE OTHER 5587 04:42:38,672 --> 04:42:43,977 PATHWAY WE CALL THE P PATH, P 5588 04:42:43,977 --> 04:42:45,145 MEANS PATH. 5589 04:42:45,145 --> 04:42:49,216 THAT'S THE CULPRIT TO CAUSE THE 5590 04:42:49,216 --> 04:42:49,449 PATH. 5591 04:42:49,449 --> 04:42:51,151 THIS STRUCTURE ALONG THIS 5592 04:42:51,151 --> 04:42:53,987 PATHWAY CAN ALSO GET A MOVIE FOR 5593 04:42:53,987 --> 04:42:57,357 THE THREE DIMENSIONAL STRUCTURES 5594 04:42:57,357 --> 04:43:02,062 AND THIS STRUCTURE P4 CAUSES 5595 04:43:02,062 --> 04:43:04,565 PATH, NEW PATH, WHAT'S GOING TO 5596 04:43:04,565 --> 04:43:05,132 HAPPEN? 5597 04:43:05,132 --> 04:43:09,069 SO WE ALSO MODEL WHEN THERE'S NO 5598 04:43:09,069 --> 04:43:14,241 PATH, P4 WILL KEEP GOING AS IT 5599 04:43:14,241 --> 04:43:16,977 ELONGATES, WILL KEEP GOING, IT 5600 04:43:16,977 --> 04:43:21,014 WILL FORM P5, P 6, P7, FINALLY 5601 04:43:21,014 --> 04:43:22,316 GO TO TAR. 5602 04:43:22,316 --> 04:43:25,385 THE PROCESS IS SUPER SLOW, VERY 5603 04:43:25,385 --> 04:43:26,253 SLOW. 5604 04:43:26,253 --> 04:43:31,391 WHEN THIS TAR IS FORMED 5605 04:43:31,391 --> 04:43:33,360 ELONGATION TRANSCRIPTION GOES TO 5606 04:43:33,360 --> 04:43:37,764 PBS OR FURTHER DOWNSTREAM. 5607 04:43:37,764 --> 04:43:41,268 WHEN THIS NATIVE TAR FORMED 5608 04:43:41,268 --> 04:43:45,372 PROTEIN BONDS THE TAR, TO 5609 04:43:45,372 --> 04:43:46,840 ACCELERATE TRANSCRIPTION THAT 5610 04:43:46,840 --> 04:43:49,042 MIGHT OCCUR, IF THAT OCCUR, THAT 5611 04:43:49,042 --> 04:43:51,044 WILL BE TOO LATE. 5612 04:43:51,044 --> 04:43:53,647 TIME IS TOO LATE. 5613 04:43:53,647 --> 04:43:57,918 SO THAT WILL LEAD TO THE 5614 04:43:57,918 --> 04:43:58,785 DETRIMENTAL CONSEQUENCES BECAUSE 5615 04:43:58,785 --> 04:44:02,956 FROM HERE TO DOWNSTREAM TO PBS 5616 04:44:02,956 --> 04:44:04,691 SLOW TRANSCRIPTION, YOU GET ALL 5617 04:44:04,691 --> 04:44:08,328 SCREWED UPU GET MULTIPLE 5618 04:44:08,328 --> 04:44:14,034 SPLICING, FOR EXAMPLE. 5619 04:44:14,034 --> 04:44:16,570 AND THE PATH TRIGGERS REROUTING 5620 04:44:16,570 --> 04:44:19,840 BECAUSE IT GET STUCK HERE, P4, 5621 04:44:19,840 --> 04:44:23,210 NO ELONGATION, IT GOES TO M4, A 5622 04:44:23,210 --> 04:44:25,178 NATIVE TAR. 5623 04:44:25,178 --> 04:44:30,984 ONCE IT FOLDS TO N4, TAR PROTEIN 5624 04:44:30,984 --> 04:44:33,153 BIND, IT GET ACCELERATED REAL 5625 04:44:33,153 --> 04:44:33,687 SLOW. 5626 04:44:33,687 --> 04:44:36,323 AND SO THE FIRST QUESTION WE 5627 04:44:36,323 --> 04:44:39,826 WANT TO ANSWER IS WHY 70-30? 5628 04:44:39,826 --> 04:44:44,164 THIS FIGURE SHOWS OUR MODEL 5629 04:44:44,164 --> 04:44:45,932 PREDICTED POPULATIONS OF EACH 5630 04:44:45,932 --> 04:44:52,339 PATHWAY, YOU CAN SEE AT 44 THE 5631 04:44:52,339 --> 04:44:53,340 PATHOCURSE, THE POPULATION 5632 04:44:53,340 --> 04:44:55,742 BETWEEN P AND N IS AROUND 80 AND 5633 04:44:55,742 --> 04:44:56,710 20. 5634 04:44:56,710 --> 04:44:59,913 IT'S VERY CLOSE TO 70 AND 30. 5635 04:44:59,913 --> 04:45:03,684 SO HAPPY ABOUT THE RESULT. 5636 04:45:03,684 --> 04:45:08,288 AND ANOTHER THING IS THAT WE 5637 04:45:08,288 --> 04:45:10,123 WANT TO UNDERSTAND WHY 22 5638 04:45:10,123 --> 04:45:11,024 SECONDS? 5639 04:45:11,024 --> 04:45:13,293 AS I EXPLAINED WHEN P 4 GOES TO 5640 04:45:13,293 --> 04:45:18,198 N 4 THE PATH GET RELEASED SO WE 5641 04:45:18,198 --> 04:45:19,032 CALL PAUSE-ESCAPE. 5642 04:45:19,032 --> 04:45:20,067 WHY 22 SECONDS? 5643 04:45:20,067 --> 04:45:27,741 TO ANSWER THAT QUESTION WE NEED 5644 04:45:27,741 --> 04:45:32,312 TO SEE HOW P4 GOES TO N4? 5645 04:45:32,312 --> 04:45:34,448 FIRST UNFOLD THIS, UNWIND THE 5646 04:45:34,448 --> 04:45:36,683 HELIX HERE. 5647 04:45:36,683 --> 04:45:38,552 AND THEN THIS SEQUENCE, THIS 5648 04:45:38,552 --> 04:45:40,854 CHAIN, THIS TAIL FOLD BACK TO 5649 04:45:40,854 --> 04:45:49,062 HERE TO THE HAIRPIN LOOP, FORM 5650 04:45:49,062 --> 04:45:49,529 PSEUDOKNOT. 5651 04:45:49,529 --> 04:45:53,900 THEY FORM BASE PAIR TO FORM 5652 04:45:53,900 --> 04:45:57,838 ANOTHER, AND UCU, PERFECT. 5653 04:45:57,838 --> 04:46:02,409 THEN KEEP GOING. 5654 04:46:02,409 --> 04:46:09,082 THIS PSEUDOKNOT PATHWAY IS 5655 04:46:09,082 --> 04:46:09,516 EXCITING. 5656 04:46:09,516 --> 04:46:12,686 AND DR. SHAW HAINES LAB DID AN 5657 04:46:12,686 --> 04:46:14,554 EXPERIMENT TO CONFIRM THESE TWO 5658 04:46:14,554 --> 04:46:18,425 BASE PAIRS, THESE TWO SETS OF 5659 04:46:18,425 --> 04:46:23,430 BASE PAIRS WHICH INITIATED 5660 04:46:23,430 --> 04:46:27,134 PSEUDOKNOT FORMATION ARE LESS 5661 04:46:27,134 --> 04:46:28,769 STABLE SHOWN FROM NMR, YOU'RE 5662 04:46:28,769 --> 04:46:31,438 EXPERTS SO I WON'T EXPLAIN 5663 04:46:31,438 --> 04:46:34,040 DETAILS. 5664 04:46:34,040 --> 04:46:36,910 BASED ON THIS PSEUDOKNOT FOLDING 5665 04:46:36,910 --> 04:46:38,078 MODEL WE DID CALCULATION, HERE 5666 04:46:38,078 --> 04:46:41,081 IS OUR RESULT. 5667 04:46:41,081 --> 04:46:43,450 SYMBOLS ARE FROM EXPERIMENT, 5668 04:46:43,450 --> 04:46:47,287 THIS LINE FROM OUR CALCULATION. 5669 04:46:47,287 --> 04:46:51,391 YOU SEE THAT AGREEMENT IS GOOD. 5670 04:46:51,391 --> 04:46:54,961 WE GOT 18 SECONDS, VERY CLOSE. 5671 04:46:54,961 --> 04:46:56,062 VERY CLOSE. 5672 04:46:56,062 --> 04:46:59,800 SO, WE'RE QUITE HAPPY ABOUT THIS 5673 04:46:59,800 --> 04:47:00,400 RESULT. 5674 04:47:00,400 --> 04:47:06,339 AND SO FINALLY I WANT TO SHOW 5675 04:47:06,339 --> 04:47:08,575 THAT THIS IS PSEUDOKNOT. 5676 04:47:08,575 --> 04:47:11,711 WE MIGHT BE ABLE TO FIND A NEW 5677 04:47:11,711 --> 04:47:13,980 DRUG TARGET HERE BECAUSE 5678 04:47:13,980 --> 04:47:16,283 PSEUDOKNOT MIGHT SERVE AS DRUG 5679 04:47:16,283 --> 04:47:22,622 TARGET AND THIS MOVIE SHOWS 5680 04:47:22,622 --> 04:47:24,724 FOLDING P4 TO N4. 5681 04:47:24,724 --> 04:47:28,795 IF WE DESIGN A DRUG MOLECULE 5682 04:47:28,795 --> 04:47:31,765 THAT CAN TARGET THE HAIRPIN, FOR 5683 04:47:31,765 --> 04:47:35,635 EXAMPLE, ANYWHERE TO PREVENT THE 5684 04:47:35,635 --> 04:47:37,637 FORMATION OF PSEUDONOT THEN 5685 04:47:37,637 --> 04:47:40,040 PROBABLY WE CAN STOP THIS 5686 04:47:40,040 --> 04:47:41,341 PATHWAY. 5687 04:47:41,341 --> 04:47:44,511 SEE, THIS IS FOR ILLUSTRATION, 5688 04:47:44,511 --> 04:47:49,049 IT'S NOT A REAL CALCULATION. 5689 04:47:49,049 --> 04:47:51,952 AND THEN PSEUDOKNOT CANNOT FORM. 5690 04:47:51,952 --> 04:47:56,690 SO THAT'S -- WE'RE VERY EXCITED 5691 04:47:56,690 --> 04:48:01,761 THIS WORK WAS JUST -- WAIT, 5692 04:48:01,761 --> 04:48:06,266 WHAT -- 5693 04:48:06,266 --> 04:48:06,800 >> RANDY? 5694 04:48:06,800 --> 04:48:08,668 THERE'S A PROBLEM. 5695 04:48:08,668 --> 04:48:11,938 YOU'RE GETTING CLOSE TO TIME 5696 04:48:11,938 --> 04:48:12,172 ANYWAY. 5697 04:48:12,172 --> 04:48:16,309 [LAUGHTER] 5698 04:48:16,309 --> 04:48:16,676 >> MAGICALLY. 5699 04:48:16,676 --> 04:48:18,044 >> ONE MORE MINUTE. 5700 04:48:18,044 --> 04:48:19,045 >> OKAY, YEAH. 5701 04:48:19,045 --> 04:48:19,946 I'M DONE. 5702 04:48:19,946 --> 04:48:23,884 SO THIS WORK WAS JUST PUBLISHED 5703 04:48:23,884 --> 04:48:25,352 A MONTH AGO. 5704 04:48:25,352 --> 04:48:28,455 AND WE'RE ALSO WORKING ON TRY TO 5705 04:48:28,455 --> 04:48:33,693 USING THIS AS PARADIGM TO STUDY 5706 04:48:33,693 --> 04:48:41,568 THE CO-TRANSFER FOLDING OF FIVE 5707 04:48:41,568 --> 04:48:45,005 PRIME REGION WITH COLLABORATION 5708 04:48:45,005 --> 04:48:48,909 WITH XIAO AND KATHLEEN'S LAB. 5709 04:48:48,909 --> 04:48:52,312 LAST SLIDE, I WANT TO THANK MY 5710 04:48:52,312 --> 04:48:58,251 GROUP AND STUDENT AND FOUR 5711 04:48:58,251 --> 04:49:00,353 HIGHLY TALENTED POSTDOC, XIAO 5712 04:49:00,353 --> 04:49:03,490 AND KATHLEEN, I JOINED THE CRNA 5713 04:49:03,490 --> 04:49:08,194 THREE MONTH, I ALREADY 5714 04:49:08,194 --> 04:49:10,697 ESTABLISHED VERY EXCITING 5715 04:49:10,697 --> 04:49:11,564 COLLABORATIONS WITH KWAKU AND 5716 04:49:11,564 --> 04:49:13,366 VICKIE AND STEVE AND NATE AND 5717 04:49:13,366 --> 04:49:14,868 MANY OTHER PEOPLE. 5718 04:49:14,868 --> 04:49:21,341 I THANK NIH FOR THE MONEY. 5719 04:49:21,341 --> 04:49:22,742 THANK YOU. 5720 04:49:22,742 --> 04:49:23,543 [APPLAUSE] 5721 04:49:23,543 --> 04:49:26,579 >> WE HAVE TIME FOR ONE QUICK 5722 04:49:26,579 --> 04:49:30,317 QUESTION. 5723 04:49:30,317 --> 04:49:33,253 5724 04:49:33,253 --> 04:49:37,524 5725 04:49:37,524 --> 04:49:41,728 AND ONE MINUTE. 5726 04:49:41,728 --> 04:49:44,764 >> HOW DO YOU BACKMAP CLOSE 5727 04:49:44,764 --> 04:49:49,269 RANGE REPRESENTATION BACK TO THE 5728 04:49:49,269 --> 04:49:49,669 REPRESENTATION? 5729 04:49:49,669 --> 04:49:51,838 GOOD QUESTION. 5730 04:49:51,838 --> 04:49:56,576 WE FIND MAPPING FROM RANDOM 5731 04:49:56,576 --> 04:49:57,677 STRUCTURE TO ALL-ATOM STRUCTURE. 5732 04:49:57,677 --> 04:50:05,418 >> ONE MORE ROUND OF APPLAUSE. 5733 04:50:05,418 --> 04:50:06,286 [APPLAUSE] 5734 04:50:06,286 --> 04:50:11,658 >> NEXT SPEAKER IS FUMIAKI ITO, 5735 04:50:11,658 --> 04:50:14,194 UNIVERSITY OF CALIFORNIA LOS 5736 04:50:14,194 --> 04:50:21,201 ANGELES. 5737 04:50:21,201 --> 04:50:24,704 >> ALL RIGHT. 5738 04:50:24,704 --> 04:50:31,378 I'M A POSTDOC, BETWEEN UCLA AND 5739 04:50:31,378 --> 04:50:36,850 USC. 5740 04:50:36,850 --> 04:50:40,053 WE'VE BEEN STUDYING APOBEC FOR 5741 04:50:40,053 --> 04:50:43,723 NINE YEARS, EXCITED TO SHARE 5742 04:50:43,723 --> 04:50:45,825 FROM THE COMPLEX TODAY. 5743 04:50:45,825 --> 04:50:51,398 DURING HIV INFECTION IN HUMAN T 5744 04:50:51,398 --> 04:50:56,770 CELL APOBEC 3 FACTORS ARE 5745 04:50:56,770 --> 04:50:58,071 EXPRESSED PACKAGED, THE PROCESS 5746 04:50:58,071 --> 04:51:01,941 IS KNOWN TO BE DEPENDENT ON RNA 5747 04:51:01,941 --> 04:51:07,680 BINDING WHICH CAN BE FROM EITHER 5748 04:51:07,680 --> 04:51:15,121 VIRUS OR HOST. 5749 04:51:15,121 --> 04:51:16,322 ALL RIGHT. 5750 04:51:16,322 --> 04:51:19,959 UPON HIV REACHING THE PROTEINS 5751 04:51:19,959 --> 04:51:24,431 TARGET THE NEGATIVE STRAND 5752 04:51:24,431 --> 04:51:26,299 cDNA FOR MUTAGENIC REACTION, AS 5753 04:51:26,299 --> 04:51:30,570 A RESULT WE SEE G TO A 5754 04:51:30,570 --> 04:51:32,439 HYPERMUTATION ON INTEGRATED 5755 04:51:32,439 --> 04:51:37,143 GENOME. 5756 04:51:37,143 --> 04:51:40,346 THIS IS ESSENTIALLY HOW APOBEC 3 5757 04:51:40,346 --> 04:51:42,415 PROTEIN RESTRICT REPLICATION. 5758 04:51:42,415 --> 04:51:50,523 HOWEVER HIV HAS EVOLVED 5759 04:51:50,523 --> 04:51:53,259 ACCESSORY PROTEIN SPECIFICALLY 5760 04:51:53,259 --> 04:51:54,661 BY APOBECS. 5761 04:51:54,661 --> 04:52:02,335 HIJACKS HOST FACTORS SUCH AS CGF 5762 04:52:02,335 --> 04:52:09,909 BETA, TO POLY UBIQUINATE 5763 04:52:09,909 --> 04:52:15,181 APOBECS, DEGRADED IN 5764 04:52:15,181 --> 04:52:15,482 PROTEASOME. 5765 04:52:15,482 --> 04:52:16,716 THIS IS CLASSIC EXAMPLE, WE'VE 5766 04:52:16,716 --> 04:52:22,288 BEEN INTERESTED IN THIS PROTEIN 5767 04:52:22,288 --> 04:52:25,492 COMPLEX ASSEMBLY ESPECIALLY THIS 5768 04:52:25,492 --> 04:52:27,460 APOBEC INTERFACE. 5769 04:52:27,460 --> 04:52:29,429 IT'S BEEN QUITE CHALLENGING TO 5770 04:52:29,429 --> 04:52:30,964 CHARACTERIZE THIS COMPLEX BUT IN 5771 04:52:30,964 --> 04:52:32,532 RECENT YEARS THERE'S BEEN 5772 04:52:32,532 --> 04:52:40,073 PROGRESS IN THE FIELD AS THE 5773 04:52:40,073 --> 04:52:41,908 FIRST APOBEC 3 COMPLEX, BY THE 5774 04:52:41,908 --> 04:52:47,747 LAB OVER HERE, MORE RECENTLY 5775 04:52:47,747 --> 04:52:51,651 THIS COMPLEX IN THREE 5776 04:52:51,651 --> 04:52:52,552 INDEPENDENTY STUDIES BY JOHN'S 5777 04:52:52,552 --> 04:52:58,324 LAB AND BY OUR LAB. 5778 04:52:58,324 --> 04:53:02,595 THE SURPRISE IN THE INTERACTION 5779 04:53:02,595 --> 04:53:07,367 WAS THAT RNA STRAND MEDIATING 5780 04:53:07,367 --> 04:53:13,373 THE A3G INTERACTION HERE, IN ALL 5781 04:53:13,373 --> 04:53:14,674 THREE STUDIES THIS SINGLE STRAND 5782 04:53:14,674 --> 04:53:21,347 RNA IS IN THE SPACE FORMED 5783 04:53:21,347 --> 04:53:21,581 BETWEEN. 5784 04:53:21,581 --> 04:53:24,317 AT THIS POINT THE COMPLEX 5785 04:53:24,317 --> 04:53:26,619 REMAINED UNRESOLVED, WE WERE 5786 04:53:26,619 --> 04:53:28,855 WONDERING IF THIS TYPE OF RNA 5787 04:53:28,855 --> 04:53:34,928 MEDIATED INTERACTION CAN BE 5788 04:53:34,928 --> 04:53:35,128 SEEN. 5789 04:53:35,128 --> 04:53:40,033 WITH THAT IN MIND WE 5790 04:53:40,033 --> 04:53:43,670 RECONSTITUTED THIS COMPLEX IN 5791 04:53:43,670 --> 04:53:46,339 VITRO, PREVIOUS STUDIES HAVE 5792 04:53:46,339 --> 04:53:48,808 SHOWN THAT FROM DIFFERENT HIV 5793 04:53:48,808 --> 04:53:55,481 STRANDS HAVE DIFFERENT EXTENT OF 5794 04:53:55,481 --> 04:53:59,686 ANTAGONISM AGAINST THIS. 5795 04:53:59,686 --> 04:54:06,492 FOR EXAMPLE, LAI STRAND DEBATES 5796 04:54:06,492 --> 04:54:08,795 A-3H MORE EFFECTIVELY, 5797 04:54:08,795 --> 04:54:09,896 DIFFERENCE ATTRIBUTED TO SINGLE 5798 04:54:09,896 --> 04:54:19,606 AMINO ACID ACID POLYMORPHISM AT 5799 04:54:19,606 --> 04:54:25,345 POSITION 48. 5800 04:54:25,345 --> 04:54:28,414 WE CONFIRMED N 48 MUTANT REDUCED 5801 04:54:28,414 --> 04:54:33,219 LEVEL OF A3H MORE EFFECTIVELY. 5802 04:54:33,219 --> 04:54:37,457 AND FOR A3H THERE WAS A STUDY 5803 04:54:37,457 --> 04:54:38,992 DEMONSTRATING HOMOLOG FROM 5804 04:54:38,992 --> 04:54:42,695 CHIMPANZEE IS MORE SENSITIVE TO 5805 04:54:42,695 --> 04:54:46,199 THIS HIV MEDIATED DEGRADATION. 5806 04:54:46,199 --> 04:54:49,369 AND THEY ALSO PINPOINTED THAT 5807 04:54:49,369 --> 04:54:51,037 AMINO ACID AT 97 DETERMINED 5808 04:54:51,037 --> 04:54:54,374 SENSITIVITY TO HIV IF MEDIATED 5809 04:54:54,374 --> 04:54:55,108 WITH DEGRADATION. 5810 04:54:55,108 --> 04:54:58,177 AND THIS RESULT WAS HIGHLY 5811 04:54:58,177 --> 04:55:02,348 REPRODUCIBLE AS YOU CAN SEE 5812 04:55:02,348 --> 04:55:02,749 HERE. 5813 04:55:02,749 --> 04:55:08,454 K9 7K MUTANT OF HUMAN A-3H 5814 04:55:08,454 --> 04:55:12,825 DEGRADED -- SORRY, THIS WAS MORE 5815 04:55:12,825 --> 04:55:17,297 SENSITIVE, MUTANT BECAME MORE 5816 04:55:17,297 --> 04:55:17,664 SENSITIVE. 5817 04:55:17,664 --> 04:55:20,400 AND A3H NEVER WAS COMPLETELY 5818 04:55:20,400 --> 04:55:25,638 DEPLETED WHEN MUTANT WAS 5819 04:55:25,638 --> 04:55:26,306 COMBINED. 5820 04:55:26,306 --> 04:55:28,374 SO THIS STRAND WAS BASICALLY THE 5821 04:55:28,374 --> 04:55:29,909 SAME IN OUR PURIFIED SYSTEM. 5822 04:55:29,909 --> 04:55:38,217 AS YOU CAN SEE THE MUTANT K9 7Q 5823 04:55:38,217 --> 04:55:42,388 AND N48H, RESULTED IN MOST 5824 04:55:42,388 --> 04:55:46,426 PREDOMINANT PEAK SHIFT IN THE 5825 04:55:46,426 --> 04:55:47,327 PROFILE. 5826 04:55:47,327 --> 04:55:51,597 WE TOOK THIS COMPLEX PEAK FOR 5827 04:55:51,597 --> 04:55:52,198 CryoEM. 5828 04:55:52,198 --> 04:55:52,632 OKAY. 5829 04:55:52,632 --> 04:55:58,137 THIS IS OUR MAIN MAP SHOWING THE 5830 04:55:58,137 --> 04:56:05,244 A-3H VIF AND CDF BETA, AS 5831 04:56:05,244 --> 04:56:13,353 EXPECTED A3H IS BOUND TO 5832 04:56:13,353 --> 04:56:20,993 DOUBLE-STRANDED RNA, AND WE 5833 04:56:20,993 --> 04:56:22,562 OBSERVED -- LARGE INTERFACE, THE 5834 04:56:22,562 --> 04:56:23,963 INTERACTION IS EXCLUSIVELY 5835 04:56:23,963 --> 04:56:26,466 PROTEIN BASED, BASICALLY THERE'S 5836 04:56:26,466 --> 04:56:28,434 NO RNA INVOLVED IN THIS 5837 04:56:28,434 --> 04:56:37,009 INTERFACE WHICH IS DIFFERENT. 5838 04:56:37,009 --> 04:56:39,846 A3H VIF INTERACTION IS MAIN LIP 5839 04:56:39,846 --> 04:56:41,814 MEDIATED BY THE ALPHA HELICAL 5840 04:56:41,814 --> 04:56:45,518 SIDE OF A3H, AND FIVE-STRANDED 5841 04:56:45,518 --> 04:56:51,991 BETA SHEET OF VIF, AND MORE 5842 04:56:51,991 --> 04:56:53,426 SPECIFICALLY A3H RESIDUES ON 3 5843 04:56:53,426 --> 04:56:59,565 AND 4 ARE INTERACTING WITH THE 5844 04:56:59,565 --> 04:57:04,570 RESIDUES AND CONNECTING LOOP. 5845 04:57:04,570 --> 04:57:08,341 AND NOTABLY AT 90 ESSENTIAL TO 5846 04:57:08,341 --> 04:57:08,808 INTERFACE. 5847 04:57:08,808 --> 04:57:10,176 ALL RIGHT. 5848 04:57:10,176 --> 04:57:12,912 THEN WE VALIDATED THIS INTERFACE 5849 04:57:12,912 --> 04:57:15,648 BY USING THE SAME DEGRADATION 5850 04:57:15,648 --> 04:57:17,083 ASSAY. 5851 04:57:17,083 --> 04:57:20,186 AND AS YOU CAN SEE POINTING 5852 04:57:20,186 --> 04:57:23,389 MUTATION ON ALPHA 3 RESIDUES, 5853 04:57:23,389 --> 04:57:25,491 ALPHA 4 RESIDUES INCREASED THE 5854 04:57:25,491 --> 04:57:31,831 RESISTANCE TO VIF. 5855 04:57:31,831 --> 04:57:38,938 AND INDEED MUTATION ALONG 90 5856 04:57:38,938 --> 04:57:41,007 RENDERED FULLY RESISTANT TO VIF 5857 04:57:41,007 --> 04:57:42,775 MEDIATED DEGRADATION. 5858 04:57:42,775 --> 04:57:50,750 SO OUR STRUCTURE SHOWS THAT THIS 5859 04:57:50,750 --> 04:57:51,684 SENSITIVITY MODULATING RESIDUE 5860 04:57:51,684 --> 04:57:59,792 AT 97 WOULD BE ACTUALLY 5861 04:57:59,792 --> 04:58:01,194 ELECTROSTATICALLY REPULSIVE WITH 5862 04:58:01,194 --> 04:58:01,461 THESE. 5863 04:58:01,461 --> 04:58:06,466 WE THINK THIS IS PARTIALLY WHY. 5864 04:58:06,466 --> 04:58:12,004 NATIVE PAIR IS SUBOPTIMAL IN 5865 04:58:12,004 --> 04:58:18,077 FORMING STABLE COMPLEX. 5866 04:58:18,077 --> 04:58:22,915 AND PREVIOUSLY VIF VARIANT FROM 5867 04:58:22,915 --> 04:58:27,687 LACK ADAPTATION STUDY AND ALSO 5868 04:58:27,687 --> 04:58:31,557 HIV ISOLATES FROM THE 5869 04:58:31,557 --> 04:58:32,625 PARTICIPANTS SHOWS THAT THERE'S 5870 04:58:32,625 --> 04:58:36,662 GAIN OF FUNCTION MUTATION ON THE 5871 04:58:36,662 --> 04:58:39,632 VIF WHICH IS K63E, OUR STRUCTURE 5872 04:58:39,632 --> 04:58:42,168 SHOWS THAT GLUTAMATE AT THIS 5873 04:58:42,168 --> 04:58:43,569 POSITION IS ELECTROSTATICALLY 5874 04:58:43,569 --> 04:58:52,245 MORE FAVORED WITH THE WILDTYPE 5875 04:58:52,245 --> 04:58:54,547 RESIDUE K97. 5876 04:58:54,547 --> 04:58:56,916 BASED ON COLLECTIVE EFFORTS, WE 5877 04:58:56,916 --> 04:58:59,785 NOW HAVE MORE COMPREHENSIVE 5878 04:58:59,785 --> 04:59:01,354 UNDERSTANDING OF VIF SURFACE 5879 04:59:01,354 --> 04:59:06,325 AREA THAT BINDS TO DIFFERENT 5880 04:59:06,325 --> 04:59:06,993 APOBECs. 5881 04:59:06,993 --> 04:59:14,000 A3H BINDING RESIDES IN 5882 04:59:14,000 --> 04:59:16,269 FIVE-STRANDED BETA SHEET, A3G 5883 04:59:16,269 --> 04:59:19,205 RESIDES AROUND THE CONNECTING 5884 04:59:19,205 --> 04:59:27,213 NERVE AND ALPHA HELIX ONE, THIS 5885 04:59:27,213 --> 04:59:28,481 HIGHLIGHTS WHICH USES DISTINCT 5886 04:59:28,481 --> 04:59:33,352 SURFACE AREA TO ANTAGONIZE 5887 04:59:33,352 --> 04:59:35,855 DIFFERENT APOBECS. 5888 04:59:35,855 --> 04:59:39,292 IN SUMMARY A-3H MODULATES THE 5889 04:59:39,292 --> 04:59:45,765 SENSITIVITY TO HIV VIF MEDIATED 5890 04:59:45,765 --> 04:59:47,199 DEGRADATION, ALSO A3H VIF 5891 04:59:47,199 --> 04:59:53,205 INTERACTION IS PROTEIN BASED 5892 04:59:53,205 --> 04:59:56,242 WITH NO INTERFACE. 5893 04:59:56,242 --> 05:00:06,752 ALPHA HELICAL BINDS IN VIF IS 5894 05:00:11,657 --> 05:00:13,225 DISTINCT WITH LIMITED OVERLAP. 5895 05:00:13,225 --> 05:00:17,597 WITH THAT, I WOULD LIKE TO THANK 5896 05:00:17,597 --> 05:00:25,237 OUR FANTASTIC TEAM FROM BOTH 5897 05:00:25,237 --> 05:00:26,005 LABS, AND ALSO COMPUTATIONAL 5898 05:00:26,005 --> 05:00:27,573 CENTER, OF COURSE FUNDING 5899 05:00:27,573 --> 05:00:27,940 SUPPORT. 5900 05:00:27,940 --> 05:00:28,207 THANK YOU. 5901 05:00:28,207 --> 05:00:33,346 [APPLAUSE] 5902 05:00:33,346 --> 05:00:38,017 5903 05:00:38,017 --> 05:00:41,220 5904 05:00:41,220 --> 05:00:42,455 >> ANY QUESTIONS FOR FUMIAKI? 5905 05:00:42,455 --> 05:00:43,289 I HAVE A QUESTION. 5906 05:00:43,289 --> 05:00:46,359 WHY DO YOU THINK IT'S SO 5907 05:00:46,359 --> 05:00:48,227 DIFFICULT TO DEVELOP INHIBITORS 5908 05:00:48,227 --> 05:00:57,069 OF THE VIF APOBEC PATHWAY, 5909 05:00:57,069 --> 05:00:58,371 PEOPLE HAVE TRIED HARD TO 5910 05:00:58,371 --> 05:00:59,939 INHIBIT, IT'S BEEN A LITTLE BIT 5911 05:00:59,939 --> 05:01:00,239 ABANDONED. 5912 05:01:00,239 --> 05:01:03,109 DO YOU HAVE ANY IDEAS? 5913 05:01:03,109 --> 05:01:05,511 >> RIGHT. 5914 05:01:05,511 --> 05:01:08,247 I THINK FOR A3G BECAUSE 5915 05:01:08,247 --> 05:01:11,217 INTERFACE IS NOT JUST PROTEIN 5916 05:01:11,217 --> 05:01:14,086 PROTEIN INTERACTION, IT'S RNAs 5917 05:01:14,086 --> 05:01:18,658 COMING INTO THE INTERFACE, IT'S 5918 05:01:18,658 --> 05:01:22,061 COMPLICATED, AND ACTUAL PROTEIN 5919 05:01:22,061 --> 05:01:24,964 PROTEIN INTERFACE FOR A3G IS 5920 05:01:24,964 --> 05:01:30,036 LIMITED, THREE OR FOUR AMINO 5921 05:01:30,036 --> 05:01:32,104 ACID, ALSO FOR A3H, YEAH, I 5922 05:01:32,104 --> 05:01:37,343 DON'T HAVE MUCH INSIGHT INTO A3H 5923 05:01:37,343 --> 05:01:40,546 INHIBITOR DEVELOPMENT BUT I 5924 05:01:40,546 --> 05:01:43,482 THINK A3H IS PLAYING A BACKUP 5925 05:01:43,482 --> 05:01:44,150 ROLE. 5926 05:01:44,150 --> 05:01:45,251 >> A MINOR ROLE. 5927 05:01:45,251 --> 05:01:47,753 >> PROBABLY MORE MINOR COMPARED 5928 05:01:47,753 --> 05:01:48,187 TO A3G. 5929 05:01:48,187 --> 05:01:50,389 >> MOST PEOPLE FOCUS ON A3G I 5930 05:01:50,389 --> 05:01:52,024 GUESS FOR DRUG DEVELOPMENT. 5931 05:01:52,024 --> 05:01:53,359 >> THAT'S RIGHT, YEAH. 5932 05:01:53,359 --> 05:01:55,461 >> OKAY, THAT MAKES SENSE. 5933 05:01:55,461 --> 05:01:56,195 THANK YOU. 5934 05:01:56,195 --> 05:02:01,867 ANY QUESTIONS IN THE MEANTIME? 5935 05:02:01,867 --> 05:02:05,805 IF NOT, LET'S GIVE ANOTHER ROUND 5936 05:02:05,805 --> 05:02:06,539 OF APPLAUSE TO FUMIAKI. 5937 05:02:06,539 --> 05:02:09,742 [APPLAUSE] 5938 05:02:09,742 --> 05:02:12,378 AND LAST BUT NOT LEAST SPEAKER 5939 05:02:12,378 --> 05:02:20,553 OF THE DAY KATE SKORUPKA FROM 5940 05:02:20,553 --> 05:02:21,420 NCI CANCER INNOVATION LABORATORY 5941 05:02:21,420 --> 05:02:31,630 AT THE NIH. 5942 05:03:02,795 --> 05:03:05,431 >> THANK YOU ALL FOR FREEZING 5943 05:03:05,431 --> 05:03:07,666 THIS LONG. 5944 05:03:07,666 --> 05:03:09,935 [LAUGHTER] 5945 05:03:09,935 --> 05:03:12,571 I'M A POSTDOC HERE AT NCI, AND I 5946 05:03:12,571 --> 05:03:19,545 WILL BE CONTINUING THE TOPIC OF 5947 05:03:19,545 --> 05:03:25,351 VIF-INDUCED YOUR BIC UBIQUITINT 5948 05:03:25,351 --> 05:03:29,555 A BRIEF OVERVIEW OF THE SYSTEM, 5949 05:03:29,555 --> 05:03:38,731 THE PROTEIN WE'RE WORKING ON, 5950 05:03:38,731 --> 05:03:45,504 THESE, AS FOR APOBEC 3H WE CHOSE 5951 05:03:45,504 --> 05:03:47,473 CHIMPANZEE, HERE WE HAVE 5952 05:03:47,473 --> 05:03:49,875 SEQUENCE ALIGNMENT, THE MAIN 5953 05:03:49,875 --> 05:03:52,411 DIFFERENCE AT POSITION 97, IN 5954 05:03:52,411 --> 05:03:54,180 CHIMP GLUTAMINE RESIDUE, THIS 5955 05:03:54,180 --> 05:04:02,488 MAKES IT MORE SENSITIVE TO 5956 05:04:02,488 --> 05:04:04,890 VIF-INDUCED UBIQUITINATION AND 5957 05:04:04,890 --> 05:04:05,624 DEGRADATION. 5958 05:04:05,624 --> 05:04:08,394 I'M SHOWING THE CryoEM 5959 05:04:08,394 --> 05:04:10,329 STRUCTURE WE SOLVED TO 3.9 5960 05:04:10,329 --> 05:04:10,629 RESOLUTION. 5961 05:04:10,629 --> 05:04:21,073 IN GREEN WE HAVE A3H, GOLD 5962 05:04:25,177 --> 05:04:26,879 STRAND RNA. 5963 05:04:26,879 --> 05:04:29,381 THIS IS DIMERIC STRUCTURE BUT WE 5964 05:04:29,381 --> 05:04:32,351 SEE A3H IS A DIMER THROUGH 5965 05:04:32,351 --> 05:04:34,253 DOUBLE-STRANDED RNA. 5966 05:04:34,253 --> 05:04:39,024 RESOLUTION IS ONLY 3.9 SO WE 5967 05:04:39,024 --> 05:04:41,861 DECIDED TO IMPOSE 2D SYMMETRY, 5968 05:04:41,861 --> 05:04:45,865 AND OUR RESULTING STRUCTURE IS 5969 05:04:45,865 --> 05:04:48,601 3.6 RESOLUTION, WITH BETTER 5970 05:04:48,601 --> 05:04:51,670 QUALITY, AND DOMAINS ARE COLOR 5971 05:04:51,670 --> 05:04:52,037 CODED. 5972 05:04:52,037 --> 05:04:56,041 AS I MENTIONED BECAUSE IT IS A 5973 05:04:56,041 --> 05:04:57,476 C2 SYMMETRY WE SEE WHO 5974 05:04:57,476 --> 05:04:59,411 DIFFERENT -- EXCUSE ME. 5975 05:04:59,411 --> 05:05:02,581 WE SEE WHO PROTOMERS, IN EACH 5976 05:05:02,581 --> 05:05:06,886 CASE THE PROTOMER IS BUILD FROM 5977 05:05:06,886 --> 05:05:11,924 DOUBLE-STRANDED RNA WITH A3H 5978 05:05:11,924 --> 05:05:15,728 THE INTERESTING PART OF OUR 5979 05:05:15,728 --> 05:05:20,065 STRUCTURE IS IT'S A DIMER 5980 05:05:20,065 --> 05:05:21,100 THROUGH VIF-VIF INTERACTION. 5981 05:05:21,100 --> 05:05:26,138 AMINO ACID AT THE INTERFACE MOST 5982 05:05:26,138 --> 05:05:36,615 ARE BULKY AROMATIC LIKE TRIP 5983 05:05:36,982 --> 05:05:37,583 FAN, HISTIDINE. 5984 05:05:37,583 --> 05:05:42,054 THESE PARTICIPATE IN BINDING 5985 05:05:42,054 --> 05:05:47,826 WITH A3G BUT WE'RE TALKING ABOUT 5986 05:05:47,826 --> 05:05:50,362 A3H, IN CONTACT WITH WHO VIF 5987 05:05:50,362 --> 05:05:52,898 MOLECULES, FIRST IS EXTENSIVE, 5988 05:05:52,898 --> 05:05:56,168 PROTEIN PROTEIN INTERACTION, 5989 05:05:56,168 --> 05:05:56,835 SECOND PROTEIN-PROTEIN 5990 05:05:56,835 --> 05:05:57,736 INTERACTING SURFACE IS MUCH 5991 05:05:57,736 --> 05:06:02,041 SMALLER AS MOLECULES ARE 5992 05:06:02,041 --> 05:06:05,778 POSITIONED FURTHER APART. 5993 05:06:05,778 --> 05:06:08,714 SO WE HEARD ABOUT THIS 5994 05:06:08,714 --> 05:06:09,148 INTERFACE. 5995 05:06:09,148 --> 05:06:11,383 WE SEE TRYPTOPHAN 90 AND 5996 05:06:11,383 --> 05:06:12,785 INTERACTION BETWEEN TWO PROTEINS 5997 05:06:12,785 --> 05:06:15,854 IS FOCUSED ON THIS ALPHA HELIX 3 5998 05:06:15,854 --> 05:06:21,360 AND 4 AND RESIDUES ON VIF ARE ON 5999 05:06:21,360 --> 05:06:22,728 THE SIDE. 6000 05:06:22,728 --> 05:06:24,830 THE SAME RESIDUES IMPORTANT ON 6001 05:06:24,830 --> 05:06:27,766 VIF HAVE BEEN DETERMINED 6002 05:06:27,766 --> 05:06:29,201 EXPERIMENTAL IN MUTAGENESIS 6003 05:06:29,201 --> 05:06:33,339 STUDIES, AND WE ALSO SEE THEM ON 6004 05:06:33,339 --> 05:06:34,707 OUR INTERFACE. 6005 05:06:34,707 --> 05:06:37,843 THE SECOND INTERFACE IS MUCH 6006 05:06:37,843 --> 05:06:38,077 SMALLER. 6007 05:06:38,077 --> 05:06:39,611 AND INTERACTION BETWEEN TWO 6008 05:06:39,611 --> 05:06:47,720 PROTEINS IS MOSTLY CONFINED TO 6009 05:06:47,720 --> 05:06:49,321 LYSINE-RICH LOOP. 6010 05:06:49,321 --> 05:06:52,358 WHAT'S INTERESTING IT SEEMS ONE 6011 05:06:52,358 --> 05:06:55,361 OF THE DOUBLE-STRANDED RNA 6012 05:06:55,361 --> 05:06:56,095 MEDIATED INTERACTION BETWEEN TWO 6013 05:06:56,095 --> 05:06:58,297 PROTEINS SO IF WE HAVE CLOSER 6014 05:06:58,297 --> 05:06:59,698 LOOK ON DOUBLE-STRANDED RNA HERE 6015 05:06:59,698 --> 05:07:01,800 ARE THE MAIN RESIDUES THAT ARE 6016 05:07:01,800 --> 05:07:04,236 IN CLOSE PROXIMITY TO ONE OF THE 6017 05:07:04,236 --> 05:07:05,170 STRANDS FROM DOUBLE-STRANDED 6018 05:07:05,170 --> 05:07:05,637 RNA. 6019 05:07:05,637 --> 05:07:11,143 AND I WOULD LIKE TO BRIEFLY TALK 6020 05:07:11,143 --> 05:07:11,877 ABOUT THE 116. 6021 05:07:11,877 --> 05:07:15,047 THIS IS A BEGINNING OF PPLP 6022 05:07:15,047 --> 05:07:18,550 MOTIF RIGHT IN HERE, AND PPLP 6023 05:07:18,550 --> 05:07:27,626 MOTIF HAS BEEN IMPLICATED IN 6024 05:07:27,626 --> 05:07:28,494 DIMERIZATION AND 6025 05:07:28,494 --> 05:07:30,662 OLIGOMERIZATION, ADDITIONALLY IN 6026 05:07:30,662 --> 05:07:34,833 PREVIOUS STRUCTURES OF VIF WITH 6027 05:07:34,833 --> 05:07:37,669 A3G THERE ARE NUCLEOTIDES THAT 6028 05:07:37,669 --> 05:07:41,073 BIND IN THIS PPLP BINDING POCKET 6029 05:07:41,073 --> 05:07:45,177 BECAUSE OF HOW WE PREPARE OUR 6030 05:07:45,177 --> 05:07:47,212 A3H, OUR DOUBLE-STRANDED RNA IS 6031 05:07:47,212 --> 05:07:49,882 EXTENSIVELY CHOPPED AND WE 6032 05:07:49,882 --> 05:07:52,584 DIDN'T DETECT ANY NUCLEOTIDES 6033 05:07:52,584 --> 05:07:54,219 INSIDE OF THIS POCKET. 6034 05:07:54,219 --> 05:07:58,023 AND NOW LET'S TALK ABOUT THE 6035 05:07:58,023 --> 05:07:58,390 UBIQUITINATION. 6036 05:07:58,390 --> 05:08:02,961 THIS IS PARTLY DONE IN 6037 05:08:02,961 --> 05:08:05,297 COLLABORATION WITH THE LAB, WE 6038 05:08:05,297 --> 05:08:10,803 PURIFIED THE COMPONENTS OF THE 6039 05:08:10,803 --> 05:08:14,473 SYSTEM, A3H HAS 11 RESIDUES THAT 6040 05:08:14,473 --> 05:08:18,243 CAN BE UBIQUINATED. 6041 05:08:18,243 --> 05:08:26,985 FROM THOSE 11 IN MASS 6042 05:08:26,985 --> 05:08:28,587 SPECTROMETRY ANALYSIS, MUTATED 6043 05:08:28,587 --> 05:08:32,691 THEM TO ARGININE, ACH IS 6044 05:08:32,691 --> 05:08:34,193 PROTECTED FROM THIS MEDIATED 6045 05:08:34,193 --> 05:08:35,194 UBIQUITINATION AND DEGRADATION. 6046 05:08:35,194 --> 05:08:37,729 WE REVERSE EACH ONE OF THE 6047 05:08:37,729 --> 05:08:39,264 MUTATIONS BACK TO THEIR ORIGINAL 6048 05:08:39,264 --> 05:08:39,998 LICENSE. 6049 05:08:39,998 --> 05:08:43,035 AND WE SEE THAT REVERSAL AT 6050 05:08:43,035 --> 05:08:47,039 POSITION 50 AND 51 IS ENOUGH TO 6051 05:08:47,039 --> 05:08:51,777 GRANT WILDTYPE LEVEL DEGRADATION 6052 05:08:51,777 --> 05:08:56,115 OF A3H. 6053 05:08:56,115 --> 05:08:58,650 THIS BRINGS TO WHETHER PROXIMAL 6054 05:08:58,650 --> 05:09:00,853 OR DISTAL IS UBIQUINATED. 6055 05:09:00,853 --> 05:09:02,821 TO ANSWER THIS QUESTION WE 6056 05:09:02,821 --> 05:09:06,992 DESIGNED AN EXPERIMENT WHERE WE 6057 05:09:06,992 --> 05:09:10,395 TRANSFECTED CELLS WITH TWO 6058 05:09:10,395 --> 05:09:11,263 WILDTYPE PLASMIDS OR MUTANT 6059 05:09:11,263 --> 05:09:16,368 PLASMIDS OR MIXTURE OF THE TWO. 6060 05:09:16,368 --> 05:09:19,338 IN PRESENCE OF THIS THE 6061 05:09:19,338 --> 05:09:20,172 WILDTYPE-WILDTYPE HETERODIMER 6062 05:09:20,172 --> 05:09:22,741 EACH ONE OF THE MOLECULES IS 6063 05:09:22,741 --> 05:09:24,343 DEGRADED TO SIMILAR LEVEL. 6064 05:09:24,343 --> 05:09:27,746 IN CASE OF THE HETERODIMER WITH 6065 05:09:27,746 --> 05:09:30,182 MUTANT THE WILDTYPE IS DEGRADED 6066 05:09:30,182 --> 05:09:32,885 BUT WE SEE ACCUMULATION OF W 90A 6067 05:09:32,885 --> 05:09:34,419 MUTANT INSIDE THE CELLS, THIS 6068 05:09:34,419 --> 05:09:38,323 STRONGLY SUGGESTS THAT ONLY THE 6069 05:09:38,323 --> 05:09:41,093 WILDTYPE PROTEIN IS UBIQUINATED 6070 05:09:41,093 --> 05:09:42,594 AND DEGRADED IN THE DEPENDENT 6071 05:09:42,594 --> 05:09:44,897 MANNER. 6072 05:09:44,897 --> 05:09:52,337 THIS BRINGS TO US A VERY GENERAL 6073 05:09:52,337 --> 05:09:59,378 MODEL WITH THE PRIME TO 6074 05:09:59,378 --> 05:10:00,279 UBIQUITIN. 6075 05:10:00,279 --> 05:10:02,314 ON THIS SIDE DOUBLE-STRANDED 6076 05:10:02,314 --> 05:10:02,948 DNA. 6077 05:10:02,948 --> 05:10:06,451 THE SECOND MOLECULE OF A3H FALLS 6078 05:10:06,451 --> 05:10:08,754 APART AT SOME LEVEL IN THE 6079 05:10:08,754 --> 05:10:10,289 TRANSFER, THAT'S ONE. 6080 05:10:10,289 --> 05:10:16,361 AND TWO IS HARD TO SEE, 6081 05:10:16,361 --> 05:10:21,133 LYSINE-RICH LOOP, PRIMARILY 6082 05:10:21,133 --> 05:10:23,802 ACCEPTORS OF THE UBIQUITIN, THE 6083 05:10:23,802 --> 05:10:26,171 TRANSFER IS FAVORABLE, THEY ARE 6084 05:10:26,171 --> 05:10:26,838 POSITIONED. 6085 05:10:26,838 --> 05:10:29,241 TO SUMMARIZE, IN OUR STRUCTURE 6086 05:10:29,241 --> 05:10:34,146 WE SEE THE VIF-VIF NOVEL 6087 05:10:34,146 --> 05:10:36,481 INTERACTION NOT SEEN BEFORE, 6088 05:10:36,481 --> 05:10:39,184 IDENTIFIED AMINO ACIDS THAT LIE 6089 05:10:39,184 --> 05:10:40,752 AT THE INTERACTION BETWEEN A3H 6090 05:10:40,752 --> 05:10:45,324 AND IF I HAVE MOLECULES. 6091 05:10:45,324 --> 05:10:47,292 WE ADDITIONALLY IDENTIFIED 6092 05:10:47,292 --> 05:10:47,726 INTERACTIONS BETWEEN 6093 05:10:47,726 --> 05:10:49,795 DOUBLE-STRANDED RNA AND VIF. 6094 05:10:49,795 --> 05:11:00,305 WE SHOW K 50 AND K 51 PRIMARY 6095 05:11:01,607 --> 05:11:05,010 ACCESSORS OF UBIQUITIN. 6096 05:11:05,010 --> 05:11:07,546 PROXIMAL A3H IS DEGRADED. 6097 05:11:07,546 --> 05:11:10,449 I WOULD LIKE TO ACKNOWLEDGE 6098 05:11:10,449 --> 05:11:11,350 THESE PEOPLE. 6099 05:11:11,350 --> 05:11:11,883 THANK YOU. 6100 05:11:11,883 --> 05:11:14,086 [APPLAUSE] 6101 05:11:14,086 --> 05:11:20,158 6102 05:11:20,158 --> 05:11:22,461 6103 05:11:22,461 --> 05:11:24,796 >> IF I UNDERSTOOD THE 6104 05:11:24,796 --> 05:11:26,131 DIFFERENCE FROM PREVIOUS SPEAKER 6105 05:11:26,131 --> 05:11:28,233 YOU'RE SEEING RNA AT THE 6106 05:11:28,233 --> 05:11:29,101 SECONDARY INTERACTION INTERFACE, 6107 05:11:29,101 --> 05:11:30,102 IS THAT CORRECT? 6108 05:11:30,102 --> 05:11:30,435 >> YES. 6109 05:11:30,435 --> 05:11:32,471 SO BECAUSE WE HAVE TWO 6110 05:11:32,471 --> 05:11:39,278 PROTOMERS, OUR INTERACTION 6111 05:11:39,278 --> 05:11:42,147 OCCURS THROUGH THE PROTOMER. 6112 05:11:42,147 --> 05:11:43,215 RNA 1 INTERACTS WITH VIF 2. 6113 05:11:43,215 --> 05:11:45,350 >> EVERYTHING IS MADE FROM 6114 05:11:45,350 --> 05:11:45,951 PURIFIED COMPONENTS INCLUDING 6115 05:11:45,951 --> 05:11:49,454 THE RNA, IS THAT RIGHT? 6116 05:11:49,454 --> 05:11:52,357 >> SO THE RNA PURIFIES WITH A3H. 6117 05:11:52,357 --> 05:11:55,294 WE DO NOT ADD IT AND CANNOT 6118 05:11:55,294 --> 05:11:56,928 REMOVE IT. 6119 05:11:56,928 --> 05:11:59,097 >> A3H MADE IN BACTERIA? 6120 05:11:59,097 --> 05:11:59,631 >> BACTERIA. 6121 05:11:59,631 --> 05:12:00,098 E. COLI. 6122 05:12:00,098 --> 05:12:08,173 >> OKAY. 6123 05:12:08,173 --> 05:12:10,275 >> I'M SORRY IF I MISSED THIS. 6124 05:12:10,275 --> 05:12:13,645 DID YOU TRY THIS IN THE CELL, IN 6125 05:12:13,645 --> 05:12:16,481 THE PRESENCE OF PROTEASOME 6126 05:12:16,481 --> 05:12:18,483 INHIBITOR? 6127 05:12:18,483 --> 05:12:21,353 >> WHICH PART? 6128 05:12:21,353 --> 05:12:22,821 >> THE UBIQUITINATION, I'M 6129 05:12:22,821 --> 05:12:23,021 SORRY. 6130 05:12:23,021 --> 05:12:26,558 >> I THINK SO BUT I'M NOT ONE 6131 05:12:26,558 --> 05:12:28,660 PERCENT SURE. 6132 05:12:28,660 --> 05:12:31,797 >> OKAY, THANK YOU. 6133 05:12:31,797 --> 05:12:34,466 >> ANY MORE QUESTIONS? 6134 05:12:34,466 --> 05:12:37,269 I ALSO HAVE A FINAL QUESTION. 6135 05:12:37,269 --> 05:12:41,773 YOU ARE SEEING RNA-RNA 6136 05:12:41,773 --> 05:12:43,842 INTERACTION WITH APOBEC 3H IS 6137 05:12:43,842 --> 05:12:48,947 THAT RIGHT OR NO? 6138 05:12:48,947 --> 05:12:50,449 BECAUSE FUMIAKI SAID IT WAS 6139 05:12:50,449 --> 05:12:51,583 PROTEIN BASED. 6140 05:12:51,583 --> 05:12:53,685 DO YOU SEE ANY RNA INTERACTIONS 6141 05:12:53,685 --> 05:12:56,188 OR NOT? 6142 05:12:56,188 --> 05:12:58,290 >> THAT BRINGS ME TO THIS POINT 6143 05:12:58,290 --> 05:13:00,292 WHERE HIS STRUCTURE IS BASICALLY 6144 05:13:00,292 --> 05:13:03,261 A MONOMER, SO HE HAS ONE 6145 05:13:03,261 --> 05:13:04,262 MOLECULE OF A3H WITH 6146 05:13:04,262 --> 05:13:04,896 DOUBLE-STRANDED RNA. 6147 05:13:04,896 --> 05:13:07,799 WE ALSO SEE IT IN OUR STRUCTURE. 6148 05:13:07,799 --> 05:13:10,135 BUT HE ONLY HAS ONE MOLECULE OF 6149 05:13:10,135 --> 05:13:10,602 VIF. 6150 05:13:10,602 --> 05:13:13,171 WE HAVE TWO MOLECULE OF VIF, 6151 05:13:13,171 --> 05:13:18,577 EVERYTHING A DIMER OF THE 6152 05:13:18,577 --> 05:13:19,411 COMPLEX. 6153 05:13:19,411 --> 05:13:20,946 THERE'S NO INTERACTION INSIDE 6154 05:13:20,946 --> 05:13:23,582 THE SAME PROTOMER, BETWEEN VIF 6155 05:13:23,582 --> 05:13:25,217 AND DOUBLE-STRANDED RNA. 6156 05:13:25,217 --> 05:13:26,651 WE SEE INTERACTION ACROSS THE 6157 05:13:26,651 --> 05:13:27,352 PROTOMERS. 6158 05:13:27,352 --> 05:13:27,953 >> OKAY. 6159 05:13:27,953 --> 05:13:30,155 >> THIS IS THE UNIQUE FEATURE OF 6160 05:13:30,155 --> 05:13:32,557 OUR VERSUS HIS STRUCTURE. 6161 05:13:32,557 --> 05:13:34,092 >> THANK YOU FOR THE 6162 05:13:34,092 --> 05:13:34,626 CLARIFICATION. 6163 05:13:34,626 --> 05:13:35,994 THAT CONCLUDES THE AFTERNOON 6164 05:13:35,994 --> 05:13:36,228 SESSION. 6165 05:13:36,228 --> 05:13:38,964 [APPLAUSE] 6166 05:13:38,964 --> 05:13:41,066 6167 05:13:41,066 --> 05:13:43,468 I'LL TAKE OUR FINAL APPLAUSE FOR 6168 05:13:43,468 --> 05:13:45,637 ALL THE SPEAKERS, ESPECIALLY FOR 6169 05:13:45,637 --> 05:13:45,837 KATE. 6170 05:13:45,837 --> 05:13:49,608 [APPLAUSE] 6171 05:13:49,608 --> 05:13:51,076 I'LL GIVE THE MICROPHONE TO 6172 05:13:51,076 --> 05:13:51,910 DAVID. 6173 05:13:51,910 --> 05:13:56,481 >> IF I COULD HAVE THE TALKS FOR 6174 05:13:56,481 --> 05:13:57,916 THE NEXT SESSIONS, MORNING TALK, 6175 05:13:57,916 --> 05:13:58,817 TWO MORNING TALKS. 6176 05:13:58,817 --> 05:14:00,685 IF YOU WANT TO TELL ME IF YOU'RE 6177 05:14:00,685 --> 05:14:11,229 GOING TO GO ON A LAPTOP, I DON'T 6178 05:14:12,097 --> 05:14:12,497 SEE ALICE. 6179 05:14:12,497 --> 05:14:16,568 I HOPE YOU WARM UP WHEN YOU GET 6180 05:14:16,568 --> 05:14:16,802 OUTSIDE. 6181 05:14:16,802 --> 05:14:26,802 [END OF PROGRAM]