1 00:00:05,960 --> 00:00:10,360 >> MY NAME'S ERIC FREED PART OF 2 00:00:10,360 --> 00:00:12,760 THE NCI PROGRAM AT FREDERICK AND 3 00:00:12,760 --> 00:00:16,720 A PLEASURE TO INTRODUCE THE 4 00:00:16,720 --> 00:00:18,440 LECTURE. 5 00:00:18,440 --> 00:00:24,000 SO A FEW WORDS ABOUT GEORGE. 6 00:00:24,000 --> 00:00:26,280 HE WAS A GRADUATE OF PRINCETON 7 00:00:26,280 --> 00:00:28,880 UNIVERSITY AND HARVARD MEDICAL 8 00:00:28,880 --> 00:00:31,720 SCHOOL AND BECAME HEAD OF THE 9 00:00:31,720 --> 00:00:33,880 LABORATORY OF MOLECULAR VIROLOGY 10 00:00:33,880 --> 00:00:35,800 AT THE NCI AND BECAME CHIEF IN 11 00:00:35,800 --> 00:00:40,480 1980 OF THAT LAB AND IN 1981 HE 12 00:00:40,480 --> 00:00:42,040 RECEIVE THE ARTHUR S. FLEMING 13 00:00:42,040 --> 00:00:44,120 AWARD FOR OUTSTANDING GOVERNMENT 14 00:00:44,120 --> 00:00:50,720 SERVICE AND IN 1987 THE YEAR OF 15 00:00:50,720 --> 00:00:52,080 HIS IN TIMELY DEATH WAS ELECTED 16 00:00:52,080 --> 00:00:55,160 TO THE NATIONAL ACADEMY OF 17 00:00:55,160 --> 00:00:55,440 SCIENCES. 18 00:00:55,440 --> 00:01:00,440 THIS IS A PICTURE OF HIM AND 19 00:01:00,440 --> 00:01:04,880 RECEIVING THE AWARD. 20 00:01:04,880 --> 00:01:08,880 GEORGE PUBLISHED OVER 140 PAPERS 21 00:01:08,880 --> 00:01:12,880 DURING HIS SHORT CAREER 22 00:01:12,880 --> 00:01:16,040 DECIPHERIDECIPHER 23 00:01:16,040 --> 00:01:24,840 DECIPHERIDECIPHER 24 00:01:24,840 --> 00:01:28,560 DECIPHERIDECIPHER 25 00:01:28,560 --> 00:01:34,560 DECIPHERING MANY MECHANISMS. 26 00:01:34,560 --> 00:01:39,360 THE GEORGE KHOURY LECTURES BEGAN 27 00:01:39,360 --> 00:01:42,880 IN 1995 AND FEATURED A SIMILARLY 28 00:01:42,880 --> 00:01:44,320 DISTINGUISHED GROUP OF SPEAKERS 29 00:01:44,320 --> 00:01:48,280 SOME OF WHOM RECEIVE THE NOBEL 30 00:01:48,280 --> 00:01:52,000 PRIZE AND THESE ARE THE MOST 31 00:01:52,000 --> 00:01:56,480 RECENT SPEAKERS. 32 00:01:56,480 --> 00:02:00,280 IT'S A PLEASURE TO INTRODUCE THE 33 00:02:00,280 --> 00:02:00,520 LECTURER. 34 00:02:00,520 --> 00:02:04,080 A NATIVE OF ARIZONA AND RECEIVED 35 00:02:04,080 --> 00:02:08,920 HER BACHELOR'S DEGREE FROM 36 00:02:08,920 --> 00:02:11,640 UNIVERSITY OF ARIZONA AND DID A 37 00:02:11,640 --> 00:02:15,440 POSTDOC AT UCSF AND IN 2006 WAS 38 00:02:15,440 --> 00:02:17,520 RECRUITED TO AN ASSISTANT 39 00:02:17,520 --> 00:02:19,880 PROFESSOR POSITION AT BERKELEY 40 00:02:19,880 --> 00:02:21,800 IN THE DEPARTMENT OF PLANT AND 41 00:02:21,800 --> 00:02:23,440 MICROBIOLOGY AND RECEIVED TENURE 42 00:02:23,440 --> 00:02:26,960 IN 2012 AND BECAME AN HHI 43 00:02:26,960 --> 00:02:27,840 INVESTIGATOR AND PULL PROFESSOR 44 00:02:27,840 --> 00:02:29,920 IN 2017 AND SINCE 2021 HAS BEEN 45 00:02:29,920 --> 00:02:32,280 ASSOCIATE CHAIR OF THAT 46 00:02:32,280 --> 00:02:33,280 DEPARTMENT AT BERKELEY. 47 00:02:33,280 --> 00:02:35,960 SHE RECEIVED A NUMBER OF HONORS 48 00:02:35,960 --> 00:02:36,880 AND RECOGNIZES. 49 00:02:36,880 --> 00:02:40,600 TOO MANY TO LIST HERE BUT IF YOU 50 00:02:40,600 --> 00:02:42,120 KNOW THE ONES THE KECK 51 00:02:42,120 --> 00:02:44,400 DISTINGUISHED YOUNG SCHOLAR 52 00:02:44,400 --> 00:02:44,720 AWARD. 53 00:02:44,720 --> 00:02:48,440 THE U.C. BERKELEY EXCELLENCE IN 54 00:02:48,440 --> 00:02:50,720 GRADUATE MENTORSHIP AND 2019 55 00:02:50,720 --> 00:02:53,280 ELECTED FELLOW OF THE AMERICAN 56 00:02:53,280 --> 00:02:54,560 ACADEMY OF MICROBIOLOGY. 57 00:02:54,560 --> 00:02:57,840 HER RESEARCH FOCUSSED ON SEVERAL 58 00:02:57,840 --> 00:03:01,280 P.O.s INCLUDING MECHANISMS AND 59 00:03:01,280 --> 00:03:04,200 CONSEQUENCES OF mRNA 60 00:03:04,200 --> 00:03:05,480 DEGRADATION AND TRANSCRIPTION 61 00:03:05,480 --> 00:03:07,920 REGULATION DURING LATE STAGES OF 62 00:03:07,920 --> 00:03:10,040 IN STAGES AND NON-CODING RNA. 63 00:03:10,040 --> 00:03:12,680 THE SEMINAR IS ENTITLED 64 00:03:12,680 --> 00:03:14,480 CONTROLLING THE MESSAGE HOW 65 00:03:14,480 --> 00:03:16,800 HERPES VIRUSES MANIPULATE THE 66 00:03:16,800 --> 00:03:19,520 GENE EXPRESSION LANDSCAPE. 67 00:03:19,520 --> 00:03:21,600 BEFORE BRITT DOES HER SEMINAR WE 68 00:03:21,600 --> 00:03:25,880 DISCOVERED AT DINNER LAST NIGHT 69 00:03:25,880 --> 00:03:28,000 SHE NEVER HEARD OF OLD BAY SO 70 00:03:28,000 --> 00:03:29,400 FOR A TOKEN OF APPRECIATION I'D 71 00:03:29,400 --> 00:03:34,480 LIKE TO PRESENT HER WITH THIS 72 00:03:34,480 --> 00:03:37,160 SMALL TIN OF GENUINE MARYLAND 73 00:03:37,160 --> 00:03:41,040 OLD BAY. 74 00:03:41,040 --> 00:03:44,440 IT'S A REAL PLEASURE TO BE HERE. 75 00:03:44,440 --> 00:03:46,760 THE FLOWERS ARE IN BLOOM. 76 00:03:46,760 --> 00:03:47,960 IT'S A BEAUTIFUL SUNNY DAY 77 00:03:47,960 --> 00:03:49,680 COMPARED TO THE RAIN STORM I 78 00:03:49,680 --> 00:03:50,840 LEFT IN CALIFORNIA YESTERDAY AND 79 00:03:50,840 --> 00:03:52,960 AS ERIC ALLUDED TO I SPENT 80 00:03:52,960 --> 00:03:55,120 PRETTY MUCH MY ENTIRE CAREER 81 00:03:55,120 --> 00:03:59,280 STUDYING VIRUS INTERACTION AND 82 00:03:59,280 --> 00:04:03,280 IT'S AN HONOR TO GIVE THIS 83 00:04:03,280 --> 00:04:06,080 LECTURE FOR GEORGE KHOURY AND 84 00:04:06,080 --> 00:04:07,720 I'M FAMILIAR WITH THE RESEARCH 85 00:04:07,720 --> 00:04:12,080 HE CARRIED OUT RELATED TO USING 86 00:04:12,080 --> 00:04:14,640 DNA VIRUSES IN PARTICULAR TO 87 00:04:14,640 --> 00:04:18,240 DISSECT FUNDAMENTAL PRINCIPLES 88 00:04:18,240 --> 00:04:19,600 OF GENE REGULATION. 89 00:04:19,600 --> 00:04:23,560 MUCH OF HIS RESEARCH PROVIDED 90 00:04:23,560 --> 00:04:24,840 THE FOUNDATION OF KNOWLEDGE THAT 91 00:04:24,840 --> 00:04:26,080 UNDER LIES MANY OF THE PROJECTS 92 00:04:26,080 --> 00:04:27,720 IN MY OWN LAB NOW. 93 00:04:27,720 --> 00:04:30,240 I FEEL LIKE IT'S A REAL DELIGHT 94 00:04:30,240 --> 00:04:31,600 TO BE ABLE TO PARTICIPATE IN 95 00:04:31,600 --> 00:04:33,840 THIS LECTURE SAYS. 96 00:04:33,840 --> 00:04:35,120 -- SERIES. 97 00:04:35,120 --> 00:04:38,600 WHEN I THINK OF VIRAL INFECTION, 98 00:04:38,600 --> 00:04:39,960 REALLY GENE REGULATION AND THE 99 00:04:39,960 --> 00:04:42,840 ABILITY TO MANIPULATE GENE 100 00:04:42,840 --> 00:04:45,280 REGULATION IS AT THE CRUX OF 101 00:04:45,280 --> 00:04:46,080 WHETHER OR NOT OR NOT AN 102 00:04:46,080 --> 00:04:47,800 INFECTION IS GOING IT TAKE OVER 103 00:04:47,800 --> 00:04:49,440 OR PETER OUT. 104 00:04:49,440 --> 00:04:51,440 AND THIS IS BECAUSE FROM THE 105 00:04:51,440 --> 00:04:53,960 HOST SIDE THE ABILITY TO ELICIT 106 00:04:53,960 --> 00:04:55,520 A PARTICULAR GENE EXPRESSION 107 00:04:55,520 --> 00:04:57,560 PROGRAM IN RESPONSE TO INFECTION 108 00:04:57,560 --> 00:04:59,960 IS GOING TO DETERMINE WHETHER OR 109 00:04:59,960 --> 00:05:02,400 NOT THE HOST CELL CAN COUNTER 110 00:05:02,400 --> 00:05:03,760 ACT AND FIGHT BACK AGAINST THE 111 00:05:03,760 --> 00:05:06,800 VIRAL INFECTION AND CONVERSELY 112 00:05:06,800 --> 00:05:11,200 THE ABILITY OF THE VARIOUS TO 113 00:05:11,200 --> 00:05:12,360 COMMANDEER THOSE GENE EXPRESSION 114 00:05:12,360 --> 00:05:19,720 MACHINERY AND SHUT DOWN THE ATS 115 00:05:19,720 --> 00:05:22,640 ASPECTS IN CONTROLLING VIRAL 116 00:05:22,640 --> 00:05:23,320 INFECTION WILL DETERMINE WHETHER 117 00:05:23,320 --> 00:05:25,160 THE VIRAL INFECTION WILL TAKE 118 00:05:25,160 --> 00:05:25,720 OUT. 119 00:05:25,720 --> 00:05:28,400 REHOSTING THE ENVIRONMENT IS 120 00:05:28,400 --> 00:05:30,720 CRITICAL IMPORTANCE FOR VIRUSES 121 00:05:30,720 --> 00:05:31,360 AND SOMETHING I'VE BEEN 122 00:05:31,360 --> 00:05:33,720 INTERESTED IN FOR A LONG TIME. 123 00:05:33,720 --> 00:05:35,920 BECAUSE IT'S CENTRAL IMPORTANCE 124 00:05:35,920 --> 00:05:39,240 TO VIROLOGY MANY CODE PROTEINS 125 00:05:39,240 --> 00:05:42,120 WITH A DEDICATED JOB OF SHUTTING 126 00:05:42,120 --> 00:05:44,040 THEM DOWN AND THEY CAPTIVATED ME 127 00:05:44,040 --> 00:05:45,040 FOR YEARS. 128 00:05:45,040 --> 00:05:47,640 THE IDEA HERE SOME OF WHICH ARE 129 00:05:47,640 --> 00:05:48,400 SHOWN IN THE DIAGRAM EXPRESSION 130 00:05:48,400 --> 00:05:52,000 OF THE PROTEINS, MANY OF WHICH 131 00:05:52,000 --> 00:05:55,600 TARGET RNA DIRECTLY LEADS TO A 132 00:05:55,600 --> 00:05:58,960 LARGE-SCALE DOWN REGULATION OF 133 00:05:58,960 --> 00:06:01,360 HOST GENE EXPRESSION. 134 00:06:01,360 --> 00:06:04,240 FIRST, IT HELPS THE VIRUS SHUNT 135 00:06:04,240 --> 00:06:05,680 TRANSLATION RESOURCES AWAY FROM 136 00:06:05,680 --> 00:06:07,240 THE HOST AND TOWARDS THE VIRUS 137 00:06:07,240 --> 00:06:09,400 BECAUSE NO VIRUS OF COURSE CAN 138 00:06:09,400 --> 00:06:11,280 ENCODE THE TRANSLATION MACHINERY 139 00:06:11,280 --> 00:06:14,280 ALL VIRUSES NEED. 140 00:06:14,280 --> 00:06:15,640 SECOND, BY CASTING A WIDE NET 141 00:06:15,640 --> 00:06:20,640 AND TARGETING MOST OF THE 142 00:06:20,640 --> 00:06:22,040 MESSENGER RNA THE VIRUS IS ABLE 143 00:06:22,040 --> 00:06:27,480 TO CAPTURE MANY OF THE IMMUNE 144 00:06:27,480 --> 00:06:28,400 STIMULATORY FACTORS IN THE 145 00:06:28,400 --> 00:06:30,120 ATTEMPT TO STOP THE VIRUS. 146 00:06:30,120 --> 00:06:33,040 IN THIS REGARD THE VIRUSES BY 147 00:06:33,040 --> 00:06:36,360 GLOBALLY SHUTTING DOWN GENE 148 00:06:36,360 --> 00:06:37,640 EXPRESSION CAN HELP COUNTER 149 00:06:37,640 --> 00:06:38,080 ACTED RESPONSE. 150 00:06:38,080 --> 00:06:40,440 WE STUDIED THE SHUT OFF FACTORS 151 00:06:40,440 --> 00:06:43,080 BUT THE MAIN VIRUS IS FOCUSSED 152 00:06:43,080 --> 00:06:43,800 ON THE HERPES VIRUS AND THAT 153 00:06:43,800 --> 00:06:48,040 WILL BE THE TOPIC OF THE VIRUS 154 00:06:48,040 --> 00:06:48,320 TODAY. 155 00:06:48,320 --> 00:06:50,280 HERPES VIRUS I WOULD ARGUE AND 156 00:06:50,280 --> 00:06:52,360 MANY WOULD AGREE ARE AMONG THE 157 00:06:52,360 --> 00:06:54,120 MOST SUCCESSFUL PATHOGENS IN THE 158 00:06:54,120 --> 00:06:58,200 PLANET IN PART BECAUSE THEY'RE 159 00:06:58,200 --> 00:07:04,600 UBIQUITIOIOUIOUIOUS -- UBIQUITOUS AND 160 00:07:04,600 --> 00:07:09,200 IT'S NOT JUST HUMANS, HERPES 161 00:07:09,200 --> 00:07:10,240 VIRUSES ARE INCREDIBLY 162 00:07:10,240 --> 00:07:12,240 WIDESPREAD ON THE PLANET. 163 00:07:12,240 --> 00:07:15,920 MANY VERTEBRATES HAVE THIS VIRUS 164 00:07:15,920 --> 00:07:23,520 ASSOCIATED WITH THEN AND OYSTERS 165 00:07:23,520 --> 00:07:26,120 HAVE THEM AND THEY'RE AROUND 200 166 00:07:26,120 --> 00:07:29,000 AND 400 MILLION YEARS OLD AND 167 00:07:29,000 --> 00:07:31,560 IT'S AN EXTENSIVE 168 00:07:31,560 --> 00:07:32,240 CO-EVOLUTIONARY HISTORY WITH US 169 00:07:32,240 --> 00:07:35,280 AND OTHER HOSTS AND ONE OF THE 170 00:07:35,280 --> 00:07:37,080 REASONS WE THINK THEY MAKE 171 00:07:37,080 --> 00:07:39,080 EXCELLENT MODEL TO DISSECT GENE 172 00:07:39,080 --> 00:07:42,680 REGULATION IN MAMMALIAN CELLS 173 00:07:42,680 --> 00:07:45,480 UNDER STRESSFUL CONDITIONS LIKE 174 00:07:45,480 --> 00:07:46,120 PATHOGEN INVASION. 175 00:07:46,120 --> 00:07:48,400 MY LAB IS FOCUSSED ON THE GAMMA 176 00:07:48,400 --> 00:07:49,960 HERPES VIRUSES THAT HAVE TWO 177 00:07:49,960 --> 00:07:52,920 HUMAN PATHOGENS, EPSTEIN BARR 178 00:07:52,920 --> 00:07:54,960 VARIETY WHICH CAUSES A NUMBER OF 179 00:07:54,960 --> 00:07:57,720 B CELL LYMPHOMAS AND IDENTIFIED 180 00:07:57,720 --> 00:08:03,920 AS A MAIN CAUSE OF MULTIPLE AND 181 00:08:03,920 --> 00:08:06,840 KAPOSI'S SARCOMA HERPES VIRUS IN 182 00:08:06,840 --> 00:08:10,600 AIDS PATIENTS AND WE WORKED ON 183 00:08:10,600 --> 00:08:14,480 MOUSE HERPES VIRUS MHV68 184 00:08:14,480 --> 00:08:17,000 COMMONLY USED IN THE FIELD FOR 185 00:08:17,000 --> 00:08:21,400 IN VIVO AND STUDYING THE 186 00:08:21,400 --> 00:08:25,240 WHITTAKER CYCLE WE'RE INTERESTED 187 00:08:25,240 --> 00:08:26,640 IN. 188 00:08:26,640 --> 00:08:40,240 DURING LYTIC AMPLIFICATION THEY 189 00:08:40,240 --> 00:08:42,440 HAVE A SHUT OFF FACTOR AND SHUT 190 00:08:42,440 --> 00:08:44,440 OFF THE NUCLEASE OUR LABS AND 191 00:08:44,440 --> 00:08:45,920 OTHER LABS HAVE SHOWN THE 192 00:08:45,920 --> 00:08:49,640 ABILITY OF THIS NUCLEASE TO 193 00:08:49,640 --> 00:08:51,480 ELICIT WIDESPREAD RNA DECAY AND 194 00:08:51,480 --> 00:08:54,240 PLAYS A VARIETY OF ROLES IN THE 195 00:08:54,240 --> 00:08:57,480 GAMMA HERPES LIFE CYCLE FROM 196 00:08:57,480 --> 00:08:59,040 PROMOTING IMMUNE EVASION TO 197 00:08:59,040 --> 00:09:00,240 ENABLING THE VIRUS TO TRAFFIC 198 00:09:00,240 --> 00:09:02,840 WHERE IT NEEDS TO GO IN THE IN 199 00:09:02,840 --> 00:09:04,160 VIVO MODEL IN A MOUSE. 200 00:09:04,160 --> 00:09:06,440 ESTABLISHING LATENCY BEING ABLE 201 00:09:06,440 --> 00:09:10,240 TO REPLICATE IN B CELLS THE MAIN 202 00:09:10,240 --> 00:09:11,800 NICHE FOR GAMMA HERPES VIRUSES 203 00:09:11,800 --> 00:09:16,640 AND BEYOND CONTROLLING THE HOST 204 00:09:16,640 --> 00:09:18,640 GENE EXPRESSION ENVIRONMENT 205 00:09:18,640 --> 00:09:20,080 SETTING THE HOST GENES VERSUS 206 00:09:20,080 --> 00:09:22,040 VIRAL GENES INDUCES VIRAL GENE 207 00:09:22,040 --> 00:09:24,000 EXPRESSION AND PROTEIN 208 00:09:24,000 --> 00:09:25,720 PRODUCTION AND WHAT GETS 209 00:09:25,720 --> 00:09:27,840 PACKAGED TO NASCENT VIRAL 210 00:09:27,840 --> 00:09:29,520 PARTICLES INFLUENCING NEW ROUNDS 211 00:09:29,520 --> 00:09:30,840 OF INFECTION. 212 00:09:30,840 --> 00:09:32,240 VARIOUS STAGES ARE INFLUENCED BY 213 00:09:32,240 --> 00:09:34,280 THE ABILITY TO CONTROL THE GENE 214 00:09:34,280 --> 00:09:35,280 EXPRESSION CASCADE AND WE 215 00:09:35,280 --> 00:09:37,680 STUDIED THIS PROCESS FOR A 216 00:09:37,680 --> 00:09:38,080 NUMBER OF YEARS. 217 00:09:38,080 --> 00:09:41,280 IN PART STUDYING THE RNA 218 00:09:41,280 --> 00:09:44,760 MECHANISMS OF THE ENZYMES THE 219 00:09:44,760 --> 00:09:47,000 SOCK PROTEIN CAN BROADLY TARGET 220 00:09:47,000 --> 00:09:50,440 CELLULAR MESSENGER RNA AND 221 00:09:50,440 --> 00:09:53,160 TARGETS ONLY TRANSCRIBED RNA AND 222 00:09:53,160 --> 00:09:56,040 THOUGH IT CAN BROADLY TARGET RNA 223 00:09:56,040 --> 00:10:00,240 IT HAS SPECIFICITY BUILT IN AND 224 00:10:00,240 --> 00:10:03,120 CLEAVES THROUGH A COMBINATION OF 225 00:10:03,120 --> 00:10:05,160 SEQUENCE AND STRUCTURE CAUSING 226 00:10:05,160 --> 00:10:07,760 AN INTERNAL CLEAVAGE EVENT 227 00:10:07,760 --> 00:10:09,360 BASICALLY IT CREATES UNPROTECTED 228 00:10:09,360 --> 00:10:12,200 FREE ENDS OF THE RNA THAT CAN 229 00:10:12,200 --> 00:10:14,720 THEN BE ATTACKED BY THE RESIDENT 230 00:10:14,720 --> 00:10:17,440 EXISTING NUCLEASES OUR OWN CELLS 231 00:10:17,440 --> 00:10:20,440 NORMALLY USE TO CONTROL RNA 232 00:10:20,440 --> 00:10:20,720 TURNOVER. 233 00:10:20,720 --> 00:10:22,800 THE TWO-STEP PROCESS OF THE 234 00:10:22,800 --> 00:10:24,440 VIRUS INDUCING AN INTERNAL 235 00:10:24,440 --> 00:10:29,760 CLEAVE CATHAGE AND CLEARING THE RNA IS 236 00:10:29,760 --> 00:10:31,080 IMPORTANT AND HOLD THAT THOUGHT 237 00:10:31,080 --> 00:10:32,480 IN YOUR MIND I'LL TOUCH ON IT 238 00:10:32,480 --> 00:10:34,920 LATER IN THE LECTURE. 239 00:10:34,920 --> 00:10:37,520 SO BEYOND STUDYING HOW THESE 240 00:10:37,520 --> 00:10:39,840 ENZYMES WORK AND TARGETING RNA 241 00:10:39,840 --> 00:10:41,360 AND WHAT SORT OF RNA FEATURES 242 00:10:41,360 --> 00:10:43,720 ARE REQUIRED FOR TARGETING OR 243 00:10:43,720 --> 00:10:45,120 ESCAPE, IN THE LAST SEVERAL 244 00:10:45,120 --> 00:10:47,560 YEARS WE'VE SHIFTED OUR FOCUS 245 00:10:47,560 --> 00:10:51,280 SOMEWHAT TO SAYING CAN WE USE 246 00:10:51,280 --> 00:10:53,680 THESE AS TOOLS TO PROBE HOW 247 00:10:53,680 --> 00:10:55,520 CELLS SENSE AND RESPOND TO LARGE 248 00:10:55,520 --> 00:10:57,240 CHANGES IN MESSENGER RNA 249 00:10:57,240 --> 00:10:57,840 ABUNDANCE. 250 00:10:57,840 --> 00:10:59,720 BECAUSE THESE ARE FEATURES 251 00:10:59,720 --> 00:11:00,760 CHARACTERISTIC OF A NUMBER OF 252 00:11:00,760 --> 00:11:03,120 PATHOGENIC SITUATIONS. 253 00:11:03,120 --> 00:11:06,240 SO I'M GOING IT TELL YOU TWO 254 00:11:06,240 --> 00:11:07,640 RELATED STORIES TODAY. 255 00:11:07,640 --> 00:11:09,520 THE FIRST STORY FOCUSES ON THE 256 00:11:09,520 --> 00:11:10,920 HOST SIDE AND JUST THAT QUESTION 257 00:11:10,920 --> 00:11:13,640 THAT I OUTLINED OF USING THE 258 00:11:13,640 --> 00:11:15,960 VIRUSES AS A TOOL TO EXPLORE 259 00:11:15,960 --> 00:11:17,520 CONNECTIONS BETWEEN SEEMINGLY 260 00:11:17,520 --> 00:11:20,440 VERY DISTAL STAGES OF THE VIRAL 261 00:11:20,440 --> 00:11:23,840 LIFE CYCLE AND HOW THOSE ARE 262 00:11:23,840 --> 00:11:25,280 INTERCONNECTED AND THE CELL USES 263 00:11:25,280 --> 00:11:27,440 THOSE IN RESPONSE TO STRESS AND 264 00:11:27,440 --> 00:11:29,000 IN THE SECOND PART OF MY TALK 265 00:11:29,000 --> 00:11:29,840 I'LL FOCUS ON THE VIRUS ITSELF 266 00:11:29,840 --> 00:11:31,440 AND HOW IT BENEFITS FROM THIS 267 00:11:31,440 --> 00:11:36,440 AND IS ABLE TO ORCHESTRATE AN 268 00:11:36,440 --> 00:11:38,240 UNUSUAL GENE EXPRESSION CASCADE 269 00:11:38,240 --> 00:11:40,440 OR REGULATION FOR THE VIRAL 270 00:11:40,440 --> 00:11:41,960 GENES. 271 00:11:41,960 --> 00:11:45,040 SO THIS FIRST PART OF THE STORY 272 00:11:45,040 --> 00:11:48,360 BEGINS WITH THE IDEA THAT CELLS 273 00:11:48,360 --> 00:11:50,320 AND THEY'RE UNINFECTED STATE 274 00:11:50,320 --> 00:11:52,840 BASICALLY ARE TRYING TO ACHIEVE 275 00:11:52,840 --> 00:11:53,160 HOMEOSTASIS. 276 00:11:53,160 --> 00:11:55,600 THEY WANT AN APPROPRIATE OVER 277 00:11:55,600 --> 00:11:58,520 ALL BALANCE OF MESSENGER RNA. 278 00:11:58,520 --> 00:11:59,960 THE IDEA OF CELLS TRYING TO 279 00:11:59,960 --> 00:12:02,960 ACHIEVE HOMEOSTASIS BY 280 00:12:02,960 --> 00:12:04,520 CONTROLLING THEIR MESSENGER 281 00:12:04,520 --> 00:12:06,200 LEVELS IS WELL ESTABLISHED IN 282 00:12:06,200 --> 00:12:08,000 YEAST AND MAMMALIAN CELLS. 283 00:12:08,000 --> 00:12:12,400 IT'S BEEN SHOWN FOR EXAMPLE IF 284 00:12:12,400 --> 00:12:16,640 YOU DECREASE THE OVER ALL RATE 285 00:12:16,640 --> 00:12:20,400 OF MESSENGER RNA SYNTHESIS, FOR 286 00:12:20,400 --> 00:12:23,920 EXAMPLE USING SLOW RNA 287 00:12:23,920 --> 00:12:25,080 POLYMERASE 2 THE CELL WILL 288 00:12:25,080 --> 00:12:26,880 RESPOND AND COMPENSATE BY 289 00:12:26,880 --> 00:12:29,000 GENERALLY STABLIZING THE 290 00:12:29,000 --> 00:12:30,720 MESSAGES THAT EXIST. 291 00:12:30,720 --> 00:12:32,480 FEWER BEING PRODUCED AND YOU 292 00:12:32,480 --> 00:12:35,640 NEED THOSE THERE STICKING AROUND 293 00:12:35,640 --> 00:12:35,840 LONGER. 294 00:12:35,840 --> 00:12:39,440 CONVERSELY FURTHER SLOW DOWN THE 295 00:12:39,440 --> 00:12:40,240 DEGRADATION BY GETTING RID OF 296 00:12:40,240 --> 00:12:42,440 KEY DECAY FACTORS, FOR EXAMPLE, 297 00:12:42,440 --> 00:12:44,360 THE CELL WILL COMPENSATE BY 298 00:12:44,360 --> 00:12:46,160 SLOWING THE RATE OF 299 00:12:46,160 --> 00:12:46,840 TRANSCRIPTION BECAUSE YOU HAVE 300 00:12:46,840 --> 00:12:48,080 THOSE STICKING AROUND LONGER AND 301 00:12:48,080 --> 00:12:50,240 DON'T NEED TO PRODUCE AS MANY TO 302 00:12:50,240 --> 00:12:52,680 ACHIEVE YOUR OPTIMAL BALANCE. 303 00:12:52,680 --> 00:12:54,600 BUT WE WONDERED WHAT ABOUT IF 304 00:12:54,600 --> 00:12:56,600 YOU WERE LOOKING IN THE OTHER 305 00:12:56,600 --> 00:13:00,200 DIRECTION INSTEAD OF STABLIZING 306 00:13:00,200 --> 00:13:02,040 RNA A MUCH MORE COMMON SITUATION 307 00:13:02,040 --> 00:13:06,040 IN VIRAL INFECTION IS GLOBAL 308 00:13:06,040 --> 00:13:07,320 DESTABILIZATION OF RNA. 309 00:13:07,320 --> 00:13:09,440 AND SO IN THIS REGARD WITH THE 310 00:13:09,440 --> 00:13:13,240 CELL ALSO TRY AND MAINTAIN 311 00:13:13,240 --> 00:13:19,200 HOMEOSTASIS WE THOUGHT NOT. 312 00:13:19,200 --> 00:13:23,840 CELLS GENERALLY WHEN FAZED TRY 313 00:13:23,840 --> 00:13:26,880 TO SLOW THING DOWN WITH PRK 314 00:13:26,880 --> 00:13:28,400 ACTIVATION AND THINGS LIKE THAT. 315 00:13:28,400 --> 00:13:32,400 WHAT WE FOUND IN THE CASE OF 316 00:13:32,400 --> 00:13:34,440 CELLULAR THREAT THE HERPES VIRUS 317 00:13:34,440 --> 00:13:38,000 INFECTION THAT ELICIT RNA DECAY 318 00:13:38,000 --> 00:13:39,960 THE BALANCE IT TIPPED FROM 319 00:13:39,960 --> 00:13:42,440 HOMEOSTASIS AND THE CELL INSTEAD 320 00:13:42,440 --> 00:13:44,760 OF AMPLIFYING TRANSCRIPTION 321 00:13:44,760 --> 00:13:46,440 RESPONDS BY SHUTTING DOWN 322 00:13:46,440 --> 00:13:52,440 TRANSCRIPTION ESSENTIALLY MAG 323 00:13:52,440 --> 00:13:56,080 MAGNIFYING THE SCREEN AND WE 324 00:13:56,080 --> 00:13:58,320 HYPOTHESIZE IS BECAUSE MANY 325 00:13:58,320 --> 00:14:00,800 VIRUSES LIKE DNA VIRUSES ARE 326 00:14:00,800 --> 00:14:01,880 RELIANT ON THE MACHINERY. 327 00:14:01,880 --> 00:14:03,280 WHEN CELLS SENSE THEY'RE 328 00:14:03,280 --> 00:14:04,560 INFECTED ARE TRYING TO FIND WAYS 329 00:14:04,560 --> 00:14:06,840 TO SHUT DOWN CORE CAN CELLULAR 330 00:14:06,840 --> 00:14:08,880 PROCESSES AS I SAID TRANSLATION 331 00:14:08,880 --> 00:14:09,920 BEING VERY WELL CHARACTERIZED 332 00:14:09,920 --> 00:14:11,720 ONE BUT WE THINK TRANSCRIPTION 333 00:14:11,720 --> 00:14:13,640 IS ANOTHER ONE PARTICULARLY FOR 334 00:14:13,640 --> 00:14:15,520 DNA VIRUSES. 335 00:14:15,520 --> 00:14:16,440 ALL RIGHT. 336 00:14:16,440 --> 00:14:19,320 SO LET ME SHOW YOU WHAT I MEAN. 337 00:14:19,320 --> 00:14:22,440 WE OBSERVE THE CONNECTION 338 00:14:22,440 --> 00:14:29,200 BETWEEN RNA DECAY AND A -- 339 00:14:29,200 --> 00:14:31,640 TRANSSCRIPTION. 340 00:14:31,640 --> 00:14:34,160 WE WERE USING A WILD TYPE VIRUS 341 00:14:34,160 --> 00:14:38,280 USING A FUNCTIONAL SOX PROTEIN. 342 00:14:38,280 --> 00:14:41,080 WHEN YOU INFECT CELLS AND WE'RE 343 00:14:41,080 --> 00:14:43,680 USING MOUSE FIBROBLAST CELLS AND 344 00:14:43,680 --> 00:14:56,760 IT INDUCES CYTO-MICK -- 345 00:14:56,760 --> 00:15:00,240 CYTOPLASMIC EFFECT AND IT 346 00:15:00,240 --> 00:15:03,920 TARGETS mRNA s FOR DECAY. 347 00:15:03,920 --> 00:15:05,240 THE VIRUSES REPLICATE SIMILARLY 348 00:15:05,240 --> 00:15:08,520 BUT THE SOX MUTANT DOES NOT 349 00:15:08,520 --> 00:15:12,920 ACCELERATE RNA DECAY IN THE 350 00:15:12,920 --> 00:15:14,600 CYTOPLASM AND THE WILD TYPE 351 00:15:14,600 --> 00:15:14,800 DOES. 352 00:15:14,800 --> 00:15:17,400 HOW DOES THE CELL RESPOND TO 353 00:15:17,400 --> 00:15:20,880 ACCELERATED RNA DECAY OR NORMAL 354 00:15:20,880 --> 00:15:23,880 RNA DECAY AT THE TRANSCRIPTIONAL 355 00:15:23,880 --> 00:15:28,400 LEVEL? 356 00:15:28,400 --> 00:15:41,240 WE LOOK AT NASCENT RNA 357 00:15:41,240 --> 00:15:43,680 DESCRIPTION AND IN THE 358 00:15:43,680 --> 00:15:46,200 UNINFECTED CELL WHEN LOOK AT THE 359 00:15:46,200 --> 00:15:50,280 PILE UP OF POLYMERASE IT'S 360 00:15:50,280 --> 00:15:52,760 CENTERED NEAR THE TRANSCRIPTION 361 00:15:52,760 --> 00:15:54,280 START SITE AS YOU'D EXPECT 362 00:15:54,280 --> 00:15:56,160 DURING INFECTION WITH WILD TYPE 363 00:15:56,160 --> 00:15:58,440 THERE'S A LARGE SCALE CLEARANCE 364 00:15:58,440 --> 00:16:00,240 OF THE POLYMERASE FROM THE HOST 365 00:16:00,240 --> 00:16:08,840 GENOME AND LINKED TO CYTOPLASMIC 366 00:16:08,840 --> 00:16:18,120 RNA DECRAY -- DECAY AND THERE 367 00:16:18,120 --> 00:16:19,960 IS SOMETHING ABOUT RNA DECAY IN 368 00:16:19,960 --> 00:16:24,440 THE CYTOPLASM THAT IS CAUSING A 369 00:16:24,440 --> 00:16:25,960 REDUCTION IN RNA SYNTHESIS IN 370 00:16:25,960 --> 00:16:26,920 THE NUCLEUS. 371 00:16:26,920 --> 00:16:28,720 SO HOW MIGHT THAT HAPPEN? 372 00:16:28,720 --> 00:16:32,400 THERE'S A COUPLE POSSIBILITIES. 373 00:16:32,400 --> 00:16:35,040 ONE, SOMETHING ABOUT TARGETING 374 00:16:35,040 --> 00:16:36,200 TRANSCRIPTS INDIVIDUALLY THAT 375 00:16:36,200 --> 00:16:36,720 MATTER. 376 00:16:36,720 --> 00:16:38,600 FOR EXAMPLE, IF YOU HAVE FACTORS 377 00:16:38,600 --> 00:16:41,280 THAT ENCODE TRANSCRIPTIONAL 378 00:16:41,280 --> 00:16:42,440 REGULATORS AND YOUR GETTING RID 379 00:16:42,440 --> 00:16:45,240 OF THEIR TRANSCRIPTS AND THE 380 00:16:45,240 --> 00:16:46,480 PROTEINS ARE LABEL YOU MAY 381 00:16:46,480 --> 00:16:49,200 CHANGE THE PROTEINS IN THE CELL 382 00:16:49,200 --> 00:16:50,840 RESPONSIBLE FOR TRANSCRIPTION 383 00:16:50,840 --> 00:16:53,560 AND DAMP ENNING TRANSCRIPTION 384 00:16:53,560 --> 00:16:53,840 THAT WAY. 385 00:16:53,840 --> 00:16:55,960 IN ADDITION, YOU CAN HAVE A 386 00:16:55,960 --> 00:16:57,840 SITUATION WHERE THE CELL IS 387 00:16:57,840 --> 00:17:01,240 SOMEHOW SENSING THE DEGRADATION 388 00:17:01,240 --> 00:17:05,360 OF THE CLEAVED FRAGMENTS THE 389 00:17:05,360 --> 00:17:06,640 ACTIVE RNA DECAY AND RESPONDING 390 00:17:06,640 --> 00:17:07,040 TO THAT. 391 00:17:07,040 --> 00:17:09,640 WHAT I'LL TELL YOU IS BOTH OF 392 00:17:09,640 --> 00:17:13,040 THESE TURN OUT TO BE TRUE AND WE 393 00:17:13,040 --> 00:17:13,760 DISCOVERED THEM IN REVERSE 394 00:17:13,760 --> 00:17:14,360 ORDER. 395 00:17:14,360 --> 00:17:17,640 THE ONE WE HIT ON FIRST WAS THE 396 00:17:17,640 --> 00:17:19,600 IDEA THAT CELLS COULD ACTUALLY 397 00:17:19,600 --> 00:17:23,080 SENSE ACCELERATED RNA DECAY. 398 00:17:23,080 --> 00:17:25,720 THE REASON WE KNEW IT WAS THE 399 00:17:25,720 --> 00:17:28,040 CASE WE COULD RECAPITULATE THE 400 00:17:28,040 --> 00:17:30,120 RESULTS IN UNINFECTED CELLS IF 401 00:17:30,120 --> 00:17:32,440 WE EXPRESSED THE VIRAL NUCLEASE 402 00:17:32,440 --> 00:17:33,560 WHICH CAUSED RNA DECAY IN THE 403 00:17:33,560 --> 00:17:35,440 OUTSIDE OF INFECTION AND IF WE 404 00:17:35,440 --> 00:17:37,760 PREVENTED DEGRADATION MUCH THESE 405 00:17:37,760 --> 00:17:40,520 CLEAVED FRAGMENTS YOU NO LONGER 406 00:17:40,520 --> 00:17:43,400 HAVE THE TRANSCRIPTIONAL 407 00:17:43,400 --> 00:17:43,720 REGRESSION. 408 00:17:43,720 --> 00:17:46,000 IT'S POST TRANSLATIONAL IN 409 00:17:46,000 --> 00:17:47,640 ACTIVATION BUT INVOLVING THE 410 00:17:47,640 --> 00:17:49,440 DETECTION SOMEHOW OF THE 411 00:17:49,440 --> 00:17:50,640 DEGRADING RNA. 412 00:17:50,640 --> 00:17:52,640 WE SAT OUT TO FIGURE OUT HOW 413 00:17:52,640 --> 00:17:55,360 DOES THAT HAPPEN? 414 00:17:55,360 --> 00:17:57,800 IN THINKING ABOUT THIS WE 415 00:17:57,800 --> 00:17:59,600 CONSIDERED WHAT HAPPENS TO AN 416 00:17:59,600 --> 00:18:02,840 RNA AS IT'S BEING DEGRADED IT 417 00:18:02,840 --> 00:18:05,000 MAY BE ABLE TO SENSE? 418 00:18:05,000 --> 00:18:07,520 ONE OF THE HYPOTHESES WE ENDED 419 00:18:07,520 --> 00:18:10,160 UP TESTING WAS THE IDEA THAT 420 00:18:10,160 --> 00:18:13,280 CELLS THE RNA IN A CELL IS NOT 421 00:18:13,280 --> 00:18:13,560 NAKED. 422 00:18:13,560 --> 00:18:15,240 IT'S CODED BY SUITES OF RNA 423 00:18:15,240 --> 00:18:16,240 BINDING PROTEIN. 424 00:18:16,240 --> 00:18:20,960 AS THE RNAs ARE BEING CLEAVED 425 00:18:20,960 --> 00:18:23,920 AND THE SUBSEQUENT CLEAVAGE 426 00:18:23,920 --> 00:18:32,840 PRODUCTS BEING DESTROYED THE PRO 427 00:18:32,840 --> 00:18:33,840 PROTEINS AND THEY'RE BEING 428 00:18:33,840 --> 00:18:36,280 EVICTED AND YOU CAN ENVISION IF 429 00:18:36,280 --> 00:18:39,880 YOU'RE RAMPING UP THE RATE WITH 430 00:18:39,880 --> 00:18:41,320 WHICH RNAs ARE BEING DESTROYED 431 00:18:41,320 --> 00:18:42,960 YOU HAVE MORE PROTEINS BEING 432 00:18:42,960 --> 00:18:44,440 RELEASED FROM THE RNAs. 433 00:18:44,440 --> 00:18:46,160 WE KNOW RNA BINDING PROTEINS 434 00:18:46,160 --> 00:18:48,120 MANY OF THEM ARE SHUTTLING 435 00:18:48,120 --> 00:18:49,840 PROTEINS THAT MOVE IN AND OUT OF 436 00:18:49,840 --> 00:18:52,760 THE NUCLEUS DEPENDING AS 437 00:18:52,760 --> 00:18:54,000 MESSENGER RNA ITSELF STARTS IN 438 00:18:54,000 --> 00:18:58,520 ONE COMPARTMENT AND ENDS IN 439 00:18:58,520 --> 00:18:58,760 ANOTHER. 440 00:18:58,760 --> 00:19:00,400 WE CONSIDERED THE POSSIBILITY 441 00:19:00,400 --> 00:19:03,080 PERHAPS RELEASE OF RNA BINDING 442 00:19:03,080 --> 00:19:05,800 PROTEINS WAS BEING SENSED BY THE 443 00:19:05,800 --> 00:19:07,680 CELL AND LEADING TO THE 444 00:19:07,680 --> 00:19:08,440 TRANSCRIPTIONAL RESPONSE PERHAPS 445 00:19:08,440 --> 00:19:12,400 BECAUSE THEY'RE MOVING WITHIN 446 00:19:12,400 --> 00:19:14,240 THE CELL DIFFERENTIALLY 447 00:19:14,240 --> 00:19:15,920 DEPENDING IF THEY'RE BOUND OR 448 00:19:15,920 --> 00:19:17,400 RELEASED FROM RNA. 449 00:19:17,400 --> 00:19:23,520 WE TEST THE HYPOTHESIS WITH A 450 00:19:23,520 --> 00:19:25,840 LAB BY ANALYZING THE PROTEIN 451 00:19:25,840 --> 00:19:26,960 DISTRIBUTION AND REDISTRIBUTION 452 00:19:26,960 --> 00:19:30,640 THAT HAPPENS IN THE CELL IN 453 00:19:30,640 --> 00:19:31,120 RESPONSE TO RNA DECAY. 454 00:19:31,120 --> 00:19:32,440 THE FIRST TIME WE DID THIS WE 455 00:19:32,440 --> 00:19:34,240 DID IT IN THE MINIMALIST SYSTEM 456 00:19:34,240 --> 00:19:36,880 WE'RE JUST TRYING TO ISOLATE THE 457 00:19:36,880 --> 00:19:41,200 RNA DECAY PHENOTYPE AND TAKING 458 00:19:41,200 --> 00:19:41,480 H 459 00:19:41,480 --> 00:19:43,720 HEXTILES OUT OF THE CONTEXT OF 460 00:19:43,720 --> 00:19:46,920 INFECTION AND WE EITHER HAD A 461 00:19:46,920 --> 00:19:47,920 WILD TYPE VERSION OF THE 462 00:19:47,920 --> 00:19:57,040 NUCLEASE, WE HAD A CATALYTICALLY 463 00:19:57,040 --> 00:19:59,160 VERSION AND WE RELEASED THE 464 00:19:59,160 --> 00:20:00,400 BINDING PROTEINS AND THAT WAS 465 00:20:00,400 --> 00:20:05,440 CARRIED OUT BY THE CELLULAR 466 00:20:05,440 --> 00:20:07,240 NUCLEASE EXERON 1 TO SLOW THE 467 00:20:07,240 --> 00:20:12,400 RATE OF DECAY OF THE ALREADY 468 00:20:12,400 --> 00:20:13,880 CLEAVED TRANSCRIPT. 469 00:20:13,880 --> 00:20:16,200 EACH OF THESE CELLS WE 470 00:20:16,200 --> 00:20:19,760 FRACTIONATED AND EXTRACTED TOLD 471 00:20:19,760 --> 00:20:23,920 PROTEIN LATED WITH TAGS AND 472 00:20:23,920 --> 00:20:34,200 IDENTIFIED BY MASS SPECTOMETRY 473 00:20:34,200 --> 00:20:35,320 AND LOOKING FOR THE SIGNATURE 474 00:20:35,320 --> 00:20:37,640 AND THAT WOULD BE THE ABUNDANCE 475 00:20:37,640 --> 00:20:40,080 OF A PROTEIN UP IN ONE 476 00:20:40,080 --> 00:20:43,680 COMPARTMENT AND DOWN IN A 477 00:20:43,680 --> 00:20:45,040 COMPENSATORY WAY IN ANOTHER 478 00:20:45,040 --> 00:20:45,800 COMPARTMENT. 479 00:20:45,800 --> 00:20:47,720 IF WE LOOK FOR THEM MOVING FROM 480 00:20:47,720 --> 00:20:52,040 THE CYTOPLASM TO NUCLEUS YOU SEE 481 00:20:52,040 --> 00:20:57,440 A DECREASE IN THE CYTOPLASMIC 482 00:20:57,440 --> 00:20:59,800 SIGNAL AND THAT WAS THE DOMINANT 483 00:20:59,800 --> 00:21:00,040 PHENOTYPE. 484 00:21:00,040 --> 00:21:03,160 WE SAW A CLEAR SIGNATURE IN 485 00:21:03,160 --> 00:21:04,400 CELLS EXPRESSING THE WILD TYPE 486 00:21:04,400 --> 00:21:07,240 NUCLEASE OF PROTEINS THAT WERE 487 00:21:07,240 --> 00:21:08,400 SHIFTING THEIR DISTRIBUTION FROM 488 00:21:08,400 --> 00:21:10,920 THE CYTOPLASM OF THE CELL TO THE 489 00:21:10,920 --> 00:21:11,520 NUCLEUS OF THE CELL. 490 00:21:11,520 --> 00:21:13,920 AND SO WHAT I'M SHOWING YOU HERE 491 00:21:13,920 --> 00:21:16,440 ARE THESE RESULTS IN A HEAT MAP 492 00:21:16,440 --> 00:21:18,960 IN WHICH YOU CAN SEE THAT 493 00:21:18,960 --> 00:21:23,920 COMPARED TO THE CATALYTICALLY 494 00:21:23,920 --> 00:21:25,640 INACTIVE VERSION OF THE NUCLEASE 495 00:21:25,640 --> 00:21:30,000 THERE'S A SET OF PROTEINS 30 OR 496 00:21:30,000 --> 00:21:33,240 SO THAT REPRODUCIBLY ACCUMULATE 497 00:21:33,240 --> 00:21:38,880 IN THE NUCLEUS IN THE CELLS AND 498 00:21:38,880 --> 00:21:42,520 70% ARE NO LONGER IN CELLS 499 00:21:42,520 --> 00:21:44,000 LACKING EXERON 1. 500 00:21:44,000 --> 00:21:46,800 WHEN THEY SHIFT WE DID AN 501 00:21:46,800 --> 00:21:48,800 ANALYSIS AND THE ONLY SIGNATURE 502 00:21:48,800 --> 00:21:51,080 THAT POPS OUT ARE THINGS RELATED 503 00:21:51,080 --> 00:21:53,440 TO MESSENGER RNA BINDING. 504 00:21:53,440 --> 00:21:54,960 AGAIN, RNA BINDING PROTEIN 505 00:21:54,960 --> 00:21:59,800 SIGNATURES LIKE POLY A AND U 506 00:21:59,800 --> 00:22:02,480 BINDING AND MESSENGER RNA, UTR 507 00:22:02,480 --> 00:22:03,840 BINDING THE SORTS OF PROTEINS 508 00:22:03,840 --> 00:22:06,520 YOU EXPECT TO BE RELEASED FROM A 509 00:22:06,520 --> 00:22:07,680 DEGRADING RNA ONCE RELEASED 510 00:22:07,680 --> 00:22:10,480 TRANSITING TO THE NUCLEUS. 511 00:22:10,480 --> 00:22:13,840 WHAT THIS TELLS US IS INDEED RNA 512 00:22:13,840 --> 00:22:16,440 DESTRUCTION IN THE CYTOPLASM IF 513 00:22:16,440 --> 00:22:21,440 YOU RAMP THAT UP CAUSES RELEASE 514 00:22:21,440 --> 00:22:26,040 OF BINDING PROTEINS ANDS WITH 515 00:22:26,040 --> 00:22:29,400 THE REDUCTION IN POL 2 OCCUPANCY 516 00:22:29,400 --> 00:22:30,960 IN THE HOST GENOME AND 517 00:22:30,960 --> 00:22:31,840 TRANSCRIPTION BUT DOESN'T TELL 518 00:22:31,840 --> 00:22:34,240 US IF THE REDISTRIBUTION OF THE 519 00:22:34,240 --> 00:22:35,640 RNA BINDING PROTEINS IS 520 00:22:35,640 --> 00:22:38,400 NECESSARY TO CONVEY THE SIGNAL 521 00:22:38,400 --> 00:22:41,840 OF CYTOPLASMIC TRANSCRIPTION. 522 00:22:41,840 --> 00:22:46,280 TO TEST THAT A FORMER STUDENT 523 00:22:46,280 --> 00:22:49,200 CHRIS DONE CAN LEWIS USED A DRUG 524 00:22:49,200 --> 00:22:52,840 THAT TARGETS THE NUCLEAR IMPORT 525 00:22:52,840 --> 00:22:53,120 MACHINERY. 526 00:22:53,120 --> 00:22:55,640 THE IMPORTANT ALPHA BETA COMPLEX 527 00:22:55,640 --> 00:22:57,560 IMPORTANT FOR RELOCALIZING 528 00:22:57,560 --> 00:22:59,360 PROTEINS TO THE NUCLEUS. 529 00:22:59,360 --> 00:23:02,840 WHAT HE COULD SEE IS IN CELLS 530 00:23:02,840 --> 00:23:08,400 WHERE HE TREATED WITH MOX A DMSO 531 00:23:08,400 --> 00:23:09,360 TREATMENT THAT EXPRESSED THE SOX 532 00:23:09,360 --> 00:23:12,440 PROTEIN WE'RE LOOKING AT POL 2 533 00:23:12,440 --> 00:23:15,080 OCCUPANCY HERE AT A COUPLE 534 00:23:15,080 --> 00:23:17,760 REPRESENTATIVE HOST PROMOTERS IN 535 00:23:17,760 --> 00:23:19,280 CELLS EXPRESSING THIS YOU HAVE 536 00:23:19,280 --> 00:23:21,480 THE DECREASE IN POL 2 OCCUPANCY 537 00:23:21,480 --> 00:23:26,680 BECAUSE OF CYTOPLASMIC RNA DECAY 538 00:23:26,680 --> 00:23:28,280 AND IF THE BLOCKS THE TRANSPORT 539 00:23:28,280 --> 00:23:32,040 BY TREATING THE CELLS BIG THIS 540 00:23:32,040 --> 00:23:35,320 THE CELL STILL UNDER GO THE SAME 541 00:23:35,320 --> 00:23:38,800 RNA DECAY IN THE CYTOPLASM BUT 542 00:23:38,800 --> 00:23:43,640 NO LONGER CAUSE THE 543 00:23:43,640 --> 00:23:46,640 TRANSCRIPTIONAL REPRESSION IT'S 544 00:23:46,640 --> 00:23:48,360 THE SIGNAL READ OUT TO THE CELL 545 00:23:48,360 --> 00:23:50,920 TO TRIGGER THIS TRANSCRIPTIONAL 546 00:23:50,920 --> 00:23:51,280 REPRESSION. 547 00:23:51,280 --> 00:23:53,240 SO ONE OF THE THINGS WE'RE 548 00:23:53,240 --> 00:23:54,520 INTERESTED IN NOW IS THIS IS 549 00:23:54,520 --> 00:23:58,320 KIND OF A LONG LIST OF PROTEINS 550 00:23:58,320 --> 00:24:00,800 HERE SHUTTLING AND IS IT THAT 551 00:24:00,800 --> 00:24:02,640 INDIVIDUAL PROTEINS WITHIN THE 552 00:24:02,640 --> 00:24:04,360 LIST ARE KEY PLAYERS EITHER 553 00:24:04,360 --> 00:24:06,360 CONTACTING OR SIGNALLING THINGS 554 00:24:06,360 --> 00:24:09,520 IN THE NUCLEUS TO ELICIT THE 555 00:24:09,520 --> 00:24:10,080 TRANSCRIPTIONAL REGRESSION 556 00:24:10,080 --> 00:24:13,240 RESPONSE OR THE MASS ACTION OF 557 00:24:13,240 --> 00:24:16,400 GENERALLY REDISTRIBUTING THE RNA 558 00:24:16,400 --> 00:24:17,520 BINDING PROTEINS THAT'S CAUSING 559 00:24:17,520 --> 00:24:20,680 A STRESS SIGNAL IN THE CELL THAT 560 00:24:20,680 --> 00:24:25,040 LEADS TO TRANSCRIPTIONAL 561 00:24:25,040 --> 00:24:25,440 REPRESSION? 562 00:24:25,440 --> 00:24:30,360 WE FAVOR THE KEY HYPOTHESIS AND 563 00:24:30,360 --> 00:24:31,680 DOING KNOCKOUT AND 564 00:24:31,680 --> 00:24:32,200 OVEREXPRESSION FOR THE 565 00:24:32,200 --> 00:24:33,240 INDIVIDUAL RNA BINDING PROTEINS 566 00:24:33,240 --> 00:24:40,240 AND FOUND A COUPLE OF EXAMPLES 567 00:24:40,240 --> 00:24:41,440 WHERE INDIVIDUAL PROTEINS LUKE 568 00:24:41,440 --> 00:24:44,360 POLY A SEEM TO PLAY IMPORTANT 569 00:24:44,360 --> 00:24:45,560 ROLES AND OTHERS I WON'T SHOW 570 00:24:45,560 --> 00:24:47,400 THE DATA FOR BECAUSE THEY SHOWED 571 00:24:47,400 --> 00:24:48,360 NO PHENOTYPE WHEN WE DID THESE 572 00:24:48,360 --> 00:24:56,040 EXPERIMENTS. 573 00:24:56,040 --> 00:24:59,160 AND CYTOPLASMIC POLY A BINDING 574 00:24:59,160 --> 00:25:00,840 PROTEINS ARE STUDIED FOR A LONG 575 00:25:00,840 --> 00:25:03,440 TIME AND THEY BIND TO THE POLY A 576 00:25:03,440 --> 00:25:06,440 TAIL OF MESSENGER RNAs AND PLAY 577 00:25:06,440 --> 00:25:10,640 IMPORTANT ROLES INSTABILITY AND 578 00:25:10,640 --> 00:25:15,960 TRANSLATIONAL COMPETENCE IN THE 579 00:25:15,960 --> 00:25:18,200 CYTOPLASM AND THEY TEND TO GET 580 00:25:18,200 --> 00:25:19,920 RELOCALIZED AND ACCUMULATE IN 581 00:25:19,920 --> 00:25:22,560 THE NUCLEUS IN RESPONSE TO LARGE 582 00:25:22,560 --> 00:25:24,160 RNA CHANGES IN THE CYTOPLASM 583 00:25:24,160 --> 00:25:25,480 THAT OCCUR DURING INFECTION. 584 00:25:25,480 --> 00:25:27,280 IT'S NOT A PHENOTYPE WE SEE ONLY 585 00:25:27,280 --> 00:25:30,680 IN HERPES VIRUS INFECTION OR IN 586 00:25:30,680 --> 00:25:34,080 GAMMA AND SEEN IN ALPHA HERPES 587 00:25:34,080 --> 00:25:40,320 AND OTHER VIRUSES AS WELL IN THE 588 00:25:40,320 --> 00:25:42,160 NORMAL LEVELS, EXPRESSION OF THE 589 00:25:42,160 --> 00:25:45,000 SOX PROTEIN CAUSE THE STRONG 590 00:25:45,000 --> 00:25:45,600 TRANSCRIPTIONAL REPRESSION 591 00:25:45,600 --> 00:25:48,040 HOWEVER, IF WE DEPLETE THE POLY 592 00:25:48,040 --> 00:25:50,840 A BINDING PROTEINS FROM CELLS 593 00:25:50,840 --> 00:25:52,240 YOU SEE THE PHENOTYPE IS 594 00:25:52,240 --> 00:25:54,400 REVERSED SUGGESTING THEY'RE 595 00:25:54,400 --> 00:25:56,320 PLAYING A ROLE IN THE REPRESSION 596 00:25:56,320 --> 00:25:56,760 PHENOTYPE. 597 00:25:56,760 --> 00:25:58,280 WE CAN ALSO ASK WHAT HAPPENS IF 598 00:25:58,280 --> 00:26:00,360 YOU JUST DRIVE THE PROTEINS INTO 599 00:26:00,360 --> 00:26:02,400 THE NUCLEUS IN THE ABSENCE OF 600 00:26:02,400 --> 00:26:04,440 RNA DECAY IN THE CYTOPLASM. 601 00:26:04,440 --> 00:26:06,480 WE CAN DO THIS THROUGH A COUPLE 602 00:26:06,480 --> 00:26:07,800 OF STRATEGIES. 603 00:26:07,800 --> 00:26:09,520 ONE OF WHICH IS A NUMBER OF 604 00:26:09,520 --> 00:26:11,120 YEARS AGO WE HAD SHOWN THE 605 00:26:11,120 --> 00:26:16,440 REASON POLY A BINDING PROTEINS 606 00:26:16,440 --> 00:26:19,720 THE REASON THEY'RE RETAINED IN 607 00:26:19,720 --> 00:26:22,080 THECYTE 608 00:26:22,080 --> 00:26:24,600 THECYTEOPLASM THEY HAVE A SIGNAL 609 00:26:24,600 --> 00:26:27,160 HIDDEN IN THE RECOGNITION MOTIFS 610 00:26:27,160 --> 00:26:30,160 AND THE SIGNAL IS MASKED AND 611 00:26:30,160 --> 00:26:32,040 KEPT IN THE CYTOPLASM AS THE RNA 612 00:26:32,040 --> 00:26:33,520 IS DESTROYED AND THEY'RE 613 00:26:33,520 --> 00:26:35,600 RELEASED, THE NUCLEAR 614 00:26:35,600 --> 00:26:37,800 LOCALIZATION SIGNAL IS EXPOSED, 615 00:26:37,800 --> 00:26:42,800 BINDS IMPORTANT ALPHAAN AND 616 00:26:42,800 --> 00:26:45,360 TRANSPORTED TO THE NUCLEUS. 617 00:26:45,360 --> 00:26:51,440 THE LEVELS ARE CAREFULLY TRI 618 00:26:51,440 --> 00:26:53,960 TRITATED AND IT GOES NOT NUCLEUS 619 00:26:53,960 --> 00:26:54,680 BECAUSE IT'S EXPOSED. 620 00:26:54,680 --> 00:27:00,360 WHEN WE DO THIS IN THE ABSENCE 621 00:27:00,360 --> 00:27:03,800 OF CYTOPLASMIC mRNA DECAY AND 622 00:27:03,800 --> 00:27:04,440 INCREASING THE CONCENTRATION IN 623 00:27:04,440 --> 00:27:09,320 THE NUCLEUS CAUSES A DECREASE IN 624 00:27:09,320 --> 00:27:11,640 POL 2 OCCUPANCY AT THE CELLULAR 625 00:27:11,640 --> 00:27:11,920 PROMOTERS. 626 00:27:11,920 --> 00:27:15,280 FOR THIS REASON WE THINK THE 627 00:27:15,280 --> 00:27:19,720 PROTEINS AS WELL AS WE SUSPECT A 628 00:27:19,720 --> 00:27:20,840 COUPLE OTHERS MAY BE KEY PLAYERS 629 00:27:20,840 --> 00:27:23,320 IN REMEDIATING THE 630 00:27:23,320 --> 00:27:23,880 TRANSCRIPTIONAL EXPRESSION. 631 00:27:23,880 --> 00:27:25,720 NOW THE QUESTION IS HOW ARE THEY 632 00:27:25,720 --> 00:27:26,840 DEGREE THAT? 633 00:27:26,840 --> 00:27:27,520 THIS IS AN ONGOING PIECE OF 634 00:27:27,520 --> 00:27:36,240 WORK. 635 00:27:36,240 --> 00:27:37,680 I'LL SHARE SHARE FAVORITE 636 00:27:37,680 --> 00:27:39,760 HYPOTHESIS GENERATED BY A 637 00:27:39,760 --> 00:27:41,000 CURRENT GRADUATE STUDENT SAM 638 00:27:41,000 --> 00:27:43,040 RYDER WHO CAME UP WITH THE IDEA 639 00:27:43,040 --> 00:27:44,400 THAT PERHAPS WHAT HAPPENS WHEN 640 00:27:44,400 --> 00:27:46,840 YOU HAVE AN INFLUX OF THE RNA 641 00:27:46,840 --> 00:27:49,680 BINDING PROTEINS IS THAT THEY 642 00:27:49,680 --> 00:27:51,240 MAY COMPETE WITH RESIDENT 643 00:27:51,240 --> 00:27:52,440 NUCLEAR BINDING PROTEINS WHICH 644 00:27:52,440 --> 00:27:56,560 OF WHICH PLAY IMPORTANT ROLES IN 645 00:27:56,560 --> 00:27:57,720 CO-TRANSCRIPTIONAL RNA 646 00:27:57,720 --> 00:27:58,880 PROCESSING ACTIVITY HAPPEN 647 00:27:58,880 --> 00:28:00,400 DURING THE PROCESS OF 648 00:28:00,400 --> 00:28:02,000 TRANSCRIPTION. 649 00:28:02,000 --> 00:28:05,080 AND INDEED EVEN THE PROCESS OF 650 00:28:05,080 --> 00:28:07,680 TRANSCRIPTION HELPS COMPETE OFF 651 00:28:07,680 --> 00:28:12,520 CHROMATIN MODIFIERIERS MAKE LOCUS 652 00:28:12,520 --> 00:28:12,800 COMPETENT. 653 00:28:12,800 --> 00:28:18,200 AND SHE HYPOTHESIZES AS THEY GO 654 00:28:18,200 --> 00:28:19,760 IN THE NUCLEUS THEY MAY 655 00:28:19,760 --> 00:28:22,800 RE-OCCUPY THE SPACE THE NORMAL 656 00:28:22,800 --> 00:28:25,520 mRNA BINDING PROTEINS OCCUPY ON 657 00:28:25,520 --> 00:28:28,720 NASCENT RNA AND THEREFORE 658 00:28:28,720 --> 00:28:31,880 DISRUPT THE RNA EARLY ON IN THE 659 00:28:31,880 --> 00:28:33,520 TRANSCRIPTION PROCESS AND ALTER 660 00:28:33,520 --> 00:28:33,840 TRANSCRIPTION. 661 00:28:33,840 --> 00:28:36,440 THESE IN THE PROCESS OF PURIFY 662 00:28:36,440 --> 00:28:40,040 PURIFYING NASCENT WITH THE 663 00:28:40,040 --> 00:28:41,640 PROTEOME ASSOCIATED WITH CELLS 664 00:28:41,640 --> 00:28:45,600 UNDERGOING ACCELERATEDD DECAY AND 665 00:28:45,600 --> 00:28:49,440 LOOKING AT WHAT IS THERE BY MASS 666 00:28:49,440 --> 00:28:53,480 SPEC 667 00:28:53,480 --> 00:28:55,480 SPECTOMETRY AND SEEING THE RVPs 668 00:28:55,480 --> 00:28:58,040 TO THOSE THAT MOVED IN THE 669 00:28:58,040 --> 00:28:59,160 CYTOPLASM. 670 00:28:59,160 --> 00:29:01,800 I'LL CLOSE THIS SECTION BY 671 00:29:01,800 --> 00:29:03,640 SAYING WE ASSUME THAT THIS 672 00:29:03,640 --> 00:29:07,360 PATHWAY WE THINK IS RELATED TO 673 00:29:07,360 --> 00:29:09,440 CELL STRESS SENSED BY REMOVAL OF 674 00:29:09,440 --> 00:29:11,800 RNA BY THE CYTOPLASM IS BEING 675 00:29:11,800 --> 00:29:14,840 USED PROBABLY DURING A NUMBER OF 676 00:29:14,840 --> 00:29:16,040 VIRAL INFECTIONS AND PATHWAYS 677 00:29:16,040 --> 00:29:18,680 TRIGGERED BY VIRUSES WE THINK 678 00:29:18,680 --> 00:29:19,840 ARE THERE FOR OTHER REASONS AS 679 00:29:19,840 --> 00:29:21,040 WELL. 680 00:29:21,040 --> 00:29:22,760 SO WE ASSUMED THAT SURELY THERE 681 00:29:22,760 --> 00:29:25,320 ARE CASES IF THIS IS A NORMAL 682 00:29:25,320 --> 00:29:29,160 CELLULAR RESPONSE TO REMOVAL OF 683 00:29:29,160 --> 00:29:31,760 RNA WHERE NON-VIRAL STRESSES MAY 684 00:29:31,760 --> 00:29:36,440 TRIGGER THE ACTIVATION. 685 00:29:36,440 --> 00:29:46,920 INDEED CHRIS DUNCAN LEWIS AND AS 686 00:29:46,920 --> 00:29:50,320 DISCOVERED BY THE FREEDMAN LAB A 687 00:29:50,320 --> 00:29:54,520 FORMER LECTURER AS A SIGNATURE 688 00:29:54,520 --> 00:29:59,400 FEATURE OF APOPTOSIS BEFORE 689 00:29:59,400 --> 00:30:05,960 THERE'S GROSS CHANGES AND A NEWS 690 00:30:05,960 --> 00:30:08,440 NUCLEUS THAT IS RELEASED AND 691 00:30:08,440 --> 00:30:10,720 CHRIS FOUND LIKE VIRAL SITUATION 692 00:30:10,720 --> 00:30:13,280 THIS CAUSES RNA BINDING PROTEIN 693 00:30:13,280 --> 00:30:14,160 RELOCALIZATION TO THE NUCLEUS IN 694 00:30:14,160 --> 00:30:17,080 THE CELLS AND A SELECTIVE PRE 695 00:30:17,080 --> 00:30:19,240 INVESTIGATION OF POL 2 ACTIVITY 696 00:30:19,240 --> 00:30:21,280 BUT NOT POL 3 ACTIVITY WHICH 697 00:30:21,280 --> 00:30:23,880 AGAIN IS SOMETHING THAT WE HAD 698 00:30:23,880 --> 00:30:25,680 SEEN IN THE VIRAL CONTEXT AS 699 00:30:25,680 --> 00:30:26,480 WELL. 700 00:30:26,480 --> 00:30:30,600 MESSENGER RNA DECAY TRIGGERING 701 00:30:30,600 --> 00:30:32,320 POL 2 TRANSCRIPTIONAL 702 00:30:32,320 --> 00:30:34,080 REPRESSION. 703 00:30:34,080 --> 00:30:34,360 OKAY. 704 00:30:34,360 --> 00:30:37,160 SO THAT'S THE FIRST PATHWAY. 705 00:30:37,160 --> 00:30:39,320 ONE IN WHICH RNA DESTRUCTION 706 00:30:39,320 --> 00:30:41,280 ITSELF IS DETECTED. 707 00:30:41,280 --> 00:30:43,280 BUT I TOLD YOU THAT THERE WERE 708 00:30:43,280 --> 00:30:44,280 TWO PATHWAYS INVOLVED. 709 00:30:44,280 --> 00:30:47,360 ALSO A CONTROL OF THE GENERAL 710 00:30:47,360 --> 00:30:48,960 PROTEIN ABUNDANCE OF 711 00:30:48,960 --> 00:30:50,720 TRANSCRIPTIONAL REGULATORS. 712 00:30:50,720 --> 00:30:51,840 THIS PATHWAY WE FOUND WHEN WE 713 00:30:51,840 --> 00:30:57,520 WENT BACK AND REPEATED THESE TMT 714 00:30:57,520 --> 00:30:59,360 BASED MASS SPECT EXPERIMENTS BUT 715 00:30:59,360 --> 00:31:00,640 IN THE CONTEXT OF INFECTION. 716 00:31:00,640 --> 00:31:03,400 THIS PATHWAY ONLY MANIFESTED IN 717 00:31:03,400 --> 00:31:04,960 INFECTED CELLS AND EXPRESSION OF 718 00:31:04,960 --> 00:31:09,200 VIRAL NUCLEASE WAS NOT ENOUGH TO 719 00:31:09,200 --> 00:31:14,160 TRIGGER IT AND WHEN REPEAT THIS 720 00:31:14,160 --> 00:31:18,040 USING MICE FIBROBLASTS INFECTED 721 00:31:18,040 --> 00:31:21,400 WITH THE SOX VERSION OR ANOTHER 722 00:31:21,400 --> 00:31:27,000 VERSION WE SAW THE SAME BINDING 723 00:31:27,000 --> 00:31:29,560 PHENOTYPE WE SAW IN THE SOX 724 00:31:29,560 --> 00:31:30,720 EXPRESSING CELLS AND SAW 725 00:31:30,720 --> 00:31:32,040 SOMETHING ELSE. 726 00:31:32,040 --> 00:31:35,240 IT WAS A GENERALIZED DECREASE IN 727 00:31:35,240 --> 00:31:41,640 MANY OF THE SUBUNITS OF mRNA 728 00:31:41,640 --> 00:31:43,240 POLYMERASE AND POL 2 IS AN 729 00:31:43,240 --> 00:31:49,040 ENZYME AND 7 OF 12 SUB UNITS OF 730 00:31:49,040 --> 00:31:52,880 RNA POLYMERASE ARE DEPLETE WITH 731 00:31:52,880 --> 00:31:54,320 THE WILD TYPE BUT NOT THE SOX 732 00:31:54,320 --> 00:31:57,680 MUTANT AND NOT JUST RELOCALIZED 733 00:31:57,680 --> 00:32:00,040 BUT SELECTIVELY DELETED FROM THE 734 00:32:00,040 --> 00:32:02,840 NUCLEUS AND NOT CYTOPLASM. 735 00:32:02,840 --> 00:32:06,240 ONE INTERESTING THING IS IT HAS 736 00:32:06,240 --> 00:32:08,720 SEVEN SUB UNITS AND FIVE SHARED 737 00:32:08,720 --> 00:32:12,240 WITH THE OTHER POLYMERASES POL 1 738 00:32:12,240 --> 00:32:14,080 AND POL 3. 739 00:32:14,080 --> 00:32:16,280 THE SHARED UNITS WAS NOT 740 00:32:16,280 --> 00:32:16,680 CHANGING. 741 00:32:16,680 --> 00:32:19,720 SOMETHING ABOUT INFECTION WAS 742 00:32:19,720 --> 00:32:22,440 SPECIFICALLY DELETING THE POL 2 743 00:32:22,440 --> 00:32:23,040 SPECIFIC SUB UNITS. 744 00:32:23,040 --> 00:32:23,480 WOE THOUGHT THAT WAS 745 00:32:23,480 --> 00:32:27,320 INTERESTING. 746 00:32:27,320 --> 00:32:30,600 A NUMBER OF VIRUS KNOWN TO 747 00:32:30,600 --> 00:32:31,640 TARGET POL II. 748 00:32:31,640 --> 00:32:36,360 GENERAL MY PEOPLE LOOK AT THE 749 00:32:36,360 --> 00:32:37,560 LARGE CATALYTIC SUBUNIT AND THIS 750 00:32:37,560 --> 00:32:40,240 IS THE FIRST DEMONSTRATION THAT 751 00:32:40,240 --> 00:32:41,640 VIRAL INFECTION COULD IN FACT 752 00:32:41,640 --> 00:32:43,680 LEAD TO DEPLETION OF MULTIPLE 753 00:32:43,680 --> 00:32:47,880 BUT NOT ALL POL II SUB UNITS. 754 00:32:47,880 --> 00:32:54,120 THIS SUBUNIT DEPLETION WE SEE IN 755 00:32:54,120 --> 00:32:58,160 KSHV INFECTED CELLS HAPPENS 756 00:32:58,160 --> 00:33:01,120 THROUGH TURN OVER. 757 00:33:01,120 --> 00:33:04,320 WE'RE TAKING B CELL, BCBL CELL 758 00:33:04,320 --> 00:33:07,320 LINE INFECTED WITH KSHV AND 759 00:33:07,320 --> 00:33:08,720 LOOKING AT WHAT THE HALF LIFE OF 760 00:33:08,720 --> 00:33:10,960 THE PROTEINS ARE IN THE CELLS IN 761 00:33:10,960 --> 00:33:14,720 THEIR LATENT STATE WITH NO HOST 762 00:33:14,720 --> 00:33:16,720 SHUT OFF HAPPENING VERSUS WHEN 763 00:33:16,720 --> 00:33:20,400 WE ENTER THEM IN THE LYTIC 764 00:33:20,400 --> 00:33:20,640 STATE. 765 00:33:20,640 --> 00:33:24,400 I'M SHOWING ONE EXAMPLE THE RPB2 766 00:33:24,400 --> 00:33:27,320 SUB UNIT AND A POL II SPECIFIC 767 00:33:27,320 --> 00:33:37,240 SUB UNIT IF WE TREAT CELLS WITH 768 00:33:37,240 --> 00:33:39,800 CYCLOHEXIMIDE YOU CAN SEE THE 769 00:33:39,800 --> 00:33:42,000 PROTEIN IN THE REACTIVATED CELLS 770 00:33:42,000 --> 00:33:43,480 IS TURNING OVER MORE RAPIDLY 771 00:33:43,480 --> 00:33:45,720 THAN IN THE UNREACTIVATED CELLS 772 00:33:45,720 --> 00:33:49,080 AND ACCELERATED PROTEIN TURNOVER 773 00:33:49,080 --> 00:33:53,800 AND SEE IT FOR THE POL II SUB 774 00:33:53,800 --> 00:33:55,640 UNITS AND WE THINK SOMETHING IS 775 00:33:55,640 --> 00:33:58,080 ACTIVATING A PROTEIN TURNOVER OF 776 00:33:58,080 --> 00:34:00,200 THE POL II SPECIFIC SUB UNITS 777 00:34:00,200 --> 00:34:03,800 AND INFECTED CELLS. 778 00:34:03,800 --> 00:34:05,040 I MENTIONED THERE'S SEVEN 779 00:34:05,040 --> 00:34:06,480 DIFFERENT POL II SUB UNITS. 780 00:34:06,480 --> 00:34:08,440 IS IT THE VIRUS HAVING A 781 00:34:08,440 --> 00:34:10,720 STRATEGY TO SOMEHOW PROMOTE 782 00:34:10,720 --> 00:34:14,760 TURNOVER OF ALL OF THESE SEVEN 783 00:34:14,760 --> 00:34:16,400 SUBUNITS SEPARATELY? 784 00:34:16,400 --> 00:34:19,320 OR ALTERNATIVELY, IS IT POSSIBLE 785 00:34:19,320 --> 00:34:22,640 IT IS TARGET SINGLE SUB UNIT AND 786 00:34:22,640 --> 00:34:27,280 WHEN HAVE YOU PROTEIN COMPLEXES 787 00:34:27,280 --> 00:34:29,120 THEIR STABILITY IS DEPENDENT ON 788 00:34:29,120 --> 00:34:30,240 THE FORMATION OF THAT COMPLEX 789 00:34:30,240 --> 00:34:31,040 ITSELF. 790 00:34:31,040 --> 00:34:34,040 IF YOU REMOVE A COMPLEX SUBUNIT 791 00:34:34,040 --> 00:34:36,440 FROM A PROTEIN COMPLEX IT CAN 792 00:34:36,440 --> 00:34:40,280 LEAD TO DESTABILIZATION OF THE 793 00:34:40,280 --> 00:34:44,360 OTHER MEMBERS OF THIS COMPLEX. 794 00:34:44,360 --> 00:34:47,200 AND PERHAPS IT'S A SINGLE SUB 795 00:34:47,200 --> 00:34:50,520 UNIT AND CO-DEPLETION OF A 796 00:34:50,520 --> 00:34:53,240 PROTEINS ALL THE POL II SPECIFIC 797 00:34:53,240 --> 00:34:55,760 SUB COMPLEX OF THAT POLYMERASE. 798 00:34:55,760 --> 00:34:58,800 WE THINK THIS IS PROBABLY THE 799 00:34:58,800 --> 00:34:59,000 CASE. 800 00:34:59,000 --> 00:35:04,040 WE COULD DO THIS BY SELECTIVELY 801 00:35:04,040 --> 00:35:09,480 DEPLETING RPB1 IN UNINFECTED 802 00:35:09,480 --> 00:35:12,840 CELLS AND WHEN WE DEPLETE THE 803 00:35:12,840 --> 00:35:19,520 SUB UNIT OF POL II WE SEE 804 00:35:19,520 --> 00:35:21,560 CO-DEPLETION OF OTHER SUB UNITS 805 00:35:21,560 --> 00:35:24,000 SUBJECT TO POLYMERASE II AND 806 00:35:24,000 --> 00:35:28,080 LESS DEPLETION OF RPB5. 807 00:35:28,080 --> 00:35:29,840 OUR HYPOTHESIS IS THE VIRUS IS 808 00:35:29,840 --> 00:35:31,160 TARGETING A PARTICULAR SUB UNIT 809 00:35:31,160 --> 00:35:34,360 OR ELICITING A STRESS TARGETING 810 00:35:34,360 --> 00:35:37,720 A PARTICULAR SUB UNIT OF POL 2 811 00:35:37,720 --> 00:35:38,840 FOR DESTRUCTION. 812 00:35:38,840 --> 00:35:42,440 IT'S LEADING TO CO-DEPLETION OF 813 00:35:42,440 --> 00:35:44,320 SUB UNITS CONTRIBUTING TO THE 814 00:35:44,320 --> 00:35:45,520 TRANSCRIPTIONAL SHUT DOWN WE SEE 815 00:35:45,520 --> 00:35:46,200 IN CELLS. 816 00:35:46,200 --> 00:35:49,680 TO SUMMARIZE WHAT I'VE SHOWN YOU 817 00:35:49,680 --> 00:35:52,360 IN THE FIRST PART IS THAT DURING 818 00:35:52,360 --> 00:35:54,600 GAMMA HERPES VIRUS INFECTION 819 00:35:54,600 --> 00:35:55,960 THERE'S A LARGE SCALE DEPLETION 820 00:35:55,960 --> 00:35:59,680 OF MESSENGER RNA FROM THE 821 00:35:59,680 --> 00:36:00,280 CYTOPLA 822 00:36:00,280 --> 00:36:02,680 CYTOPLASM INITIATED FROM THE 823 00:36:02,680 --> 00:36:05,840 SPECIFIC NUCLEASE CALLED SOX. 824 00:36:05,840 --> 00:36:07,360 THIS RNA DECAY PHENOTYPE SIGNALS 825 00:36:07,360 --> 00:36:10,240 IN AT LEAST TWO DIFFERENT WAYS 826 00:36:10,240 --> 00:36:17,640 FROM THE CYTOPLASM TO CAUSE 827 00:36:17,640 --> 00:36:18,960 REPRESSION BY POLYMERASE II AND 828 00:36:18,960 --> 00:36:21,600 THE BINDING PROTEINS FROM THE 829 00:36:21,600 --> 00:36:23,640 DEGRADING RNA FRAGMENTS AND THE 830 00:36:23,640 --> 00:36:25,000 SECOND MECHANISM WE THINK I 831 00:36:25,000 --> 00:36:31,760 DIDN'T SHOW THE DATA BUT A 832 00:36:31,760 --> 00:36:33,840 PROTEOSOME UNIT SPECIFIC TO POL 833 00:36:33,840 --> 00:36:36,400 II NOT SHARED WITH THE OTHER 834 00:36:36,400 --> 00:36:37,400 POLYMERASES. 835 00:36:37,400 --> 00:36:40,040 SO YOU MIGHT BE WONDERING, WELL, 836 00:36:40,040 --> 00:36:42,040 THESE VIRUSES ARE 837 00:36:42,040 --> 00:36:44,400 DOUBLE-STRANDED DNA VIRUSES. 838 00:36:44,400 --> 00:36:48,400 THEY'RE TRANSCRIPTION DEPENDS ON 839 00:36:48,400 --> 00:36:50,160 RNA POLYMERASE II LIKE THE HOST 840 00:36:50,160 --> 00:36:50,640 DOES. 841 00:36:50,640 --> 00:36:52,360 HOW ARE THEY ABLE TO GET AROUND 842 00:36:52,360 --> 00:36:53,320 THE PHENOTYPE? 843 00:36:53,320 --> 00:36:54,920 OBVIOUSLY THEY DO SINCE THEY'RE 844 00:36:54,920 --> 00:36:55,720 SUCCESSFUL VIRUSES. 845 00:36:55,720 --> 00:36:58,960 SO I'M GOING IT TURN MY 846 00:36:58,960 --> 00:37:00,360 ATTENTION NOW TO THE SECOND 847 00:37:00,360 --> 00:37:02,320 QUESTION OF VIRAL GENE 848 00:37:02,320 --> 00:37:02,640 REGULATION. 849 00:37:02,640 --> 00:37:05,840 SO OF COURSE WHILE HOST 850 00:37:05,840 --> 00:37:08,200 TRANSCRIPTION IS BEING REPRESSED 851 00:37:08,200 --> 00:37:12,200 DURING LYTIC INFECTION 852 00:37:12,200 --> 00:37:14,480 PARTICULARLY LATER, VIRAL 853 00:37:14,480 --> 00:37:16,680 EXPRESSION AND OTHER PROCESS 854 00:37:16,680 --> 00:37:18,880 HUMMING ALONG NICELY IN THE 855 00:37:18,880 --> 00:37:20,920 NUCLEUS IN VIRAL REPLICATION 856 00:37:20,920 --> 00:37:21,240 COMPARTMENTS. 857 00:37:21,240 --> 00:37:24,400 THESE ARE SUBCOMPARTMENTS OF THE 858 00:37:24,400 --> 00:37:30,520 NUCLEUS NOT MEMBRANE BOUND BUT 859 00:37:30,520 --> 00:37:31,640 CONCENTRATE REPLICATING VIRAL 860 00:37:31,640 --> 00:37:33,560 DNA BUT EXCLUDE HOST DNA. 861 00:37:33,560 --> 00:37:36,960 THE VIRAL DNA IN THE REPLICATION 862 00:37:36,960 --> 00:37:38,720 COMPARTMENTS IS LARGELY THOUGHT 863 00:37:38,720 --> 00:37:45,680 TO BE UNCHROMATINIZED AND 864 00:37:45,680 --> 00:37:47,320 HUNDREDS OF COPIES AND THINK OF 865 00:37:47,320 --> 00:37:50,960 IT AS A CHAOTIC PLACE BECAUSE IT 866 00:37:50,960 --> 00:37:52,360 LACKS THE ORGANIZATIONAL 867 00:37:52,360 --> 00:37:55,680 PRINCIPLES USED BY THE HOST DNA 868 00:37:55,680 --> 00:37:59,600 AND YET ON THAT CHAOTIC 869 00:37:59,600 --> 00:38:03,800 REPLICATING DNA MILIEU THERE'S 870 00:38:03,800 --> 00:38:05,760 SOPHISTICATED GENE REGULATORY 871 00:38:05,760 --> 00:38:07,640 PROCESSES AND JUST DNA PROCESSES 872 00:38:07,640 --> 00:38:11,480 THAT ARE HAPPENING INCLUDING 873 00:38:11,480 --> 00:38:14,640 VIRAL DNA REPLICATION AND THE 874 00:38:14,640 --> 00:38:17,040 DNA IS GETTING PACKAGED. 875 00:38:17,040 --> 00:38:20,840 THE LOCALIZATION OF THE VIRAL 876 00:38:20,840 --> 00:38:22,640 GENOME WITHIN THE COMPARTMENTS 877 00:38:22,640 --> 00:38:25,680 IS WHAT WE THINK PROTECTS THE 878 00:38:25,680 --> 00:38:27,960 VIRAL GENOME FROM THE 879 00:38:27,960 --> 00:38:28,640 TRANSCRIPTIONAL REPRESSION THAT 880 00:38:28,640 --> 00:38:30,560 IS LEVIED ON THE HOST GENOME. 881 00:38:30,560 --> 00:38:32,760 THIS PROVIDES A PROTECTIVE 882 00:38:32,760 --> 00:38:34,440 ENVIRONMENT WHERE THE REMAINING 883 00:38:34,440 --> 00:38:37,000 POL II FROM THE CELL IS 884 00:38:37,000 --> 00:38:37,800 RECRUITED STRONGLY TO VIRAL 885 00:38:37,800 --> 00:38:39,440 REPLICATION COMPARTMENTS 886 00:38:39,440 --> 00:38:41,520 PROBABLY BECAUSE OF THE OPEN 887 00:38:41,520 --> 00:38:43,760 NATURE OF THE VIRAL DNA. 888 00:38:43,760 --> 00:38:47,520 NON CHROMATINIZED DNA IS LIKE A 889 00:38:47,520 --> 00:38:50,680 SPONGE AND HELPS RECRUIT FACTORS 890 00:38:50,680 --> 00:38:54,080 FROM THE REST OF THE NUCLEOPLASM 891 00:38:54,080 --> 00:38:55,840 IT NEEDS TO COORDINATE ITS OWN 892 00:38:55,840 --> 00:38:56,480 GENE EXPRESSION. 893 00:38:56,480 --> 00:39:00,000 WHAT I'LL TELL YOU ABOUT TODAY 894 00:39:00,000 --> 00:39:03,520 IS HOW IN THAT RECRUITMENT 895 00:39:03,520 --> 00:39:07,520 PROCESS THERE IS LATENT 896 00:39:07,520 --> 00:39:09,960 INFECTION, SPECIALIZED VIRAL 897 00:39:09,960 --> 00:39:12,280 REGULATORS TO HELP COORDINATE ON 898 00:39:12,280 --> 00:39:15,240 PROMOTERS AND WE FOUND CONNECT 899 00:39:15,240 --> 00:39:16,200 EACH OF THESE PROCESSES IN SOME 900 00:39:16,200 --> 00:39:20,040 SURPRISING WAYS. 901 00:39:20,040 --> 00:39:21,960 LET ME FIRST REMIND YOU THAT FOR 902 00:39:21,960 --> 00:39:25,440 ALL DNA VIRUSES IT DOESN'T 903 00:39:25,440 --> 00:39:28,440 MATTER IF BACTERIA, PHAGE OR 904 00:39:28,440 --> 00:39:32,840 HERPES VIRUS YOU HAVE SETS OF 905 00:39:32,840 --> 00:39:34,000 GENES EXPRESSED PRIOR TO THE 906 00:39:34,000 --> 00:39:36,400 VIRAL DNA REPLICATION CALLED THE 907 00:39:36,400 --> 00:39:36,840 EARLY GENES. 908 00:39:36,840 --> 00:39:39,760 THEY BY AND LARGE LOOK LIKE HOST 909 00:39:39,760 --> 00:39:40,280 PROMOTERS. 910 00:39:40,280 --> 00:39:41,440 MANY WERE USED AS MODEL SYSTEMS 911 00:39:41,440 --> 00:39:58,440 TO UNDERSTAND THE BASICS OF HOW 912 00:39:58,440 --> 00:40:00,280 EUKARYOTIC FACTORS WORK LIKE OUR 913 00:40:00,280 --> 00:40:03,240 OWN PROMOTERS DO. 914 00:40:03,240 --> 00:40:06,320 LATE GENES ARE THE SET OF GENES 915 00:40:06,320 --> 00:40:08,600 WHOSE EXPRESSION INITIATES ONLY 916 00:40:08,600 --> 00:40:10,600 AFTER THE VIRUS STARTS 917 00:40:10,600 --> 00:40:12,320 REPLICATING ITS DNA AND THIS 918 00:40:12,320 --> 00:40:13,440 MAKES SENSE. 919 00:40:13,440 --> 00:40:14,960 LIKE CAPSID PROTEINS, ETCETERA, 920 00:40:14,960 --> 00:40:18,240 THE THING THE VIRUS DOESN'T NEED 921 00:40:18,240 --> 00:40:21,000 UNTIL THERE'S REPLICATED DNA TO 922 00:40:21,000 --> 00:40:22,320 PACKAGE INTO NEW CAPSIDS. 923 00:40:22,320 --> 00:40:25,480 THESE GENES ARE THE ONES THAT 924 00:40:25,480 --> 00:40:28,160 ARE BEING EXPRESSED AT THIS 925 00:40:28,160 --> 00:40:29,440 PARTICULARLY CHAOTIC TIME IN THE 926 00:40:29,440 --> 00:40:30,480 VIRAL REPLICATION COMPARTMENTS 927 00:40:30,480 --> 00:40:32,640 WHERE THERE'S A LOT GOING ON IN 928 00:40:32,640 --> 00:40:35,280 THE DNA AND THEY'RE EXPRESSION 929 00:40:35,280 --> 00:40:37,440 STRATEGIES ARE UNIQUE COMPARED 930 00:40:37,440 --> 00:40:39,560 TO VIRAL EARLY GENES AND HOST 931 00:40:39,560 --> 00:40:40,560 GENES. 932 00:40:40,560 --> 00:40:41,640 FIRST OF ALL THEY HAVE 933 00:40:41,640 --> 00:40:44,040 SIMPLISTIC PROMOTERS THAT HAVE A 934 00:40:44,040 --> 00:40:47,000 LITTLE MORE THAN A MODIFIED BOX 935 00:40:47,000 --> 00:40:48,240 AND THEIR TRANSCRIPTION IS 936 00:40:48,240 --> 00:40:49,520 COORDINATED BY A COMPLEX OF 937 00:40:49,520 --> 00:40:52,080 VIRALLY ENCODED PROTEINS OR SIX 938 00:40:52,080 --> 00:40:54,840 IN THE BETA AND GAMMA HERPES 939 00:40:54,840 --> 00:40:56,800 VIRUS CALLED THE VIRAL 940 00:40:56,800 --> 00:41:00,400 TRANSCRIPTION ACTIVATORS THAT 941 00:41:00,400 --> 00:41:01,920 DIRECTS TRANSCRIPTION OFF THE 942 00:41:01,920 --> 00:41:02,840 LATE GENES. 943 00:41:02,840 --> 00:41:04,480 WE STUDIED THEM 10 YEARS AND 944 00:41:04,480 --> 00:41:05,960 MAPPED THE GENERAL ORGANIZATION. 945 00:41:05,960 --> 00:41:08,920 IT'S A FASCINATING COMPLEX AND A 946 00:41:08,920 --> 00:41:10,400 TOPIC FOR ANOTHER SEMINAR. 947 00:41:10,400 --> 00:41:14,640 ONE IS A MIMIC OF CELLULAR 948 00:41:14,640 --> 00:41:16,440 PROTEIN NOT AT THE SEQUENCE 949 00:41:16,440 --> 00:41:18,800 LEVEL BUT STRUCTURALLY AND 950 00:41:18,800 --> 00:41:20,840 MIMICS TVP AND DOES SOMETHING IT 951 00:41:20,840 --> 00:41:24,440 DOESN'T DO WHICH ACTS LIKE A 952 00:41:24,440 --> 00:41:27,800 BACTERIAL SIGNAL FACTOR IT CAN 953 00:41:27,800 --> 00:41:29,240 BIND DIRECTLY IT POL II AND 954 00:41:29,240 --> 00:41:32,240 BRING IT TO VIRAL PROMOTERS. 955 00:41:32,240 --> 00:41:35,360 SO TVP DOES NOT MAKE CONTACT 956 00:41:35,360 --> 00:41:37,760 LIKE THE VIRAL VERSION DOES. 957 00:41:37,760 --> 00:41:40,280 SO WE WONDERED DESPITE THE FACT 958 00:41:40,280 --> 00:41:42,360 WE HAVE A DECENT IDEA OF THE 959 00:41:42,360 --> 00:41:43,240 ORGANIZATION OF THIS COMPLEX AND 960 00:41:43,240 --> 00:41:45,240 THE PROMOTER ELEMENTS INVOLVED 961 00:41:45,240 --> 00:41:51,360 WE DON'T REALLY KNOW HOW IT'S 962 00:41:51,360 --> 00:41:57,040 REGULATE 963 00:41:57,040 --> 00:42:00,000 REGULATED AND WHAT TRIGGERS THE 964 00:42:00,000 --> 00:42:02,880 LATE TIME POST INFECTION AND 965 00:42:02,880 --> 00:42:05,120 THIS IS WORK FROM A GRAD STUDENT 966 00:42:05,120 --> 00:42:06,840 FIGURING OUT WHAT ARE THE SETS 967 00:42:06,840 --> 00:42:08,280 OF THINGS THERE AT THE SCENE OF 968 00:42:08,280 --> 00:42:09,160 THE CRIME. 969 00:42:09,160 --> 00:42:12,840 THE CELLULAR AND VIRAL FACTORS 970 00:42:12,840 --> 00:42:14,240 LOCALIZED TO THESE LATE GENE 971 00:42:14,240 --> 00:42:16,200 TRANSCRIPTION COMPLEXES TO THE 972 00:42:16,200 --> 00:42:18,640 LATE PROMOTERS AT THE POINT IN 973 00:42:18,640 --> 00:42:19,880 TIME WHERE LATE GENE EXPRESSION 974 00:42:19,880 --> 00:42:21,600 IS HAPPENING. 975 00:42:21,600 --> 00:42:23,640 THIS SOUNDS SIMPLE BUT IT'S NOT 976 00:42:23,640 --> 00:42:25,400 BECAUSE TRANSCRIPTION COMPLEXES 977 00:42:25,400 --> 00:42:27,920 ARE INHERENTLY DYNAMIC. 978 00:42:27,920 --> 00:42:30,560 YOU GENERALLY CAN'T STUDY THEM 979 00:42:30,560 --> 00:42:32,000 USING STANDARD IP ASSAYS. 980 00:42:32,000 --> 00:42:33,120 YOU'RE NOT GOING TO BE ABLE TO 981 00:42:33,120 --> 00:42:34,040 SEE THE INTERACTION. 982 00:42:34,040 --> 00:42:36,720 THEY'RE JUST HAPPENING IN A 983 00:42:36,720 --> 00:42:38,600 SNAPSHOT OF TIME MOVING SHAPE 984 00:42:38,600 --> 00:42:42,320 SHIFTING AS THEY GO ALONG. 985 00:42:42,320 --> 00:42:44,360 SHE WANTED TO USE A PROXIMITY 986 00:42:44,360 --> 00:42:46,440 LABELLING APPROACH TO CAPTURE 987 00:42:46,440 --> 00:42:47,440 TRANSIENT INTERACTIONS HAPPENING 988 00:42:47,440 --> 00:42:49,240 AT A PARTICULAR SNAPSHOT IN TIME 989 00:42:49,240 --> 00:42:50,960 AND PLACE. 990 00:42:50,960 --> 00:42:59,360 AND SO FOR THIS SHE USED 991 00:42:59,360 --> 00:43:01,080 PROMISCUOUS LIGASE CALLED TURBO 992 00:43:01,080 --> 00:43:03,360 I.D. AND FUSES ENDOGENOUSLY IN 993 00:43:03,360 --> 00:43:07,680 THE VIRAL GENOME TO THE LATE 994 00:43:07,680 --> 00:43:09,160 TRANSCRIPTION ACTIVATORS. 995 00:43:09,160 --> 00:43:22,680 IF YOU BLOW BIOTIN IT WILL GO TO 996 00:43:22,680 --> 00:43:28,360 WHERE LATE GENE TRANSCRIPTION IS 997 00:43:28,360 --> 00:43:35,720 HAPPENING AND YOU CAN PULL OUT 998 00:43:35,720 --> 00:43:38,520 THE BIOTIN PROTEINS AND IDENTIFY 999 00:43:38,520 --> 00:43:49,280 THEM BY MASS SPECTOMETRY AND IT 1000 00:43:49,280 --> 00:43:51,440 TOOK A TON OF WORK PROBABLY A 1001 00:43:51,440 --> 00:43:53,520 YEAR OR MORE OF TRYING DIFFERENT 1002 00:43:53,520 --> 00:43:54,720 LOCATIONS FOR THE TAG AND 1003 00:43:54,720 --> 00:43:55,680 EXPRESSION AND CONTROLS TO LAYER 1004 00:43:55,680 --> 00:43:56,160 IN. 1005 00:43:56,160 --> 00:43:57,840 BE AWARE. 1006 00:43:57,840 --> 00:44:01,640 IT WAS A TON OF WORK. 1007 00:44:01,640 --> 00:44:13,680 NONETHELESS, CHLOE LOOK FOR 1008 00:44:13,680 --> 00:44:15,080 CELLS IN ASSOCIATION WITH THE 1009 00:44:15,080 --> 00:44:20,800 COMPLEX BUT NOT ANY OF HER 1010 00:44:20,800 --> 00:44:22,440 NEGATIVE CONTROLS AND CAME UP 1011 00:44:22,440 --> 00:44:24,640 WITH A LIST OF 45 PROTEINS AND 1012 00:44:24,640 --> 00:44:27,840 THESE ENCOMPASSED THE KNOWN 1013 00:44:27,840 --> 00:44:30,600 COMPONENTS OF THE COMPLEX IN POL 1014 00:44:30,600 --> 00:44:30,760 II. 1015 00:44:30,760 --> 00:44:31,720 EVERYTHING WE KNEW SHOULD BE 1016 00:44:31,720 --> 00:44:35,120 THERE WAS PICKED UP BY THE 1017 00:44:35,120 --> 00:44:35,360 SCREEN. 1018 00:44:35,360 --> 00:44:38,000 THE 45 LIST OF PROTEINS DOESN'T 1019 00:44:38,000 --> 00:44:39,640 TELL YOU WHETHER THEY'RE 1020 00:44:39,640 --> 00:44:41,320 INVOLVED IN LATE GENE PROTEINS 1021 00:44:41,320 --> 00:44:43,480 AND RAN THEM THROUGH FUNCTIONAL 1022 00:44:43,480 --> 00:44:45,200 ASSAYS TO TEST IF THEY WERE 1023 00:44:45,200 --> 00:44:47,280 IMPORTANT FOR THE TRANSCRIPTION 1024 00:44:47,280 --> 00:44:47,520 PROCESS. 1025 00:44:47,520 --> 00:44:49,600 FOR THIS SHE NEEDED ADDITIONAL 1026 00:44:49,600 --> 00:44:54,600 TOOL BUILDING AND ENGINEERED ON 1027 00:44:54,600 --> 00:44:58,080 THE KSHG GENOME ON THE BACKBONE 1028 00:44:58,080 --> 00:44:59,440 A REPORTER THAT WAS DRIVEN 1029 00:44:59,440 --> 00:45:01,280 EITHER BY AN EARLY GENE PROMOTER 1030 00:45:01,280 --> 00:45:04,360 OR A LATE GENE PROMOTER. 1031 00:45:04,360 --> 00:45:07,360 SHE DEPLETED EACH OF THESE THEN 1032 00:45:07,360 --> 00:45:09,800 USING A CRISPR SCREEN CAN ASK 1033 00:45:09,800 --> 00:45:12,320 WHETHER THE DELETION SELECTIVELY 1034 00:45:12,320 --> 00:45:16,520 IMPACTED LATE GENE EXPRESSION 1035 00:45:16,520 --> 00:45:18,640 AND WHETHER THEY WERE 1036 00:45:18,640 --> 00:45:20,240 FUNCTIONALLY INVOLVED AND RAN 1037 00:45:20,240 --> 00:45:22,040 THROUGH A SECOND ROUND OF 1038 00:45:22,040 --> 00:45:26,640 SCREENING TO AN ORTHOGONAL PRECH 1039 00:45:26,640 --> 00:45:27,800 APPROACH TO GET ACCURATE HITS. 1040 00:45:27,800 --> 00:45:30,640 SHE DID ALL THIS AND PICKED UP 1041 00:45:30,640 --> 00:45:31,960 EVERY KNOWN FACTOR THAT WAS 1042 00:45:31,960 --> 00:45:36,400 REQUIRED FOR LATE GENE 1043 00:45:36,400 --> 00:45:36,720 TRANSCRIPTION. 1044 00:45:36,720 --> 00:45:41,960 THAT WAS GREAT. 1045 00:45:41,960 --> 00:45:44,760 AND ONE NEW HIT. 1046 00:45:44,760 --> 00:45:46,080 SHE WAS VERY NERVOUS. 1047 00:45:46,080 --> 00:45:48,360 THIS IS A MEETING IN THE OFFICE 1048 00:45:48,360 --> 00:45:50,200 WHERE YOU'RE SPENDING A LOT OF 1049 00:45:50,200 --> 00:45:52,080 TIME SAYING IT'S GOING TO BE 1050 00:45:52,080 --> 00:45:53,440 OKAY BECAUSE WHEN YOU DO SCREENS 1051 00:45:53,440 --> 00:45:54,760 YOU HOPE TO GET ONE TRUE HIT AND 1052 00:45:54,760 --> 00:45:58,680 YOU HAVE ONE HIT AND SHE'S LIKE 1053 00:45:58,680 --> 00:46:00,280 WHAT IF SOMETHING ELSE TELLS ME 1054 00:46:00,280 --> 00:46:01,720 THIS HIT ISN'T RIGHT AND I'VE 1055 00:46:01,720 --> 00:46:04,240 DONE ALL THIS WORK FOR YEARS AND 1056 00:46:04,240 --> 00:46:05,920 NOTHING COMES FROM IT. 1057 00:46:05,920 --> 00:46:10,600 THOUGH THE ONE HIT WAS NOT A 1058 00:46:10,600 --> 00:46:11,120 PROTEIN WE EXPECTED QUITE 1059 00:46:11,120 --> 00:46:11,440 INTERESTING. 1060 00:46:11,440 --> 00:46:12,120 THIS IS TO SHOW YOU THE DATA FOR 1061 00:46:12,120 --> 00:46:19,320 THE HIT BUT THE HIT IS A PROTEIN 1062 00:46:19,320 --> 00:46:22,840 CALLED ORPH29 THE LATE GENE 1063 00:46:22,840 --> 00:46:27,320 DEPLETER AND IT RESIDES WITH THE 1064 00:46:27,320 --> 00:46:29,520 OTHER LATE GENE ACTIVATORS AND 1065 00:46:29,520 --> 00:46:30,920 HAVE A REDUCTION IN LATE GENE 1066 00:46:30,920 --> 00:46:31,640 SIGNAL. 1067 00:46:31,640 --> 00:46:35,040 IF WE DO IT WITH THEY WILL EARLY 1068 00:46:35,040 --> 00:46:38,040 GENE REPORTER LIKE ORPH30 1069 00:46:38,040 --> 00:46:42,440 THERE'S NO REPRESSION AND IT'S 1070 00:46:42,440 --> 00:46:43,080 THE PHENOTYPE THAT WE'RE LOOKING 1071 00:46:43,080 --> 00:46:45,080 FOR. 1072 00:46:45,080 --> 00:46:47,280 BUT THE PROTEIN WAS CONFUSING TO 1073 00:46:47,280 --> 00:46:51,200 US BECAUSE IT HAS NOTHING TO DO 1074 00:46:51,200 --> 00:46:52,760 WITH TRANSCRIPTION AS FAR AS WE 1075 00:46:52,760 --> 00:46:57,080 CAN TELL IT'S THE CATALYTIC PART 1076 00:46:57,080 --> 00:47:04,800 OF THE TERMINASE AND ESTABLISHED 1077 00:47:04,800 --> 00:47:13,320 ROLES IN PACKAGING DNA. 1078 00:47:13,320 --> 00:47:15,640 SO SHE TRIPLE CHECKED HERSELF 1079 00:47:15,640 --> 00:47:24,400 AND MADE THE MUTANT IN KSHV AND 1080 00:47:24,400 --> 00:47:28,400 IF YOU DON'T HAVE IT YOU CANT 1081 00:47:28,400 --> 00:47:31,200 HAVE PRODUCTION OF NEW 1082 00:47:31,200 --> 00:47:32,400 INFECTIOUS VIRON AND THE VIRUS 1083 00:47:32,400 --> 00:47:36,400 IS COMPLETELY DEAD. 1084 00:47:36,400 --> 00:47:41,720 INDEED GETTING RID OF THE 1085 00:47:41,720 --> 00:47:43,720 TERMINASE GOES TO PRODUCTION AND 1086 00:47:43,720 --> 00:47:48,200 WE LOOKED AT LATE OR EARLY 1087 00:47:48,200 --> 00:47:51,640 PRODUCED PROTEINS AND COULD 1088 00:47:51,640 --> 00:47:54,440 RECAPITULATE LATE PROTEINS LIKE 1089 00:47:54,440 --> 00:48:00,200 K.1 WERE SIGNIFICANTLY REDUCED 1090 00:48:00,200 --> 00:48:01,280 IN THE ORF VIRUS AND OTHERS WERE 1091 00:48:01,280 --> 00:48:08,440 NOT REDUCED AT ALL. 1092 00:48:08,440 --> 00:48:12,360 WHAT IS IT GETTING RID OF THE 1093 00:48:12,360 --> 00:48:19,800 TE 1094 00:48:19,800 --> 00:48:20,240 TERM 1095 00:48:20,240 --> 00:48:20,640 TERM 1096 00:48:20,640 --> 00:48:21,040 TERMINASE WILL DO. 1097 00:48:21,040 --> 00:48:22,920 HAVE YOU A PORTAL THROUGH WHICH 1098 00:48:22,920 --> 00:48:26,800 THE VIRAL DNA REPLICATED IN A 1099 00:48:26,800 --> 00:48:30,720 ROLLING CIRCLE MECHANISM TO HAVE 1100 00:48:30,720 --> 00:48:33,120 THESE PUNCTUATED BY TERMINAL 1101 00:48:33,120 --> 00:48:36,040 REPEAT AND THE COMPLEX BINDS 1102 00:48:36,040 --> 00:48:40,400 THESE AND IN AN ATP DEPENDENT 1103 00:48:40,400 --> 00:48:44,920 WAY PUMPS THEM IN THE CAPSIS AND 1104 00:48:44,920 --> 00:48:47,080 AS THE NEARLY LIQUID CRYSTAL IN 1105 00:48:47,080 --> 00:48:47,320 DENSITY. 1106 00:48:47,320 --> 00:48:49,400 IT'S A POWERFUL MOTOR TO BE ABLE 1107 00:48:49,400 --> 00:48:51,640 TO GET THESE IN AND THEN IT HAS 1108 00:48:51,640 --> 00:48:54,480 A NUCLEASE ACTIVITY THAT 1109 00:48:54,480 --> 00:48:55,600 SUBSEQUENTLY CLEAVES AT A 1110 00:48:55,600 --> 00:48:56,760 TERMINAL REPEAT WITH A FULL 1111 00:48:56,760 --> 00:48:59,720 GENOME PACKAGE TO THEN ALLOW THE 1112 00:48:59,720 --> 00:49:01,640 PORTAL TO SEAL AND THE VIRAL 1113 00:49:01,640 --> 00:49:04,360 PARTICLE TO UNDER GO THE REST OF 1114 00:49:04,360 --> 00:49:04,840 ITS MATURATION PROCESS. 1115 00:49:04,840 --> 00:49:06,840 SO ONE OF THE THINGS WE THOUGHT 1116 00:49:06,840 --> 00:49:08,720 WAS PERHAPS IF YOU'RE PREVENTING 1117 00:49:08,720 --> 00:49:12,360 THIS PROCESS YOU'RE MESSING UP 1118 00:49:12,360 --> 00:49:17,400 THE PACKAGING REACTION AND GOING 1119 00:49:17,400 --> 00:49:21,040 TO A CHAOTIC ENVIRONMENT AND 1120 00:49:21,040 --> 00:49:21,680 CREATING TOO MUCH SOMETHING IN 1121 00:49:21,680 --> 00:49:23,720 THE REPLICATION COMPARTMENT AND 1122 00:49:23,720 --> 00:49:26,320 IT'S NOT A DIRECT EFFECT BUT 1123 00:49:26,320 --> 00:49:28,000 INDIRECT EFFECT OF FAILURE TO 1124 00:49:28,000 --> 00:49:29,360 PACKAGE YOUR DNA. 1125 00:49:29,360 --> 00:49:31,040 WE COULD TEST THIS HYPOTHESIS 1126 00:49:31,040 --> 00:49:33,560 BECAUSE A COUPLE OF YEARS 1127 00:49:33,560 --> 00:49:36,440 EARLIER A FORMER POST DOCK IN 1128 00:49:36,440 --> 00:49:40,280 THE LAB IDENTIFIED ANOTHER 1129 00:49:40,280 --> 00:49:44,200 CRITICAL PACKAGING FACTOR CALLED 1130 00:49:44,200 --> 00:49:45,720 ORF68 WHOSE ACTIVELY IS 1131 00:49:45,720 --> 00:49:51,360 ESSENTIAL FOR PACKAGING BUT 1132 00:49:51,360 --> 00:49:54,040 UNRELATED TO THE TERMINASE 1133 00:49:54,040 --> 00:49:55,120 PACKAGING AND WE CAN BLOCK THIS 1134 00:49:55,120 --> 00:49:58,320 AND SEE IF WE GET THE SAME 1135 00:49:58,320 --> 00:50:01,760 REACTION AND YOU GET RID OF ORF6 1136 00:50:01,760 --> 00:50:04,880 YOU DON'T PACKAGE YOUR DNA. 1137 00:50:04,880 --> 00:50:09,400 IF YOU GET RID OF ORF68 YOU 1138 00:50:09,400 --> 00:50:11,440 EXPRESS K.1 JUST FINE. 1139 00:50:11,440 --> 00:50:14,800 STO STOPPING PACKAGES DOES NOT 1140 00:50:14,800 --> 00:50:16,880 HAVE THE BYPRODUCT OF REDUCING 1141 00:50:16,880 --> 00:50:19,800 GENE EXPRESSION AT LATE TIMES. 1142 00:50:19,800 --> 00:50:23,240 THERE'S SOMETHING SPECIFIC ABOUT 1143 00:50:23,240 --> 00:50:26,240 THE VIRAL TERMINASE IS DOING. 1144 00:50:26,240 --> 00:50:28,760 THIS IS MY LAST DATA SLIDE. 1145 00:50:28,760 --> 00:50:32,000 WHAT CHLOE WANTED TO DETERMINE 1146 00:50:32,000 --> 00:50:34,040 IS IT THE CATALYTIC PROTEIN 1147 00:50:34,040 --> 00:50:35,240 NECESSARY FOR THIS FUNCTION AND 1148 00:50:35,240 --> 00:50:37,200 MADE POINT MUTATIONS IN THE 1149 00:50:37,200 --> 00:50:40,120 WALKER A OR WALKER B MOTIFS 1150 00:50:40,120 --> 00:50:43,760 IMPORTANT FOR ATP BINDING AND 1151 00:50:43,760 --> 00:50:46,080 HYDROLYSIS FOR THE PACKAGING 1152 00:50:46,080 --> 00:50:48,600 REACTION AND IMPORTANT FOR 1153 00:50:48,600 --> 00:50:51,240 CLIPPING THE DOMAIN FOR FINISH 1154 00:50:51,240 --> 00:50:55,240 THE REACTION AND ASK CAN THE 1155 00:50:55,240 --> 00:50:56,480 MUTANTS COMPLEMENT WHEN SHE 1156 00:50:56,480 --> 00:50:59,640 TRANSDUCES THESE INTO CELLS A 1157 00:50:59,640 --> 00:51:02,440 FIRST WITH LAX ORF29 AND 1158 00:51:02,440 --> 00:51:04,560 COMPARED THE MUTANTS TO WILD 1159 00:51:04,560 --> 00:51:05,720 TYPE. 1160 00:51:05,720 --> 00:51:07,240 I'M SUMMARIZE THE DATA BECAUSE 1161 00:51:07,240 --> 00:51:08,440 THE MUTANTS ALL LOOK THE SAME 1162 00:51:08,440 --> 00:51:12,360 AND IF YOU MUTATE ANY ASPECT OF 1163 00:51:12,360 --> 00:51:14,840 THE CATALYTIC UNLIKE THE WILD 1164 00:51:14,840 --> 00:51:23,560 TYPE PROTEIN YOU'RE UNABLE TO 1165 00:51:23,560 --> 00:51:25,800 COMPLETE THE DETECT AND IT'S 1166 00:51:25,800 --> 00:51:27,440 CRITICAL FOR POTENTIATING GENE 1167 00:51:27,440 --> 00:51:28,520 EXPRESSION LATE IN INFECTION. 1168 00:51:28,520 --> 00:51:31,800 LET ME BRING THIS TOGETHER FOR 1169 00:51:31,800 --> 00:51:32,560 YOU. 1170 00:51:32,560 --> 00:51:34,880 THIS IS WHY THIS IS INTERESTING 1171 00:51:34,880 --> 00:51:35,840 AND IMPORTANT. 1172 00:51:35,840 --> 00:51:37,680 FIRST, FOR A LONG TIME IT'S BEEN 1173 00:51:37,680 --> 00:51:40,440 KNOWN FOR ALL DNA VIRUSES THAT 1174 00:51:40,440 --> 00:51:42,080 THIS ACTIVE DNA REPLICATION IS 1175 00:51:42,080 --> 00:51:45,440 REQUIRED IN SOME WAY TO LICENSE 1176 00:51:45,440 --> 00:51:48,000 TRANSCRIPTION OF GENES LATE IN 1177 00:51:48,000 --> 00:51:49,000 INFECTION BUT WHAT WE'RE SHOWING 1178 00:51:49,000 --> 00:51:50,600 NOW IS THAT INDEED IT IS NOT 1179 00:51:50,600 --> 00:51:54,240 JUST THIS CRITICAL ACTIVITY BUT 1180 00:51:54,240 --> 00:51:56,160 DOWN STREAM EFFECT THE PACKAGING 1181 00:51:56,160 --> 00:51:58,200 REACTION ALSO SOMEHOW FEEDS BACK 1182 00:51:58,200 --> 00:52:00,400 TO POTENTIATE GENE EXPRESSION AS 1183 00:52:00,400 --> 00:52:00,600 WELL. 1184 00:52:00,600 --> 00:52:02,760 AND HOW MIGHT THIS BE HAPPENING? 1185 00:52:02,760 --> 00:52:08,480 WE HAVE A COUPLE OF IDEAS. 1186 00:52:08,480 --> 00:52:10,240 BECAUSE THE ATP ACTIVITY IS 1187 00:52:10,240 --> 00:52:14,280 REQUIRED ONE POSSIBILITY IS THE 1188 00:52:14,280 --> 00:52:15,480 TERMINASE IS FUNCTIONING IN -- 1189 00:52:15,480 --> 00:52:18,000 WE DON'T THINK IT'S JUST A 1190 00:52:18,000 --> 00:52:19,440 SCAFFOLDING FACTOR BUT MAYBE 1191 00:52:19,440 --> 00:52:22,280 SCANNING AND CLEARING THE DNA TO 1192 00:52:22,280 --> 00:52:27,480 MAKE IT EASIER TO TRANSCRIBE. 1193 00:52:27,480 --> 00:52:28,880 ALTERNATIVELY WE THINK THE 1194 00:52:28,880 --> 00:52:31,080 PACKAGING REACTION ITSELF IS 1195 00:52:31,080 --> 00:52:34,240 YOUR TWISTING THE DNA IN THE 1196 00:52:34,240 --> 00:52:36,520 CAPSID AND CREATING TORSIONAL 1197 00:52:36,520 --> 00:52:40,360 STRAIN ON DNA. 1198 00:52:40,360 --> 00:52:43,720 THE SAME THING WHEN OUR DNA IS 1199 00:52:43,720 --> 00:52:45,400 BEING REPLICATED YOU NEED TO 1200 00:52:45,400 --> 00:52:47,800 RELEASE THE STRAIN OTHERWISE IT 1201 00:52:47,800 --> 00:52:49,560 WILL CAUSE TRANSCRIPTIONAL 1202 00:52:49,560 --> 00:52:50,040 INHIBITION. 1203 00:52:50,040 --> 00:52:53,680 WE FAVOR THE IDEA AT PRESENT 1204 00:52:53,680 --> 00:52:57,280 PERHAPS THE TERMINASE THROUGH 1205 00:52:57,280 --> 00:52:59,200 PACKING IS RELIEVING THE STRAIN 1206 00:52:59,200 --> 00:53:01,400 DURING THE PACKAGING REACTION 1207 00:53:01,400 --> 00:53:02,360 NECESSARY TO ALLOW TRANSCRIPTION 1208 00:53:02,360 --> 00:53:04,960 TO CONTINUE TO HAPPEN ON THE 1209 00:53:04,960 --> 00:53:09,040 NEWLY REPLICATED GENOMES. 1210 00:53:09,040 --> 00:53:10,720 IT'S ALSO INTERESTING BECAUSE IT 1211 00:53:10,720 --> 00:53:13,120 SUGGESTS IF INHIBITERS CAN BE 1212 00:53:13,120 --> 00:53:15,000 DEVELOPED AGAINST THE PACKAGING 1213 00:53:15,000 --> 00:53:17,440 COMPLEX THE INHIBITORS MAY BE 1214 00:53:17,440 --> 00:53:19,760 POTENT NOT JUST BECAUSE THEY 1215 00:53:19,760 --> 00:53:22,000 BLOCK PACKAGING BUT ALSO POISON 1216 00:53:22,000 --> 00:53:24,120 LATE GENE TRANSCRIPTION CRITICAL 1217 00:53:24,120 --> 00:53:25,640 FOR COMPLETION OF THE VIRAL LIFE 1218 00:53:25,640 --> 00:53:26,800 TIME. 1219 00:53:26,800 --> 00:53:27,440 SO I'LL END THERE. 1220 00:53:27,440 --> 00:53:28,800 I'LL MENTION THE FIRST PART OF 1221 00:53:28,800 --> 00:53:32,880 MY TALK WAS CARRIED OUT LARGELY 1222 00:53:32,880 --> 00:53:34,680 BY THE FORMER Ph.D. STUDENTS WHO 1223 00:53:34,680 --> 00:53:36,760 START THE WORK. 1224 00:53:36,760 --> 00:53:39,880 THE POL II PROTEIN SUB TURNOVER 1225 00:53:39,880 --> 00:53:42,560 WORK IS CARRIED OUT BY A CURRENT 1226 00:53:42,560 --> 00:53:44,960 Ph.D. STUDENT AND THE LATE GENE 1227 00:53:44,960 --> 00:53:48,240 WORK THE STORY WAS LARGELY 1228 00:53:48,240 --> 00:53:55,160 CARRIED OUT BY CHLOE McCALLUM 1229 00:53:55,160 --> 00:53:56,680 AND WAS HELPED TO GET THE TOOLS 1230 00:53:56,680 --> 00:53:59,000 AND SYSTEM SET UP. 1231 00:53:59,000 --> 00:54:00,680 ALWAYS GRATEFUL FOR OUR FUNDING 1232 00:54:00,680 --> 00:54:03,240 AND I'M MORE THAN HAPPY TO TAKE 1233 00:54:03,240 --> 00:54:04,160 QUESTIONS I'LL LEAVE UP OUR 1234 00:54:04,160 --> 00:54:05,680 MODEL TO HELP YOU THINK OF ANY 1235 00:54:05,680 --> 00:54:07,720 IF THERE'S TIME. 1236 00:54:07,720 --> 00:54:08,560 THANKS. 1237 00:54:08,560 --> 00:54:10,600 >>WE DO HAVE A QUESTION 1238 00:54:10,600 --> 00:54:10,840 ONLINE. 1239 00:54:10,840 --> 00:54:12,320 I'LL TAKE THAT FIRST BEFORE WE 1240 00:54:12,320 --> 00:54:14,160 GO. 1241 00:54:14,160 --> 00:54:17,280 SO OUR FRIEND HOWARD YOUNG UP AT 1242 00:54:17,280 --> 00:54:19,400 NCI FREDERICK ASKED DO 1243 00:54:19,400 --> 00:54:24,040 INTERFERONS PLAY ANY ROLE IN 1244 00:54:24,040 --> 00:54:24,920 YOUR DATA? 1245 00:54:24,920 --> 00:54:26,880 >> I GUESS I WONDER -- I'M 1246 00:54:26,880 --> 00:54:27,920 GUESSING HE'S TALKING ABOUT THE 1247 00:54:27,920 --> 00:54:28,600 FIRST PART. 1248 00:54:28,600 --> 00:54:31,280 >> THE QUESTION CAME IN PRETTY 1249 00:54:31,280 --> 00:54:31,480 EARLY. 1250 00:54:31,480 --> 00:54:34,400 >> AN IDEA IS THAT ONE OF THE 1251 00:54:34,400 --> 00:54:37,200 ROLES OF HOST SHUT OFF IS TO 1252 00:54:37,200 --> 00:54:38,440 RESTRICT PRODUCTION OF 1253 00:54:38,440 --> 00:54:40,040 INTERFERON STIMULATED GENES. 1254 00:54:40,040 --> 00:54:41,280 THAT'S BEEN PRETTY WELL 1255 00:54:41,280 --> 00:54:43,720 ESTABLISHED FOR A NUMBER OF HOST 1256 00:54:43,720 --> 00:54:45,680 SHUTOFF FACTORS IN THESE AND 1257 00:54:45,680 --> 00:54:46,800 OTHER VIRUS. 1258 00:54:46,800 --> 00:54:49,640 SO COUNTER ACTING THE INTERFERON 1259 00:54:49,640 --> 00:54:51,880 RESPONSE IS AN IMPORTANT ROLE OF 1260 00:54:51,880 --> 00:54:53,360 HOST SHUTOFF THOUGH WE DON'T 1261 00:54:53,360 --> 00:54:56,840 HAVE EVIDENCE THAT INTERFERONS 1262 00:54:56,840 --> 00:55:05,320 ARE PREFERENTIALLY TAR GUEST GETED FOR 1263 00:55:05,320 --> 00:55:06,000 DISRUPTION. 1264 00:55:06,000 --> 00:55:07,000 >> GREAT SCIENCE AND GREAT 1265 00:55:07,000 --> 00:55:08,000 PRESENTATION AS WELL. 1266 00:55:08,000 --> 00:55:10,080 I HAVE A QUESTION ABOUT ON YOUR 1267 00:55:10,080 --> 00:55:11,960 MODEL SLIDE THERE OF POLY 1268 00:55:11,960 --> 00:55:14,120 BINDING PROTEINS BEING IMPORTED 1269 00:55:14,120 --> 00:55:15,600 INTO THE NUCLEUS. 1270 00:55:15,600 --> 00:55:20,400 COULD THAT POTENTIALLY INHIBIT 1271 00:55:20,400 --> 00:55:22,440 TRANSLATION OF VIRAL GENES OR 1272 00:55:22,440 --> 00:55:23,920 COULD THAT BENEFIT TRANSLATION 1273 00:55:23,920 --> 00:55:28,600 OF VIRAL GENES BECAUSE I'M NOT 1274 00:55:28,600 --> 00:55:30,680 REMEMBERING WHETHER THE BINDING 1275 00:55:30,680 --> 00:55:33,600 PROTEINS RELATE TO THE NUCLEUS 1276 00:55:33,600 --> 00:55:35,160 OR THE CYTOPLASM. 1277 00:55:35,160 --> 00:55:36,800 >> YOU'RE NOT REMEMBERING 1278 00:55:36,800 --> 00:55:37,880 BECAUSE IT'S NOT BEEN WELL 1279 00:55:37,880 --> 00:55:38,200 ESTABLISHED. 1280 00:55:38,200 --> 00:55:41,720 THE THINKI INING IS IT PROBABLY 1281 00:55:41,720 --> 00:55:44,320 BINDS THEM AND SHUTTLING PROTEIN 1282 00:55:44,320 --> 00:55:46,200 AND PROBABLY BINDS IN THE 1283 00:55:46,200 --> 00:55:48,120 NUCLEUS AND ESCORTED IN THE 1284 00:55:48,120 --> 00:55:49,600 CYTOPLASM AND CYCLES BACK TO THE 1285 00:55:49,600 --> 00:55:51,800 NUCLEUS. 1286 00:55:51,800 --> 00:55:53,440 YES, I WOULD THINK PAB IS 1287 00:55:53,440 --> 00:55:54,280 IMPORTANT FOR VIRAL TRANSLATION 1288 00:55:54,280 --> 00:55:56,120 AND LOOKED AT WHERE IT IS IN THE 1289 00:55:56,120 --> 00:55:56,440 NUCLEUS. 1290 00:55:56,440 --> 00:55:58,560 YOU CAN SEE IT IN VIRAL 1291 00:55:58,560 --> 00:56:00,360 REPLICATION COMPARTMENTS AS 1292 00:56:00,360 --> 00:56:00,920 WELL. 1293 00:56:00,920 --> 00:56:04,000 SO WE ORIGINALLY THOUGHT IT'S 1294 00:56:04,000 --> 00:56:05,200 EXCLUDED AND THIS IS MAYBE THE 1295 00:56:05,200 --> 00:56:07,000 WAY VIRUSES ESCAPE BUT IT'S 1296 00:56:07,000 --> 00:56:09,040 POSSIBLE IT'S USED BY THE VIRUS 1297 00:56:09,040 --> 00:56:11,400 IN THE REPLICATION COMPARTMENTS 1298 00:56:11,400 --> 00:56:12,920 AN PROCESSING THE RNAs SO THEY 1299 00:56:12,920 --> 00:56:14,640 CAN BE EXPORTED EFFICIENTLY AND 1300 00:56:14,640 --> 00:56:16,400 TRANSLATED EFFICIENTLY AS WELL. 1301 00:56:16,400 --> 00:56:18,440 THE VIRUS NEEDS IT TOO. 1302 00:56:18,440 --> 00:56:20,120 >> GREAT. 1303 00:56:20,120 --> 00:56:20,560 >> THAT WAS LOVELY. 1304 00:56:20,560 --> 00:56:23,400 THANK YOU. 1305 00:56:23,400 --> 00:56:32,160 I COULD ASK THE SAME THING. 1306 00:56:32,160 --> 00:56:34,080 IN THE LAST BIT THE TORSIONAL 1307 00:56:34,080 --> 00:56:34,920 RELEASE, DO YOU THINK YOU NEED 1308 00:56:34,920 --> 00:56:38,200 THE PACKAGE AT THE END? 1309 00:56:38,200 --> 00:56:40,160 IS THAT WHAT IT'S FIGHTING 1310 00:56:40,160 --> 00:56:40,440 AGAINST? 1311 00:56:40,440 --> 00:56:44,000 OR DID YOU DIDN'T HAVE SOMETHING 1312 00:56:44,000 --> 00:56:45,000 GOING ON THERE -- 1313 00:56:45,000 --> 00:56:47,400 >> THERE WAS NO PACKAGING WOULD 1314 00:56:47,400 --> 00:56:49,600 YOU STILL NEED -- THAT IS AN 1315 00:56:49,600 --> 00:56:51,080 EXCELLENT QUESTION AND THE WAY 1316 00:56:51,080 --> 00:56:53,080 WE COULD ADDRESS THAT WOULD BE 1317 00:56:53,080 --> 00:56:58,600 TO MAKE THE DOUBLE MUTANT OF AN 1318 00:56:58,600 --> 00:57:03,920 ORF68 TO BLOCK PACKAGING AND 1319 00:57:03,920 --> 00:57:05,920 ORF28 IN THE BACKGROUND OF 1320 00:57:05,920 --> 00:57:07,680 PACKAGING DO YOU STILL NEED TO 1321 00:57:07,680 --> 00:57:08,800 RELIEF THE TORSIONAL STRAIN? 1322 00:57:08,800 --> 00:57:09,440 I'LL BRING IT UP TO MY STUDENT. 1323 00:57:09,440 --> 00:57:11,560 THANK YOU. 1324 00:57:11,560 --> 00:57:12,360 SHE'S PROBABLY ALREADY THOUGHT 1325 00:57:12,360 --> 00:57:15,240 OF IT. 1326 00:57:15,240 --> 00:57:19,320 >> THERE WAS A PROTEIN IN YOUR 1327 00:57:19,320 --> 00:57:19,600 THIRD. 1328 00:57:19,600 --> 00:57:20,600 WHAT IS THAT AND WHAT DO YOU 1329 00:57:20,600 --> 00:57:22,800 THINK IS GOING ON WITH THAT? 1330 00:57:22,800 --> 00:57:24,360 >> SO IT TURNS OUT THERE'S A 1331 00:57:24,360 --> 00:57:25,880 NUMBER OF FACTORS INVOLVED IN 1332 00:57:25,880 --> 00:57:28,560 RNA DECAY AND THAT'S ONE OF THEM 1333 00:57:28,560 --> 00:57:30,240 THAT TEND TO SHUTTLE INTO THE 1334 00:57:30,240 --> 00:57:31,400 NUCLEUS AND PARTICULARLY THINGS 1335 00:57:31,400 --> 00:57:34,680 IN DECAPPING. 1336 00:57:34,680 --> 00:57:35,880 WE DELVED INTO THAT WHEN WE WERE 1337 00:57:35,880 --> 00:57:39,000 FIRST WORKING INTO THE DATA. 1338 00:57:39,000 --> 00:57:40,080 AND I'LL SAY I DON'T HAVE 1339 00:57:40,080 --> 00:57:42,120 ANYTHING SATISFYING TO TELL YOU 1340 00:57:42,120 --> 00:57:44,480 THE OUTCOME EXCEPT WE NOTICED A 1341 00:57:44,480 --> 00:57:47,400 NUMBER OF THINGS INVOLVED IN 1342 00:57:47,400 --> 00:57:49,000 MESSENGER RNA DE CAPPING AND 1343 00:57:49,000 --> 00:57:51,400 OTHER REGULATORS OF RNA DECAY 1344 00:57:51,400 --> 00:57:53,400 THAT HAPPEN TO SHIFT 1345 00:57:53,400 --> 00:57:54,200 LOCALIZATION IN THE FACE OF RNA 1346 00:57:54,200 --> 00:57:54,400 DECAY. 1347 00:57:54,400 --> 00:57:56,880 YEAH. 1348 00:57:56,880 --> 00:57:58,480 >> DO YOU HAVE A QUESTION? 1349 00:57:58,480 --> 00:58:04,440 >> IS THERE ANY MODIFICATION 1350 00:58:04,440 --> 00:58:05,720 AFTER THIS INFECTION? 1351 00:58:05,720 --> 00:58:08,000 WHY I'M ASKING THE QUESTION -- 1352 00:58:08,000 --> 00:58:11,360 >> GET CLOSER TO THE MICROPHONE. 1353 00:58:11,360 --> 00:58:23,320 >> AND BACK IN 1967 OR '68 THE D 1354 00:58:23,320 --> 00:58:27,200 FOR ATTACHES TO THE CELL THE 1355 00:58:27,200 --> 00:58:30,240 POLYMERASE GETS ENACTIVATED 1356 00:58:30,240 --> 00:58:32,720 BECAUSE OF THE ISOLATION OF THE 1357 00:58:32,720 --> 00:58:35,680 SIGMA FACTOR AND THAT MODIFIED 1358 00:58:35,680 --> 00:58:38,600 ONLY TRANSCRIBES THE D4 DNA. 1359 00:58:38,600 --> 00:58:40,920 I THINK IT MUST BE VERY 1360 00:58:40,920 --> 00:58:44,520 IMPORTANT SOME MODIFICATION 1361 00:58:44,520 --> 00:58:47,600 TAKES PLACE THAT THEY'RE ACTIVE 1362 00:58:47,600 --> 00:58:50,200 WITH RESPECT TO THE ALTERNATIVE. 1363 00:58:50,200 --> 00:58:53,480 >> YOU'RE ASKING WHETHER THERE'S 1364 00:58:53,480 --> 00:58:57,000 MODIFICATION OF THE POL II SUB 1365 00:58:57,000 --> 00:58:59,120 UNITS THAT UNDER GO DECAY? 1366 00:58:59,120 --> 00:58:59,640 >> YES. 1367 00:58:59,640 --> 00:59:01,000 >> GREAT QUESTION. 1368 00:59:01,000 --> 00:59:06,400 I DON'T KNOW BUT WE SUSPECT 1369 00:59:06,400 --> 00:59:17,720 THERE'S UBIQUITINATION AND SOME 1370 00:59:17,720 --> 00:59:18,880 TARGETS THESE FOR TURN OVER AND 1371 00:59:18,880 --> 00:59:20,720 THE OTHER HYPOTHESIS IS IT'S 1372 00:59:20,720 --> 00:59:22,320 LINKED TO CELL STRESS. 1373 00:59:22,320 --> 00:59:26,840 THERE'S OTHER STRESSORS, DNA 1374 00:59:26,840 --> 00:59:30,360 DAMAGE AND COLLISION THAT WILL 1375 00:59:30,360 --> 00:59:39,400 TRIGGER REMOVAL OF POLE -- 1376 00:59:39,400 --> 00:59:44,920 POLYMERASE AND TYPICALLY THROUGH 1377 00:59:44,920 --> 00:59:52,800 MODIFICATION IS THE TURNOVER. 1378 00:59:52,800 --> 00:59:54,600 >> I HAVE A QUESTION ON THE 1379 00:59:54,600 --> 00:59:57,320 CLOSE. 1380 00:59:57,320 --> 01:00:03,440 YOU SHOWED DATA IN THE MOUSE AND 1381 01:00:03,440 --> 01:00:12,200 WE KNOW THE CODE ENDS. 1382 01:00:12,200 --> 01:00:14,920 >> IT'S THE IDEA SO YOU HAVE 1383 01:00:14,920 --> 01:00:18,160 THIS HYPER ABUNDANT NUCLEAR RNA 1384 01:00:18,160 --> 01:00:23,000 AND THE INFECTED CELLS IT'S 1385 01:00:23,000 --> 01:00:25,600 BINDING THE PAB C FROM THE 1386 01:00:25,600 --> 01:00:26,280 CYTOPLASM. 1387 01:00:26,280 --> 01:00:28,200 THE QUESTION IS IN THE CONTEXT 1388 01:00:28,200 --> 01:00:31,640 OF KSAB IS MAYBE HELPING RETAIN 1389 01:00:31,640 --> 01:00:33,360 PAD IN THE NUCLEUS. 1390 01:00:33,360 --> 01:00:35,600 CERTAINLY IT'S INFLUENCING 1391 01:00:35,600 --> 01:00:37,440 PRESUMABLY PAN STABILITY THERE. 1392 01:00:37,440 --> 01:00:39,440 WE DON'T THINK THAT'S WHAT'S 1393 01:00:39,440 --> 01:00:46,920 BRINGING PAP INTO THE NUCLEUS. 1394 01:00:46,920 --> 01:00:52,920 WE THINK IT'S SERVING AS A 1395 01:00:52,920 --> 01:00:58,880 SPONGE IN THE NUCLEASE AND I 1396 01:00:58,880 --> 01:00:59,440 THINK IT'S INTERESTING 1397 01:00:59,440 --> 01:01:00,640 POSSIBILITY I DON'T KNOW THE 1398 01:01:00,640 --> 01:01:00,920 ANSWER TO. 1399 01:01:00,920 --> 01:01:01,120 YEAH. 1400 01:01:01,120 --> 01:01:05,160 >> THANK YOU. 1401 01:01:05,160 --> 01:01:06,360 >> BEAUTIFUL TALK. 1402 01:01:06,360 --> 01:01:08,680 SO YOU SPOKE TO THE CHAOS OF 1403 01:01:08,680 --> 01:01:10,440 REPLICATION AND I THINK 1404 01:01:10,440 --> 01:01:12,280 PACKAGING'S BEEN MORE OF A BLACK 1405 01:01:12,280 --> 01:01:12,560 BOX. 1406 01:01:12,560 --> 01:01:14,240 YOU HAVE REPLICATION, LATE GENE 1407 01:01:14,240 --> 01:01:15,760 EXPRESSION AND PACKAGING WHICH 1408 01:01:15,760 --> 01:01:18,600 SEEM TO NOW BE LINKED. 1409 01:01:18,600 --> 01:01:20,960 ONE QUESTION I HAD MAYBE A 1410 01:01:20,960 --> 01:01:23,160 SIMPLE HYPOTHESIS FOR THE DEFECT 1411 01:01:23,160 --> 01:01:25,960 IS IF YOUR NOT PACKAGING THE 1412 01:01:25,960 --> 01:01:27,680 GENOMES DO YOU JUST HAVE MORE 1413 01:01:27,680 --> 01:01:29,360 GENOMES OVER ALL AND A 1414 01:01:29,360 --> 01:01:32,160 DISTRIBUTION OF POL II WITH THE 1415 01:01:32,160 --> 01:01:33,120 LATE COMPLEXES AND WHY YOU'RE 1416 01:01:33,120 --> 01:01:34,120 GETTING LATE GENE EXPRESSION? 1417 01:01:34,120 --> 01:01:37,280 >> I THINK THAT WAS A 1418 01:01:37,280 --> 01:01:39,600 POSSIBILITY THAT WE CONSIDERED 1419 01:01:39,600 --> 01:01:45,360 BUT THAT I THINK IS DISPROVEN WE 1420 01:01:45,360 --> 01:01:47,560 CAN BLOCK PACKAGING AND NOT 1421 01:01:47,560 --> 01:01:49,080 IMPAIR EXPRESSION OF LATE GENES 1422 01:01:49,080 --> 01:01:50,800 WHICH SUGGESTS JUST HAVING MORE 1423 01:01:50,800 --> 01:01:54,040 DNA AROUND IS NOT THAT YOU'VE 1424 01:01:54,040 --> 01:01:58,400 GOT A LIMITING AMOUNT OF POL II 1425 01:01:58,400 --> 01:02:00,400 AND SAMPLING THE DNA ALL OVER 1426 01:02:00,400 --> 01:02:02,560 THE PLACE WITH OTHER NUCLEIC 1427 01:02:02,560 --> 01:02:04,680 ACID PROTEINS. 1428 01:02:04,680 --> 01:02:06,360 >> ERIC, ANY CLOSING THOUGHTS. 1429 01:02:06,360 --> 01:05:22,880 >> THANK YOU VERY MUCH.