LOOKS LIKE WE HAVE A PRETTY GOOD
TURNOUT AND HOPE NOT TOO MANY
ARE STRUCK IN SECURITY AND GET
GOING ON THE MEETING AND WELCOME
BACK.
WELCOME TO NEWCOMERS.
I HOPE YOU SEE THAT WE ALL LOVE
TO TALK ABOUT SCIENCE.
THAT'S WHAT WE'RE HERE TO DO AND
WELCOME ESPECIALLY TO THE
VIDEOCAST MEMBERS ONLINE THAT
COULDN'T MAKE IT TO THE MEETING.
SO WE'RE HERE IS TO EVALUATE THE
WILL HIV CENTER FOR STRUCTURAL
BIOLOGY AND TALK ABOUT THE
SCIENCE AND SEE WHAT CUTTING
EDGE SCIENCE LOOKS LIKE AND WHAT
LARGE GROUPS OF GOOD SCIENTISTS
CAN GET DONE.
SO, I DIDN'T NOTICE UNTIL I PUT
THIS ON THE SLIDE TOGETHER THE
OTHER DAY, WE'VE GOT THEM SET UP
THREE A DAY.
YOU I'M NOT SURE IF YOU'VE SEEN
THIS BUT WE HAVE THREE ONE-HOUR
SESSIONS AND SET THEM UP WEST TO
EAST BUT THEN ALSO REALIZED I
SET UP A GENDER BALANCE AND THIS
MEETING HAS BEEN GOING ON 18
YEARS THIS PROGRAM IS IN ITS
18th YEAR AND BEEN AT NI AD AND
THE RO1s AROUND GRANTS
REPRESENTED IN THE ROOM AND IN
THE BASIC SCIENCE PORTFOLIO
REGARDING MOLECULAR AND
STRUCTURAL BIOLOGY ARE KIND OF
BOY
B
BOEYED AND TO THE CENTERED I
WANT TO TALK ABOUT THIS IS
SPONSORED BY THE BASIC SCIENCE
PROGRAM.
THIS IS BASIC SCIENCE WE'LL BE
TALKING ABOUT TODAY.
ORGANIZERS MYSELF AND UDI AND
MODERATORS BRIGITA AND ROGER
WILL TAKE OVER THE AFTERNOON
SESSIONS.
THE REASON I'M LOSING IT IS HERE
IS BECAUSE I WAS GOING TO RUN A
SCRIPT AND THEN FORGOT.
SO I'M GOING TO GO BACK AND
START BY THANKING OUR LEADERSHIP
AND BRINGING TOGETHER TALENTED
SCIENTISTS FROM DIVERSE FIELDS
AND THE MAGIC THEY DO WHAT I WAS
TRYING TO DESCRIBE POORLY BEFORE
IS PUT TOGETHER THE
COLLABORATIONS AND ANSWER BIG
QUESTIONS.
FIRST I WANT TO ACKNOWLEDGE THE
PEOPLE THAT GOT OUT HERE.
THIS IS A PICTURE FROM THE '90s
ON THE NIH CAMPUS WHEN THE
EPIDEMIC IN THE U.S. WAS
PRIMARILY AMONG YOUNG URBAN
WHITE GAY MEN AND THEY WERE
DYING AND ANGRY THE GOVERNMENT
WASN'T RESPONDING WELL.
THERE'S A LOT OF FACES YOU
SHOULD RECOGNIZE UP HERE AND
THREE IMPORTANT LEADERS IN THIS
ACTIVISM WERE PETER STALEY,
LARRY KRAMER, FAMOUSLY WROTE IN
THE VILLAGE VOICE AND MARTIN
DELANEY AND THESE WITH OTHERS
THAT WERE AFFECTED BY THE
EPIDEMIC AT THE TIME CAME TO THE
NIH AND PROTESTED.
YOU AGAIN RECOGNIZE SOME OF THE
FACES.
I THINK DR. FAUCI IS BACK HERE
AND HIS WILL EFFAGY BEING BURNED
AND DR. FAUCI EMBRACED THE
IDEALS OF THE ACTIVIST
COMMUNITIES.
THEY WANTED ACCESS TO CARE AND
LIFE-SAVING MEDICINES AND CHANGE
THE WAY THERAPIES WERE TESTED
AND TREAT ED AND THIS GUY,
PRESIDENT BUSH ASKED DR. FAUCI
FOR ADVICE ON WHAT TO DO ABOUT
THE AIDS PANDEMIC AND
RECOMMENDED THE CONGRESSIONALLY
MANDATED SET ASIDE FOR HIV
FUNDING AND ESTABLISH NEXT
OFFICE OF AIDS RESEARCH.
THAT FUNDING STILL STANDS 30
YEARS LATER AROUND $5 BILLION A
YEAR FOR INTRAMURAL AND
EXTRAMURAL IT NIH PROGRAMS AND
IT'S CHANGED THE PANDEMIC WE
REALIZE IT'S A WORLDWIDE
PANDEMIC HITTING MIDDLE AND
LOW-INCOME COUNTRIES AT THIS
TIME.
THE PROBLEM IS PEOPLE INFECTED
WITH HIV WHO HAVE ACCESS TO CARE
CAN BE HEALTHY.
WE HAVE OVER 30 APPROVED
MEDICATIONS THAT REVERSE AND
REDUCE VIRAL LOAD.
IF YOU TAKE THEM IN COMBINATION
YOU BECOME UNDETECTABLE WHICH
MEANS YOU'RE BOTH FREE FROM HIV
DISEASE AND SYMPTOMS AND YOU'RE
UNTRANSMISSIBLE.
YOU WON'T TRANSMIT THE VIRUS TO
OTHERS.
THE OTHER THING IS YOU MIGHT
NOTICE THE DIVERSITY OF THE HIV
COMMUNITY IS GREATLY WILL
INCREASED AND ENHANCE.
SO THE PANDEMIC STILL HERE BUT
FORTUNATELY WE HAVE ACCESS TO
CARE FOR A LOT OF PEOPLE.
THERE'S ABOUT $40 MILLION PEOPLE
GIVE OR TAKE INFECTED WORLDWIDE
AND THERE ARE ABOUT 30 MILLION
UNDER CARE TO RETROVIRAL THERAPY
BUT STILL ABOUT 10 MILLION LEFT.
SO WE NEED OUR COMMUNITY MORE
THAN EVER AND TO COMMUNICATE TO
US AND COMMUNICATE OUT TO THEIR
IT COMMUNITY TO BRING THEM INTO
EDUCATION AND CARE.
IT BRINGS ME TO THIS.
DR. MARAZZO PUT OUT A STATEMENT
LAST WEEK THE INCLUSIVE
FIRST-PERSON USE LANGUAGE IN ALL
NIAID SPONSORED EVENTS AND THE
COMMUNITY SAID THEY DON'T WANT
TO BE CALLED PEOPLE INFECTED BUT
PEOPLE LIVING WITH HIV AND WE'RE
NOT GOING TO POLICE THE LANGUAGE
BUT ASK EVERYBODY BE RESPECTFUL
AND UNDERSTAND THAT IT'S AN
IMPORTANT ASPECT OF OUR LANGUAGE
THAT WE COMMUNICATE TO OTHERS.
WE INVITE ALL THE COMMUNITY INTO
OUR TALKS AND PRESENTATIONS FROM
BASIC SCIENCE ALL THE WAY TO
CLINICAL AND TRANSLATIONAL WORK.
AND WE ASK -- I THINK IF WE GET
THROUGH THE FIRST TWO -- IT DOES
FEEL UNCOMFORTABLE, CLUMSY AND
DIFFICULT AT TIMES TO CHANGE.
I'M SURE I'VE MADE THIS MISTAKE
ALREADY IN THIS TALK AND SOME
MAY HAVE NOTICED THAT BECAUSE WE
DO.
IT'S HARD TO CHANGE THE WAY YOU
TALK BUT WE THINK THIS WAY.
SO THE BIGGEST ONE I THINK IS
PERSON-FIRST LANGUAGE AND PEOPLE
LIVING WITH HIV INSTEAD OF HIV
INFECTED PEOPLE.
PEOPLE THAT INJECT DRUGS AND
IT'S A FLIP DRUGS AND IT'S
SIMPLE AND RESPECTFUL.
ANOTHER HOT BUTTON ARE SUBJECTS
AND PATIENTS AND COMMUNITY WOULD
PREFERS TO BE PARTICIPANT OR
VOLUNTEER.
THIS IS ALSO TRICKY BECAUSE
HUMAN SUBJECTS IS WRITTEN INTO
OUR CONTRACTS AT NIH NOT ALL
THESE WILL CHANGE RIGHT AWAY BUT
WE ASK TO BE RESPECTFUL AND YOU
CAN READ THE REST AND THERE'S A
LOT OF INFORMATION.
I PUT DOWN THREE WILL ONES THAT
IMPACTED ME A LOT OVER THE IT 30
OR MORE YEARS AND ENCOURAGE YOU
TO LEARN A HISTORY TO LEARN
ABOUT THE IMPORTANCE OF
COMMUNITY IN EVERYTHING WE DO.
SO DETAILS.
I HOPE EVERYBODY KNOWS FOR LUNCH
WILL THE CAFETERIA IS OPEN AND
DON'T RECOMMEND WALKING ACROSS
THE CAMPUS AND IT'S COOL IN HERE
BUT HOPE IT WILL WARM UP WITH
THE BODIES AND HAVE THIS CHOP
OPTION THIS YEAR DELIVERED TO
THE AREA IN FRONT OF THE
AUDITORIUM AND YOU CAN ORDER
THOSE UP TO 9:30.
YOU HAVE A LITTLE BIT OF TIME
LEFT FOR TODAY.
AGAIN THE LAYOUT OF THE MEETING
I DON'T HAVE THE AGENDA IN FRONT
OF ME.
WE'LL DO THREE ONE-OUR SECT --
ONE-HOUR SESSIONS AND
PRESENTATIONS AND Q&A AND BREAKS
IN THERE AT THE END OF THE THIRD
WE'LL HAVE A LONG LUNCH AND
POSTERS.
WE HAVE 135 POSTERS THIS YEAR.
THAT'S MORE THAN I'VE SEEN EVER.
WE SPLIT THEM INTO TWO DAYS TO
GIVE RELIEF TO OUR PRESENTERS
AND ALLOWS EVERYONE TO GO TO
EVERYONE ELSE'S POSTERS.
I ENCOURAGE EVERYONE TO VIEW THE
POSTERS.
THIS IS REALLY EXCITING SCIENCE
AND WHAT'S GOING TO BE HAPPENING
IN THE NEXT 10 YEARS IN THIS
CENTER PROJECTS.
SO WILL AGAIN WE APPRECIATE THAT
THERE'S AS MANY SENIOR AND VERY
JUNIOR PEOPLE AT THE MEETING AND
LOVE THE MIX AND HOPE WE CAN
SUPPORT OUR JUNIOR FOLKS YOU,
LOOK AT THEIR POSTERS, ASK
QUESTIONS AND ENJOY.
AND THEN IN THE AFTERNOON AS
USUAL WE'VE SET UP SOME ABSTRACT
SELECTED TALKS AND WE'LL RUN IT
AS IT GOES AND TRY TO STAY ON
TIME.
YOU GUYS HAVE TWO MINUTES,
ENJOY, EVERYBODY.
[APPLAUSE]
>> OKAY.
GOOD MORNING, EVERYONE.
I'M WES SUNDQUIST AND IT'S MY
PRIVILEGE TO INTRODUCE AND GIVE
THE FIRST TALK FOR THE CHEETAH
CENTER.
LIKE ALL THE CENTERS, OUR
SUCCESS IS REALLY PREDICATED ON
A GREAT GROUP OF PEOPLE.
NIS IS
THIS IS A PICTURE THE ONE-DAY
MEETING RUN BY TRAINEES.
THE CENTER'S ORGANIZED AROUND
THREE PROJECTS.
THE FIRST IS AIMED AT
UNDERSTANDING THE FIRST HALF OF
THE VIRAL LIFE CYCLE IN
MOLECULAR DETAIL AND THE SECOND
AT HOW CELLS CAN DEFEND
THEMSELVES AGAINST HIV AND THE
THIRD PROJECT IS AIMED AT
OBSERVING AND MANIPULATING VIRAL
DYNAMICS ACROSS THE LARGE RANGE
OF SPATIAL AND TEMPORAL SCALES.
YOU'LL HEAR FROM THREE GROUPS OF
PEOPLE FROM OUR CENTER.
I'LL TELL YOU ABOUT OUR EFFORTS
THAT ARE AIMED AT DEVELOPING A
CELL-FREE SYSTEM WITH PURE
RE-AGENTS TO DO REVERSE
TRANSCRIPTION AND INTEGRATION.
THEN YOU'LL HEAR FROM THOMAS
SCHWARTZ AND THEY'RE LOVELY WORK
AIMED AT UNDERSTANDING HOW NANO
BODIES CAN HELP CONTROL THE
STRUCTURE AND FUNCTION OF THE
CAPSID AND THEN JEREMY LUBEN
WILL TALK ABOUT THEIR DISCOVERY
OF A MOLECULE FOR CELLS TO
SENSED UNSPLICED HIV RNA.
I'M JUMP INTO MY PART OF THE
TALK.
THIS IS IT THE FIRST HALF OF THE
VIRAL LIFE CYCLE.
I WANT TO MAKE A COUPLE POINTS
ABOUT IMPORTANT TRANSFORMATIONS
THAT HAVE TO OCCUR.
OF COURSE THE VIRAL RNA SINGLE
STRANDED HAS TO BE CONVERTED TO
DOUBLE-STRANDED DNA COPY.
AND THE IT DOUBLE-STRANDED DNA
COPY HAS TO BREACH THE CAPSID TO
GET OUT.
THAT'S THE SECOND TRANSFORMATION
AND THE THIRD IS INTEGRATION
INTO THE HOST CHROMOSOME.
IF WE'RE GOING TO RECONSTITUTE
THOSE ARE THE STEPS.
AND WORK A LOT OF IT BY PEOPLE
HERE IN THE AUDIENCE OVER THE
LAST FEW YEARS HAS TAUGHT US THE
TRANSFORMATION ARE OCCURRING
DEEP IN THE CELL LARGELY IN THE
NUCLEUS AND THAT'S GREAT
PROGRESS BUT MEANS IT'S HARD TO
STUDY BECAUSE SINGLE VARIANTS
ARE UNCODING AND REPLICATING IN
THE NUCLEUS AND THAT'S PART OF
THE MOTIVATION FOR US TO DEVELOP
A CELL-FREE SYSTEM TO STUDY
THESE PROCESSES IN MOLECULAR AND
STRUCTURAL DETAIL IN A BULK
EXAMINE.
SINCE I'M THE FIRST SPEAKER I'LL
GIVE A BRIEF PRIMER ON THE CORE
PARTICLE.
THE OUTER SHELL IS CALLED THE
CAPSID AND COMPOSED OF ABOUT 250
COPIES OF THE CA HEXAMER AND 12
COPIES OF THE DEXAMER.
AND THE HEXAMER HAS BASIC
RESIDUES REPULSING TOGETHER AND
TWO LAYERS EXCEPT COMBINE THE
PHOSPHATE WHICH NEUTRALIZES
THEM.
THAT'S STABILIZES THE HEXAMER
AND CAN DO CORE PARTICLES WITH
CAPSIDS AND A LAB DISCOVERED THE
CENTER OF THE HEXAMER HAS A
SPECIAL PROPERTY OF BEING ABLE
TO PASS NUCLEOTIDES IN AND OUT
OF THE CAPSID.
NOW WE CAN THINK OF REVERSE
TRANSCRIPTION OF TAKING PLACE
INSIDE A CLOSED CONTAINER.
THIS IS A REMINDER THE CORE
PARTICLE INCLUDES THE VIRAL
REVERSE TRANSCRIPTASE AND TWO
COPIES OF THE SINGLE STRANDED
RNA AND THOUSANDS OF THE NC
PROTEIN.
SO A FEW YEARS AGO WE REPORTED
WE WERE ABLE TO DO REVERSE
TRANSCRIPTION AND INTEGRATION IN
A CELL FREE SYSTEM AND THAT'S
WHAT IT LOOKED LIKE AND START
WITH PURE VIRAL PARTICLES.
AND THE PEPTIDE GIVES ACCESS TO
THE CORE PARTICLE AND IP6 AND
NTPs THE CORE PARTICLE IS STABLE
AND IN THE PRESENCE OF DNTPs
ENDOGENOUS REVERSE TRANSCRIPTION
STARTS AND CAN BE EFFICIENT TO
REPLICATE THE ENTIRE GENOME.
THEN IF WE HAVE A TARGET DNA
PRESENT LIKE A PLASMID WE
OBSERVE INTEGRATION BUT IT
IMPLIES THE REQUIREMENT FOR HOST
FACTORS.
IT AND THERE WERE IMPORTANT
CONTRIBUTIONS ON A NUMBER OF
FRONTS BUT IMPORTANTLY IN OUR
UNDERSTANDING OF INTEGRATION
SITE SELECTION AND OTHERS HAVE
SPEARHEADED THE ELEMENTS AND
I'LL SHOW SOME LATER IN MY TALK.
WE CONCLUDED IT ENDOGENOUS
TRANSCRIPTION WAS REQUIRED TO
GET EFFICIENT REVERSE
TRANSCRIPTION IMPLYING THE
CAPSID WAS AN IMPORTANT PART OF
THE REPLICATION COMPLEX.
AND AS I TOLD YOU WE OBSERVED
INTEGRATION BUT REQUIRED EXTRACT
AND THEREFORE HOST FACTORS AND
SO YOU INTEGRATION THAT OCCURRED
IN THE SEQUENCE PREFERENCES.
IT TOOK US A WHILE BUT WE
FIGURED OUT HOW TO QUANTIFY THE
INTEGRATION EFFICIENCY AND WHEN
WE DID THAT THE NUMBER WAS A
LITTLE BIT EMBARRASSING.
SO OVER ALL THE EFFICIENCY WAS
.03%.
WE PUT IN 10,000 CORES AND GOT
THREE TO REPLICATE COMPLETELY
AND INTEGRATE.
SINCE WE'RE INTERESTED IN USING
THIS TO WATCH THE PROCESS IN
STRUCTURAL STUDY WE FELT WE
NEEDED TO DO BETTERS ORE ELSE WE
WOULDN'T KNOW FOR SURE WE WERE
LOOKING AT PARTICLES THAT WERE
ON PATHWAY.
THEY OPTIMIZED THE BUFFERS I
WON'T TALK ABOUT BUT HAS BEEN
ABLE TO REDUCE CONTAMINATING
EXOSOMES AND NUCLEIC ACIDS AND
USING EVERYTHING NOT INSIDE THE
VARIANT AND AN OLD TRICK TO GET
RID OF EXOSOMES BY TREATING AND
WHEN HE DOES THAT THIS IS OUR
OLD PREP AND A MARKER FOR
EXOSOMES AND WE NOW HAVE EXOSOME
FREE PREPARATIONS THIS IS A
NUCLEIC ACID AND STARTING WITH
PURER VIRAL PARTICLES.
MEANWHILE DEVON CHRISTIANSON WAS
INVESTIGATING HOW WE COULD GET
CORES FROM THE VIRUS TO DO WITH
PURE CORE PREPARATIONS AND USES
MELTON BUT MAKES BIGGER HOLES IN
THE MEMBRANE AND IT WILL AT
ABOUT THIS TIME THEY WERE ABLE
TO PURIFY CAPSID ASSEMBLY TUBES
ON GEL FILTRATION COLUMNS IF
THEY DID THAT IN THE PRESENCE OF
IP6.
WE THOUGHT WE'D TRY THAT WITH AU
THEN AUTHENTIC CORES AND CAN DO
THIS WITH SUFFICIENT IP6
PRESENT.
THIS PEAK NUMBER 2 IS THE PEAK
IN WHICH THE CORES COME OUT.
YOU CAN SEE IT CONTAINS AALL THE
EXPECTED PROTEINS AND IT'S QUITE
PURE IN TERMS OF PROTEIN CONTENT
AND WHEN YOU LOOK AT THAT YOU
CAN SEE IT CONTAINS IN FACT CORE
PARTICLES.
THE ABILITY TO MAKE LARGE
QUANTITIES OF CORE PARTICLES
WILL BE IMPORTANT FOR IMAGING.
AND THEY CAN NOW LITERALLY IMAGE
THOUSANDS AND EVEN TENS OF
THOUSANDS OF VIRAL CORE
PARTICLES BY CRYO EM AND TREAT
THE LATTICE TO GET SUB 3
RESOLUTION AND ALSO WORKS FOR
LIGANDS.
THEY ADDED THE CAPSID INHIBITOR
AND OBSERVED THAT THE LATTICE IS
FLATTENED AND THE SITES THAT
WERE PENTAMERS ARE STILL MISSING
AND STILL HEXAMERS AND CAN USE
THE SINGLE PARTICLE TRICK TO END
UP WITH RECONSTRUCTION OF THE
CAPSID HEXAMER AND YOU CAN SEE
WILL LENACAPAVIR AND INTERPRET
IT IN MOLECULAR DETAIL.
OUR LAB'S BEEN WORKING ON CAPSID
30 YEARS AND I WOND HAVE
BELIEVED IF YOU YOU WOULD HAVE
TOLD ME IT WOULD BE POSSIBLE FOR
THE FIELD AND GET THIS
RESOLUTION AND THERE'S INHIBITOR
YOU MAY HAVE SEEN JUST THIS WEEK
GILEAD ANNOUNCED THAT
LENACAPAVIR IS PROTECTING
AFRICAN AMERICAN WOMEN AND
THERE'S REVERSE TRANSCRIPTION
AND INTEGRATION.
I WOULDN'T HAVE KNOWN THAT WAS
POSSIBLE BUT IT'S AN EXCITING
TIME TO BE WORKING ON CORE
PARTICLES.
THIS REMINDS ME TO TELL YOU
THERE ARE MANY STEPS IN THE
VIRAL REPLICATION GOING FROM A
SINGLE STRANDED RNA TO DOUBLE
STRANDED DNA AND CAN MEASURE
DIFFERENT STEPS USING DIFFERENT
PCR PRIMERS TO MEASURE THE
INITIATION OF REVERSE
TRANSCRIPTION AND MEASURE LATE
DOUBLE STRANDED DNA PRODUCTION
AFTER SECONDS-STRAND TRANSFER
AND MEASURE INTEGRATION.
WE DO ALL OF THAT QUANTITATIVELY
USING DROPLET PCRs AND THE LAB
DID A SIMILAR EXPERIMENT
STARTING WITH PURE CORES AND
THIS IS THE NUMBER OF DNA COPIES
PER CORE AND IT'S QUITE GOOD FOR
INITIATION OF REVERSE
TRANSCRIPTION AND LATE ERT.
DEVON STARTED TO FRACTIONATE OUR
CELL AND THIS STEP DOESN'T
REQUIRE EXTRACT BUT YOU CAN SEE
WE ONLY SEE INTEGRATION IN THE
PRESENCE OF NUCLEAR EXTRACT.
SO IT'S NUCLEAR FACTORS THAT ARE
REQUIRED FOR THIS.
AND YOU'LL ALSO SEE BECAUSE OF
THESE IMPROVEMENTS WE'VE
INCREASED EFFICIENCY ABOUT 100
FOLD SO NOW IT'S NOT SO
EMBARRASSING ANYMORE.
WE'D LOVE TO KNOW ALL THE
FACTORS AND I WANT TO HIGHLIGHT
A FEW THINGS.
THE INTEGRASE PROTEIN INTEGRATES
BINDING DOMAIN AND THE VIRUS USE
TO TETHER TO CHROMATIN.
AND WE HAVE A STRUCTURE FROM THE
LAB AND KNOW WHAT THAT LOOKS
LIKE AND IDENTIFIED WHAT KNOCKS
OUT THE INTERACTION AND THE
VIRUS IS USING THAT TO TETHER
THE INTOSOME TO CHROMATIN AND
THEY'RE TRI METHYLATED ON
LYESENE 36 AND YOU CAN SEE THE
EFFICIENT REACTION AND WE EXPECT
ONE DNA COPY FOR CORE AND FOR
THE FIRST TIME IN THE PURE
RECOMBINANT SYSTEM WE CAN
MEASURE INTEGRATION AND DO THAT
IN THE PRESENCE OF CHROMATINIZED
DNA AND THIS IS MIXED CHROMATIN
AND YOU CAN SEE WE CAN KNOW GET
UP TO ABOUT 10% OVERALL
REPLICATION AND INTEGRATION
EFFICIENCY AND THAT REQUIRES AN
ACTIVE IT-LEDGF MOLECULE WITHOUT
INTEGRATION.
THIS NEXT SLIDE IS A RECENT
OBSERVATION AND A SURPRISE TO US
AND THE OBSERVATION IS THAT THE
INTEGRATION STEP GOES WELL AT 4
DEGREES CENTIGRADE.
WE CAN DO OUR NORMAL EXPERIMENT
THIS IS A 10 HOUR INCUBATION AND
GET DOUBLE-STRANDED DNA AND THEN
TAKE THAT SAMPLE AND EITHER
LEAVE IT AT 37 FOR 10 MORE HOURS
OR SHIFT IT TO 4 DEGREES.
WE CAN STOP INTEGRATION WHEREVER
WE WANT BY ADDING WILL
REGTAGLAVIRE AND IN THE FIRST
CONDITION HE GOES FOR 10 HOURS
AND 37 AND SHIFTS TO FOUR
DEGREES FOR ANOTHER 10 HOURS.
YOU SEE THE SLIGHT INCREASE IN
ERT AND TO BE EXPECTED AT 37.
THE REAL SURPRISE IS WE ONLY
OBSERVE INTEGRATION ESSENTIALLY
UNDER THE 4 DEGREE CONDITIONS.
THAT MEANS WE'RE DOING
INTEGRATION.
THERE'S SOME STEP BETWEEN THE
COMPLETION OF REVERSE
TRANSCRIPTION AND INTEGRATION
THAT IS PROMOTED BY 4 DEGREES.
I'M HAPPY TO SPECULATE THAT IN
QUESTIONS IF YOU WANT BUT WE
DON'T COMPLETELY UNDERSTAND IT
YET.
THE FINAL THING I WANT TO TELL
YOU TO MAP THE SEQUENCE AND
THAT'S WHAT THIS IS, WE'RE NOW
EFFICIENT ENOUGH WE DON'T HAVE
TO USE PCR, WE CAN DIRECTLY
SEQUENCE ALL THE INTEGRATION
SITE AND WHEN WE DO THAT WE SEE
THE EXPECTED PALINDROME
PREFERENCE AND THAT MATCHES THE
SEQUENCE THAT WE HAVE PREVIOUSLY
SEEN IN OUR CELL EXTRACTS AND
OTHERS HAVE SEEN IN INFECTED
CELLS AND PATIENTS.
THERE WAS AN INTERESTING
OBSERVATION THE PALINDROME IS
EMBEDDED WITHIN A LARGER
PALINDROME AND SEE IT ON THE
INTEGRATION SITE AND SO FORTH.
WE THINK THAT CAN ONLY BE
EXPLAINED THE INTOSOME SEES 50
BASE PAIRS AND THAT'S OF
INTEREST FROM THE STRUCTURES
THAT HAVE COME UP FOR
LENTIVIRUSES.
SO JUST TO FINISH, WE NOW HAVE
QUITE EFFICIENT ERT.
IT GOES WITH PURE CORES.
THE PURE CORES ARE STILL VISIBLE
AT THE END OF THE REACTION.
WE THINK EVERYTHING'S TAKING
PLACE INSIDE AN INTACT CAPSID
AND USE LEGGF BUT HAS A LARGE
PALINDROMIC PIECE OF DNA AND
INTEGRATION IS STRONGLY FAVORED
AT 4 AND OUR OVER ALL YIELDS ARE
NOW ABOUT 10%.
WITH THAT I'LL FINISH BY
THANKING THE PEOPLE ONCE MORE
WHO DID ALL THE WORK I TALKED
ABOUT.
THANKS.
OF
>> OKAY.
HELLO.
MY NAME IS THOMAS SCHWARTZ.
I'M ALSO AT THE CENTER AND I'LL
TELL YOU TODAY ABOUT NANO BODIES
AGAINST CAPSIDS WE GENERATED IN
THE LAB.
THE MOTIVATION FOR THIS IS NANO
BODIES THE CAPSID WOULD BE A
USEFUL TOOL SIMPLY BECAUSE NANO
BODIES ARE WONDERFUL SMALL
PROTEINS THAT TYPICALLY CAN HAVE
VERY HIGH AFFINITY AND WE WANTED
TO GENERATE THOSE TO TRACK
CAPSIDS AND TO LABEL IT OR
ANCHOR IT AT CERTAIN POSITIONS
IN THE PASSAGE THROUGH THE CELL.
AND WE INTRODUCED THE HIV
CAPSID.
SO ALL WE NEED TO KNOW FOR THIS
PURPOSE IS IF YOU WANT TO FIND
AN ANTIBODY THAT BINDS TO THE
CAPSID IT NEEDS TO BIND TO THE
CA PROTEIN AND THE MONOMER IS
ORGANIZED IN PENPENTAMERS AND
HEXAMERS.
AND THE CAPSID HAS A SMALL
PORTION OF THE PORTION TO BIND
TO NORD TO WORK.
-- IN ORDER TO WORK.
AND THE ONES PUBLISHED ARE
INCOMPATIBLE WITH THE CAPSID.
AND SOME BIND TO THE HEXAMER AND
AREN'T OF USE TO US.
AND WE TEAMED UP IN GERMANY AND
COLLABORATED WITH VARIOUS THINGS
AND PRODUCED NANOBODIES IN THE
ALPACA FARM AND WE THOUGHT LET'S
TRY THIS.
THEY HAD ALREADY IMMUNIZED THEM
BEFORE WE STARTED AND THOUGHT WE
CAN SCREEN AGAINST THE HEXAMER.
WE DID THIS AND GOT A LIBRARY OF
13 DIFFERENT NANO BODIED AND
SEQUENCE TO FIVE GROUPS.
WE TESTED THESE NANOBODIES AND
SEE THEY ALL BIND WITH HIGH
AFFINITY.
AND THIS HAS ONE THAT'S ALMOST
NEGLIGIBLE AND WE DID THIS ASSAY
WHERE WE INCUBATED THEM WITH THE
MONOMER AND SUGGESTED A 1:1
BINDING AND WE CAN SEE THEY
DON'T ALL BIND THE SAME WAY
BECAUSE SOME HAVE THIS
INTERESTING BEHAVIOR WHEN THEY
BIND TO THE MONOMER IT SHIFTS
SUGGESTING THEY ASSEMBLE THE CA
PRESUMABLY INTO A HEXAMER AND
DIFFERENT IN THE BINDING AND
STRUCTURE AND WE WANT TO KNOW
THE ATOMIC DETAILS OR RESIDUE
DETAILS.
NANOBODIES AND THE STRONGEST
BINDER WAS CRYO EM AND THE OTHER
CRYSTALS GREW QUITE NICELY WITH
GOOD RESOLUTION AND WHAT WE CAN
SEE FROM THIS IS THEY ACTUALLY
ALL BIND TO THE INTERNAL DOMAIN
BUT IN DIFFERENT WAYS AS YOU CAN
SEE MOST DRAMATICALLY HERE AND
THESE TWO CAN BIND TOGETHER AND
EVEN HAVE A CRYSTAL STRUCTURE OF
BOTH OF THEM BOUND.
ALL RIGHT.
SO IT GIVES US DETAILS AND HOW
DOES THIS RELATE TO THE
PHENOMENA WE SAW?
WE CAN NICELY EXPLAIN WHY SOME
BIND MONOMER AND SOME BIND AND
FORCE THE ASSEMBLY.
THE ONES THAT REALLY BIND 1:1
BIND JUST ONE CA AND HAVE A
BINDING SITE AND WHAT YOU CAN
APPRECIATE HERE IS THAT THE
BINDING SITE IS PRETTY MUCH
OVERLAPPING.
THEY ARE COMPETITIVE BINDERS.
BUT REALLY HOW THEY RECOGNIZE
THIS IS QUITE SIGNIFICANTLY
DIFFERENT.
AND WHAT HAPPENS IN THE OTHER
CASE?
WHAT HAPPENS IN THE CASE WHERE
WE SEE THE SHIFT INFILTRATION IS
HERE THE BINDING SITE IS
ACTUALLY COMPOSED OF THE TWO
NEIGHBORING CAs AND BRINGS THE
TWO TOGETHER AND STABILIZES THE
HEXAMER.
ALL RIGHT.
SO THAT'S ALL GOOD PRELIMINARY
DATA SUGGESTING THESE NANOBODIES
BIND HEXAMERS OR SHOWING THEY
BIND HEXAMERS BUT WE WANT IT
THEM TO BIND CAPSIDS.
WE FIRST CHECK THESE IN SILICO
AND IMPOSED WHAT THEY THE CA.
-- CAPSID WOULD LOOK LIKE AND
THESE TWO THERE'S NO CLASH WE
CAN SEE AND SUGGESTS THEY SHOULD
BE COMPATIBLE WITH BINDING TO
THE CAPSID AND WE SEE CLASH WITH
A NEIGHBORING ANTIBODY
SELF-CLASH OR CLASH OF THE
NEIGHBORING IT CAPSID PROGRAM
AND LATTICE CLASH AND THESE ARE
CASES WHERE THEY WERE IN THE
FIELD OF THE CAPSID OR BIND WITH
STABILITY OR WE WOULD SEE WILL
THE SUBBINDING AND TESTED THIS
AGAIN IN AN ASSAY WHERE WE
STABILIZE AND FORM CRPs AND THEN
LOOKED AND SAW THE TWO PRIME
ANTIBODIES AOA AND AO5 WITH THE
TWO CLPs AND THE OTHER THREE
ALSO BIND BUT YOU CAN CLEARLY
SEE WILL THEY'RE DIFFERENT AND
CONTROL ANTIBODIES DO NOT AS
EXPECTED.
THEN WE WANTED TO LOOK AT THIS
STRUCTURE AS WELL AND ASSEMBLED
WITH THE RESPECTIVE NANOBODIES
AND MADE TWO DIFFERENT CRYO EM
STRUCTURES ONE OF THE STRONGEST
BINDER AS IT BINDS A SINGLE CA
YOU CAN DO THIS IN THE CONTEXT
OF THE CLP AND CAN DECORATE THE
PENTAMER AS WELL AS THE HEXAMER
AND EVERY CA SEEMS TO BE
OCCUPIED BY THE NANOBODY.
IF YOU DO THIS WITH PO5 WHICH
BINDS BETWEEN THE TWO CA MONOMER
AND HEXAMER AND BINDS AND ALSO
DOES NOT BIND TO PENTAMER
BECAUSE IN THE PENTAMER IT
CANNOT PROPERLY BIND BUT IT CAN
DECORATE THE CAPSID.
ALL RIGHT.
SO NOW I SAID SOMETHING IN THE
TITLE ABOUT USING THESE
NANOBODIES AS A THERAPEUTIC.
WHAT IS THE IDEA HERE?
THE INTERESTING PART ABOUT THE
NANOBODIES IS THEY INTERESTINGLY
BIND CLOSE AND SOME OVERLAPPING
FASHION WITH THE BINDING SITE ON
C
CA AWE CAN SEE IT BINDS AND
BLOCKED IF IT COINCUBATE WITH
THE DIFFERENT ANTIBODIES.
WHY IS THIS IMPORTANT?
THE FG BINDING IS IMPORTANT IN
THE IT HIV LIFE CYCLE AS THE
CAPSID TRAVELS SO THE CYTOPLASM
AND THE NUCLEUS IT HAS TO PASS
THE NUCLEAR CORE COMPLEX AND
THAT CHANNEL IS ESSENTIALLY A
CLOUD FULL OF FGs AND WHAT IT'S
ENGAGED WITH.
SO WE BASICALLY SHOWED EARLIER
WE PUBLISHED EARLIER THIS YEAR
TOGETHER WITH A GROUP IN BACK TO
BACK PUBLICATION HOW THE IS
ASSEMBLED CAPSID CAN GO THROUGH
THE COMPLEX AND THE CORE OF THE
NUCLEAR CORE COMPLEX IS FILLED
WITH THIS THIN MESH WORK OF
PROTEINS RICH IN FG REPEATS AND
CAN PASS AND THE KEY ASSAY AWAY
USED HERE WAS AN FG HYDROGEL
ASSAY DONE IN THIS LAB AND THEY
CAN MIMIC WHAT HAPPENS IN THE
NUCLEAR CORE COMPLEX IN A SMALL
HYDRO SHELL AND YOU CAN TAKE FG
PEPTIDES OR PROTEINS AND FORM A
MICROFG DROPLETS WITH THE
PROPERTIES OF THE NUCLEAR CORE
COMPLEX MEANING CARGO THAT IS
BOUND BY NUCLEAR TRANSPORT
RECEPTOR CAN PENETRATE THE
DROPLETS.
IF YOU LABEL IT AND BIND IN WITH
THE RECEPTOR IT GOES IN THE
DROPLETS AND PAINT THEM GREEN
WHERE IN EXCLUSION MODELS THEY
STAY OUTSIDE.
IN THE PAPER WE HAVE SHOWN THIS
WITH CLPs WE ASSEMBLED FOR THE
NANO METER SPHERES OR THE THOSE
FILLED WITH THE IT MARKER AND
YOU CAN SEE THE IMPORT OF THE OF
THESE HYDROGELS AND IT GOES IN
JUST A NUCLEAR TRANSPORT
RECEPTOR AND NOW OF COURSE THE
QUESTION IS WHAT DO THE
NANOBODIES DO?
NOW IF YOU DO THE EASY IN THE
PRESENCE OF THESE -- ASSAY IN
THE PRESENCE OF THE NANO MARKERS
AND THEY'RE FILLED WITH GFP AND
IF YOU CONTROL OUT GET THE GFP
SIGNAL INSIDE THE DROPLETS AND
COME IN WITH THE DIFFERENT
NANOBODIES AND YOU CAN SEE
BASICALLY IN ALL OF THEM IT'S
COMPLETELY BLANK AND THEY BLOCK
THE IMPORT INTO THE NUCLEAR CORE
AND IN A CONTROL EXPERIMENT WITH
A POINT MUTATION THAT CANNOT
BIND THE CAPSID ANYMORE THEN OF
COURSE YOU CAN RESCUE.
SO THEY COULD WORK IN PRINCIPLE
AS A COMPETITOR FOR NUCLEAR CORE
IMPORT BUT OF COURSE WE ALSO
KNOW THERE IS THIS STABILITY
ISSUE IF YOU BIND SOMETHING INTO
CAPSID THAT AFFECTS POTENTIAL
THE CAPSID STABILITY MOST
FAMOUSLY SHOWN BY THE
LENACAPAVIR AND WANTED TO TEST
WITH THE STABILITY OF CAPSIDS
AND AND THIS IS THE ST. LOUIS IS
ASSAY WHERE WE TAKE PURIFIED
CAPSIDS THAT UNDER GO EN DODGE
NEWS REVERSE TRANSCRIPTION --
ENDOGENOUS REVERSE TRANSCRIPTION
AND ASK HOW STABLE IT IS IF WE
COME IN WITH NANOBODIES OR
LENACAPAVIR AND COLLECT FOR THE
RELEASE OF DOUBLE-STRANDED DNA
AND CONVERT ATP AND GTP TO CGAMP
AND LYDIA DID THIS WONDERFUL
EXPERIMENT IN THE LAB.
YOU CAN SEE HOW THE CAPSID IS DE
STABILIZED IN A CONCENTRATION
DEPENDENT MANNER.
IF WE DO THIS WITH DIFFERENT
ANTIBODIES, SOME HAVE A SMALL
EFFECT.
OUR PREFERRED BINDER HAS NO
EFFECT AND THEN WE HAVE ONE WITH
A STRONGER EFFECT SO WHEN WE
LOOK AT THIS WE HAD THE
STRUCTURE AND DID SOME STRUCTURE
GAZING AND MODIFIED THIS AND NOW
HAVE A NANOBODY THAT REALLY IS
BASICALLY AS STRONG AND
DESTABILIZING AS LENACAPAVIR IS.
AND LAST BUT NOT LEAST, WE
TESTED THESE NANOBODIES CAN
RESTRICT SO WE TURNED TO JEREMY
LUBEN'S LAB AND ASKED IN AN
INFECTION ASSAY WHETHER THESE
NANOBODIES COULD RESTRICT AND SO
IN THIS ASSAY OF GFP IS
BASICALLY A READ OUT FOR
INFECTION.
AND YOU CAN LOOK AND GET A
CERTAIN SIGNAL WITH A KNOWN
RESTRICTION FACTOR YOU CAN GET A
REDUCTION IN INTEGRATION AND IF
YOU DO THE SAME ASSAY WITH OUR
MODIFIED NANO BODY THEN THE
INFECTION GOES EVEN FURTHER DOWN
AND IF YOU TAKE A SINGLE POINT
MUTATION WITH THE INTERACTION
WE'RE BACK TO CONTROL.
[RINGING]
>> SORRY.
[LAUGHTER]
SO THEY CLEARLY OPERATE IN A
RESTRICT ITCH MANNER.
SO IN SUMMARY WE'VE DEVELOPED A
NUMBER OF NANOBODIES.
SOME WE CAN NICELY SAY CAN BIND
THE CAPSID WITH VERY HIGH
AFFINITY AND DON'T SEEM TO
INTERFERE WITH THE STABILITY AND
USE THEM IN MARKERS AND TRACERS
AND WE HAVE ENGINEERED IN A WAY
THAT DESTROY THE CAPSID AND CAN
BE USED IN A THERAPEUTIC WAY.
I'LL END WITH MY
ACKNOWLEDGEMENTS.
I TOLD YOU ABOUT OUR WONDERFUL
COLLABORATIONS.
THIS WORK IS PRIMARILY DONE BY A
FANTASTIC POSTDOC IN MY LAB WHO
HAS DONE ALL THE STRUCTURES AND
ASSAYS AND NICK SWANSON A CRYO
EM EXPERT AND WAS HEAVILY
INVOLVED IN THAT ASPECT OF THE
PROJECT.
THANK YOU.
IT
>> THERE'S A COLLABORATION OF
THE CHEETAH CENTER.
IT'S AN INCREDIBLE PRIVILEGE TO
BE HERE WITH YOU TODAY.
AND I'M GOING TO TELL YOU A
STORY ABOUT DETECTION OF THE
UNSPLICED RNA BY MD85.
BRIEFLY I'M GOING TO REMIND YOU
THAT -- MDA5.
I'M GOING TO REMIND YOU THERE'S
30 YEARS OF RESEARCH LARGELY
FUNDED BY A.I., I'D SAY TO
IDENTIFY AND CHARACTERIZE
RESTRICTION FACTORS OF HIV-1 AND
IN PARALLEL FACTORS THAT COUNTER
ACT AND AND CHEETAH AND THE
OTHER CENTERS HAVE BEEN
CHARACTERIZING THESE LARGELY
PROTEIN-PROTEIN INTERACTIONS FOR
SOME IN THE FIELD.
IN FACT, SIGNATURES OF POSITIVE
SELECTION ARE CONSIDERED A
DEFINING FEATURE OF RESTRICTION
FACTORS.
SO CHEETAH HAS BEEN INVOLVED IN
HOW THEY INTERACT WITH VIRAL
PROTEINS AND HOW THEY COUNTER
ACT AND HIGHLIGHT A COUPLE
PEOPLE IN OUR CENTER.
THESE TWO VERY TALENTED PEOPLE
CAN TURN THEIR HEADS COMPLETELY
180 DEGREES BACKWARDS.
WE'VE HEARD A BIT ABOUT JARED
WORK AND MOLLY AND SIMILARLY AS
A GIANT IN OUR CENTER SCREENING
AND IDENTIFYING NEW HOST
FACTORS.
WE HAVE THESE SENIOR MEMBERS
WELL KNOWN IN OUR COMMUNITY.
THEY HAVE A LARGE NUMBER OF
ABSTRACTS THAT WILL BE APPEARING
AT THE MEETING.
AT THE SAME TIME FOR A NUMBER OF
YEARS WE'VE BEEN WONDERING
WHETHER THE CELL HAS THE ABILITY
TO DISTINGUISH BETWEEN
RETROVIRAL RNA AND GENERIC mRNAs
ARE CAPPED AND SAME OF
RETROVIRAL RETROVIRAL RNAs AND
THEY HAVE UNSPLICED FORMS OF THE
RNA THAT HAVE TO GET TO THE
CYTOPLASM AS A GENOMIC RNA AND
ENCODE STRUCTURAL PROTEINS WE'VE
WONDERED WHETHER THE CELLS HAS
TOOLS TO DISTINGUISH BETWEEN THE
RNAs.
OVER A NUMBER OF YEARS MY LAB
HAS BEEN FOCUSSING ON TWO
PROJECTING WHERE WE THINK WE
HAVE CONVINCING EFFORTS THAT
RETAINED UNSPLICED FORMS OF
THESE ELEMENTS ARE DETECTED AS A
DANGER SIGNAL AND CONTINUAL SAN
FRANCISCO THEY'VE SEEN A SIMILAR
PATTERN IN CRYPTO CAUCUS WHERE
THEY DETECTED AND THE PATHOGENIC
FUNGUS.
ONE PROJECT WE HAVE WHICH I
WON'T TALK ABOUT CONCERNS CORE
MUCH A CAUSING AN EPIDEMIC IN
KUALAS ALONG THE EAST COAST OF
AUSTRALIA AND WE'RE INTERESTED
BECAUSE IT GOES IN GERM LINE AND
IT'S ENDODGENIZING AND THEY HAVE
AN ADAPTIVE IMMUNE CELL THE
PIRNA SYSTEM AND THE INITIAL
EVENT INVOLVES DETECTION OF THE
UNSPLICED RNA AND THE SPLICED
STRIPS SEEM TO BE IGNORED.
I'LL GET TO THE DETECTION OF THE
UNSPLICED RNA AND ONE MADE BY
RALPH STEINMAN WHO DISCOVERED
THEM AND NAMED THEM DENDRITIC
CELLS.
NOW I'M CRASHING.
AH, OKAY.
SO THIS PROJECT BEGINS WITH A
GRADUATE STUDENT, SHAWN, WHO SET
UP THE SYSTEM MAKING DENDRITIC
CELLS FROM BLOOD AND THE DATA IS
FROM PRIMARY HUMAN BLOOD WHERE
YOU GENERATE AND ISOLATE
MONOCYTES FROM BLOOD.
SHAWN DEVELOPED A PROTOCOL WITH
HOMEMADE HUMAN PRODUCED IL4 AND
IT HUMAN SERUM AND WHEN YOU
CHALLENGE WITH HIV-1 IN THIS
CASE FOR ALL THE ASSAYS I'LL
SHOW YOU WE'RE USING A SINGLE
CYCLE VIRUS WITH REPORTER GENE
AND YOU GET MATURATION AND OUT
CAN LOOK LIKE THIS AND THIS
COMPARES THE RESPONSE OF THIS
ESSENTIALLY FULL-LENGTH HIV
VECTOR WITH A MINIMAL LENGTHY
VECTORS AND THEY GET ACTIVATED
BY THE VIRUS AND NOT THE VECTOR
AND WHEN YOU DO RNA SEQ YOU CAN
SEE PATTERNS THAT LOOK LIKE A
TYPE 1 INTERFERON RESPONSE.
SO SEAN FOR HIS THESIS PROJECT
WANTED TO FIGURE OUT WHAT WAS
BEING DETECTED.
AND TRANSCRIPTION WAS BLOCKED
WITH MUTANTS AND DRUGS AND THEY
WOULD PREVENT TYPE 1 INTERFERON
INDUCTION.
TRANSCRIPTION WAS REQUIRED.
TAD WAS NOT REQUIRED AND YOU CAN
USE A HETEROLOGOUS SYSTEM TO
PRODUCE THE FULL LENGTH RNA.
THE ONLY REQUIRED WAS REF AND HE
FOUND LEPTOMYCIN WOULD BLOCK
WITH THE HIV CHALLENGE BUT THE
VIRUS REPLICATES IN THE
CYTOPLASM DIDN'T SEEM TO CARE
AND THESE EXPERIMENTS CONCLUDED
THE UNSPLICED RNA IS LIKELY THE
DANGER SIGNAL AND SHOWED REM
CRM1 WAS REQUIRED AND WILL THEN
THEY WANTED TO IDENTIFY WHAT THE
RECEPTOR WAS AND THEY MODIFIED
THE PROTOCOL A LITTLE BIT SO
WHEN THE MONOCYTES ARE FIRST
ISOLATED HE CHALLENGES WITH A
THAT DELIVERED A MICRORNA TO
KNOCK DOWN GENES OF INTEREST.
HE MAKES DCs AND ASKS WHETHER
THE KNOCK DOWN HAS AN EFFECT.
HE AND SHAWN PICKED A NUMBER OF
CANDIDA
CANDIDATES THERE ARE ADDITIONAL
ONES BUT THIS COMES FROM RNA
BINDING PROTEINS WE TESTED AND
OF THE AND XPL1 PREVENTED TYPE 1
INTERFERON INDUCTION.
AND THE GENE THAT ENCODES MDA5
AND IN CONTRAST THE DDX58 WHICH
ENCODES DID NOT HAVE AN EFFECT.
AND SO THIS IS JUST A LITTLE BIT
MORE DETAIL.
EACH DOT IS AN INDIVIDUAL BLOOD
DONOR.
YOU PUT HI ON A CONTROL,
KNOCKOUT DOWN AND GET ACTIVATION
AND YOU KNOCK DOWN REGI IT DOES
NOT PREDICT AND KNOCK DOWN MDA5
YOU PREVENT IT.
AND THERE WAS AN ALL IN ONE
SINGLE TRANSCRIPT VECTOR THAT
DOES A KNOCK DOWN AND RESCUE
WITH THE CDNA OR CDNA ENCODING
MDA5 WITH A MUTATION.
SO IT'S NOT TARGETED BY THE
KNOCK DOWN.
YOU CAN SEE BY WESTERN BLOT IF
YOU KNOCK DOWN MDA5, IFIH1
PROTEIN YOU LOSE THE PROTEIN AND
CAN PUT IT BACK IN THE CONTEXT
OF THE KNOCK DOWNS.
AND THE ISG15 TRACKS WITH THIS.
INDEED IT'S NOT AN OFF TARGET
EFFECT BUT SEEMS THE MDA5
PROTEIN IS REQUIRED.
AND IN KNOCK DOWN OF MDA5
EXPRESSES THE INDUCTION OF THE
MAJORITY OF THE ISGs.
WE'RE VERY INTERESTED IN WHAT
THE OTHER GENES ARE THAT ARE
HANGING AROUND WHETHER IT'S JUST
THIS IS A KNOCK DOWN AND NOT A
KNOCKOUT OR WHETHER THERE ARE
OTHER PATHWAYS INVOLVED.
SO WE WANTED TO SEE IF WE CAN
DETECT DIRECT INTERACTION AND
SHE CHALLENGED BETWEEN MDA5 AND
THE RNA AND CROSS LINKED WITH
FORMALDEHYDE AND DID IPs AND DID
PRIMERS FOR SPLICED AND
UNSPLICED RNAs.
SHE CAN PULL OUT RIGGI HERE OR
MDA5 TO UNSPLICED RNA PRIMERS OR
SPLICED PRODUCTS AND WHEN SHE
QUANTITATED THESE BY RTQPCR
FINDS RNAs SPLICED AND IF SHE
PULLS WITH RIGGI SHE DOESN'T SEE
THE SAME.
THERE'S A NUMBER OF OTHER
CONTROLS SHE'S DONE.
SO WE THINK THE UNSPLICED RNA
SEEMS TO BE THE PATH AND GOING
FORWARD WE'RE INTERESTED IN WHAT
THE STRUCTURAL REQUIREMENTS ARE
FOR RECOGNITION OF THE UNSPLICED
RNA WITH ASSAYS LIKE THESE AND
AS PART OF CHEETAH WE'RE HOPING
TO DEVELOP AN IN VITRO SYSTEM
USING PROTEINS PRODUCED BY HEIDI
AND CHRIS IN SALT LAKE CITY AND
MARY HAS SOME AMAZING PREPS OF
FULL LENGTH IN VITRO TRANSCRIBE
HIVs PUT IN THE SYSTEM.
THESE ARE THE FOLKS IN MY LAB
WHO PRODUCED THE DATA I SHOWED
YOU.
THESE GRADUATED AND THOMAS HAS
AN ABSTRACT AND WE HAVE A BRAND
NEW STUDENT HERE.
SO THANK YOU.
>> I DON'T THINK YOU'RE LIVE.
>> CONGRATULATIONS.
BEAUTIFUL PRESENTATION.
FANTASTIC DATA.
ONE QUESTION ABOUT THE ASSAYS
AND COMPARING TO LENACAPAVIR AND
IN THAT ASSAY IT SEEMS
LENACAPAVIR WAS WORKING HIGHLY
EFFECTIVE.
10 NANO MOLAR AND MAYBE I MISSED
IT.
10 NANO MOLAR IS MORE THAN WE'D
EXPECT FOR LENACAPAVIR AND
WONDER IF YOU HAVE COMMENTS FOR
THAT.
>> I SHARE ON THE EXACT ASSAY
MEAN AND WE LOOK AT IT AS A
COMPARISON TO THE NANO BODY AND
COMPARISON.
>> IT'S WORTH ASKING JARED THE
QUESTION BUT THEY SEE A BIPHASIC
BEHAVIOR AND IF YOU GET HIGH
ENOUGH FOR REVERSE TRANSCRIPTION
BUT IT'S ABOUT 10 NANOMOLE AR.
>> GREAT TALKS.
I'M FROM THE DANA FAR BER
INSTITUTE AND WE HAVE YOU IN THE
DID CLARIFY WHETHER THAT FULLY
REPLACES THE NEED FOR NUCLEAR
EXTRACT.
>> YEAH, WE GET HIGHER
EFFICIENCIES SO IN THAT SENSE IT
REPLACES IT BUT I THINK THERE
ARE ADDITIONAL FACTORS IN OUR
EXTRACT THAT I THINK IT ACCOUNTS
FOR THE 4 DEGREES AND WE'RE
MISSING AN ENCODING FACTOR BUT
BECAUSE WE CAN DRIVE THE
REACTION WITH LEGF IT REPLACES
IT COMPLETELY BUT I DON'T THINK
WE HAVE PRECONSTITUTED
EVERYTHING YESTERDAY.
>> WONDERING ABOUT COLD
TEMPERATURE.
CAN TO YOU PONTIFICATE MORE?
>> I THINK AND MAYBE OTHERS
THINK THE SAME SOME EVIDENCE
THAT IT'S NOT JUST ENOUGH FOR
THE CAPSID TO FALL A PART BUT
ONE STEP IS FAVORED REACTION
WHERE THE EQUILIBRIUM IS BEING
DRIVEN AT 4 DEGREES BUT WE
HONESTLY DON'T KNOW THAT.
>> IT'S CALLED BREWED COFFEE.
>> AND IN THE CELL THERE'S SOME
PROTEIN COMPLEX OR INTERACTION.
>> YES.
WE SEE LESS OF A TEMPERATURE
DEPENDENCE WHEN WE USE THE
NUCLEAR VERSUS THE LEGF BUT
HAVEN'T REALLY NAILED IT YET.
>> AND THOMAS IN TERMS OF THE
WILL CELL INFECTION OF THE
PRESENCE OF THE ANTIBODIES OR
HAVE YOU INFECTED WITH N74D?
>>
>> THE LATTER NOT YET.
THESE ARE BRAND NEW.
WE'RE DOING SPREADING.
SPREADING INFECTIONS WE CAN'T --
CAN SEE EVERYTHING.
IT'S SPECTACULAR.
>> THANK YOU.
GREAT TALKS, REALLY IMPRESSIVE.
I HAVE A QUESTION.
THIS ENHANCED THE INTEGRATION
FOR ENCHROMATINIZED CONTEXT BUT
ON THE LEFT ID LOOKED LIKE NAKED
DNA AND LOOKED BETTER THAN
BEFORE.
>> IT STIMULATES BETTER ON
CHROMATIN AND HAS THESE A.T.
HOOKS AND THINK IT COULD BE A
DNA BINDING ELEMENT.
>> SO IT'S NOT JUST CHROMATIN.
>> THAT'S CORRECT.
IT'S BETTER ON CHROMATIN BUT
STIMULATES IN MAKING DNA.
>> THEN MY LAST QUESTION TO
JEREMY DOES MDA5 HAVE AN EFFECT
IN SPREADING INFECTIONS IN
DENDRITIC CELLS?
>> MDA5?
>> RIGHT.
>> SHAWN SPREAD INFECTIONS IN
THE POSITIVE T CELLS IN CASES OF
ACTIVATION.
THIS BRA WE KNEW ABOUT MDA35.
FAFRZ SPREADING INFECTION AND --
IN TERMS OF SPREADING CELLS WE
DON'T HAVE MEANINGFUL DATA YET.
>> OKAY.
>> GOOD QUESTION.
>> THANK YOU.
>> ADDING ON WITH THE QUESTION
IS THERE A TIME TO ADD AT 10
HOURS?
>> WE ADD IT FROM THE START.
I BET DEVON HAS DONE THAT AND
THINK IT WOULD BE CORRELATED
WITH THE INTEGRATION TIME BUT
DON'T REMEMBER FOR SURE.
DEVON CHRISTIANSON'S HERE AND
HAS A POSTER SO YOU SHOULD ASK.
>> IF YOU INCREASE TEMPERATURE
AT A CERTAIN POINT DOES IT
INCREASE INTEGRATION?
>> THE TEMPERATURE DEPENDENCE IN
THE NUCLEAR EXTRACT IS LESS
DRAMATIC.
THERE'S STILL SOME TEMPERATURE
DEPENDENCE AND WITH THE LEGF YOU
CAN GO TO 72 HOURS AND KEEPS
GETTING BETTER AND BETTER.
>> THANK YOU.
>> KELLY LEE, UNIVERSITY OF
WASHINGTON.
THE CORES THAT ARE ICE LAID ARE
BEAUTIFUL.
CAN YOU SEE REVERSE
TRANSCRIPTASE OR RNA OR
INTEGRASE?
>> I THINK OWEN AND BARBI ARE
LOOKING AT THAT BUT THAT'S A
GOAL.
>> MIKE SOMMERS AT UNBC.
>> I KNOW THAT.
>> I THINK IT'S INTERESTING YOU
HAVE THE PRIMARY AT THE
PALINDROME AND PALINDROME
STRUCTURE DNA CAN BE SENSITIVE
TO TEMPERATURE A HAIR PIN VERSUS
DUPLEX.
HAVE YOU THOUGHT ABOUT THAT AS A
POSSIBILITY?
>> THEY WERE PUSHING US TO
REMIND US IT WAS TEMPERATURE
SENSITIVE AND THAT'S POSSIBLE
AND THE INTOSOME IS TWOFOLD
SYMMETRIC AND THE SURPRISE IS
IT'S SUCH A BIG FOOTPRINT THAT
GIVES CONSENSUS.
YEAH.
>> I'M ALICE FROM THE UNIVERSITY
OF MICHIGAN.
HI, WES.
SO I KNOW I'VE ASKED THIS
QUESTION OF YOU AND JARED BEFORE
AND WENT FAST SO I MAY HAVE
MISSED IT BUT SHOWED THE PROTEIN
GELS SHOWED NO PROTEASE IN THE
CORES?
>> THAT'S A GOOD QUESTION.
>> I'M NOT SURE WE LOOKED
CAREFULLY ENOUGH.
IT'S NOT -- THAT WAS A PRO STAIN
SO THAT'S LIKE THE STOCHIOMETRIC
AND WOULD WANT TO DO THAT IN A
METRIC.
>> I'M POINTING THIS OUT TO
EVERYBODY THAT'S POTENTIALLY A
COOL QUESTION HOW THERE WOULD BE
INSIDE THE CAPSID.
>> WE DON'T SEE IT AS A
PROMINENT PROTEIN.
THAT'S TRUE.
>> I'M LOUISA FROM THE
UNIVERSITY OF MINNESOTA.
ABOUT THE MDA5 DETECTION HAVE
YOU CHECKED OTHER CELL LINES
THAT ARE.
METRICS TO FOR THE CELL.
>> MDA5 SPECIFICALLY WE ONLY
LOOKED AT PRIMARY MYELOID CELLS
SO DCs AND MACRO PHAGES.
I KNOW SOME HAVE LOOKED AT SOME
CELL LINES AND SEEN THE SAME
THING.
WE'VE LOOKED AT INNATE
ACTIVATION OF OTHER CELL TYPES.
WE HAVE SOUGHT EYE TRANSFORMED
CELL LINE DO THE BIO CHEMICAL
EXPERIMENTS AND SO FAR WE'VE
BEEN UNSUCCESSFUL FINDING THE
CELL LINE THAT BEHAVES THE WAY
WE LIKE SO WE COULD DO THE BIO
CHEMISTRY EASIER TO REMIND YOU
EVERYTHING I SHOWED YOU IS
PRIMARY BLOOD CELL DERIVED FROM
A DIFFERENT DONOR AND IT'S A LOT
OF WORK.
>> MY QUESTION IS FOR WES.
I WONDER IF YOU CAN COMMENT MORE
WHY DO YOU THINK THERE'S AN
EXPANDED SEQUENCE SPECIFICITY
AND WHY IT WASN'T OBSERVED IN
PREVIOUS STUDIES?
>> THIS IS BRAND NEW DATA IN
WHAT WE OBSERVED.
I DIDN'T MAKE IT CLEAR BUT
THAT'S NOT THE CHROMATINIZED
SEQUENCE.
IT'S POSSIBLE THE LEGF IS SO
STRONG YOU DON'T SEE THE REST OF
THE SEQUENCE SIGNAL SO THIS IS
LINEAR -- WELL, NAKED DNA.
I THINK IT'S POSSIBLE.
I WAS TRYING TO FIGURE OUT
WHETHER YOU THINK THE STRUCTURE
COULD COVER 50 BASE PAIRS BUT
THAT'S WHAT WE OBSERVED BUT IT'S
ON NAKED DNA.
>> THANK YOU.
THAT MAKES SENSE.
IT COULD ALSO BE THE BIPHYSICAL
STRUCTURE AND IT ONLY COVERS
FIVE OR SIX NUCLEOTIDES SO COULD
BE THE BENABILITY OF THE DNA.
>> AGREE BUT WHEN I LOOK AT YOUR
STRUCTURE THERE WERE EMPTY
SEQUENCE THAT LOOK LIKE THEY
COULD BE BOUND TO DNA AND NOT
NOT IN YOUR STRUCTURE.
>> WE CAN DISCUSS FURTHER.
>> LESLIE FROM PENN STATE
COLLEGE OF MEDICINE.
A QUESTION FOR YOU, IF I
UNDERSTAND CORRECTLY YOU USED
CMNCA BUT DIDN'T MENTION IF
ANTIBODY WAS DETECTED OR THE
FULL LENGTH GAG PROTEIN.
DID YOU LOOK FOR THAT?
>> NO, WE DIDN'T SCREEN FOR IT
BUT THE WAY IT CAME ABOUT IS
THEY HAD THE LIBRARY AND THEN WE
WERE INTERESTED IN HEXAMER AND
CAPSID SO THAT'S WHAT WE
SCREENED.
>> THERE COULD BE HIDDEN GEMS IN
THERE.
>> THERE COULD BE.
>> THE SO-CALLED PALINDROME HAS
BEEN REPORTED BY A COUPLE
GROUPS.
FOR EXAMPLE, GO BACK AND LOOK AT
SOME WORK AND THERE'S A
SECONDARY PIECE OF INFORMATION
THAT'S USEFUL AND THAT'S
ESSENTIALLY A PALINDROME AND TWO
HALVES.
THE INDIVIDUAL SITES ARE NOT
PALINDROMIC.
>> I DIDN'T REALIZE THE EXTENDED
FLANKING SEQUENCE.
>> IT'S BEEN REPORTED AS
APPROXIMATELY 100 BASE PAIRS.
>> THANK YOU.
I DIDN'T KNOW THAT.
I SHOULD HAVE REFERENCED THAT.
THANKS.
FIELD LIKE WES' INTEGRATION
CONDITION.
>> IN THE PROJECTION BOOTH CAN
WE GET SLIDES UP?
THANK YOU.
>> OKAY.
GOOD MORNING, EVERYBODY.
SO B-HIVE STANDS FOR BEHAVIOR OF
HIV IN ENVIRONMENT ENVIRONMENTS
AND ONE OF THE CENTERS OF
SEVERAL OF US HAVE BEEN SINCE
EARLY ON AND MORE THAN A QUARTER
CENTURY SINCE THE EVOLUTION OF
ALL THESE CENTERS HAVE SUPPORTED
HIV STRUCTURAL BIOLOGY.
AND B-HIVE IS CO-DIRECTED BY
BRUCE CORBETT AND MYSELF AT
EMORY UNDERSTAND AND THIS YEAR
IS MY TURN.
LAST YEAR WAS BRUCE.
AND NEXT YEAR AGAIN BRUCE.
SO COMPRISED WE HAVE A CENTER
THAT HAS APPROXIMATELY 31
INVESTIGATORS THAT COME FROM 16
DIFFERENT INSTITUTIONS
THROUGHOUT THE WORLD.
AND THEN THIS FORMAT HAS ENABLED
INCREDIBLE OPPORTUNITIES FOR
COLLABORATIONS THROUGHOUT THE
YEARS.
AND WE MEET TWICE A YEAR IN FACE
TO FACE MEETINGS AND HERE'S THE
LATEST IN FEBRUARY '79 AND MEET
AGAIN IN A COUPLE DAYS FROM
TODAY.
AND WE HAD ABOUT 100 PEOPLE THAT
ATTENDED.
THE STRUCTURE OF THE CENTER IS
BASED IN THREE SCIENTIFIC
PROJECT.
THE FIRST STUDIES THE DYNAMICS
OF HIV CORE AND SPECIFICALLY THE
INTERACTIONS OF CAPSIDS WITH
SMALL MOLECULE AND WITH HOST
FACTORS.
THE SECOND PROJECT STUDY THE
DYNAMICS OF HIV NUCLEAR
INTERACTIONS.
AND THEN AGAIN WE STUDY HOST
FACTORS THERE AND HOW THEY
AFFECT EVENTS IN THE NUCLEUS AS
WELL AS NUCLEIC ACID HIV
INTERACTION AND THIRD IS LATER
STAGES OF THE INFECTION,
PACKAGING AND ASSEMBLY.
WE ALSO HAVE FIVE CORES.
THE FIRST IS LED BY GREG BOOTH
AND STEPHAN AND THE SECOND IS ON
STRUCTURAL BIOLOGY AND DIMITRY
AND ROB DICK AND THEN BIO
PHYSICS WITH GREG AND CORINNE
AND DNA SEQUENCING WITH WILL
DEVELOPMENT AND OUTREACH.
IN TERMS OF THE ADMINISTRATION
IT'S RUN BY THE EXECUTIVE
COMMITTEE IN ADDITION TO THE TWO
CO-DIRECTORS WE HAVE TWO NIH
COLLEAGUES RACIAL AND ERIC FREED
AND CHRISTIAN WHO WORKS ON THE
NUTS AND BOLTS OF EVERYDAY
OPERATIONS.
WE'RE HONORED TO HAVE GREAT
SCIENTIFIC ADVISORY GROUP AND
CAROL CARTER AND SHOUT OUT TO
HER SHE BECAME A MEMBER OF THE
NATIONAL ACADEMIES THIS YEAR AND
DOUG RICHMOND.
WE HAVE TWO PROGRAMS AND WE WORK
ON ARTIFICIAL INTELLIGENCE AND
MEGAN KEN AND GUOCUHN JIANG
WORKING ON HIV LATENCY RESERVOIR
IN THE HUMAN BRAIN AND MYELOID
CELLS.
PREVIOUS CDP VEEP -- EVEN AFTER
FUNDING WAS FINISHED THEY
CONTINUED TO PARTICIPATE IN ALL
COLLABORATION WITH OTHER MEMBERS
AND SOPHI HARVEY AND DANIEL FROM
OHIO STATE UNIVERSITY AND THE
UNIVERSITY OF MINNESOTA.
LAST YEAR WE'VE BEEN BUSY.
SEVERAL PUBLICATIONS.
ONE INTERESTING NEW THING WE
HAVE IN THE WEBSITE WHERE WE
HIGHLIGHT SOME HIGH IMPACT
PUBLICATIONS AND IN ADDITION TO
THE SCIENCE WE HIGHLIGHT THE
POST-DOCTORAL FELLOW IN THIS
CASE WE HAVE INTERVIEWS AND
DISCUSSION OF THE SCIENTIFIC
TRAJECTORY AND CAREER PATH WHERE
THEY COME FROM AND WHERE THEY'RE
GOING.
AND THERE'S CONTINUED OUTREACH
AND TRAINING EFFORTS AND DAVID
GOODSELL CONTINUED TO WORK ON
THE PDP WEBSITE AND WE HAVE
MENTORING AND TEACHING IN PERSON
COMPUTATIONAL METHODS AND
WORKSHOP.
THIS TIME IT WAS DELIVERED BY
STE
STEFAN FORLY AND GREG BOOTH AND
WE HAVE A PROGRAM IN SAN DIEGO
FOR UNDER REPRESENTED MINORITY
STUDENTS AND TO PROMOTE
DEVELOPMENT OF SCIENTIFIC
INTEREST AND ACTIVE
PARTICIPATION IN S.T.E.M.
THERE'S ABOUT 30 POSTERS OUT
THERE AND THERE'S ENTHUSIASTIC
YOUNG PEOPLE PRESENTING THEM AND
WE APPRECIATE ALSO IF YOU STOP
BY TO ENCOURAGE THEM AND LEARN
ABOUT WHAT THEY DO.
IS FINALLY THE PRESENTATIONS
FROM ON THE LONG TRANSCRIPTION
PRODUCTS FOR HIV ENCODING AND
THE SECOND IS LENACAPAVIR BASED
AND DELIVER BY KAREN AND ROBERT
TAG TEAM AND THE LENACAPAVIR
WILL WOULD BE BY KAREN.
ON THE M 6I MUTATIONS AND
COMPOUNDS THAT WORK WELL AGAINST
IT.
ROB IS GOING TO PRESENT WITH
IMAGERY LATTICE AND GREG VOTH
WILL PRESENT ON COMPUTER
MODELLING.
WITH THAT WE'RE READY FOR THE
FIRST SPRINGS.
-- FIRST PRESENTATION.
>> OKAY.
THANK YOU VERY MUCH, STEPHAN.
IT'S AN HONOR TO HAVE THE
OPPORTUNITY TO TALK TO YOU ABOUT
OUR RECENT WORK.
SO THE TRADITIONAL VIEW OF HIV
REPLICATION UNTIL RECENTLY --
I'M HAVING TROUBLE SEEING THE
POINTER.
THE CODING IN THE CYTOPLASM IN
COORDINATION WITH REVERSE
TRANSCRIPTION AT THE NUCLEAR
ENVELOPE DURING IMPORT.
ENCODING HAS BEEN TRADITIONALLY
DIFFICULT TO STUDY BECAUSE
QUANTITATIVE ASSAYS TO DETERMINE
CAPSID PROTEIN AMOUNTS
ASSOCIATED WITH COMPLEXES WERE
NOT UNTIL RECENTLY AVAILABLE.
VERY FEW CORES THAT ENTERED THE
CELLS LEAD TO PRODUCTIVE
INFECTION AND BIO CHEMICAL
ASSAYS THAT DETERMINE THE
PROPERTIES OF THE POPULATION OF
VIRUSES LEAD TO INFORMATION
ABOUT POST THE PARTICLES THAT
ARE DEFECTIVE.
IN 2020 WE STATED REVERSE
TRANSCRIPTION OCCURS WITHIN THE
INTACT CAPSID AND COMPLETED IN
THE NUCLEAR.
THE VIRAL CORES LOSE THEIR
INTEGRITY AND SEVERAL STUDY IN
THE LAST FEW YEARS HAVE STRONGLY
SUPPORTED VARIOUS ASPECTS OF
THIS MODEL SUCH AS INTACT CORES
ENTERING THE NUCLEUS AND THE
NEED FOR CAPSIDS TO INTERACT TO
ENTER THE NUCLEUS.
SO A KEY FEATURE OF THIS MODEL
IS THAT INFECTIOUS VIRAL CORES
ENCODE IN THE NUCLEUS AND WANTED
TO LOOK AT THE MOLECULAR
TRIGGER.
RYAN BURDIC VISUALIZED THE
CODING AND LABELLED THE PROTEINS
AND INFECTS THEM AND ALLOWS THE
CORE TO ENTER THE NUCLEUS AND
DID TIME LAPSED IMAGING TO
DETERMINE THE FATE OF THE CORES.
AND THIS LABELLING METHOD HAS
BEEN CHARACTERIZED AND LESS THAN
A TWO-FOLD EFFECT ON VIRUS
INFECTIVITY AND FOR THOSE
INTERESTED IN THIS METHOD GO TO
THE POSTER NUMBER 56.
AND HE'LL HAVE MORE DETAILS
ABOUT THE CHARACTERIZATION.
SO RYAN OBSERVED PARTICLES THAT
EITHER DISAPPEARED IN THE
OBSERVATION TIME OF 24 HOURS AND
LOSS OF THE GFPC SIGNAL
INDICATES CAPSID DISASSEMBLY AND
PARTICLES DID NOT ENCODE AND
STAYED IN THE END OF THE
OBSERVATION PERIOD.
HE QUANTIFIED THE EFFICIENCY AND
ABOUT 60% OF THE CORES IN THE IT
NUCLEUS ENCODE WITH MEDIAN TIME
OF ABOUT 10.6 HOURS AFTER
INFECTION.
HOWEVER, IF YOU BLOCK REVERSE
TRANSCRIPTION OR USE THE
CATALYTIC SITE MUTANT THE
EFFICIENCY DROPS TO A BASELINE
OF ABOUT 13% AND THE KINETICS
ARE SLOWER AND OCCUR WITHIN THE
MEDIAN TIME OF ABOUT 16, 17
HOURS.
IMPORTANTLY IF WE ADD IT WILL
AFTER IB INFECTION 75% OF THE
LATE PRODUCTS HAVE BEEN
SYNTHESIZED AND TELLS US IT MUST
BE SYNTHESIZED TO TRIGGER
EFFICIENT ENCODING.
NEXT WE WANTED TO WE WANTED TO
LOOK AT THE RELATIONSHIP BETWEEN
KINETICS OF UNCODING AND REVERSE
TRANSCRIPTION AND ADDS THE
CAPSID WILL INHIBITOR PF74 AND
LOOKS AMOUNT THE LOSS OF
SENSITIVITY OF PARTICLES AND IT
OCCURS WHEN ENCODING HAS TAKEN
PLACE.
RYAN ADDED THE THERAPY AT THE
BEGINNING OF THE INFECTION TO
INHIBIT REVERSE TRANSCRIPTION
INITIATION AND WASHED IT OUT TWO
HOURS LATER THAT ALLOWED RT TO
PROCEED.
AND THEN ASKED WHAT HAPPENS TO
ENCODING.
HE FINDS IF YOU DELAY THE
REVERSE TRANSCRIPTION BY TWO
HOURS AND SAME DELAY WAS
OBSERVED AND INDICATES A
RELATIONSHIP BETWEEN KINETICS OF
THE REVERSE TRANSCRIPTION AND
ENCODING.
NEXT WE WANTED TO ASK THE EFFECT
OF THE GENOME SIZE ON ENCODING
EFFICIENCY WE LOOK AT THE
VECTORS WITH THE ELEMENTS
REQUIRED FOR PACK ANDING AND
REVERSE TRANSCRIPTION.
THEY'RE TRANSFERRED WITH THE
CAPSID AND WILD TYPE AND
ENCODING EFFICIENCY WAS
DETERMINED IN THE IMAGING ASSAY
AND THE ENCODING EFFICIENCY
CORRELATES WITH GENOME SIZE AND
AS IT APPROACH APPROACHES THE
EFFICIENCY REACHES THE MAXIMUM
OF 60% IN ADDITION THE MINIMUM
GENOME SIZE IS REQUIRED FOR
ENCODING SINCE GENOMES LESS THAN
6.5GV DID NOT ENCODE MORE
EFFICI
EFFICIENTLY THAN THOSE THAT
CONTAINED NO GENOME AT ALL AND
THEY WERE REVERSE TRANSCRIBED
INDICATING THE SMALL GENOMES THE
FAILURES ARE NOT DUE TO THEIR
FAILURE TO REVERSE TRANSCRIBE
THEIR DNA AND WANTED TO SEE IF
THEY'RE CAPABLE OF INTEGRATION
AND GENE EXPRESSION AND THERE
WAS A DIFFERENT CONSTRUCT THAT
CONTAINED MOSTLY HIV SEQUENCES
THAT EXPRESSED GFP IN THE
PROTEIN AND FULL LENGTH THAT
ALSO EXPRESSED GFP AND FOUND
SHORT VECTORS WERE EFFICIENTLY
REVERSE TRANSCRIBED AND
TRANSPORT THE INTO THE NUCLEUS
AND WERE EFFICIENT OF ENCODING
AND DIDN'T ENCODE MORE THAN THE
GENOME CONTROL AND IT WAS LOW
COMPARED TO THE CONTROL VECTOR
WE THINK BECAUSE THE DNA WAS
TRAPPED INSIDE THE CORES THAT
ARE INTACT BECAUSE THEY DIDN'T
ENCODE.
WE WANTED TO ASK CAN WE RELEASE
THE SHORT DNA WITH CAPSID
INHIBITORS AND CAN IT EXPRESS
THE REPORTER GENE AND HE FOUND
THAT WHEN YOU SIS ASSEMBLE --
DISASSEMBLE THE CORES THE DNA IS
DEGRADED.
MOST THE DNAs IS DEGRADED AND
SOME SURVIVES.
SO THE QUESTION IS DOES THE
SURVIVING DNA EXPRESS AND
INTEGRATES WITH THE T ONE HALF
OF 77 MINUTES AFTER TRIGGERING
ENCODING AND THE DNA THAT
SURVIVES IS INTEGRATED AS
EFFICIENTLY WITHOUT THE PF74
TREATMENT.
IN ADDITION -- IF WE TREAT WITH
THE LENACAPAVIR 10 HOURS LATER
AND ASK HOW MANY CELLS ARE
EXPRESSING GFP WE FIND COMPARED
TO THE UNTREATED CELLS ADDING
PF74 INCREASES THE EXPRESSING
CELLS SO IT MEANS THE SHORT DNAs
CAN INTEGRATE AND EXPRESS THE
REPORTER.
TO SUMMARIZE MOST SHORT DNA
REMAINS TRAPPED IN THE CORES AND
IT'S NOT DEGRADED AFTER PF74
INDUCED CAPSID AND CAN EXPRESS
THE GFP REPORTING.
NEXT WE WANTED TO LOOK AT THE
ROLE OF CAPSID STABILITY IN
ENCODING.
WE ENVISIONED THE DNA THAT IS
SYNTHESIZED CREATES AN OUTWARD
PRESSURE I'LL SAY MORE ABOUT
THAT LATER AND THE OUTWARD
PRESSURE IS COUNTER ACTED BY THE
INWARD PRESSURE CREATED BY THE
CAPSID STABILITY.
YOU LOOK AT THE CAPSID STABILITY
WE MANIPULATED THE CORE BINDING
TO AND INCREASES THE STABILITY
OF THE CA MUTANT AND THE BINDING
HAS MANY AFFECTS ON IT POWER
REPCATION AND PROTECT THE VIRAL
CORES AND SLOWS THE KINETIC OF
IMPORT AND INCREASES CAPSID
STABILITY.
THEY'D BE FOUND IT WAS MORE
EFFICIENT THAN THE CONTROL
VECTOR.
SIMILARLY IF THE CELLS WERE
TREATED TO INHIBIT AND THIS
EFFICIENCY WAS INCREASED
COMPARED TO THE CONTROL.
AND WE DON'T GET ENCODING IN THE
ABSENCE OF THE BINDING.
THEREFORE LOW CAPSID STABILITY
INFLUENCES THE EFFICIENCY OF
ENCODING AND SYNTHESIS OF DOUBLE
STRAND DNA REMAINING A TRIGGER
FOR ENCODING.
NEXT WE WANTED TO LOOK AT THE
EFFECT S OF CYCLOPHILIN BINDING
ON THE KINETICS OF REVERSE
TRANSCRIPTION ENCODING AND WE
COMPARED THE KINETICS IN THE
TREATED CELLS AND FOUND REVERSE
TRANSCRIPTION IS COMPLETED AT
ABOUT THE SAME TIME IN THE CELLS
HOWEVER, THE ENCODING OCCURS
MUCH FASTER IN THE CSA TREATED
CELLS.
TO SUMMARIZE IN THE PRESENCE OF
BINDING THE CAPSIDS ARE MORE
STABLE AND ENCODING OCCURS TWO
AND A HALF HOURS AFTER REVERSE
TRANSCRIPTION IS COMPLETED AND
BINDING ENSURES ENCODING OCCURS
AFTER COMPLETION OF THE REVERSE
TRANSCRIPTION.
WHEN THE IT CODING OCCURS FASTER
WHEN THEY'RE UNSTABLE AND SAME
AS REVERSE COMPLETION AND
ENCODING STILL REQUIRES REVERSE
TRANSCRIPTION INDICATING THE
PRODUCT IS A MAJOR TRIGGER FOR
ENCODING.
FINALLY WE'RE COLLABORATING WITH
MARK WILLIAMS AND THE LAB STO
UNDERSTAND HOW SYNTHESIS
TRIGGERS ENCODING.
BASED ON PREVIOUS STUDIES WE
THINK IT'S MORE RIGID AND
RESISTS COMPRESSION AND
THEREFORE IT THE LONG STRANDED
DNA INSIDE THE CAPSID INSERTS
INTERNAL PRESSURE ON THE CAPSID
AND HOWEVER, THE DOUBLE STRANDED
DNA IS EXPRESSED OR CONDENSED BY
THE HIV NUCLEAR CAPSID PROTEIN.
THIS ATOMIC FORCE MICROSCOPY
PERFORMED BY MIKE MORRIS SHOWED
A 7.5KB DOUBLE STRAND DNA
COMPARED TO THE CONTROL IS
SIGNIFICANTLY CONDENSED WHEN NC
IS ADDED IN A CONCENTRATION
DEPENDENT MANNER AS THERE'S MORE
CONDENSATION.
IN THE SECOND EXPERIMENT HE
ADDED 16 NUCLEOTIDE FRAGMENTS TO
MIMIC POWER GENOME DEGRADATION
THAT MAY BE REVERSED INSIDE THE
CORE AND FOUND IF WE ADD THE RNA
FRAGMENTS THEY RESULT IN LESS.
DNA CONDENSATION AND BASED ON
THE OBSERVATIONS AND SOME
INFORMATION OF REVERSE
TRANSCRIPTION WE THINK THE
CAPSID IS INCREASED BY 150 TO
200% AND THE AMOUNT OF NC
AVAILABLE IN THE CAPSID MAY NOT
BE EFFICIENCY TO FULLY CONDENSE
THE DNA AND AS A RESULT THE FULL
LENGTH STRANDED DNA DOES NOT
REMAIN CONDENSED AND THIS
INCREASES THE PRESSURE INSIDE
THE CORE LEADING TO ENCODING.
TO SUMMARIZE THE LONG BINDED DNA
IS REQUIRE TO INDUCE EFFICIENCY
ENCODING AND THAT DO NO REDUCE
DEFISH IN THE ENCODING AND COULD
BE RELEASED WITH PF74 AND MOST
THE DNA IS DEGRADED AND THE DNA
THAT SURVIVES IS CAPABLE OF
REPORTER GENE EXPRESSION.
WE THINK OVER ALL IT BALANCES
THE PRESSURE WITH CAPSID
STABILITY AND ENSURES THE
SYNTHESIS IS COMPLETED IN THE
INTACT CAPSID AND IT CAN ONLY
OCCUR AFTER COMPLETION OF
REVERSE TRANSCRIPTION.
I WANT TO THANK RYAN BURRDOCK
WHO CONDUCTED MOST OF THE
SCIENCES AND MY COLLABORATORS
AND WILL OTHERS.
THANK YOU SO MUCH.
>>
>> GOOD MORNING.
ROB AND I WILL PRESENT OUR
RECENT RESULTS ON ONGOING CAPSID
RELATED PROJECTS BUT FIRST I
WANT TO MENTION THAT WE HAVE
SEVERAL COLLEAGUES IN THE B-HIVE
CENTER THAT WORK ON VARIOUS
ASPECTS OF CAPSID AND I DON'T
HAVE TIME TO RECOGNIZE THEM ALL
TODAY BUT YOU SEE THE PEOPLE WHO
WORKED DIRECTLY ON THE PROJECTS
YOU WILL HEAR ABOUT TODAY AND
ENCOURAGE YOU TO VISIT OUR
TRAINEES WHO HAVE RELATED CAPSID
POSTERS TOMORROW RELATED TO THIS
TALK.
SO AS I WAS INTRODUCED BY THE
CHEETAH CENTER EARLIER,
LENACAPAVIR TARGETS VALIDATING
CAPSID AS A THERAPEUTIC TARGET
AND POTENT AND A LONG ACTING
FORMATION AND MANY GROUPS OVER
THE YEARS DEVELOPING LENACAPAVIR
AND OTHER ANTIVIRALS AND
APOLOGIES FOR NOT CAPTURING
EVERYBODY HERE FOR TIME
CONSTRAINTS AND IT BIND THE
CAPSID CORE SHOWN IN RED AND IN
DOING SO IT BLOCKS NUCLEAR
TRANSPORT BECAUSE IT COMPETES OR
BLOCKS BINDING OF NUCLEAR AND
OTHER HOST FACTORS NEEDED FOR
NUCLEAR TRANSPORT.
IT'S ALSO KNOWN TO EFFECT
ASSEMBLY IN MATURATION AND AT
THE BEGINNING OF THE TALK I'M
GOING TO TALK ABOUT RESISTANCE
MUTANTS THAT AFFECT LENACAPAVIR
ABILITY TO BLOCK TRANS ORDER
AND ROB WILL TALK ABOUT THE
IMMATURE CAPSID LATTICE.
THOUGH IT'S BEEN A BREAKTHROUGH
IN THE CLINIC, RESISTANCE
MUTATIONS HAVE BEEN IDENTIFIED
IN VITRO AND IN THE CLINIC.
AMONG THEM M66I IS THE MOST
DEVASTATING CAUSING HIGH
RESISTANCE TO LENACAPAVIR.
FIRST IT WAS IDENTIFIED IN VITRO
AND THEN ALSO SEEN IN THE CLINIC
WITH OTHER DRUG RESISTANCE
MUTATIONS.
SO THE BIG QUESTION IS HOW CAN
LENACAPAVIR RESISTANCE BE
ADDRESSED?
FROM DECADES OF RESEARCH ON
REVERSE TRANSCRIPTASE WE KNOW
COMBINATIONS HAVE BEEN
SUCCESSFUL TREATMENTS AND
SUCCESSFUL STRATEGIES FOR
OVERCOMING DRUG RESISTANT
MUTATIONS.
TRADITIONALLY COMBINATIONS USE
DIFFERENT MOLECULES THAT TARGET
DIFFERENT TARGETS IN THE LIFE
CYCLE.
MOST OF THOSE ARE REVERSE
TRANSCRIPTASE AND PROTEASE ARE
USING COMBINATION WITH EACH
OTHER.
AND MOST OF THEM HAVE ONE SINGLE
BINDING SITE PER ENZYME BUT IF
YOU THINK IN TERM OF THE CAPSID
CORE YOU HAVE SO MANY BINDING
SITES, 1500 AND WE KNOW FROM THE
STRUCTURE OF THE CAPSID CORE
THAT THESE ARE ALL LIKELY TO BE
DIFFERENT.
WE ASKED THE QUESTION CAN
LENACAPAVIR BE COMBINED WITH
OTHER ANTIVIRALS?
WE USED A TECHNIQUE IN
COLLABORATION WITH SOPHI HARVEY
AT OHIO STATE UNIVERSITY.
SOPHI BEING A FORMER CVPs AND
USED NATIVE MASS SPECTOMETRY AND
USED A COMPOUND PREVIOUSLY
IDENTIFIED 57D WE COULD STE THE
AND WHAT WOULD SEE WHAT WAS
BINDING AND THE NEW WAY TO LOOK
AT THE MOLECULE BOUND TO THE
SAME SITE WITHIN ONE CAPSID
HEXAMER.
TO FURTHER UNDERSTAND IF WE CAN
USE DIFFERENT FG MOLECULES IN
TERMS OF USING COMBINATIONS WE
DID CELL-BASED ANTIVIRAL ASSAYS
AND TITRATED DIFFERENT
CONCENTRATIONS OF LENACAPAVIR
AND 1261 AND FOUND THEY'RE NOT
ANTAGONISTIC.
THEY'RE AT LEAST ADDITIVE AT
SOME CONCENTRATIONS.
THIS IS PROOF OF PRINCIPLE WE
CAN USE COMBINATIONS OF
MOLECULES THAT TARGET THE FG
BINDING SITE AS FEASIBLE.
TO FURTHER ADDRESS THIS WE HAVE
BEEN WORKING TO DO A
STRUCTURE-RELATIONSHIP ACTIVITY
STUDY.
WE EXAMINED SEVERAL CRYSTAL
STRUCTURES, BIO PHYSICAL AND
VIROLOGICAL DATA TO
STRATEGICALLY COMBINED COMPOUNDS
TO BIND THE I6 MUTATION WITH THE
GOAL OF OVERCOMING THE
LENACAPAVIR RESISTANCE AND USING
THESE IN THE FUTURE IN
COMBINATION WITH LENACAPAVIR AND
THIS IS WORK DONE WITH THE
UNIVERSITY OF MINNESOTA.
SO WE INITIALLY TESTED THE
COMPOUNDS IN THERMAL SHIFT
ASSAYS AND THIS PRESENCE
LENACAPAVIR LOSES BINDING
SIGNIFICANTLY AND WHEN WE TEST
OUR NEW COMPOUNDS WE HAVE
THEY'RE BINDING BETTER TO M66I
THAN TO WILD TYPE YOU CAN SEE BY
THE THIRD COLUMN AND THE
DIFFERENCE IN BINDING BETWEEN
THE M66I AND THE WILD TYPE.
TO FURTHER UNDERSTAND THIS
BINDING, WE ALSO DID BY LAYER
ASSAY TO UNDERSTAND THE BINDING
KINETICS AND I'M SHOWING THE
FULL CHANGE OF THE MUTATION
COMPARED TO THE WILD TYPE.
AS EXPECTED.
LENACAPAVIR BINDING THE KV
INCREASES AND IN THE PRESENCE OF
THE COMPOUNDS THERE'S STRONGER
BINDING TO M66I CAPSID HEXAMER.
AND WE TESTED THEM IN CELL-BASED
ANTIVIRAL ASSAYS USING FULLY
INFECTIOUS VIRUS.
AGAIN FOR SIMPLICITY AND TIME
SAKE I'M SHOWING THE FULL CHANGE
IN M66I COMPARED TO THE WILD
TYPE VIRUS.
SO LENACAPAVIR AS PREVIOUSLY
REPORTED IS HIGHLY RESISTANT AND
DETRIMENTAL TO LENACAPAVIR
INHIBITION WITH THE COMPOUNDS.
IN SOME CASES THEY INHIBIT M66I
BETTER THAN WILD TYPE.
AND ADDITIONALLY WE USE OUR
NATIVE FULL LENGTH CAPSID
CONSTRUCT WITH ATLANTIC OR M66I
MUTATION.
UNFORTUNATELY I CANNOT SHOW THE
COMPOUNDS TODAY BECAUSE OF
INTELLECTUAL PROPERTY CONCERNS
BUT WE HAVE STRUCTURES AVAILABLE
AND WE'RE USING THOSE FOR FUTURE
DESIGN.
QUITE HIGH RESOLUTION STRUCTURES
PROVIDING A LOT OF INFORMATION
ON HOW THE COMPOUNDS ARE BINDING
AND GIVING INSIGHTS INTO HOW WE
CAN OVERCOME RESISTANCE.
AND IN THE FUTURE BECAUSE THESE
COMPOUNDS -- I FORGOT TO MENTION
ON THE PREVIOUS SLIDE, THEY'RE
NOT QUITE AS POTENT IN WILD TYPE
AS LENACAPAVIR.
SO WE'RE IMPROVING IN THE NEXT
ROUND OF DESIGN.
WE ARE TRYING TO IMPROVE THE
POTENCY AS WELL AS TRYING TO
TEST AGAINST OTHER MUTATIONS AND
SEE IF WE CAN DEVELOP COMPOUNDS
THAT WILL BE EFFECTIVE OR ACTIVE
AGAINST SEVERAL MUTATIONS.
AND I ALSO ENCOURAGE YOU TO SEE
MY COLLEAGUE CHRIS' POSTER
NUMBER 127 RELATED TO THE CRYO
EM STRUCTURE WITH M66I.
AND SO JUST IN SUMMARY, WE
STRATEGICALLY DESIGN COMPOUNDS
SUCCESSFULLY IN THE FIRST ROUND
OF COMPOUNDS UNDER THE NEW
DESIGN THAT WE HAVE THAT CAN BE
EFFECTIVE AGAINST M66I COMPARED
TO WILD TYPE VIRUS AND FUTURE
STUDIES WILL FOCUS ON IMPROVING
POTENCY AND DESIGN TOWARDS
HOPEFULLY POTENTIAL STUDIES WITH
LENACAPAVIR AND WITH THAT I'LL
TURN THE MIC OVER TO ROB.
>> THANK YOU, KAREN.
SO WE'VE BEEN USING A METHOD IN
THE LAB CALLED TEMPLATING METHOD
TO DETERMINE THE STRUCTURE
MATURE HIV LATTICE UNDER A
NUMBER OF CONDITIONS AND WE'VE
BEEN ABLE TO LEVERAGE THIS TO
STUDY SOME OF THE EFFECTS OF THE
MUTATIONS.
HERE'S AN EXAMPLE OF WHERE THE
STRUCTURE OF THE M66I MUTATION
AND ABOUT WHAT WAS INTERESTING
IN THE CONTEXT OF THE STRUCTURE
WAS THE LARGEST CHANGE WE SAW IN
M66I WAS THE POSITION OF THE K70
LYESENE AND USED CRYO EM TO
DETERMINE THE STRUCTURE OF THE
IMMATURE LATTICE AND ADDED
LENACAPAVIR AND FIND IT BINDS
WELL TO THE IMMATURE LATTICE IN
VITRO AND WONDERED IF WE CAN
FIND THESE IN CELLS TREATED WITH
LENACAPAVIR AND THESE ARE
RELEASED CELLS AT 10 NANO MOLAR
LENACAPAVIR WE SEE MATURE
LATTICE AND THEN NO LONGER
DETECT AND ALL THE LATTICE IS
MATURE.
THE MATURE LATTICE IS IN THE
CONTEXT OF FULL LENGTH GAG AND
IN THE CONTEXT OF 50 NANO MOLAR
LENACAPAVIR SO ALL LATTICE DI
DETECTED IS IMMATURE AND HERE
YOU CAN SEE THE PROXIMITY OF THE
LAYER TO THE PLASMA MEMBRANE AND
SEE LENACAPAVIR BOUND WELL AND
DO NOT SEE THE FORMATION OF THE
CLASSIC AND TERMINAL BETA TERM
SO THAT PROTEIN IS EXTENDED UP
TOWARDS THE MEMBRANE.
IN COLLABORATION WITH DANIEL A
NUMBER OF LENACAPAVIR ANALOGS
HAVE BEEN MADE AND TESTED.
AND MIA VISITED MY LAB AND
LEARNED OUR IN VITRO IS ASSEMBLY
REACTION AND DONE THIS IN THE
CONTEXT OF THE ANALOG AND WE
HAVE DONE PRELIMINARY CRYO EM
SCREENING AND THIS LOOKS LIKE
WE'RE ABLE TO COLLECT NICE DATA
SETS.
ONE OF THE PRELIMINARY DATA SETS
WITH WAS THE MOLECULE AND HERE'S
AN EXAMPLE HOW WE CAN SEE THE
DETAILED CHANGE BETWEEN THE
DENSITY BETWEEN THE LENACAPAVIR
ANALOG AND IF WE ZOOM IN THAT'S
THE CHANGE IN THE MOLECULE
MISSING THE MT AND YOU SEE THE
CRYO EM DENSITY NOT PRESENT FOR
THAT PART OF THE MOLECULE.
WE CAN CAPTURE THE DETAILS OF
THESE MOLECULES AND THE BINDING.
AND THIS IS NOW BEING DONE FOR
BOTH IMMATURE AND MATURE
LATTICES IN THE CONTEXT OF
MUTATIONS.
WITH THAT I'D LIKE TO THANK
CURRENT AND PAST LAB MEMBERS AS
WELL AS THE GRADUATE STUDENT
WITH THE PRIMERATIZATION OF THE
MOLECULE.
THANK YOU.
>> OKAY.
I'M GOING TO TALK ABOUT B-HIVE
AND IF I FREEZE UP IT'S BECAUSE
I FORGOT MY PARKA WHILE IT'S 100
OUT SIDE AND I'LL TALK ABOUT
CAPSID ASSEMBLY AND THE OTHER
HAS TO DO WITH NUCLEAR IMPORT.
IN MY GROUP WE SPECIALIZED FOR
ABOUT 20 YEARS IN THE CORES
GRAIN MODELLING AND TAKE
STRUCTURE INTERACTIONS AND
SIMPLIFY THEM TO GET A
COMPUTATIONALLY EFFICIENT
METHODOLOGY.
FOR EXAMPLE, ON THE CAPSID IF
YOU WANT TO SIMULATE JUST A
SINGLE CAPSID IT'S CLOSE TO 100
BILLION ATOMS AND DEMANDING AND
YOU HAVE TO DO THE METHODS WE
HAVE DEVELOPED.
I WANT TO TELL YOU ABOUT THE IP6
STORY AND I THINK IT GOES BACK
TO THIS PAPER FIRST YOU LOOK AT
THE IN VITRO EXPERIMENTS IF YOU
DON'T HAVE IP6 YOU TEND TO GET
LOTS OF TUBULES.
IF YOU ADD A BIT OF IP6 YOU
START GETTING CIGARS AND YOU CAN
START TO SEE CORES AND SMALLER
STRUCTURE AND THE QUESTION IS
HOW DOES THAT HAPPEN?
WELL, WE WERE ABLE TO TRANSLATE
THIS INFORMATION INTO THE CORES
GRAIN MODELS AND IF YOU DO AT
ATOMISTIC CALCULATIONS IT BINDS
BETTER THAN TO HEXAMERS AND THIS
IS CRITICAL.
I'LL SHOW YOU IS FROM A PAPER
YOU MAY NOT HAVE READ THE
SUPPORTING INFORMATION.
THE SUPPORTING INFORMATION HAS
MOVIES IN IT BUT WHAT I'LL SHOW
YOU IS TIME AND THIS IS THE SIZE
OF THE ASSEMBLY UP TO 1,000 THE
GRAY HASHING IS OVER MULTIPLE
TRAJECTORIES AND AN INITIAL SEED
YOU'LL SEE A CRITICAL GROWTH OF
A SHEET AND THE ROLE OF IP6
AROUND STABILIZING THAT FORMING
A CAP AND THEN A RAPID GROWTH OF
THE CAPSID AND THIS IS
PRODUCIBLE.
WHAT YOU WANT TO LOOK FOR IN THE
MOVIE IS THE IP6'S EARNING
SPHERES AND THE PENTAMERS AND
THIS IS THE ACTUAL SIMULATION
AND THIS IS TAKEN OUT OF THE
CONDENSED PHASE.
THE PENTAMERS COME AND GO AND
IP6 NAILS THEM AND WANTS TO GROW
A TUBULE BUT CAN'T AND YOU FORM
THE LONG BODY AND THEN CLOSE AND
THIS HAPPENS OVER AND OVER AGAIN
IN THE SIMULATIONS.
IF YOU LOOK AT THIS PLOT THERE'S
A FINICKY THING I'M STEPPING
ON -- OH, NO.
THAT'S THE LAST THING WE NEEDED.
SORRY.
OKAY.
THIS IS A VERY INTERESTING PLOT.
THIS IS IT TIME.
THIS IS ASSEMBLY SIZE AND LOOK
AT THE PENTAMERS THAT FORM AND
STABILIZE BY IP6 IS 2 OF 4 OF 12
AND THIS IS A KINETIC SIGNATURE
OF THE CAPSID GROWTH.
IF YOU LOOK AT A CONTROL
SIMULATION WHERE YOU DON'T HAVE
IP6 -- I'M GOING TO FORM TUBULES
OR MAYBE THEY'RE CAPPED A LITTLE
BIT.
YOU GET A LITTLE BIT OF THAT
SIGMOIDAL BEHAVIOR BUT NOT MUCH.
IT DOES NOT HAVE THE SAME
KINETIC SIGNATURE AND OF COURSE
KEEPS GROWING.
YOU CAN KEEP GROWING A TUBE --
TUBULE AND PENTAMERS COME AS
DEFECTS IN THE LATTICE AND COME
AND HEAL AND THERE'S NOTHING
LIKE IP6 TO STABILIZE THEM AND
YOU'LL END UP WITH A TUBULE.
SO THIS IS FASCINATING IF YOU
THINK ABOUT IT FROM A PHYSICAL
POINT OF VIEW.
YOU GET THESE ARE JOHN BRIGGS
TOMOGRAMS WE INVERTED AND TURNED
INTO BACK MAPPING METHODS AND
MOLECULAR STRUCTURES AND YOU GET
THESE PLEAO MORPHIC CAPSIDS AND
SOMETIMES YOU GET THE PERFECT
ONE AND YOU CAN GET THESE PILLS
AND THEY ALL HAVE DIFFERENT
BEHAVIOR AND I'M LED TO CONCLUDE
THESE ARE KINETICALLY ARRESTED
AND WOULD HAVE TO BE A MINNINA
TO GET KINETICALLY ARRESTED.
THE COMPETITION BETWEEN
LENACAPAVIR AND IP6.
THE EXPERIMENTS APPEARED.
AND THIS IS HARD TO SEE.
WHAT HAPPENS IS THIS IS THE 1:1.
IP6 WOULD LOVE TO STABILIZE
PENTAMERS AND IT CAN'T.
THE LENACAPAVIR IS BINDING AND
BINDING THE HEXAMERIC
ENVIRONMENT AND DOES NOT SUCCEED
AND THIS BEGINS TO STABILIZE
PENTAMERS.
THIS SOMETHING DIFFERENT ABOUT
THE STIMULATION LARGER PIECES OF
HEXAMER FORM FROM THE CONDENSED
SIMULATION AND IP6 IS ABLE TO
HELP STITCH THEM TOGETHER AND
YOU END UP WITH A CAPSID.
WE HAVE BEEN ASKING OURSELVES
THIS IS INTERESTING FOR IN VITRO
ASSEMBLY BUT IN THE VIRUS YOU
NEED TO INTEGRATE THE RNP IN THE
STRUCTURE.
THESE ARE EXPLORATORY SIMULATION
AND THERE'S A MODEL IN THE
STRENGTH OF SIMULATION IS TO
TAKE MODELS AND DO EXPLORATION.
WHAT WE CAN DO IS MODULATE THIS
RNP STRUCTURE TO EXPRESS MORE OR
LESS IN THE STRUCTURE AND YOU
FIND IT'S SENSITIVE TO THE
AMOUNT ON THE SURFACE ON THE RNP
AND IF YOU HAVE RELATIVE FEW
YOU'LL HAVE WHAT I TOLD YOU
ABOUT AND IF YOU HAVE TOO MANY
YOU BEGIN TO MESS UP THE
CAPSIDS.
SOMETIMES YOU GET WEIRD CAPSID
LOOKING THINGS.
THIS IS VERY FASCINATIFASCINATI.
ARE AND THERE'S THIS RELEVANT TO
THE SWITCH AND THE OTHER IS FROM
A LABORATORY SHOWING THE
IMPORTANCE OF PENTAMERIC BINDING
AND WE UNDERSTAND THERE'S A
COUPLE ISSUES.
ONE IS OF THE CAPSID
INTO THE NUCLEAR CORE AND WHAT
INTERFERES AND WHAT HAPPENS TO
IT AND PASSING THROUGH THE
NUCLEAR BASKET AND GETTING INTO
THE NUCLEUS.
I'M GOING TO TALK ABOUT THE
FIRST THING.
WHAT WE WERE LUCKY TO HAVE WAS A
STRUCTURE OF THE NUCLEAR CORE.
THERE'S BEEN SEVERAL COME OUT
FROM BECK'S GROUP IS THE ONE WE
USE THE MOST AND ARE ABLE TO
PERFORM A BOTTOM-UP CORE SCREEN
MODEL AND THOUSANDS OF PROTEINS
YOU DON'T HAVE TO WORRY ABOUT
THE MATHEMATICS RIGHT NOW BUT
BELIEVE WE DO IT SYSTEMATICALLY
AND GET A NUCLEAR CORE MODEL.
AND WE CAN START SIMULATING
DOCKING OF CAPSIDS INTO THIS
NUCLEAR CORE.
YOU TOOK MOST OF MY TIME SO YOU
SHOULD CHILL FOR A MINUTE, OKAY.
SORRY.
YEAH, CHILL OUT LITERALLY.
SO WHAT YOU FIND IN THE NUCLEAR
CORE ENTRY I HAVE TO SHOW YOU
THIS MOVIE BECAUSE IT'S
IMPORTANT.
IT'S AN ELECTROSTATIC RATCHET
AND IT RACH ETS INTO THE NUCLEAR
IN A STOCHASTIC WAY AND DEPENDS
ON THE ORIENTATION.
IF YOU'RE ORIENTED THE WRONG WAY
YOU GET SPIT OUT THE PILL LOOKS
LIKE IT CAN GO THROUGH THE CORE
BUT HAS OTHER VULNERABILITIES TO
IT.
SOMETIMES YOU CRACK THE NUCLEAR
CORE.
THE PAPER JUST CAME OUT AND WE
SEE THESE NATURALLY IN OUR
SIMULATIONS.
THEY MAKE A BIG DEAL OUT OF IT.
BUT NOW, THE PUNCH LINE THIS
SHOWS HOW IMPORTANT IT IS IN THE
ENTRY.
IT OBSCURES THE VIEW.
LET ME FINISH UP STEFAN.
THE CAPSID CAN COMPRESS AND
CONTRACT.
IT CAN OBSERVE PUNISHMENT AND
MAKE THE ENTROPY GO UP SO MINUS
TS GOES UP AND ENERGY GOES DOWN
AND HAS AN ABILITY TO WITHSTAND
THE NUCLEAR CORE ENTRY AS IT'S
GETTING SQUEEZED AND WHEN YOU
COVER IN LENACAPAVIR IT LIKES TO
FIND DISORDERED REGIONS.
I DON'T MEAN TOTALLY DISORDERED,
I MEAN DISTORTED REGIONS AND
REGIDIFIES IT AND IT WILL
RUPTURE.
THIS IS A MOVIE.
WE LOVE THEM AND WORK HARD TO
GET THEM.
THIS WILL RUPTURE BUT THAT
PERTURBATION PROPAGATES RAPIDLY
UP HERE AND YOU'LL START TO SEE
PENTAMERS FAIL AND BREAK A PART
AND WHAT'S FASCINATING IS I'LL
CALL IT ALOESTERY AND IT GOES
QUICKLY.
AND YOU BEGIN TO SEE THIS AND
THEN WE BEGIN TO SEE A SEAM
BREAK UP AND WAY FROM THE
PENTAMER AND SEEMS TO MY EYE
TRACK WHERE LENACAPAVIR IS
BINDING AND SEE IT RUPTURE.
SO WE GOT BEAUTIFUL EXPERIMENTS
THAT SHOW CAPSID DOCKED IN THE
NUCLEAR CORE.
IT'S ALWAYS A BUMMER TO BE AT
THE END OF THE GRAVY TRAIN.
SO DNA RESULTS CONFIRM THAT.
SO ANYWAY, THE LAST THING IS
WE'RE CURRENTLY WORKING ON
CYCLOPHILIN A AND I WILL THANK
THE CREW WHO DID THIS AND
ENCOURAGE YOU TO GO TO THEIR
POSTER AND THAT'S IT.
>> REALLY GREAT TALKS.
AND I'M NOT SURE YOU'RE THE BEST
PERSON BUT THIS WAS MOST RELATED
TO YOUR TALK AND MIGHT BE BETTER
PEOPLE IN THE AUDIENCE TO
ADDRESS THIS AS WELL.
WHAT IS KNOWN ABOUT THE FUNCTION
OF AN RNA GENOME BEING PACKAGED
AND THE SIZE OF THE CORE THAT IS
THEN BUILT AROUND THAT RNA OR
THE RNA THAT'S NOT THERE BECAUSE
AN IMPORTANT DENOMINATOR IN YOUR
ARGUMENT IS THAT YOU HAVE A
SMALLER DNA SO THAT DOESN'T
BREAK IT.
YOU'RE ASSUMING THAT THE CAPSID
IS THE SAME SIZE ACROSS ALL
THOSE DIFFERENT GENOMES.
HAVE YOU LOOKED AT THAT
DIRECTLY?
>> NO, WE HAVE NOT.
BUT WE GET ESSENTIALLY NORMAL
SIZE CONES EVEN IN THE ABSENCE
OF PACKAGEABLE GENOMIC RNA.
SO WE DON'T THINK THAT GENOME
SIZE MAKES MUCH DIFFERENCE TO
THE SIZE OF THE CAPSID.
IT'S JUST DESIGN TO ASSEMBLE
INTO WHATEVER IT LOOKS LIKE.
AND THAT'S MY VIEW.
OTHERS MAY COMMENT.
>> I THINK THAT'S CONSISTENT
WITH YOUR DATA.
IT COULD BE INTERESTING TO LOOK
DIRECTLY.
AND SECOND PART IS THAT UNDER
SOME CONDITIONS WHERE YOU
MANIPULATED THE POSITIONS OF
ENCODING EITHER BY P90A OR
CYCLOPHILIN KNOCKOUT WE AND
OTHERS PUBLISHED IT ALTERS SITE
WHERE THE INTEGRATION TAKES
PLACE HAVE YOU LOOK AT
INTEGRATION SITES FOR YOUR
GENOMES?
WE'RE STARTING TO LOOK AT THAT
AND RYAN'S GOING TO TALK ABOUT
THAT IN THE B-HIVE MEETING IN A
COUPLE DAYS.
HE PRESENTED SOME OF IT AT COLD
SPRING HARBOR THIS YEAR.
INTERESTINGLY THE SHORT VECTORS
IF YOU BLOW THEM UP WITH
LENACAPAVIR IT ALTERS THE SITES
OF INTEGRATION WHICH WE THINK IS
INTERESTING.
>> SO THIS QUESTION IS FOR VINAY
AND KAREN.
I'M WITH STONY BROOK.
THEY'RE KNOWN TO PACKAGE A LOT
OF RNA DO YOU THINK PERHAPS
EVOLUTIONARY OR WHATEVER THAT IS
ACTUALLY PLAYING AN IMPORTANT
ROLE IN THE STABILIZATION
FUNCTION?
>> STABILIZATION OF THE CORE?
>> YEAH.
>> I THINK RNA PLAYS A CRITICAL
ROLE IN THE ASSEMBLY.
IN ABSENCE OF THE GENOMIC RNA
THEY PACKAGE RANDOM RNAs BUT I
THINK HAVING RNA IS CRITICAL FOR
FORMING A PROPERLY SHAPED CAPSID
WITH THE RIGHT STABILITY.
THAT'S WHAT I THINK.
>> I'M DRAWN TO THE QUESTION
BECAUSE OTHER VIRUSES,
NON-RETROS WILL PACKAGE A LOT OF
SMALL RNAs AND WONDERING IF YOU
HAVE AN ACROSS THE BOARD MOTION
OF THEIR ROLE.
THANK YOU.
>> THANK YOU.
>> SO FOR KAREN, SO YOU SHOWED A
DISTRIBUTION OF WHEN YOU WOULD
USE LEN IN COMBINATION WITH
OTHER MOLECULES YOU GET A
DISTRIBUTION.
SO DO YOU THINK YOU MAY BE ABLE
TO DESIGN EXPERIMENTS THAT WOULD
ACTUALLY TARGET WHAT THE
FREQUENCY WILL BE FREQUENCY SO
YOU'LL CONTROL HIGH OR LOW
FREQUENCY A LOT OF LEN VERSUS A
LITTLE OF THE OTHER AND VICE
VERSA.
>> YES, I THINK WE CAN
DEFINITELY DO THAT.
I THINK WE CAN -- YEAH, WE CAN
DO THAT.
I'M SORRY, THE VIROLOGY
EXPERIMENTS WERE DONE BY OTHERS
IN THE LAB SO I'M NOT AS
FAMILIAR BUT YES, IT SHOULD BE
ABLE TO BE DONE.
>> I HAVE A QUESTION FOR VINAY.
DO YOU THINK YOUR EXTENDS TO
RETROVIRAL VECTORS IN GENERAL
BECAUSE IN MY EXPERIENCE THEY'RE
OFTEN BETTER IF THEY HAVE NO
INSERT, IN OTHER WORDS, THEY'RE
PRETTY SHORT THAN INSERT
CONTAINING VECTORS.
>> YEAH, THAT'S A VERY
INTERESTING QUESTION.
THE GENOMES FOR HIV THE LIMIT OF
THE SIZE THAT WE'RE TALKING
ABOUT THE 2. 8, 3.5KV ARE ALL
THE ELEMENTS WITHOUT INSERTS IN
THEM.
I THINK MOST RETROVIRAL ARE
LIKELY TO BE THIS SIZE AND
SECONDLY, THE SHORTER GENOMES
SEEM TO REVERSE TRANSCRIBE MORE
EFFICIENTLY THAN THE LARGER
ONES.
THOUGH THEY MAY LOSE SOME OF
EFFICIENCY IN ENCODING THEY MAY
MAKE UP FOR IT BY ENHANCING
THEIR ABILITY TO COMPLETE
REVERSE TRANSCRIPTION.
SO IT MAY BE I THINK A BALANCE
OF THOSE TWO PROCESSES.
>> THANKS.
>> SORRY.
I LOVE THE IDEA OF THE MINIMAL
SIZE.
WE SPENT YEARS TRYING TO MAKE
TINY, TINY VECTORS AND WE SEEM
TO HIT A LIMIT NEAR YOUR DATA
OFFERS AN EXPLANATION.
ALSO WONDER CERTAIN ELEMENTS
LIKE THE WOOD CHOP WPRE ELEMENTS
CAN HELP THE ENCODING.
BIG CHUNKS OF DNA THAT RESULT.
I WAS CURIOUS, HAVE YOU IN YOUR
EXPERIMENTS HAVE YOU LOOKED
DIRECTLY AT WHETHER CYCLOPHILIN
A IS BINDING TO THE CORE IN
CYTOPLASM IN TRACKING WITH THE
CORE INTO THE NUCLEUS WITH THE
SAME MOLECULES REMAINING
ATTACHED?
>> YOU KNOW, THAT'S A GREAT
QUESTION.
WE TRIED A LITTLE BIT WITH
THEFUL CADS RED.
WE THINK THE CYCLOPHILIN IS
REMOVED DURING THE PROCESS AND
YOU MAY HAVE A BETTER IDEA BIS
THIS BUT I WONDER WHETHER THE
CYCLOPHILIN A CAN GET IMPORTED
AND REBIND THE CAPSIDS ONCE THEY
GO IN AND THAT'S ONE OF THE
THINGS WE'RE THINKING ABOUT BUT
WE HAVEN'T DIRECTLY VISUALIZED
BOUND TO THE CORE AND NUCLEUS.
WE HAVE TO DO THAT.
>> I HAD A QUESTION FOR GREG.
DO YOU THINK YOU CAN START TO
ADDRESS ALL THE CAPSID SHAPES
YOU SEE OF MODELLING OF WHICH
WILL GENERATE PRODUCTIVE
INFECTION?
>> THAT'S THE IDEA.
WE LOOKED AT WHICH ONES MAY BE
ABLE TO DOCK WELL IN THE NUCLEAR
CORE.
WE HAVE OTHER ELEMENTS BUT
THAT'S A GOAL, I THINK IN HOW
IMPORTANT THAT PERFECT CONE OR
NEARLY PERFECT CONE WE'VE BEEN
LOOKING AT SINCE 1999.
SO FAR IT LOOKS IMPORTANT TO
DOCK PROPERLY.
THIS PILL SHAPED THINGS AND IT'S
FRAGILE AND BREAKS A PART
EASILY.
YEAH.
>> THANK YOU.
[APPLAUSE]
>> WE'LL BE UP NEXT AND
DR. KROGAN WILL KICK IT OFF.
>> IT'S A GREAT PLEASURE TO GIVE
YOU AN UPDATE ON THE HARC CENTER
STAND FOR HIV ACCESSORY AND
REGULATORY COMPLEX.
WE HAVE 18 P.I.s PRIMARILY BASED
IN CALIFORNIA.
13 P.I.s ARE AT UCSF, GLADSTONE
AND BERKELEY AND WE INCLUDED THE
HUTCH UP THE COST AND LILLY AND
MICHAEL IN SEATTLE, JUD AT
NORTHWEST AND NORTH CAROLINA.
IN OUR MORE RECENT CARNATION WE
HAVE STRUCTURE AND EVOLUTION ON
DEGRADATION AND EVOLUTION AND IN
THE JUST RESTRICTION BUT
PACKAGING.
WE'RE LOOKING AT PACKAGING WITH
RESPECT TO THIS INTERACTION IN
THE COMPLEX.
PROJECT 2 IS REGULATION OF HIV
TRANSCRIPTION AND LATENCY AND
WE'RE LOOKING AT TRANSCRIPTION
AND DIFFERENT TRANSCRIPTIONAL
FACTORS.
AIM 2 LOOK AT EXPORT.
THE STUDY OF REV AND AIM 3
LATENCY SO BIG FORCE ON
CHROMATIN MODELLING AND PROJECT
3 IS GENETICS.
COHORTS INVOLVED AND
EVOLUTIONARY GENETICS LED BY
ALEX AND TRYING TO PUSH CRISPR
TECHNOLOGY AND MACROPHAGES
LOOKING AT DIFFERENT HOST
FACTORS WITH RESPECT TO POOLED
APPROACHES AND RATE APPROACHES
AND SOME PROGRESS ON THAT FRONT
TODAY.
AND IF YOU KNOW HARC YOU KNOW AT
TO THE HART OF HARC IS
TECHNOLOGY.
AND WE HAVE FOUR CORES THAT
WE'VE STITCHED TOGETHER THE
HERE'S THE CHARACTERS ASSOCIATED
WITH EACH ONE OF THE CORES.
FOR ME PERSONALLY IT'S BEEN SO
INTERESTING TO SEE HOW THE
TECHNOLOGY HAS BEEN INTEGRATED
TOGETHER AND PROVIDING
UNPRECEDENTED INSIGHT TO HIV
BIOLOGY AND PROTEIN-PROTEIN
INTERACTIONS AND COMPLEXES AND
NOW THAT A.I.s BEING WOVEN IN
I'M EXCITED TO SEE THE BIOLOGY.
HIGHLIGHTS IN THE LAST YEAR
WE'RE WORKED ON TARGETS MANY
YEAR.
AND THERE WAS A STRUCTURE ON THE
COMPLEX WITH RNA LED BY JOHN
GROSS AND A KEY DISCOVERY IS RNA
IS A KEY GLUE WITH REGARD TO
THIS COMPLEX.
THIS HAS BEEN PUBLISHED RECENTLY
AND HERE WE'RE LOOKING AT THE
COMPLEX THAT I THINK THESE ARE
WHY THEY'RE IMPORTANT AND THE
CENTERS ALLOW FOR RUNWAY AND THE
COLLABORATIVE INFRASTRUCTURE TO
SOLVE THESE IMPORTANT PROBLEMS.
AND THESE ARE LONG STANDING
PROBLEMS.
WE STARTED WORKING ON THIS
COMPLEX 17 YEARS AGO WHEN THESE
WERE INSTEAD OF U54s P50s AND I
COMPLEX ALEN'S WORKED ON OVER 20
YEARS.
TO ME WE NEED THE PROGRAMS TO
BRING COLLABORATORS TOGETHER TO
SOLVE IMPORTANT PROBLEMS LIKE
THIS.
WE HAVE OTHER BREAKTHROUGHS HERE
OVER THE LAST YEAR.
WE SHOWED THE HOST FACTOR
REQUIRED FOR HIV TRANSCRIPTION
AND LATENCY AND IDENTIFIED HIV
REGULATOR AND CD4 POSITIVE T
CELLS AND IT F1 INVOLVED IN HIV
LATENCY.
WHAT YOU'LL HEAR TODAY IS
FURTHER WORK WE'RE CARRYING OUT
LOOKING AT THE COMPLEXES FROM
CAROLYN AND MICHAEL EMERMAN'S
LAB LOOKING AT THE EVOLUTIONARY
CONSTRAINTS OF THE KEY
CONNECTION AND WE'LL ALSO HEAR
FROM WILLIAM TARGETING
TRANSCRIPTIONAL ELONGATION
LOOKING AT THE COMPLEX AND THESE
ARE CONNECTIONS THE HARC CENTER
UNCOVERED AND WE'RE SHOWING
TARGETING PROMOTES HIV-1 LATENCY
REVERSAL.
WE MADE BREAKTHROUGHS
TECHNOLOGICALLY INCLUDING WILL
KAS 9 T CELLS AND TISSUES.
WE HAVE SOMETHING WE ARE ABOUT
TO ANNOUNCE TIED WITH THE HARC
CENTER CALLED THE READY CENTER
AND WE'RE DEVELOPING A PIPELINE
WHERE WE CAN CRISPR MANIPULATE
IPSC CELLS PUTTING IN MUTATIONS
AN TAGS AND DIFFERENTIATING TO
PRIMARY T CELLS AND PRIMARY
MACROPHAGES PURIFYING THE
COMPLEXES AND LOOKING AT AT MASS
SPECT AND CRYO EM AND ET AND
FOCUSSED ALMOST EXCLUSIVELY IN
CRYO ET IN THE BAY AREA.
AND A SCIENTIST THAT WORKS WITH
MYSELF UJJWAL WILL TELL YOU
ABOUT THE FIRST CD4 IN T CELLS
AND THERE'S BIOLOGY UNCOVERED
HE'LL TELL YOU ABOUT.
I WANT TO TELL YOU ABOUT OUR
EFFORTS LOOKING AT ALPHA FOLD.
EVERYONE IS USING IT NOWADAYS
AND FOR SOMEONE LIKE MYSELF THAT
WORKED ON PROTEIN-PROTEIN
INTERACTIONS FOR DECADES IT'S
REVOLUTIONIZING THE WORK.
SO AS YOU KNOW WE'VE BEEN
HEAVILY INVOLVED IN GENERATING
PROTEIN-PROTEIN DATA SETS.
THE FIRST WAS IN HIV AND WE HAVE
EXTRACTED FROM THAT MAP AS IN
OTHERS AND USED WHAT WE LEARNED
IN HIV AND APPLIED IT TO OTHER
PATHOGENS OVER THE LAST MANY
YEARS BUT THE CHALLENGE IS YOU
LOOK AT THE MAPS AND SAY WHICH
HOST FACTORS SHOULD WE BE
LOOKING AT AND LOOKING AT WHAT
IS TOUCHING THE PROTEIN OF
INTEREST THAT IS RELEVANT.
WE SUBJECT THIS TO ALPHA FOLD TO
IDENTIFY THE 101 OF THE
INTERACTIONS DIRECTLY TOUCHING.
BE AND OF AND NOW WE'RE MARCHING
THROUGH THE HUNDREDS OF
CONNECTIONS AND THEY'RE HIGHLY
ENRICHED FOR BEING RESTRACTABLE
AND THERE'S A NUMBER --
RETRACTIBLE AND THERE'S
STRUCTURES WE'RE CIRCLING BACK
ON AND ONE THING WE'RE CARRYING
OUT IS TAKING THE 15 PROTEINS
DEPENDING HOW YOU CARVE IT UP
AND LOOK AT ALL 20,000 HUMAN
PROTEINS USING ALPHA FOLD.
WE'LL SEE IF THE JUICE IS WORTH
THE SQUEEZE.
IT'S NOT CHEAP COMPUTATIONALLY
AND THIS IS DATA WE'LL RELEASE
TO THE COMMUNITY AND I THINK
THERE WILL BE NOVEL INSIGHTS WE
CAN LOOK INTO IN THE FUTURE.
WITH THE HARC DEVELOPMENTAL
AWARDS WE HAVE GREAT YOUNG
SCIENTISTS AND WE HAVE JEANNETT
NOW A PROFESSOR AT UCSF LOOKING
AT MODELLING TO HOOK AT HIV
HUMAN INTERACTIONS.
AND MEDHI IS LOOKING AT
IMPACTING SIGNALLING IN
POLYNEUOPATHY IN PATIENTS
INFECTED WITH HIV AND AMANDA
LOOKING AT CD4 POSITIVE T CELLS
AND ERICA SANCHEZ AND WE HAVE
JACQUELINE AND SHE'S LOOKING AT
EPIGENETIC AND PROTEIN EXCHANGES
IN HIV GENES AND HOST GENES WITH
AN INTERESTING COHORT.
SO PLEASE FOLLOW THESE PEOPLES'
WORKS IN THE CONTEXT OF THE HARC
CENTER AND TWO SCIENTIST AWARDS
LOOKING AT A LIGASE WE FOUND
INTERACTING WITH VIF AND
STRUCTURIZING THAT CONNECTION
AND MY GROUP IS TRYING TO SET UP
THIS PIPELINE PUR FIGE CON NEXT
INTO CELLS ED WITH HIV AND GO TO
CRYO EM AND MASS SPEC TOM METRY.
WE HAD ROBUST OUTREACH OVER THE
LAST YEAR AND ALMOST EVERYTHING
IN PERSON NOW WHICH IS GREAT TO
SEE AND AND WE HAVE AN
INTERNSHIP PROGRAM HIGHLY
REPRESENTATIVE OF THE FIVE
SPEAKERS NINE WERE AIATEDD --
AFFILIATED AND WE RECENTLY
SOLVED THE STRUCTURE OF RIV RRE.
WE WENT BACK AND TALK TO HIM
ABOUT A 1989 PAPER WHERE HE
SHOWED RIF INVOLVED AND I WAS IN
ELEMENTARY SCHOOL WHEN THE PAPER
CAME OUT AND I EVIDENTLY READ IT
IN 8th GRADE AND I CA CAN SAY
THIS AND THAT'S THE LAST TIME A
CANADIAN WON THE STANLEY CUP SO
I ENCOURAGE YOU TO LOOK AT THIS
AND THIS IS IN COLLABORATION
WITH INFECTIOUS DISEASE AND
RECOGNIZE CHARACTERS HERE AND
THIS WAS INITIATED WITH A VISIT
WE HAD BACK IN 2019 AND WE HAVE
A TWO-DAY SYMPOSIUM WHERE A
NUMBER OF AFRICAN TRAINEES
AROUND WORKSHOPS WITH GRANT
WRITING AND MICROBIOLOGY AND
CLOTH AND SITE VISIT IN CLONALAL
WITH THE EPICENTER OF EBOLA AND
CARS COV2 AND HAVE A PODCAST SO
ENCOURAGE YOU TO LOOK AT THE
INFORMATION.
LOOK ONLINE AND REACH OUT TO US
IF YOU'RE INTERESTED.
THE MOST IMPORTANT STLIEDZ --
SLIDES ARE THOSE INVOLVED AND
THEY'RE ALL HERE ALONG WITH THE
WORK ON THE HEART CENTERS.
HERE'S A PICK TOUGHER US AT THE
PRESIDIO RECENTLY TWO MONTHS AGO
AND IT'S GREAT WE HAVE SUCH A
FANTASTIC IT SIB AND I WANT TO
THANK THEM FOR THEIR HELP AND
INTEREST OVER THE YEARS.
WE HAVE A NUMBER OF POSTERS OVER
THE NEXT COUPLE DAYS.
I ENCOURAGE YOU TO LOOK AT WITH
ONGOING PROGRESS AND THINGS
DOESN'T DISCUSS HAPPENING AT THE
HARC CENTER AND WE'RE HEAVILY
EMPHASIZING TRAINEES AND HAVE
THREE GREAT TALKS.
THE FIRST IS FROM CAROLINE
STRAIN SPECIFIC DIFFERENCES IN
HIV-1 MUTATIONAL TOLERANCE IN
THE CONTEXT OF APOEOF BC3
RESTRICTION.
>> I'LL BE TALKING ABOUT MY
DISSERTATION WORK IN THE VIF
PROTEIN.
WE KNOW THE PROTEINS LIKE A3G
ACT AS ANTIVIRAL TRANSCRIPTION
FACTORS AND THIS IS SO AG3 CAN
GET PACKAGED INTO THESE VIRONES.
THEN A3G IS DELIVERED TO THE
TARGET CELL WHERE IT INTRODUCES
MUTATIONS TO THE VIRAL GENOME
GOING THROUGH REVERSE
TRANSCRIPTION.
THE SPECIFIC MOTIF AG3 MUTATES
IN IS PREMATURE STOP CODES AND
OTHER MUTATIONS WHICH CULMINATES
IN SUCCESSFUL VIRAL INFECTION.
BUT THIS IS NOT WHAT HAPPENS IN
THE TYPICAL HIV INFECTION.
AND THAT'S BECAUSE OF THE LENTI
VIRAL VIF PROTEIN BINDS A3G AND
PREVENTS IT FROM GETTING
PACKAGED TO GO THROUGH MUTATION
WITHOUT THESE BEING INTRODUCED.
AND VIF BINDS AND THE EXACT
INTERFACE WAS A MYSTERY IN THE
FIELD FOR SOME TIME WHICH IS WHY
IT WAS EXCITING WHEN JOHN GROSS
FROM UCSF HIS LAB SOLVED THE
CRYO EM STRUCTURE OF THE VIF IN
GREEN BOUND TO FULL LENGTH GENE
IN BLUE AND I WAS LUCKY TO GET
TO BE A PART OF THE STORY THEY
TOLD AND I'M SUMMARIZE A COUPLE
KEY FINDINGS.
FIRST OF COURSE WE PARSED OUT
THE EXACT MOLECULAR PLAYERS AT
THE INTERFACE AND WHICH VIF
RESIDUE INTERACTING DIRECTLY AND
WE LEARNED THE INTERFACE IS
SPECIES SPECIFIC.
AND THEN THE BIGGER SURPRISE IS
THAT RNA IS BEING USED AS A
MOLECULAR GLUE TO BRING THESE
PROTEINS TOGETHER.
AND WHAT I'LL TALK ABOUT TODAY
IS AIMED AT TRYING TO
COMPREHENSIVELY CHARACTERIZE THE
MUTATIONAL CONSTRAINT IN AG3.
SO A GOOD WAY OF ANSWERING THESE
KINDS OF QUESTIONS IS USING A
TECHNOLOGY CALLED MUTATIONAL
SCANNING OR DMS FOR SHORT.
AND WHAT THIS ALLOWS US TO DO IS
ASSAY THE EFFECTS OF ALL OF
AMINO ACID MUTATIONS IN A
PROTEIN SEQUENCE OF INTEREST IN
A SINGLE EXPERIMENT.
SO THE WAY THIS WORKS IS YOU
TAKE THE PROTEIN SEQUENCE IN MY
CASE THE VIF SEQUENCE AND PULL
THEM IN EQUAL OPPORTUNITIES AND
IT BECOMES YOUR DMS LIBRARY.
I LIGATED MINE TO MAKE INFECTION
HOUSE VIRUS WITH THE LIBRARY OF
VARIANTS.
THE NEXT IS TO HAVE A SELECTION
THAT ALLOWS US TO PARS OUT
NON-FUNCTIONAL VERSUS FUNCTIONAL
VARIANTS.
TO DO THIS, I FIRST INFECT SUB
T1 CELLS AT A LOW MOI AND
THEY'RE EXPRESSING AG3 AT
PHYSIOLOGICAL LEVELS.
BECAUSE WE KNOW AG3 HAS TO BE
PACKAGED AND DELIVERED TO HAVE
ANY SORT OF ANTIVIRAL PHENOTYPE
WE THEN TAKE THAT FIRST ROUND
AND DO A SECOND INFECTION AND
IT'S THE SUPER NATENT OFF THE
SECOND ROUND THAT'S FUNCTIONAL
VARIANCE.
WE CAN SEQUENCE THE AND USE THIS
TO GENERATE LOG ENRICHMENT
RATIOS FOR THE EFFECT OF
FITNESS.
AS A CONTROL IN PARALLEL TO MY
FULL SIZED LIBRARIES I PASSAGED
A MINI LIBRARY WHICH IS A
LIBRARY ONLY AT A SINGLE SITE IN
VIF POSITIONED Y40 WHICH WE
UNDERSTAND FROM THE STRUCTURE IS
IMPORTANT IN FACILITATING THE
INTERACTION WITH RNA.
ON THE Y AXIS WE LOOK AT THE
PERCENT OF CODONS AND ON THE
INPUT AND PRE-SELECTION AND
FIRST SUPER NATENT AND THIS
SHOWS THE SELECTION IS HAPPENING
BETWEEN THE PRE AND POST
SELECTION SUPER NATENTS WHICH IS
WHAT WE EXPECT OF AG3
RESTRICTION.
SO NOW LOOKING AT THE FULL SIZE
LIBRARY WE CAN VISUALIZE THE
DATA IN A NUMBER OF WAYS BUT
WHAT WE'RE LOOKING AT HERE IS
THE SITE LEVEL WHERE I'VE TAKEN
THE ENRICHMENT RATIO OF ALL
VARIANTS AND AVERAGED THEM TO
GIVE US AN IDEA OF WHAT THE OVER
ALL CONSTRAINT AT EACH SITE IS.
SO WE HAVE THE SITES ON THE X
AND LOG ENRICHMENT ON THE Y WITH
ANYTHING BELOW ZERO MEANING
DEPLETED MEANING THE SITE IS
PROBABLY HEAVILY CONSTRAINED BUT
THE VALUE IS ABOVE 0 IT MAY HAVE
MANY VARIANTS IMPROVING THE
FUNCTION IN A3G RESTRICTION.
TO TRY TO UNDERSTAND THIS
BETTER, I WANTED TO HIGHLIGHT
SOME SITES WE CHARACTERIZED VEE
JA THE STRUCTURE.
SO ANY SITE THAT HAS THIS
TRIANGLE WAS INTERACTING WITH
RNA THEN WE HAD A COUPLE THAT
INTERACTED WITH RNA WITH A G3
THE BLUE FILL.
FOR THIS TALK I WANTED TO FOCUS
ON THE SITES IN THE IT
INTERFACE.
THE ONES CIRCLES WHICH ARE ONLY
INTERACTING WITH AG3 AND NOT
RNA.
WE HAVE SITE W70 AND Q803.
I WANT TO HIGHLIGHT THE SITE
Q803 BECAUSE IT WAS ONE OF 70
SITES WITH AN ENRICHMENT RATIO
ABOVE 0 WHICH WAS SUPER
INTERESTING TO BECAUSE IT'S AN
IMPORTANT ARMS RACE INTERFACE
SITE.
TO TAKE ONE STEP CLOSER FROM THE
SITE LEVEL ANALYSIS WE JUST
LOOKED AT WE CAN LOOK AT HOW
EACH OF THE INDIVIDUAL VARIANTS
IN THE LIBRARY THAT PASSED OUR
COVERAGE THRESHOLDS IN THE
PRESELECTION POOL HOW EACH OF
THOSE FARES AT THE SPECIFIC SITE
A IT 83.
ONE WAY I FOUND HELPFUL TO
CONTEXTUALIZE THE INFORMATION
WAS TO LOOK AT THE SEQUENCE
FORMATION BOTH AMONG HIV SUB
TYPES AND THE SUB CURSER AND
AMONG BROAD SIVs.
THE REASON IT'S HELPFUL IS IN
NATURAL IN HIV AND CBC IT'S Q
AND IN THE WILD TYPE IN THE
LIBRARY IS A Q I WANTED TO LOOK
AT HOW THE H FARED AND IT
BEHAVES AS WELL AS WILD TYPE
WHICH MAKES SENSE.
FURTHERMORE IF WE LOOK AT HOW
THE Y VARIANT FARED THIS IS
IMPORTANT BECAUSE WE KNOW IT'S
ONE OF THE CHANGES THAT HAD TO
HAPPEN IN SIV VIF AND FACILITATE
THE TRANSMISSION IN OLD WORLD
MONKEYS AND SEE THE Y RES
DEPLETED.
WHAT WAS SURPRISING WAS THERE
WERE A NUMBER RESIDUES IF
ENRICHED IN THE LIBRARY NEVER
SAMPLED IN NATURAL PERFECT.
ONE OF WHICH IS THE IS VARIANT
WHICH WAS CHARACTERIZED IN IN
VIVO AND CONFERRED RESISTANCE.
LOG ENRICHMENT RATIOS IS ONE WAY
OF CHARACTERIZING BUT THE SECOND
CAME TO US IN THINKING HOW IN
PROVIRUSES AMPLIFIED FROM
INDIVIDUALS LIVING WITH HIV WE
OFTEN SEE THESE MUTATIONS
CHARAC
CHARACTERISTIC OF AG3 ACTIVITY.
CAN WE MEASURE THE NUMBER OF
WILL MUTATIONS IN EACH DMS
VARIANT TO GIVE US A SECONDARY
WAY OF CHARACTERIZING THE
EFFECTS OF THE VARIANT ON OVER
ALL FITNESS.
SO WHAT WE'RE LOOKING AT HERE IS
EACH ONE OF THE POINTS IS A
SPECIFIC VARIANT AT A SPECIFIC
SITE WITH THE LOG ENRICHMENT
RATIO ON THE Y AND THE
PERCENTAGE OF THREE OR MORE
MUTATIONS ON THE Y AXIS. AND THE
OF THREE OR MORE MUTATIONS ON
THE Y AXIS.X AND THE
PERCENTAGE OF THREE OR MORE
MUTATIONS ON THE Y AXIS.
THE MORE IN ENRICHED IT IS THE
MORE THERE'S VARIANTS AND
PREVENTS IT FROM GETTING
PACKAGED.
IT'S INTERESTING TO LOOK AT THE
DATA BECAUSE WE CAN USE IT TO
HIGHLIGHT SPECIFIC VARIANTS.
ALL THE RED DOTS DOTS ARE
CYSTEINE VARIANTS AND HERE ON
THE PLOT ENRICHED AND HAVING A
VERY SMALL AMOUNT OF WILL READS
WITH THIS VARIANT HAVING THREE
OR MORE MUTATIONS.
I ALSO THOUGHT IT WAS CURIOUS
THERE WERE A NUMBER OF SITES
WITH SIS CYSTEINE VARIANTS THAT
FIT THE PROFILE.
I WAS ABLE TO LOOK AT THE
STRUCTURE WHERE WE SEE VIF AND
GREEN AND BLUE AND THEY'RE
LOCALIZED IN RED AND WE THOUGHT
IT WAS INTERESTING THAT A LOT OF
THESE SITES ARE LOCALIZED TO THE
INTERFACE WHICH MAY INDICATE TO
US THE INTRODUCTION OF A
CYSTEINE BROADLY WOULD IMPROVE
THE BINDING IN RESTRICTION OF
AG3.
AND SO UP CONCLUSION WE'VE SHOWN
THAT CHALLENGING DMS LIBRARIES
REVEAL MUTATIONS THAT IMPROVE
THE FUNCTION THAT MAY NOT HAVE
BEEN SAMPLED IN NATURE BEFORE.
WE'RE CURRENTLY WORKING ON
VALIDATING THAT Q83C VARIANT AS
WELL AS OTHERS TO TRY AND
UNDERSTAND THE TRADE-OFFS AND
WHY THESE ARE NOT BEEN SAMPLED
IN NATURE AND THOUGH I ONLY
SHOWED DATA WITH ONE LIBRARY AND
ONE AVA VECTS WE HAVE DONE THIS
MULTIPLE STRAINS AND HAVING THE
STRUCTURAL INFORMATION MAKES IT
MORE POWERFUL TO GIVE VALUABLE
CONTEXT TO TRY TO UNDERSTAND THE
DATA.
AND FINALLY SOME NEWER FACET
WE'RE EXCITED ABOUT IS THAT WILL
WILL ENRICHMENT RATIOS AND IT
MUTATIONS GIVE US A SECONDARY
WAY TO CHARACTERIZE THE FITNESS
EFFECT OF THESE VARIANTS.
AND THANK YOU TO MY LAB AND THE
HARC CENTER AND I THINK WILLIAM
IS UP NEXT.
>> HI, EVERYONE.
I'M WILL CISNEROS I JUST FINISH
YOU HAD UP I Ph.D. AT NORTHWEST
UNIVERSITY AND GIVE WILL GIVE A
BRIEF VIGNETTE AND IT WILL OB ON
TARGETING TRANSCRIPTION IN
ELONGATION IN HHS LATENCY
REVE
REVERSAL.
THE CELLS PRODUCE VIRUS AND
PRESENT ANTIGEN AND CAN UNDER GO
CELL DEATH THROUGH PERIPHERAL
CYTOTOXIC QUALITIES OR INNATE
IMMUNE FUNCTION AND THE CELLS
CAN ENTER A LATENT STATE
ESTABLISHING THE RESERVOIR AND
ANOTHER INTERESTING DETAIL IS
HIV SOMETIMES CAN ENTER ALREADY
LATENTLY INFECTED CELLS AND HIDE
FROM THE IMMUNE SYSTEM.
THE LATENT RESERVOIR IS A MAJOR
OBSTA
OBSTACLE TOWARDS FINDING A
FUNCTIONAL THERAPY AND IF YOU
TRACK THE KINETICS OF A PATIENT,
ONE PATIENT UNDER GOES ANTRI
VIRAL THERAPY AND ONCE THE LOAD
IS SUPPRESSED THEY CAN FALL TO
UNDETECTABLE AND THE VIRUS CAN
REREBOUND -- REBOUND AND
STRATEGIES ARE NEEDED TOO DELAY
THE REBOUND OR TRANSITION AND
EVEN IN SUPPRESSED PATIENTS WHO
DON'T UNDER GO REBOUND THERE'S
ADDITIONAL RISK OF DEVELOPING
COMORBIDITIES MAYBE BY THE
PERSISTENT VIRAL REPLICATION.
SO FUTURE CURATIVE THERAPIES
LOOKED HOW TO TURN ON OR OFF THE
AN TA GOOD -- ANTIGENS AND
MOLECULES CAN HAVE PATHWAY TO
REACTIVATE THE VIRUS AND UNDER
EPIGENETICS AND AT EVERY STAGE
AND POST TRANSCRIPTION AND OUR
LAB HAS BEEN FOCUSSED ON
TRANSCRIPTIONAL ELONGATION
BLOCKS.
SIMPLISTIC TRANSCRIPTION FOR
THOSE UNFAMILIAR YOU HAVE
TRANSCRIPTION INITIATION WITH
SITES AND TRAVELS 20 TO 60 BASE
PAIRS IN THE PROMOTER AND
PAUSES.
AT THAT POINT YOU NEED
ADDITIONAL LICENSING THAT COMES
IN THE FORM OF TRANSCRIPTIONAL
ELONGATION WHICH I'LL DISCUSS
HERE FURTHER.
TRANSCRIPTIONAL ELONGATION IS
LICENSED AND THE PAUSING IS
RELIEVED AND IT CONTAINS A
CYCLIN AND IT DOESN'T KNOW WHERE
TO GO AND SHCHAPERONED AND AS MY
ARE FAMILIAR WITH THE VIRUS
ITSELF IS VERY SMART AND HAS
ENCODED A PROTEIN THAT CAN
SPECIFICALLY ENGAGE AND PROVIDE
THE LICENSING FOR ELONGATION
ALLOWING THE VIRUS TO CONTROL
ITS OWN REPLICATION.
P-TEFB IT WILL SIN MANY
COMPLEXES AND THERE'S BEEN
STRATEGIES TO RELEASE THEM FOR
LATENCY REVERSAL.
THE FIRST COMPLEX HAS NOT BEEN
SEEN AS A POTENTIAL RESERVOIR
TAR GET AND MEDIATED RELEASE AND
SUBSEQUENT RECRUITMENT TO VIRAL
INTEGRATION SITES.
TO EXPLORE THE IDEA THIS COULD
BE A POTENTIAL RESERVOIR WE HAD
TO ESTABLISH TWO GROUND TRUTHS
IT'S NOT NEEDED FOR HIV
TRANSCRIPTION AND CAN LEAVE THEM
INTACT FOR TRANSCRIPTION.
OUR LAB HAS A TOOLBOX TO
INTERROGATE FUNCTIONS IN PRIMARY
T CELLS.
FOR THIS PROJECT WE INITIATED A
MULTIPLEX CRISPR KAS 9 APPROACH
TARGETING EVERY SEC PROTEIN
ENCODING GENE.
WE WERE ABLE TO IT ACTIVATE CD4
T CELLS AND VALIDATE PROTEIN
KNOCKOUT AND IN PARALLEL
CHALLENGE THE CELLS WITH
REPLICATION COMPETENT VIRUS AND
LOOK AT CHANGES OF INFECTION.
FIRST WE'RE ABLE TO KNOCK OUT
EVERY PROTEIN WITHOUT MAJOR
VIABILITY DEFECTS WHICH IS
EXCITING AND ONCE WE VERIFIED
THE KNOCKOUT WE'RE ABLE TO
CHALLENGE THESE WITH VIRUS.
SO WHAT YOU'LL NOTICE ON THE Y
AXIS IS PERSON INFECTED CELLS TO
OUR NON-TARGETING CONTROL AND ON
THE X ARE DIFFERENT GENES TAR
GUESTED AND SEE WHEN WE KNOCKED
THESE OUT THEY'RE EXOTROPHIC AND
IF YOU KNOCK OUT THE REST OF THE
SUPER ELONGATION COMPLEX YOU
DON'T SEE A SIGNIFICANT EFFECT.
THAT'S THE FIRST GROUND TRUTH
MAYBE IT'S NOT NECESSARY FOR HIV
TRANSCRIPTION.
MAYBE PFB IS THE MOLECULE AND WE
PARTNERED AT NORTHWESTERN
UNIVERSITY TO DEVELOP A MOLECULE
THAT CAN DISRUPT THE SUPER
ELONGATION COMPLEX INTERACTION.
THE COMPOUND DEVELOPED KNOWN AS
KL-2 AND MIMICS THE BINDING
INTERFACE.
AND WE TOOK PRIMARY CD4 T CELLS
AND TREATED THEM WITH THE
COMPOUND AND CHALLENGED THEM
WITH THE COMPETENT VIRUS AND
LOOKED AT WHAT WOULD HAPPEN WITH
INFECTION.
WE WERE ABLE TO SEE INCREASED
INFECTION BETWEEN TREATED AND
YOU CAN DISRUPT THE INTERACTIONS
WITHOUT DISRUPTING PTFB
INTERACTIONS THEMSELVES.
THAT'S ACTIVE INFECTION.
THINGS DIFFER SIGNIFICANTLY IN
LATENCY AND WANTED TO TRY THIS
IN A LATENT MODEL AND LOOK AT IN
A PROVIRUS AND TREATED THE CELLS
WITH EITHER A PANEL OF LRAs ALL
VERY DIFFERENT SO JQ1 OR AGONIST
AND THE EXCITING NON-CANONICAL
KAPPA B AGONIST IN THE PRESENCE
OF KL-2.
WE DON'T SEE MUCH SINGLE AGENT
ACTIVITY OF K L-2 MAYBE A SMALL
BLIP BUT NOT STATISTICALLY
SIGNIFICANT.
WHAT YOU CAN SEE WITH OUR PANEL
OF LRAs IS SIGNIFICANT SYNERGY
ACROSS THE DIFFERENT ACTING
LRAs.
WE FURTHERMORE CALCULATED
SYNERGY AND PICKED OUT THE DATA
FROM IN COMBINATION WITH KL2 AND
SEE DOSE DEPENDENT SYNERGY WITH
A LOCKED CONCENTRATION OF A WITH
KL-2 IN COMBINATION.
SO TO LOOK DEEPER INTO THE
PROVIRUS ITSELF, WE WANTED TO
SEE WHAT IS ACTUALLY HAPPENING.
EITHER WITH PFT AND ON THE LEFT
IS THEP LITTLE AT THE
INTEGRATION SITE MAYBE SOME AT
THE PROMOTER.
WHEN YOU ADD KL-2 YOU GET MORE
BUT COMBINED YOU GET THIS HUGE
SYNERGISTIC EFFECT AND THE SAME
EFFECT WE CAN SEE WITH RNA POLL
2 AND SEE THE CONTROL LOOKED IN
AT THE PROMOTER RENALON.
WHEN KL-2 HAPPENS AROUND
THERE'LL BE FURTHER STUDIES WE
SEE RNA POL 2 STARTING TO
PROCEED BUT WEREN'T SEEING
PROTEIN EXPRESSION SO MAYBE YOU
NEED AN INDUCER NOT JUST
TRANSCRIPTIONAL ELONGATION AND
YOU SEE MORE PROTEIN EXPRESSION
WITH THE COMPOUND.
TOGETHER YOU SEE GREAT POL
OCCUPANCY ACROSS THE GENOME.
NEXT WE WANTED TO SEE IF IT IT'S
INTEGRATION SITE SPECIFIC.
WE CHOSE A COUPLE CLONES AND
HE'S ARE SOME WE TESTED 6.3 AND
11.1 WITH THE SAME PRO VIRUS AND
DIFFERENT INTEGRATION SITES AND
SEE SYNERGY WITH KL-2.
OBVIOUSLY IT'S STOCHASTIC AND I
THINK THAT AIDS IN SHOWING HOW
HARD THE LATENT RESERVOIR IS
HERE AND WE WANTED TO LOOK INTO
THE REAL DEAL PMBMs FROM
EXPRESSED PATIENTS LIVING WITH
HIV.
WE WORKED WITH STEVE WOLLINSKI
AND WE HAD UNDETECTABLE VIREMIA.
WE SAW VERY LITTLE RESULTS THERE
MAYBE A LITTLE OF A THREE-FOLD
INCREASE BUT WHEN WE LOOKED INTO
AZD35582 WE SEE A 54 FOLD
INCREASE OF GAG TRANSCRIPT IN
THIS CASE AND COMPARED TO OTHER
TRANSCRIPTIONAL ELONGATION
PROMOTERS AND IPI THE SAME AS
AZD582 WE SAW AN INCREASE AND
LEADS TO THIS IDEA THAT THEY'RE
NOT CREATED EQUAL AND THE
DIFFERENT RESERVOIRS CONTAIN
INNATE DIFFERENCES AND NEED TO
UNDERSTAND WHICH ONES ARE MORE
IMPORTANT THAN OTHERS.
IN CONCLUSION WE FUNCTIONALLY
VALIDATED THE RESERVOIR FOR
VIRAL TRANSCRIPTION SITES.
WE CLARIFIED THE ROLES OF THE
COMPLEX IN HIV TRANSCRIPTION AND
FURTHER DEMONSTRATED THAT THESE
RELEASERS ARE A POTENT AND
SYNERGISTIC STRATEGY.
THEY LOOK AT DIFFERENT PFBs AND
DETERMINE THE REGULATED GENES
AND MEDICINAL CHEMISTRY TO
IMPROVE OUR COMPOUND FOR IN VIVO
STUDIES AND I'D LIKE TO
ACKNOWLEDGE SOME FOLKS AND THE
HARC CENTER AND PASS THE MIC TO
UJJWAL.
>> HI, EVERYONE.
IT'S BEEN A FEW YEARS OF
DISCOVERY.
HOW IT TOOK AROUND 20 YEARS TO
DISCOVER THE FACTOR AGAINST HIV.
THIS WAS A BEAUTIFUL STUDY THAT
SHOWED IT THE RIF VIRUS WAS
AFFECTING CEM AND THE WAS
PERMISSIVE TO WILD TYPE
INFECTION.
THIS INDICATED IT CONTAINS
ANTIVIRAL HOST PROTEIN THAT
RESTRICTS INFECTION IF NOT
PRESENTED AND COUNTER ACTS THE
ANTIVIRAL FACTOR.
I WAS BLOWN AWAY BOUGHT IT
INDICATED IT'S AN IMPORTANT TO
DO HIV INFECTION STUDIES IN
DIFFERENT CELL TYPES AND THAT'S
WHAT THEY DID IN THE PAPER.
THEY TOOK A BUNCH OF PERMISSIVE
AND NON-PERMISSIVE CELL LINES
AND SHOWED THE NON-PERMISSIVE
CELL TYPES HAVE PROTEIN NOT IN
THE PERMISSIVE CELL LINES AND
LATER IDENTIFIED AS APOE 3G AND
IF YOU PERFORM KNOCKOUT STUDIES
IN CONDITIONS IN WHICH PROTEIN
IS NOT EVEN EXPRESSED YOU CAN'T
UNDERSTAND IT IN THE CONTEXT OF
HIV INFECTION.
WHAT WE DECIDED TO DO WAS DO TWO
GENOME WIDE CRISPR SCREENS IN
PRIMARY CELLS FROM MULTIPLE
DONORS.
IN ONE EXPERIMENT WE DID ALL
HUMAN GENES AND IN ANOTHER
INDEPENDENT GENES WE TOOK
EXPRESSING GENES AND INFECT THE
THOSE WITH REPLICATION
COMPETENCE HIV VIRUS.
BUT PERFORMING THIS TO
UNDERSTAND THE ROLE OF ALL HUMAN
GENES POSTINFECTION.
ON THE X AXIS IS THE LAWFUL
CHANGE IN HIV INFECTION AND WHAT
YOU SEE IS IN RED I'M SHOWING
TOP THE CD4 AND THIS TO ME
SHOWED THE SCREENS WORKED
CLEARLY WELL AND ALSO IDENTIFIED
FACTORS LIKE THIS ONE HERE AND
IN BLUE YOU SEE THE ANTIVIRAL
FACTORS IN THE SCREEN AND YOU
SEE THE LIST AND SIMILARLY FROM
AN ACTIVATION SCREEN AND
IDENTIFIED A NUMBER OF KNOWN AND
NOVEL ANTIVIRAL FACTORS.
WHAT I NOTICED WHEN I WAS
LOOKING AT THE RESULTS
ESPECIALLY FROM THE SCREEN WHEN
YOU OVER EXPRESS THE RECEPTORS
WE SEE AN INCREASE IN HIV
INFECTION.
SIMILAR WHEN WE OVEREXPRESS CD
48 PART OF THE SIGNALLING
COMPLEX YOU SEE AN INCREASE IN
ANTI-HIV INFECTION SHOWING SOME
IDENTIFIED MAY BE MODULATING HIV
INFECTION THROUGH REGULATING THE
B CELL STATE OR THROUGH CHANGING
THE LEVEL OF HIV RECEPTOR AND
NOT INTERACTING WITH THE VIRUS.
TO DELINEATE DIRECT EFFECTS FROM
HIV INFECTION WE TOOK A
SECONDARY LIBRARY OF THE GENES
TARGETING ONLY THE GENES SHOWN
IN COLOR HERE SO A FEW HUNDRED
FROM A GENOME WIDE SCREEN AND
DELETED THOSE IN A SEPARATE
EXPERIMENT.
THE IDEA WAS YOU DELETE OR OVER
EXPRESS THE FEW HUNDRED GENES
AND THEN DIVIDE YOUR CELLS INTO
DIFFERENT POOLS AND STAIN THE
CELLS WITH THE SELECT MARKERS
AND HIV VIRUS AND THIS WILL HELP
UNDERSTAND DIRECT EFFECTS AND
INDIRECT EFFECTS.
TO SHOW YOU A SNAPSHOT OF DATA
FROM THE CRISPR SCREENS WHAT YOU
SEE IS YOU SEE AN INCREASE
EXPRESSION OF AND THIS INCREASES
HIV INFECTION AND WHEN WHY YOU
OVEREXPRESS YOU SEE THE CD4 AND
INFECTION WITH CD4.
HOWEVER SOME FACTORS IDENTIFIED
IN THIS CASE HAD MORE COMPLEX
PHENOTYPE.
SO WHAT HAPPENS HERE IS WHEN YOU
OVEREXPRESS YOU SEE AN INCREASE
IN THE LEVELS BUT YOU SEE A
DECREASE IN CD4 LEVELS AND THEN
YOU SEE IT CAN SEE AN INCREASE
IN HIV AND INFECTION AND WE SEE
DIFFERENT VIRAL FACTORS.
WHEN WE OVER EXPRESS IT'S A GENE
THAT PRODUCES MUSIN.
YOU SEE CELLS ARE LESS ACTIVATED
BECAUSE THE LEVEL ARE DECREASED.
BUT AT THE SAME TIME YOU ALSO
GET DECREASED IN HIV INFECTION
SO CELLS ARE LESS ACTIVATED AND
GETTING LESS INFECTED WITH HIV.
WHEREAS SOME OTHER ANTIVIRAL
FACTORS WE SEE WHEN YOU
OVEREXPRESS THERE'S NO CHANGE IN
CD 25 LEVEL BUT YOU SEE A
DECREASE IN HIV INFECTION BOTH
THE WILD TYPE VIRUS AS WELL AS
FOR OTHER ANTIVIRAL FACTORS YOU
SEE THOUGH THEY CAN RESTRICT THE
VIRUS BUT NOTE THE INFECTION
INDICATING THESE FACTORS MAY BE
WORKING AT THE SITE OF HIV ENTRY
AND THE RESTRICTION GOES TO THE
HIV ENVELOPE AND WE HAVE
IDENTIFIED A NUMBER OF NEW
ANTIVIRAL FACTORS AND WANTED TO
SEE HOW STRONGLY THEY RESTRICT
THE HIV INFECTION.
WE DELETED OR OVER EXPRESSED
THESE FACTORS IN CELLS FROM
DONORS AND WHAT WE FOUND WAS
THAT HERE IN GRAY YOU SEE THE
PERCENT OF HIV INFECTION FROM A
CONTROLLED SENSE AND SEE WHEN WE
KNOCK THIS OUT YOU SEE AN
INCREASE INFECTION AND THESE
WERE IDENTIFIED AS ANTIVIRAL
FACTORS AND YOU SEE AN INCREASE
IN INFECTION.
BECAUSE WE WERE PERTURBING THE
GENES AND THIS MADE THE CELL
MORE ACTIVATED AND SEE THE
INCREASE IN HIV UP INFECTION AND
SAW THE ANTIVIRAL FACTORS TESTED
WERE SUPER STRONG IN CONTROLLING
HIV INFECTION.
AND WE GOT REALLY INTERESTED IN
SOME OF THE VECTORS HERE WHICH
COULD CONTROL HIV INFECTION FOR
MANY DAYS.
AND I'M GOING TO FIRST TALK
ABOUT SOME OF THESE WHICH WE
FOUND THROUGH PROTEIN
EXPRESSION.
SOME OF THESE, 50% OF THE
FACTORS IDENTIFIED IN THE
SCREENS ARE EXPRESSING D CELLS
AND THEY COME FROM A GAIN OF
FUNCTION SCREAM.
WE SAW THE PROTEASE INHIBITER IS
NOT EXPRESSED IN T CELLS.
HOWEVER, WHEN WE LOOK AT CELLS
AT SINGLE-CELL LEVEL WE FIND
THAT IT A SMALL TRACTION OF B
CELLS CAN EXPRESS PL16 AND SOME
CAN EXPRESS AT HIGH LEVELS.
THIS WAS INTERESTING.
THIS INDICATED MAYBE THERE MAY
BE CELLS IN THE BODY A SMALL
FRACTION WHICH ARE NATURALLY
RESISTANT TO HIV INFECTION.
WE WANTED TO UNDERSTAND HOW THIS
IS AFFECTING HIV INFECTION AND
WE STAINED THE CELLS TO SEE WHAT
PERCENTAGE OF THE CELLS
EXPRESSED THIS PROTEIN.
WHAT WE SAW WAS VERY
INTERESTING.
THEY EXPRESSED THE PROTEIN AND
HOWEVER, THE EXPRESSION GOES
DOWN WITH ACTIVATION.
IF YOU ARTIFICIALLY EXPRESS THIS
PROTEIN ON T CELLS WHAT YOU SEE
IS WHEN PI16 AT THE LEADS TO HIV
INFECTION AND IT COMES DOWN TO
1% WHEN YOU OVEREXPRESS THIS
PROTEIN IN T CELLS.
AND WE FOUND OUT THAT THE PI16
THE EXPRESSION DOESN'T AFFECT
BINDING TO THE CELL PHASE BUT
WHEN YOU OVEREXPRESS YOU WILL
PREVENT FUSION AND THE
OVEREXPRESSION PREVENTS VIRAL
ENTRY AND BY COLLABORATING WITH
THE PROTEOMICS CORE WE FOUND
PI16 INTERACTS WITH A LARGE
NUMBER OF HOST FACTORS AND FOR
EXAMPLE MOLECULES FOR SIGNALLING
AND THIS WAS A GREAT
COLLABORATION ON GENOMICS TO
UNDERSTAND HOW IT WAS AFFECTED
BY THE COHORT AND HOW IT WAS
WORKING.
THE QUESTION WAS THIS IS NOT
NORMALLY EXPRESSED T CELLS.
AND BY LOOKING AT THE DATA FROM
THOSE WITH ACUTE VIREMIA WE
FOUND THE T CELLS WITH HI
NEGATIVE HIGHER FRACTIONS OF
POSITIVE COMPARED TO IM POSITIVE
CELLS.
IT'S NOT WHAT IT LOOKS LIKE AND
CELLS THAT HAVE PI16 ARE MORE
RESISTANT TO INFECTION AND
IDENTIFIED THESE AND THE REASON
BEING PPA CAME UP IN BOTH
SCREENS AROUND STRONG ANTIVIRAL
FACTOR AND IT BELONGS TO THE
CYCLIN FAMILY OF PROTEINS AND
YOU SEE THERE'S AROUND 60%
SEQUENCE IDENTIFY AND WHEN YOU
LOOK AT THE STRUCTURE AND YOU
FIND OUT THAT IT CAN ACTUALLY
SUPER IMPROSE WITH PPIA AND
THERE'S STRONG POSITIVE
SELECTION.
IT'S POSSIBLE IT MAY BE
INTERACTING WITH CAPSID.
TO CONCLUDE I THINK WE HAVE
FOUND THINGS WHICH CAN AFFECT
THE HIV INFECTION IN MULTIPLE
STAGES AND WE NEED TO MAKE
FRIENDS TO STAY FOR FOM SIME IN
B-HIVE AND LOOK AT HOW THEY
WORK.
THANK YOU.
IT
>> GREAT TALKS, EVERYBODY.
I HAVE A MILLION QUESTIONS FOR
ALL OF YOU.
THE MOST RECENT I WANTED TO ASK
YOU THE SCREENS YOU'RE DOING
WITH EXPO TROPIC VIRUS WONDERING
IF YOU COULD COMMENT BETWEEN X4
AND R5.
CLEARLY IT'S TECHNICALLY HARDER
AND YOU TALKED ABOUT THE
DIFFERENT SUBSETS.
I'M CURIOUS, DO YOU HAVE SOME
COMMENTS ABOUT IT?
>> ABSOLUTELY.
SO WE INITIALLY STRUGGLED A LOT
TO GET HIGH INFECTION RATES OF
THE VIRUS TO DO THE SCREENS BUT
LATER WE WERE ABLE TO DO THAT.
NOW I UNDERSTAND WHICH VECTORS
UNDERSTAND THE VIRUS.
TO GIVE AN EXAMPLE, DBX21 THE
TRANSCRIPTION FACTOR IS A STRONG
PROTECTIVE SCREEN AGAINST THE
VIRUS BUT WE FOUND IT ACTUALLY
IS A HYPOTROPHIC VIRUS AND YOU
INCREASE THE LEVELS AND LOOKS
LIKE INFECTION OF THE VIRUS.
SO WE HAVE A LOT OF SITES FROM
THE DATA WE ARE GENERATING.
>> COOL.
AND DO YOU HAVE ANY EVIDENCE
PPID THE PROTEIN ENCODED IS
EXISTING INDEPENDENT OF
MITOCHONDRIA?
IT'S SUPPOSED TO BE IN THE
MITOCHONDRIAL MEMBRANE?
>> IT'S ALSO REPRESENTED IN THE
CYTOPLASM AND CAN HAVE
NUCLEARIZATION.
>> YOU WONDER IF IT HAS
SOMETHING TO DO WITH THE
MITOCHONDRIA.
>> WHAT WE'RE DOING RIGHT NOW IS
A SOMEWHAT SIMPLISTIC
EXPERIMENT.
WE LOOK AT THE STRONG POSITIVE
SELECTION AND TESTING PPID
PROTEINS FROM DIFFERENT SPECIES
TO FIGURE OUT IF ALL OF US ARE
MEDIATING THE FUNCTION.
A PART FROM THE PSYCYCLOPHILIN
ACTIVITY THERE'S SHEPHERDING
ACTIVITY AS WELL BUT IT'S AN
INTERESTING SITUATION.
>> THERE'S ALSO LITERATURE
FACTORS LIKE ARSENIC AND OTHER
DRUGS THAT AFFECT THE STATUS AND
THOSE AFFECT RESTRICTION FACTORS
IN WAYS WE DON'T UNDERSTAND.
>> VERY GOOD SESSION.
THANK YOU.
>> A QUESTION FOR NEF -- NEVIN
IF YOU DON'T MIND.
YOU PRESENTED THE INFORMATION
AND LOOKED LIKE IT IDENTIFIED
TWO DIFFERENT INTERFACES THAT
FORM THAT WILL AND YOU SAW THE
STRUCTURE.
HOW CLOSE WAS THE STRUCTURE TO
THE PREDICTED?
>> IT WAS -- I SHOULD HAVE SHOWN
THE PREDICTION WITH THE ACTUAL
STRUCTURE.
IT PREDICTED BOTH INTERFACES.
>> AND THEN THAT'S AWESOME.
WHEN IT WORKS IT WORKS.
AND WHAT ABOUT THE SECOND, THIRD
AND FOURTH COMPLEX?
>> WE'RE ON TWO, THREE AND FOUR
AND LOOKS LIKE NUMBER TWO IS
WORKING.
>> MAYBE 50/50.
GOOD.
>> WE CAN REPORT BACK SOON DAVID
ON THE SUCCESS RATE.
>> I HAVE A QUESTION ABOUT THE
TL2.
YOU FOUND WHEN YOU ADD IT YOU
FOUND POLYMERASE ALL OVER THE
PROVIRUS, YES?
SO DO YOU ANALYZE ABOUT THE
CELLULAR GENOME DOES THE SAME
PREDICT?
>> WE DEFINITELY SEE THE
CONSIDERATION OF IT IS A TOOL
COMPOUND AT HIGH MICROMOLAR
CONCENTRATIONS IT'S DEFINITELY
CYTOTOXIC.
ADDITIONALLY A LOT OF SEC
REGULATED GENES ARE ALONG THE
CELL CYCLE AND YOU SEE A REDUCED
CAPACITY TO PROLIVERATE BUT --
PROLIFERATE BUT REDUCING THE
PROLIFERATORY CAPABILITY.
>> SO HAVE YOU TRIED TO LOOK AT
THE MONOCYTIC CELL LINE AND THE
ENTROPIC VIRUSES?
I'LL GIVE BACKGROUND ON THAT.
SO I HAD A HARC PILOT GRANT AND
WE JUST PUBLISHED A FEW MONTHS
BACK.
THIS PBMCs RESTRICT HIV AND
ESPECIALLY THE ACTIVATED ONE AND
HAVE RESTRICTION FACTORS
UPREGULATED.
NOW, IT THE REVIEWS FOR THE DATA
AND WHEN WE DID THAT, WE FOUND
THAT SICKLE CELL AND WONDER IF
THE T CELL CONTRIBUTE TO THE
RESTRICTION AND WONDERED IF YOU
LOOKED AT THAT PART?
>> WE DID GAIN OF FUNCTION
SCREENS WHICH MEANS YOU CAN
REVEAL PHENOTYPES NOT NATIVE TO
D CELLS.
WE FOUND THINGS LIKE CYCLIC 1
WHICH HAVE NO CONTEXT IN HIV
INFECTION AND WHEN YOU
OVEREXPRESS THOSE THEY SHOW A
SIMILAR PHENOTYPE ON DENDRITIC
CELLS WHEN YOU OVEREXPRESS AND
SEE THE SAME PHENOTYPE WE FOUND
WE'RE IDENTIFYING MACROPHAGES
AND MONOCYTES AND CAN DISTRIBUTE
THEM BY DOING SCREENS THROUGH
GAIN OF FUNCTION SCREENS.
>> THANK YOU.
>> HI.
YOU SAID PI16 IS INHIBITING THE
FUSION OF HIV.
DOES IT INHIBIT X4 AND R5?
>> SO NO.
SO IT SPECIFICALLY INFECTION FOR
TROPIC VIRUS.
I WAS INITIALLY SURPRISED TO SEE
THAT BUT WE FOUND OUT THROUGH
PROTEOMICS THE MOLECULES ARE
BELOW SIGNALLING AND DIFFERENT
HOW SFLALING WORKS.
-- SIGNALLING WORKS.
IT MAKES SENSE BUT DOESN'T
COMPARE TO PPID.
>> AND WE KNOW AT WHAT STAGE OF
HIV INFECTION IT INHIBITS THE
INFECTION.
PI16 NOT EXPRESSED IN MACK
REFATHERS BUT IN FIBROPLASTS AND
MISS EN -- MESSENCHIMAL CELLS.
>> DID YOU MEAN ESTABLISHMENT IN
INFECTION OR LOOKING AT THE
ENTIRE REPLICATION PROCESS?
IN OTHER WORDS, WERE VIRUS
PARTICLES BEING MADE IN THE
CELLS?
>> YES, SO THESE CELLS WE SAW --
YEAH.
NO AFFECT OF THE KNOCKOUT OF THE
SUPER ELONGATION AND THE
ACTIVATED CELLS WERE INFECTED
WITH VIRUS AND NO CULTURE LIKE
NO ARC SO IT IT COULD HAVE BEEN
PRODUCING MORE VIREONES BUT
DIDN'T LOOK LIKE MORE WILL
REPLICATION FOR THE CULTURES.
[APPLAUSE]
>> ALL RIGHT.
SO THAT'S OUR MORNING SESSION.
PLEASE GO AHEAD AND GET YOUR
LUNCH AND MAKE SURE YOU MAKE IT
TO YOUR POSTERS.
WE HAVE POSTER PRESENTERS TO BE
AT THEIR POSTER AT 1:00.
NEXT SESSION SPEAKERS CAN EITHER
COME UP NOW, PREFERABLY NOW OR
BEFORE THE 2:30 RESTART TO GET
YOUR TALKS LOADED.
PLEASE TRY TO DO SO SO WE DON'T
HAVE TO TRACK YOU DOWN AT 2:30.
IS THAT GOOD?
OKAY.
JUST AS A BRIEF INTRODUCTION,
I'M SURE MANY OF YOU ARE AWARE
HIV BUDS FROM THE SURFACE OF THE
CELL, UNDERGOES A PROCESS CALLED
MATURATION.
ONCE IT'S DONE THIS, ONLY ONCE,
CAN IT FUSE WITH THE HOST CELL
MEMBRANE.
IN OTHER LAB WE THINK OF THIS ON
THREE LEVELS.
FIRST IS PROTEOLYTIC MATURE, SO
GAG IS CLEAVED INTO CONSTITUENT
DOMAIN, AND TWO PEPTIDES,
ACCOMPANIED BY STRUCTURAL
CHANGES AND THE BEST STUDIED IS
CAPSID.
THEN THERE'S ALSO ARCHITECTURAL
MATURATION, SO ARRANGEMENT OF
PROTEINS IN THE VIRUS ALSO
CHANGES.
SO, KERN IN OUR LAB DISCOVERED
LIKE CAPSID, MATRIX FORMS A
LATTICE IN VIRUSES.
OF PARTICULAR IMPORTANCE IS THE
FACT THAT THIS LATTICE ALSO
APPEARS TO UNDERGO STRUCTURAL
MATURATION.
SO IN THE IMMATURE VIRUS YOU GET
THE IMMATURE LATTICE, IN MATURE
WE GET MATURE LATTICE.
IT WAS OBSERVED A LIGAND WAS
BOUND TO MATRIX, ABSENT IN THE
IMMATURE LATTICE.
THIS IS A VIDEO THAT SUMMARIZES
THAT.
SO, I WILLING AND IS BOUND IN
THE MATURE FORM.
SO THERE'S SOME OPEN QUESTIONS
LEFT OVER.
FIRST IS WHAT IS THE
RELATIONSHIP BETWEEN CLEAVAGE OF
GAG AND MATURATION OF THE
LATTICE.
THE LAST THING HE LEFT US WAS
WITH OBSERVATION IT APPEARS
SIMPLE CLEAVAGE IS NOT THE
TRIGGER.
HE OBSERVED MAFP 1 IN LICK THE
NUCLEOCAPSID IS CLEAVED, LATTICE
WAS MATURE COMPLETE WITH BOUND
LIGAND.
NEXT HYPOTHESIS WAS ALLOSTERIC
NETWORK ACROSS SP 1 AND CAPSID,
FOUND NO EVIDENCE OF ALLOSTERIC
NETWORK.
MY JOB WAS TO INVESTIGATE THE
STATE OF MATRIX, FIRST USING
CO-ANALYSIS TO CLASSIFY GAG
LAYER AND DIRECTLY LOOK AT THE
STATE OF THE LATTICE.
SECOND WAY TO PERFORM FOURIER
ANALYSIS AS OBSERVED IN 2D
CLASSES, AS THE SPACINGS OF THE
TWO LATTICES ARE DISTINCT,
GIVING DISTINCT PROFILES.
IF WE START WITH IMMATURE VIRUS,
WE OBSERVE ELASTIC SPACE
CONSISTENT AND CAN GET A 3D.
IF WE CLEAVE THE BOTTOM DOMAIN
OF GAG P 6, WE DON'T HAVE 3D
CONSTRUCTION, BUT FOURIER
ANALYSIS LIKELY IMMATURE
ANALYSIS.
IF WE CLEAVE THE NEXT TWO, P6
AND SP2 WE SEE STRIKING CHANGE,
WE SEE A MATURE LATTICE.
IF WE KEEP GOING WE AGAIN
OBSERVE MATURE LATTICES, UP TO
THE MATURE VIRUS.
SO THIS START MAKING US THING
SP2 MIGHT BE IMPORTANT FOR
REGULATION OF MATURATION OF
MATRIX.
AND SHOULD WE GENERATE MUTE
RANTS WHERE SP2 WASN'T RELEASED,
HELD IN THE BUNDLE, THIS WAY WE
COULD APPEAR TO SUPPRESS THE
MATURATION OF THE MATRIX
LATTICE.
SO TO SUMMARIZE, WE BELIEVE
REALLY SP2 IS IMPORTANT FOR
MATRIX MATURATION.
INTERESTINGLY, SP2 IS BASICALLY
THE ONLY COMPONENT OF HIV WITH
NO ASSIGNED FUNCTION.
I ENCOURAGED THIS WHERE YOU CAN
SEE SP2 ANNOTATED WITH THE
FUNCTION, HIGHLY CONSERVED ALSO.
NEXT THING I DID WAS TRY TO
SOLVE THE HIGH RESOLUTION
STRUCTURE OF THESE LATTICES.
AND TO DO THIS USING SIMILAR
APPROACH MANY LABS ARE USING AT
THE MOMENT, I STARTED TO REALIZE
I COULD EXTRACT HIGH RESOLUTION
FEATURES USING THIS APPROACH, A
LOT OF HARD WORK BUT MANAGED TO
GET VERY HIGH QUALITY
RECONSTRUCTIONS OF THE MATRIX
LATTICE WITHIN VIRUSES.
AND THIS IS WHAT THEY LOOK LIKE.
THIS RECONSTRUCTION IS THREE
ANGSTROMS.
RESOLUTION WHERE WE CAN SOLVE
CHAINS.
FIRST THING WE DID WAS DESCRIBE
INTERFACES THAT HOLD THE MATURE
LATTICE TOGETHER, SEEMED TO BE
MADE OF VERY SPECIFIC
ELECTROSTATIC CONTACTS.
SO AS PREVIOUSLY MENTIONED
MATURE MAT RICKS LATTICE WAS
OBSERVED TO BIND A LIGAND AND WE
COULD SEE THIS LIGAND, COULD SEE
NEW FEATURES IN THE LIGAND AND
HAS VERY DEFINED SHAPE.
SO WHAT IS THIS LIGAND?
SO, BASED ON PREVIOUS STRUCTURAL
WORK THAT SHOWED MATRIX COMBINED
PIT 2 AND BASED ON SIZE OF THE
LIGAND IT WAS HYPOTHESIZED THAT
THE IDENTITY OF THE LIGAND WAS
PIT 2.
WHEN WE TRIED TO FIT THE
STRUCTURE IN THE NEW MAP WE
FOUND IT DIDN'T FIT, WE COULDN'T
GET IT TO BIND.
WE TRIED ALL THE OTHER LIPIDS,
INEFFICIENT QUANTITIES, WE
COULDN'T GET LIPIDS TO BIND NO
MATTER HOW HARD WE TRIED.
SO PREVIOUS OBSERVATION SP2
APPEARS TO REGULATE MATURATION
OF LATTICE, ATTEMPTED TO FIT SP2
INTO THIS DENSITY.
AND WE FOUND THAT THE C-TERMINAL
FIVE RESIDUES FIT INTO THIS
DENSITY QUITE WELL.
IN A CONFIRMATION THAT SUGGESTED
REMAINING TEN RESIDUES OF
PEPTIDE FIT ABOVE THE MATRIX
LAYER AND CONTACT THE MEMBRANE.
WHEN WE PERFORMED AUTOMATIC
MODEL BUILDING, WITHOUT INPUT
SEQUENCE WE FOUND THAT IT AGREED
WITH US IN OUR DETERMINATION,
AND QUITE CONSISTENTLY PREDICTED
THESE RESIDUES WERE SP2
RESIDUES.
DOMINICK IN OUR LAB WORKED ON
ASSEMBLING MATRIX LATTICES IN
VITRO, AND THEN ON LIPID
MONOLAYERS AND IMAGED ON GRIDS.
WHEN I INITIALLY TRIED HE FOUND
IT DIDN'T WORK VERY WELL, DID
GET SMALL AMOUNTS OF IMMATURE
AND MATURE LATTICE.
WHEN HE ADDED SP2 TO REACTION
MIXTURES SUDDENLY SAW
SIGNIFICANT AMOUNTS OF ONLY THE
MATURE LATTICE.
2D CLASSIFICATION OF 2D CRYSTALS
SHOWED BINDING OF SP2, IN THE
TOP ROW HOPEFULLY YOU CAN SEE WE
ACTUALLY DIRECTLY OBSERVE THE
BINDING OF A LIGAND, AND WHEN WE
STIMULATE OUR RESOLUTION 2D
CLASSES OF SP2 AND NOT SP 2
BOUND MATRIX IT MATCHES OUR DATA
WELL.
WE BELIEVE THE REMAINDER OF THE
PEPTIDE IS ABOVE MATRIX AND SEE
IN OUR RECONSTRUCTIONS AT LOW
THRESHOLD.
APPEARS TO BE CONTACTING THE
INTERFACE TO THE MEMBRANE.
WITH THAT WE COULD DESCRIBE THE
STRUCTURE OF SP2 BOUND MATRIX IN
THE VIRUS.
THIS IS THE MATURE LATTICE.
WE FOUND SP2 IS LIKELY
CRITICALLY INVOLVED IN THE
INTERTRIMERIC INTERFACE BETWEEN
MATRIX TRIMERS, SPECIFICALLY IN
THIS REGION HERE, WHERE THE
C-TERMINAL GROUP MUTUALIZES
ARGININE 22, ALLOWS THEM TO BE
IN CLOSE PROXIMITY TOGETHER.
THIS IS A SUMMARY OF WHAT WE
BELIEVE IS HAPPENING.
CLEAVAGE OF GAG, RELEASE OF SP2
WHICH BINDS MATRIX LAYER AND
INDUCES MATURATION OF MATRIX.
ALSO I'D LIKE TO POINT OUT WE
BELIEVE THIS MECHANISM IS QUITE
SIMILAR TO CAPSID, REGULATED BY
CLEAVAGE OF SP1, THE OTHER SPACE
PEPTIDE, IN THIS CAUSE IP2 IS
RELEASED, MATURE CAPSID LATTICE,
IN A SIMILAR WAY TO THE WAY SP2
TRIGGERS FORMATION OF MATURE
MATRIX.
I'D LIKE TO THANK MY LAB AND
Ph.D. SUPERVISOR JOHN BRICKS
AND COLLABORATOR DOMINICK AND
ALL THE COLLABORATORS THAT
HELPED US MAKE THE VIRUS.
THANKS VERY MUCH.
[APPLAUSE]
>> THANK YOU VERY MUCH, JAMES.
DO WE HAVE ANY QUESTIONS?
WE HAVE THREE TO FOUR MINUTES.
>> THAT WAS REALLY AN IMPRESSIVE
TALK.
CONGRATULATIONS.
HAVE YOU THOUGHT ABOUT WHETHER
OR NOT A PEPTIDE LIKE THAT MIGHT
BE PLAYING A ROLE IN OTHER
RETROVIRUSES?
LET'S SAY MURINE RETROVIRUSES?
>> YEAH, SO WE KNOW HIV-2 AT THE
VERY LEAST ALSO HAS THIS SP2
PEPTIDE, SLIGHTLY DIFFERENT
SEQUENCE, BUT I BELIEVE IT'S
PERFECTLY POSSIBLE BECAUSE IF
YOU LOOK AT THE GAG, GAG, THEY
HAVE SMALL DOMAINS, MANY OF
WHICH ARE NOT ASSIGNED TO ANY
PARTICULAR FUNCTION BUT WE DON'T
KNOW IF OTHER RETROVIRUSES
BASICALLY UNDERGO MATRIX
MATURATION, SOMETHING WE'RE
INTERESTED IN AND MIGHT TRY TO
EXPLORE, I DON'T KNOW.
>> VERY NICE TALK.
>> CAN YOU SHOW NAME TAG.
THANK YOU.
>> OH, YEAH, HERE.
DOES YOUR MODEL SUGGEST THAT SP2
REPLACES OR DISPLACES PIP 2?
>> I THINK THE ANSWER IS NO.
WE WOULD EXPECT TO OBSERVE PIP 2
WITHIN THE POCKET IN THE
IMMATURE MATRIX LATTICE WITH YOU
WE DON'T OBSERVE ANY LIGAND IN
THAT POCKET IN THE IMMATURE
LATTICE.
SO I DON'T THINK IT DISPLACES
IT, NO, BUT -- OR THE ROLE OF
PIP 2, I'M NOT SURE.
>> WOULD YOUR STRUCTURE ALLOW
YOU TO DESIGN A POINT MUTANT
THAT BREAK THIS, INDUCE
REPLICATION DEFECT THEN
COMPENSATED BY MUTATION IN
MATRIX?
>> SO THE PROBLEM WITH DESIGNING
MUTATIONS IN SP2 IS ACTUALLY THE
SEQUENCE OF SP2 OVERLAPS WITH
THE RIBOSOMAL SLIPPAGE SITE THAT
CONVERTS GAG TO GAG POLL.
WE'RE TRYING BUT IT'S TRICKY
BECAUSE WE RELY ON (INAUDIBLE).
>> ANY OTHER QUESTIONS?
IF NOT, NEXT SPEAKER IS
CHRISTIAN FRENIERE FROM YALE
UNIVERSITY.
ANOTHER APPLAUSE FOR JAMES.
[APPLAUSE]
>> ALL RIGHT.
AWESOME.
YEAH I'M CHRISTIAN FROM YALE,
FIFTH YEAR GRADUATE STUDENT IN
YONG JUNG'S LAB.
BEFORE I GET GOING I WANT TO
GIVE A QUICK SHOUT OUT TO THE
PEOPLE WHO MADE THIS WORK
POSSIBLE, SO MATT HAS BEEN MY
PARTNER IN CRIME, WE WORK VERY
INTIMATELY EVERY STOP ON THE
PROJECT.
CHARLIE TAUGHT ME EVERYTHING
ABOUT THE ELECTRON MICROSCOPE,
IMPORTANT.
FAITH IS A TALENTED
UNDERGRADUATE WORKING ON
ASSEMBLY AND CO-SEDIMENTATION
ASSAYS AND THE BOSS WHO MAKES
EVERYTHING POSSIBLE, ALSO REALLY
HELPS WITH DATA COLLECTION, DATA
PROCESSING, STUFF LIKE THAT.
ALSO VERY MUCH INVOLVED IN THE
WORK.
SO LET'S JUMP IN.
WE'LL DO HIV-2 FROM 50,000 FEET.
SO AROSE FROM DISTINCT SIMIAN
RETROVIRUS, CAN PROGRESS TO AIDS
OVER TIME, FEATURES SLOWER
PROGRESSION TO AIDS.
AND BURDEN OF THE DISEASE IS
SORT OF BORN ON SUB-SAHARAN WEST
AFRICA AND SORRY COUNTRIES HAD
COLONIAL EXCHANGE WITH THAT
REGION OF THE WORLD.
SO, YEAH, A LITTLE BIT ABOUT THE
CAPSID, TWO CAPSID PROTEINS, 80%
IDENTICAL, SO DESPITE THIS WE
KNOW THERE ARE DIFFERENT
PROVIRAL FACTORS THAT RECOGNIZE
THESE CAPSIDS DIFFERENTLY,
BINDING TO HIV-1 VERSUS HIV-2.
LIKEWISE RESTRICTION FACTORS
SUCH AS NONO HAVE DIFFERENT
BINDINGS, THERE'S REASON TO
BELIEVE DESPITE SIMILARITIES
THERE'S SOME DIFFERENCES, AND
ALTHOUGH THANKS TO MANY PEOPLE
IN THIS ROOM WE HAVE AWESOME
TOOLKIT FOR INVESTIGATION OF
HIV-1, FROM STRUCTURAL
STANDPOINT.
WE DON'T HAVE TOOLS FOR THE IN
VITRO ASSEMBLY AND STRUCTURAL
STUDY OF HIV CAPSID.
MOTIVATION OF THIS PROJECT IS
KIND OF TO TRY TO BEGIN BUILDING
A SIMILAR TOOLKIT FOR HIV-2.
ALL RIGHT.
SO, APPROACH WE TOOK IS INSPIRED
HEAVILY BY ROB DICK'S LAB, A BIG
SHOUT OUT TO ROB TO SAY THIS
METHOD IS EXTREMELY POWERFUL FOR
THE TEMPLATING OF DIFFERENT
CAPSID LATTICES.
MAKES USE OF LIPOSOMES WHICH
FEATURE A SMALL PERCENTAGE OF
NTA LIB IDENTITY THAT PROVIDES A
POINT, USING THE VARIANT OF
HIV-2 PROVIRUS, AND WE HIS TAG
ON THE C-TERMINAL TO ASSOCIATE
WITH THE NICKEL LIPOSOME,
ALLOWING FOR NUCLEATION, THE
EFFICIENT TEMPLATING OF LATTICE
AROUND THIS NUCLEATION POINT, AN
EXTREMELY POWERFUL TOOL.
SO WHAT DOES THIS LOOK LIKE WITH
HIV-2?
YOU CAN SEE CLEARLY WE GET
PRETTY DISTINCT CAPSULE-LIKE
PARTICLES, ON THE INSIDE OF THE
CAPSID-LIKE PARTICLE.
OH, THIS IS -- I'M GOING ALL
OVER THE PLACE.
ON THE INSIDE YOU CAN SEE
THERE'S LIPOSOME AND CLEARLY YOU
CAN SEE CAPSID TEMPLATED AROUND
IT INDICATING TO US THIS IS
WORKING.
WE CAN DO CLASS AVERAGING
SIMILAR TO WHAT OTHER PEOPLE
MENTIONED, TREATING THIS AS A
SINGLE PARTICLE.
WE SEE CLEAR TOP VIEWS OF THE
LATTICE, AND WE ALSO SEE AREAS
WHERE WE CAN SEE THE LIPOSOME
UNDERNEATH AS WELL.
SO THIS ALLOWS US TO GET HIGH
RESOLUTION RECONSTRUCTION OF CA
HEXAMER, ALLOWS FOR CONFIDENT
MODEL BUILDING.
SHOUT OUT TO MATT FOR THE MODEL
BUILDING BUT PERHAPS EVEN MORE
EXCITINGLY IN EARLY
HETEROGENEOUS ASSIGNMENTS
NOTICED PRESENCE OF PENTAMER
ADJACENT TO HEXAMER, SLIGHTLY
HIGHER RESOLUTION ALLOWS TO
BUILD VERY CONFIDENT MODELS.
AGAIN BIG SHOUT OUT TO MATT.
YOU MIGHT HAVE NOTICED THERE'S A
SMALL PERCENTAGE OF PARTICLES
WHICH DON'T LOOK SIMILAR.
NORMAL CAPSID-LIKE HAVING THE
MORPHOLOGY, REMINISCENT TO
PENTAMER WHICH WAS RECENTLY
PUBLISHED.
WE SAW TO SEE IF T EQUALS 1, WE
GET HIGH RESOLUTION, AGAIN WE'RE
ABLE TO BUILD MODELS.
I HAVE TO APOLOGIZE TO MATT FOR
THE PAIN OF HAVING SUCH HIGH
RESOLUTION.
YOU'RE ABLE TO SEE THE HOLES IN
THE AMINO ACID, AN EXCITING TIME
TO WORK ON CAPSID ASSEMBLY AND
USING THIS TECHNIQUE.
WE HAVE HIGH RESOLUTION
STRUCTURES OF LATTICE.
WHAT HAVE WE LEARNED?
ONE OF THE FIRST THINGS THAT WAS
IMMEDIATELY APPARENT TO US WAS
THIS LOOKS LIKE A HANDLE ON A
BUCKET YOU CAN PICK UP BUT IF I
SHOW YOU A SIMILARLY SHARPENED
MAP FROM HIV-1, SUV TEMPLATED
ASSEMBLY WE DON'T SEE THE SAME
AMOUNT OF CYP 1, SUGGESTING
BINDING OF HOSE FACTORS TO THE
CYP-A COULD BE DIFFERENT.
ANOTHER INTERESTING POINT OF
DIVERGENCE AND CONFIRMATION OF
WORK IS THAT THIS N-TERMINAL
BETA HAIRPIN, POSE TO PH SENSOR,
MAY HAVE ROLE A NUCLEOTIDE
IMPORT.
IN HIV-2 WE ALWAYS OBSERVE IT IN
THIS OPEN STATE, WHEREAS AT THIS
PH IN HIV-1 WOULD HAVE OBSERVED
IN THE CLOSED STATE.
THIS MAY HAVE SOME IMPLICATION
ON NUCLEOTIDE IMPORT.
ALL RIGHT.
SO, ONE OF THE OTHER SORT OF
DIVERGENCES WE SAW WITH HIV-2 IS
IN THE STRUCTURAL SWITCHING.
SO THE TVDG LOOK WHICH NOW IS
BECOMING FAMOUS FOR HIV-2
STRUCTURAL SWITCHING, ACTUALLY
IS A CVGD LOOP IN HIV-1, AND WE
SEE SOMETHING DIFFERENT AS WELL.
IN HIV-1 AGAIN THIS IS WORK BY
OWEN, JOHN, AND ROB'S GROUP THAT
ELEGANTLY ELIMINATED THE
SWITCHING IN THE TVGG LOOP,
PRONOUNCED DIFFERENCE BETWEEN
THE HEXAMER AND PENTAMERIC
FORMS.
IN HIV-2 WE DON'T HAVE AS
DRAMATIC A REARRANGEMENT.
HIV-2 HEXAMER LOOP ALREADY LOOKS
LIKE THE PENTAMER LOOP FOR
HIV-1, AND PENTAMER LOOP IS
SIMILAR STORY, RIGHT?
LESS OF A CHANGE THERE.
SO THIS IS LEADING US TO
THINKING THAT IN HIV-2 THIS IS
LESS OF A SWITCH AND MORE OF A
SLIDER IN THIS SORT OF AREA.
SO MORE SUBTLE CHANGE.
WHAT WE DO SEE IN TERMS OF NEW
CONTEXT BETWEEN THE HEXAMER AND
PENTAMER IS IS ACTUALLY IN THE
HELIX 2-3 AREA, AND THE
JUNCTION.
WE HAVE APPEARANCE OF -- OR
SLIDING OF DIFFERENT POLAR
CONTEXT BETWEEN HELIX 2 AND 3
BETWEEN HEXAMERRIC AND
PENTAMERIC FORM.
I'LL SHOW YOU A MOVIE.
WE HAVE THIS INTERCHANGE OF
POLAR CONTACT IN THE NTD.
SIMILARLY WE HAVE A SIMILAR
STORY GOING ON IN THE NTD-CTD
JUNG WHERE POLAR CONTACTS ARE
SLIGHTLY DIFFERENT BETWEEN THE
HEXAMER AND PENTAMER, WE BELIEVE
THERE'S A SLIDING OF THE POLAR
CONTACTS ACHIEVED IN THESE
FORMS.
SO, ONE POINT OF SIMILARITY I
FOCUSED MOSTLY ON DIFFERENCES
HERE IS BINDING OF FG PEPTIDES,
WERE ABLE TO RESOLVE STRUCTURES
WITH BOTH, THEY ALIGNED CLOSELY
WITH THE HIV 1 STRUCTURES, AND
SIMILARLY WE ONLY OBSERVED
DENSITY FOR THESE FG PEPTIDES IN
THE HEXAMER.
WE DON'T SEE IT IN THE PENTAMER.
THIS IS SLIGHTLY INTRIGUING TO
US BECAUSE IN THE HEXAMER THERE
IS NO LONGER -- SORRY, IN THE
PENTAMER THERE'S NO LONGER
STERIC OCCLUSION, SOME OTHER
MECHANISM IS PREVENTING THE FG
PEPTIDES FROM BINDING TO THE
PENTAMER.
OKAY.
SO, FINAL TAKEHOME POINTS, WHAT
HAVE WE LEARNED IN COMMON
FEATURES, SOME I DIDN'T TALK
ABOUT, YOU CAN COME TO MY POSTER
TOMORROW.
THE OVERALL TERTIARY IS SIMILAR,
IP6 I DIDN'T TOUCH ON TODAY,
IT'S ALSO VERY SIMILAR.
FG POCKET BINDING SEEMS TO BE
NEARLY IDENTICAL.
SO, CYP- A, THIS COULD HAVE
IMPLICATIONS FOR HOST FACTORS,
POTENTIALLY NUCLEAR IMPORT.
WE DO SEE SUBTLE DIFFERENCE IN
THE GATING OF THAT PH SENSITIVE
LOOP STUCK IN OPEN CONFIRMATION,
AND OUR MECHANISM OF ACHIEVING
THE PENTAMER-HEXAMER TRANSITION
IS SUBTLY DIFFERENT.
WE HAVE JUST THE SLIDING OF
POLAR CONTACTSES RATHER THAN
REARRANGEMENT OF THIS LOOP
REGION.
SO, YEAH, AGAIN, I WILL JUST
HIGHLIGHT THE PEOPLE THAT DID
THE WORK AND I'VE GOT A MINUTE
OR SO FOR QUESTIONS.
[APPLAUSE]
>> THANK YOU VERY MUCH,
CHRISTIAN.
I REALLY DO HAVE A QUESTION
MYSELF.
A COMPOUND, IT'S A CYCLOSPORIN
ANALOG, IT ONLY WORKS FOR HIV-1.
DOESN'T WORK FOR HIV-2.
MAYBE THAT HAS SOMETHING TO DO
WITH THE CA LOOP RIGIDITY.
CAN ANYONE COMMENT ON THAT?
THIS MECHANISM OF CYCLOCHLORINE
BINDING ONLY WORKS FOR HIV-1, IS
THAT CORRECT?
DOESN'T WORK FOR HIV-2, THAT'S
MY UNDERSTANDING.
>> PARTICULARLY INTERESTING,
MANY PEOPLE IN THE FIELD SUGGEST
HANDOFF BETWEEN CYP A MIGHT BE
IMPORTANT, IF ENCOURAGING MORE
BINDING THE INTERACTION OF HOST
FACTORS AND TIMING OF THE
DIFFERENT STEPS COULD BE, YEAH,
DIFFERENT JUST BECAUSE OF THE
CYP A BINDING.
>> IT'S HIV-1 SPECIFIC SO
THERE'S DIFFERENCES BETWEEN
HIV-1 AND 2 WHICH MAKES IT
HARDER TO TEST THOSE COMPOUNDS
IN AN ANIMAL MODEL, FOR EXAMPLE.
>> SO THE ROLE OF CYCLOPHILIN IS
MORE COMPLICATED.
THE READOUT FOR THE DUG EFFECT,
DIFFERENT SIVs RESPOND
DIFFERENTLY IN TERMS OF BINDING.
>> MAJOR RESTRICTION IN MICE, IS
THAT RIGHT?
I MEAN, ONE OF THE REASONS HIV
DOESN'T INFECT MICE, I MEAN,
CYCLOPHILIN, CAPSID ALTERS
BINDING, SPECIFIC IN ACTIVITIES,
SO EVEN HIV 1 IN SOME NON-HUMAN
PRIMATE SPECIES, CYCLOPHILIN
PROMOTES BINDING SO IT DEPENDS.
IT'S VERY DYNAMIC.
>> COMPLICATED.
>> THANK YOU.
NEXT QUESTION?
>> GREAT TALK.
DID YOU LOOK AT THE LOOP WITH
DIFFERENT PHs?
>> THE ASSEMBLIES AT DIFFERENT
PHs?
>> UH-HUH.
>> YES, ASSEMBLED EFFICIENTLY AT
ALL PHs, SO WE GO LOW LIKE PH
6 SOME MESSINESS BUT WE CAN GO
AS LOW AS PHYSIOLOGIC 7- 2 AREA.
>> AND YOUR T1s DO YOU SEE
INDICATION OF A ROLE FOR HIS TAG
IN FORMATION OF THESE?
>> SO, THAT'S AN INTERESTING
QUESTION.
SO THERE IS -- I SURE SAY WE
DO -- I BELIEVE THE T EQUALS 1
FORM EVEN WITHOUT THE MICELLE
TEMPLATING, THE PENT MORE IS
MORE EASILY FORMED BUT THERE'S
SOME INDICATION, VERY C-TERMINAL
WE HAD RESIDUES THAT APPEAR TO
BE IN AN ODD CONFIRMATION,
PROBABLY SEEING THEM PINNED DOWN
BY THE HISTAG.
I DON'T KNOW IF THAT FULLY
ANSWERS YOUR QUESTION.
>> LAST QUESTION IN
HETEROGENEITY REFINEMENTS DO YOU
SEE INDICATIONS OF PENTAMERS
NEXT TO PENTAMERS?
>> I'VE NEVER SEEN THAT BEFORE.
>> THANK YOU.
>> IF NO OTHER QUESTIONS ANOTHER
APPLAUSE FOR CHRISTIAN.
[APPLAUSE]
>> NEXT IS WILLIAM ARNDT FROM
UNIVERSITY OF MINNESOTA.
>> WEIR
>> WE'LL MOVE TO THE NEXT
SPEAKER AND PUT WILLIAM LAST.
NEXT SPEAKER IS JOSHUA HOPE,
UNIVERSITY OF OXFORD.
GO AHEAD, JOSHUA.
>> OKAY.
TODAY I'M GOING TO TALK TO YOU
ABOUT OUR PROJECT TO
CHARACTERIZE -- SORRY, TO
CHARACTERIZE THE INTERACTION OF
LENT VIRAL INTEGRATION MACHINERY
AND CHROMATIN.
MULTIMERIC, UPON HOST DNA
DOCKING CAUSE COVALENT INSERTION
OF THE VIRAL DNA INTO THE HOST
DNA TO FORM A COMPLEX STRAND
TRANSFER.
AFTER WHICH TIME THE HOST
MACHINERY TAKES OVER COMPLETELY
TO REPAIR THE SCARS OF
INTEGRATION AND STABLE
TRANSCRIPTION OF THIS PROVIRUS
CAN TAKE PLACE.
WE KNOW A LOT STRUCTURALLY ABOUT
INTASOMES, NVV FORMS STABLE
NUGGETS.
NOW, THE VAST MAJORITY OF
RESEARCH IS FOCUSED ON
INTEGRATION INTO NAKED DNA
SUBSTRATES.
THE TRUE SUBSTRATE FOR
INTEGRATION IS IN FACT
CHROMATIN, 80% OF MAMMALIAN
GENOME IS FOOTPRINTED BY
NUCLEOSOMES.
MANY RETROVIRUSES HAVE EVOLVED
MECHANISMS TO INTERACT WITH THE
CHROMATIN, STRIKINGLY NOT
CONSERVED.
PFV INTERACTS WITH HISTONES,
OTHERS SUV AS HIV AND MLV SEEM
TO USE THESE TETHERING FACTORS
TO TETHER THE PRE-INTEGRATION
COMPLEX TO THE CHROMATIN.
THIS HAS IMPLICATIONS FOR
INTEGRATION SITE TARGETING AND
ALSO POTENTIALLY
POST-INTEGRATION SITE REPAIR AND
SUBSEQUENT TRANSCRIPTION.
WORK IN OUR LAB IN 2015 FOUND
THAT INTEGRATION TAKES PLACE
REALLY IN THE CORE OF THE
NUCLEOSOME BY THE PROTEASOMIC
VIRUS INTERACTING STRONGLY WITH
H2A AND H2V IN THE CONTEXT OF
THE NUCLEOSOME.
IT'S LESS CLEAR WITH
LENTIVIRUSES BUT ONE KEY
CANDIDATE IS THE HOST FACTOR
LEDGER, TWO DOMAIN PROTEIN WITH
INTEGRATED BINDING DOMAIN AND
PWWP DEMAIN, IT BINDS TO
INTEGRASE, PWWP DOMAIN BINDS
STRONGLY TO LYSINE, THIS DIRECTS
INTO THESE ACTIVELY TRANSCRIBED
REGIONS THAT BEAR THIS
POST-TRANSLATIONAL MODIFICATION.
SO WE WANTED TO STUDY THIS.
AND WHILE THERE'S A WEALTH OF
DATA IN CELLS OF LEDGF, THE
PICTURE IS NOT WELL UNDERSTOOD.
SO TO TAKE A STEP BACK IN VITRO,
WE WANTED MODELS OF CHROMATIN.
WE TURNED TO POLY NUCLEOSOMAL
ARRAYS WHICH WE DEPOSITED
HISTONES ONTO 12 REPEATS, YOU
CAN SEE THIS BEAUTIFUL BEADS ON
A STRING OF THE NUCLEOSOMES.
WE HAVE HAVE THEM UNMODIFIED OR
TRIMETHYLATED, BECOMING THE TRUE
SUBSTRATE OF LEDGF.
SO, IF I EXTRACT THESE
PRE-INTEGRATION COMPLEXES FROM
ACUTELY INFECTED CELLS I GET AN
INCREASED PREFERENCE FOR THIS
TRIMETHYLATED ARRAYS, IN
AGREEMENT WITH WORK PUBLISHED BY
THE DASH LAB.
AND IF I EXTRACT THESE PICs,
THAT PREFERENCE IS JUST
NON-EXISTENT.
ADDING BACK RECOMBINANT LEDGF WE
CAN RESCUE THIS PREFERENCE FOR
TRIMETHYLATED ARRAYS.
WE WANTED TO DO THIS WITH
INTOSOMES.
BY USING INTEGRASE, LEDGF AND
SHORT VIRAL DNA MIMICS WE CAN
PURCHASE PHI AND ASSEMBLE.
WE GET INCREASED PREFERENCE, IF
YOU CHOP OFF THE PWWP DOMAIN.
AND STRIKINGLY WHEN YOU ADD
LEDGF TO THE REACTION,
DOSE-RESPONSE, WE SEEM TO GET
SELECTIVE INHIBITION INTO
UNMODIFIED CHROMATIN,
TRIMETHYLATED STAGE REASONABLY
CONSTANT.
WITH THAT WE TURNED TO
INTEGRATION SITE SEQUENCING TO
SEE HOW EXACTLY THIS INTOSOMES
IS REACTION.
WE USE PCR 3 APPROACH WITH
NANOPORE SEQUENCING TO GET LONG
READS OF INTEGRATION ACROSS THE
ENTIRE ARRAY.
COMPRESSING OUR DATA OF
INTEGRATION SITES ONTO A SINGLE
NUCLEOSOME HERE, WE HAVE THE
NUCLEOSOME FOOTPRINT IN THE GRAY
BOX, THEN THE FLANKING WHITE
SPACE WILL BE THE LINKAGE THE
NUCLEOSOME.
WHAT WE SEE IS -- WHAT WE EXPECT
FOR NAKED DNA SUBSTRATE HIGHLY
PROMISCUOUS INTEGRATION ACROSS
THE BODY OF THE NAKED DNA.
BUT WITH STILL SOME SEQUENCE
SPECIFICITY EXPECTED.
WHEN WE WRAP THESE IN
NUCLEOSOMES WE SEE PUSHING OUT
OF INTEGRATION FROM THE CORE OF
THE NUCLEOSOME TO LINKERS
BETWEEN THE NUCLEOSOMES.
IF WE AGGREGATE DATA TO LOOK AT
INTEGRATION JUST INTO THE LINKER
OF THE TOTAL INTEGRATION, WHAT
WE SEE IS INCREASED INTEGRATION
INTO THE LINK 2007 TRISOMY
MARKER IS PRESENT, GREATLY
RESISTED WHEN YOU RAMP UP TO
LEDGF.
AND THIS KIND OF SUMPED US.
WHAT IS GOING ON?
HOW IS INTEGRATION MOVING INTO
THE CORE OF THE NUCLEOSOME WHEN
THIS MARKER IS NOT PRESENT?
THERE'S SOME SITES WHICH I'LL
TRY AND POINT OUT.
HERE WHICH WOULD BE COMPLETELY
REFRACTORY TO INTEGRATION IN THE
CONTEXT OF THE NUCLEOSOME
THERE'S NO WAY THAT INTASOME CAN
GET IN THERE.
NO REACTION IS 100%.
MAYBE THERE'S A SMALL AMOUNT OF
NAKED DNA IN OUR SAMPLE, AND
ACTUALLY INTEGRATION IS GOING
THERE.
WE BUILT EXPERIMENTS WITH NAKED
DNA, DIFFERENT SEQUENCE, RUN
ALIGNMENT OF WHERE INTEGRATION
GOES.
WHEN THE CHROMATIN IS
UNMETHYLATED WE GET SPARING
INTEGRATION INTO THE UNMODIFIED
CHROMATIN BUT WHEN IT'S
TRIMETHYLATED IT REALLY GOES
INTO THAT CHROMATIN BUT STILL IN
THAT LINKER BETWEEN THE
NUCLEOSOMES.
WE EXPAND THIS DATASET ACROSS
INTEGRATION ACROSS THE WHOLE
ARRAY AND SEE SOMETHING REALLY
COOL.
SO, WHEN THERE'S NO MARK PRESENT
THERE'S NO PREFERENCE FOR WHICH
NUCLEOSOME IT GOES INTO ACROSS
THE ARRAY, STILL TARGETING THAT
NAKED LINKER BUT DOESN'T CARE
WHICH NUCLEOSOME IT IS.
WHEN WE HAVE THE LEDGF AND
MARKER PRESENT THERE'S A PUSHING
OF INTEGRATION INTO THE BODY OF
THE NUCLEOSOME WHICH WE THINK IS
PROBABLY EXPLAINED BY THE FACT
THAT THIS HIGHLY DISORDERED
LEDGF PROTEIN CAN MAKE SEVERAL
CONTACTS ACROSS SEVERAL
NUCLEOSOMES, TOWARDS THE CENTER
YOU'RE LIKELY TO GET MORE THAN
AT THE ENDS.
I'M GOING TO SUMMARIZE WITH
REALLY HOT OFF THE PRESS
STRUCTURES THAT A POSTDOC IN OUR
LAB HAS MANAGED TO SOLVE.
THIS IS THE CHROMATIN BINDING
PLATFORM OF THE INTOSOME.
WE USE LEDGF BUT CAN ONLY SEE
RESOLUTION FOR LEDGF INTEGRASE
BINDING DOMAINS WHEN SATURATED
WITH LEDGF.
WE SEE A MORE LEDGF BINDING
MOLECULES ON THIS, THE TARGET
DNA FACING SIDE, AND THAT HELPS
TO EXPLAIN THESE MULTIVALENT
INTERACTIONS THAT CAN OCCUR
BECAUSE THE INTOSOME CAN BIND.
WHEN THIS TARGET DNA BINDING
GROUP, SO THESE LEDGF MOLECULES
FRAME THE SITE WHERE THIS TARGET
DNA COMBINED, AND REALLY
RESTRICT THAT THE DNA CAN BE IN.
WHEN WE'VE LOOKED AT THIS, AND
TRY DOCK WHERE A NUCLEOSOME
WOULD FIT INTO THIS STRUCTURE
WHEN IT'S STAT RATED WITH LEDGF
YOU JUST CAN'T FIT IT IN.
WHEN WE HAVE THE PWWP PRESENT,
YES IT'S TARGETING CHROMATIN,
BUT WHEN THE LEDGF IS PART OF
THIS KIND OF CHROMATIN BINDING
PLATFORM IBDs ARE RESTRICTING,
PREVENTS DIRECT INTERACTION WITH
NUCLEOSOME, TARGETS NAKED
SUBSTRATES.
WITH THAT I'D LIKE TO THANK MY
SUPERVISOR, MENTOR IN PETER'S
LAB, AND A LOT OF THE STRUCTURAL
WORK FOR THIS PROJECT, AND
NICOLA COLLECTED DATA.
THANK YOU.
[APPLAUSE]
>> ANY QUESTIONS FOR JOSHUA?
>> HI, JOSHUA.
BEAUTIFUL WORK.
ESPECIALLY THE STRUCTURES AT THE
END.
I WANTED TO ASK ABOUT THE
INITIAL SEQUENCING DATA.
SO YOU SEEM TO HAVE A PREFERENCE
FOR THE EXIT SITE OF THE
NUCLEOSOME BUT NOT THE ENTRY
SITE INTEGRATION.
DO YOU HAVE ANY EXPLANATION FOR
THAT?
>> SO, WE THINK THAT THE
SEQUENCE SPECIFICITY, YOU KIND
OF HAVE MORE SPECIFICITY FOR
THAT EXIT SITE BECAUSE IT'S
SYMMETRIC, RIGHT?
THERE SHOULDN'T BE A DIFFERENCE.
WE'RE WORKING ON GETTING
INTEGRATION SITE DATA FOR --
WITHOUT ANY SEQUENCE
SPECIFICITY, JUST RANDOM
SEQUENCES TO AVERAGE THAT OUT.
>> DO YOU THINK IT MIGHT HAVE TO
DO WITH NORMAL MALLEABILITY OF
THE DNA, HOW THE SEQUENCE -- IS
THIS W 6 O 1?
>> IT IS.
MAYBE IT'S FLOPPIER AT EXIT
SITE, YEAH, THAT COULD BE THE
CASE.
>> THANK YOU.
>> DO YOU HAVE ANY ESTIMATE HOW
MANY LEDGFs ARE IN YOUR
ISOLATED PICKS?
>> OH, WE DON'T HAVE ANY
ESTIMATE.
BUT THEY ARE NUCLEAR EXTRACTED
PITS, SO THERE'S A LOT OF IT.
AND WHEN WE GET IT FROM THESE
KNOCKOUT PICKS, LEDGF KNOCKOUT
PICKS, WE START AT ONE NANOMOLAR
TO KICK THAT PREFERENCE, YOU
STILL GET REALLY HIGH TARGETING
TO TRIOMY REGIONS SO I DON'T
THINK YOU NEED A LOT BASICALLY
TO TARGET THIS.
>> OKAY.
NO MORE QUESTIONS, APPLAUSE
AGAIN FOR JOSHUA.
[APPLAUSE]
SO SLIGHT CHANGE IN SCHEDULE,
ONE MORE SPEAKER AND THEN TAKE
THE BREAK EARLY AND GO BACK TO
WILLIAM TO MAKE SURE HIS SLIDES
ARE WORKING.
NEXT SLEEKER IS AVIK BISWAS,
SALK INSTITUTE FOR BIOLOGICAL
STUDIES.
TWO MORE SPEAKERS AND THEN
BREAK.
ONE, TWO, THREE.
>> YES, THANK YOU FOR THE
INVITATION TO SPEAK.
SORRY ABOUT THE SLIDE ISSUE.
I'LL TALK ABOUT OUR STRUCTURAL
ANALYSIS OF THE HW 1 IMMATURE
CAPSID LATTICE, I'M A GRADUATE
STUDENT, CO-MENTORED, UNIVERSITY
OF MINNESOTA.
SO HUMAN T CELL TYPE 1 INFECTS
10 TO 15 MILLION PEOPLE
WORLDWIDE.
AND IS THE CAUSATIVE AGENT OF
ADULT T CELL LEUKEMIA LYMPHOMA
AND MYELOPATHY AND A
NEUROLOGICAL DISORDER.
DIFFERENT RETROVIRUSES HAVE
DISTINCT MORPHOLOGIES, IT OFTEN
HAS FLAT REGIONS OF THE LATTICE
AND REGIONS THAT DON'T ALWAYS
FOLLOW THE CURVE CURVATURE.
THESE ARE 2D CRYOIMAGES, HOW
DOES THIS LOOK IN THREE
DIMENSIONS?
COLLECTED TOMOGRAPHY DATASET,
YOU CAN SEE GAPS IN THE LATTICE
AS WELL AND FLAT REGIONS.
WE USED SUBTOMOGRAM AVERAGING,
GAVE IT TO A SCIENTIST AT THE
UNIVERSITY WHO WROTE A PROGRAM
WHERE HE CAN CALCULATE WITH
LOCAL CURVATURE AT PER-HEXAMER
LEVEL.
THOSE IN BLUE TEND TO HAVE MORE
FLAT LATTICE, LARGER RADIUS, UP
TO 75 NANOMETERS.
HEXAMERS IN RED HAVE HIGHER
CURVATURE OR MORE CURVED AND
SMALLER RADIUS DOWN TO 28
NANOMETERS.
EVEN WITH THE THE SAME PARTICLE
YOU HAVE DRASTIC DIFFERENCES,
NOT JUST FOLLOWING SIMPLE
SPHERE.
ANOTHER THING WE'VE NOTICED IS
THAT EVEN WITHIN THE SAME
PARTICLE WE'RE ON THE RIGHT
COLORING THEM BASED OFF DISTANCE
FROM CAPSID TO MEMBRANE, YOU CAN
HAVE DIFFERENT DISTANCES EVEN
WITHIN THE SAME HEXAMER.
WITHIN THE SAME PARTICLE.
SO OVERALL THIS IS SHOWING
HTLV-1 PARTICLES ARE
HETEROGENEOUS, RECENT
bioRxiv PUBLICATION DID A
NICE JOB SHOWING ARC SEC TIRE OF
IMMATURE LATTICE AND GOT HIGH
RESOLUTION USING HELICAL TUBES.
THE CTD IN RECONSTRUCTION WASN'T
AS WELL RESOLVED DUE TO
FLEXIBILITY.
WE WANTED TO USE SINGLE PARTICLE
ANALYSIS ON IMMATURE PARTICLES
THAT WERE PRODUCED BY
TRANSFECTING GAG INTO CELLS AND
PURIFYING THEM.
SO ON THE LEFT IS JUST SCHEMATIC
OF WHAT THE OVERALL
PARTICLE-PICKING SCHEME WOULD
BE, ESSENTIALLY PICKING ALL
PARTS OF THE LATTICE UNDER THE
VIRAL MEMBRANE.
WE CAN GET SIDE VIEWS AND MOST
IMPORTANTLY RECOVER TOP VIEWS OF
THE LATTICE AS WELL.
AND SO WITH THAT WE'RE ABLE TO
ACHIEVE A 3.4-ANGSTROM
RECONSTRUCTION, TOP VIEW,
CENTRAL HEXAMER, SURROUNDING
HEXAMER.
ON ITS SLIDE IN BLUE IS
N-TERMINAL DOMAIN, GOLD IS
C-TERMINAL DOMAIN.
NO SPACER PEPTIDE REGION,
STRAIGHT TO NUCLEOCAPSID.
THERE'S DENSITY BUT THAT'S
MOSTLY ATTRIBUTED TO NUCLEOCAP
C
APSID.
TOP RIGHT IS ESTIMATE, IN THE
N-TERMINAL DOMAIN ABLE TO
ACHIEVE HIGHER RESOLUTION THAN
C-TERMINAL DOMAIN, APPEARS TO BE
MORE FLEXIBLE.
THIS MOVIE PLAYING IN THE BOTTOM
RIGHT IS A GREAT DEPICTION OF
WHY, PICKING UP ON
HETEROGENEITY.
THE MAIN HETEROGENEITY IT'S
PICKING UP IS DIFFERENT
CURVATURE STATES OF THE LATTICE.
THE TOP OF THE NTD IS ACTING AS
HINGE POINT ALLOWING FOR THE CTD
TO MOVE OUT AND GET VARYING
CURVATURE.
LIKELY WHY WE'RE SEEING WORSENED
RESOLUTION.
WITHOUT ABSENCE OF SIX HELIX
BUNDLE IT'S MORE FLEXIBLE TO
MOVE.
HERE IS A MODEL OF WHAT OUR
LATTICS LOOK LIKE.
COLOR RECONSTRUCTION BASED ON
HEXAMER, SIDE VIEW, BACK TO THE
TOP, FIT INTO A MODEL.
AND CROSS-SECTION THROUGH THE
MODEL TO LOOK AT TRIMERIC NTD
INTERFACE, CTD WITH DIAPER
INTERFACE, ROTATE ON ITS SIDE
AND DO A CROSS-SECTION, AGAIN TO
SHOW NTD AND CTD.
HERE SHOWING THE MODEL IN THE
CENTRAL HEXAMER, BLUE IS NTD,
GOLD CTD.
I WON'T GO INTO DETAIL BECAUSE
THAT'S THE MAIN FOCUS OF DR.
FULLER'S PUBLICATION.
WE'RE IN GOOD AGREEMENT WITH THE
TUBE ASSEMBLY WITH VIRAL-LIKE
PARTICLES WHERE WE'RE SEEING
TRIMERIC NTD INTERFACE
STABILIZED BY HELIX 4
INTERACTIONS.
LOOKING AT CTD IT FORMS A DIMER
INTERFACE.
WE DON'T SEE INTERACTION BETWEEN
CTD MONOMERS WITHIN THE SAME
HEXAMER.
IT'S ONLY FORMING INTERACTIONS
WITH MONOMER ON THE ADJACENT
HEXAMER THROUGH THE DIMER
INTERFACE, MOSTLY COMPOSED OF
HELIX 8 AND HELIX 9, HELIX 9
INTERACTIONS.
IT HAS HYDROPHOBIC POCKET, AND
THEN ALSO TRYPTOPHAN 133, A
MUTATION OF RESIDUES TO ALANINE
DECREASES IMMATURE PARTICLE
PRODUCTION.
SIMILARLY AT THE BOTTOM OF HELIX
8 AND 9 HYDROGEN BONDING,
DIMINISHED PARTICLE PRODUCTION.
THE INTERFACE IS ACTUALLY QUITE
CRITICAL FOR PARTICLE ASSEMBLY,
MOSTLY UNDERAPPRECIATED, MOSTLY
NTD THAT IS IMPORTANT FOR
PARTICLE ASSEMBLY.
ANOTHER THING WE NOTICED WAS IN
THE N-TERMINAL DOMAIN WE HAVE
THIS SMALL EXTRA DENSITY,
CENTRAL HEXAMER, ADJACENT
HEXAMERS AS WELL.
I'LL COLOR IT SO YOU CAN SEE IT
BETTER.
IF YOU FOCUS ON WHAT THAT
DENSITY IS WE HAVE A RING OF
LYSINE 18, ANALOGOUS TO WHERE
YOU EXPECT TO SEE IP6 IN THE
MATURE STRUCTURE.
WE USE THESE KNOCKOUT CELL
LINES, IPPK IS KINASE THAT
CONVERTS TO IPK, AND WE ALSO
TRANSFECT IN CP 1, A PHOSPHATASE
THAT CONVERTS IP6 BACK TO IP 5.
WHEN WE TRANSFECT, IT DOES NOT
AFFECT IMMATURE PARTICLE
PRODUCTION, WE'RE USING THE
CONTROL, AS YOU'D EXPECT WE GET
DEPRESSED PARTICLE PRODUCTION.
OVERALL IT SEEMS LIKE IP6 IS NOT
REQUIRED FOR IMMATURE PARTICLE
PRODUCTION.
LAST THING WAS THE LATTICE WAS
INTACT.
SO WE TOOK CRYOIMAGES OF
PARTICLES FROM THE KNOCKOUT
CELLS, OVEREXPRESSING
PHOSPHATASE, IT'S NOT AFFECTING
PARTICLE MORPHOLOGY EITHER.
SO, OVERALL CONCLUSIONS THAT
IMMATURE HTLH-1 PARTICLES, THE
LATTICE IS STABILIZED BY
INTERFACE AND DISRUPTION CAN
DIMINISH PARTICLES PRODUCTION.
IP6 IS NOT REQUIRED FOR IMMATURE
PARTICLE ASSEMBLY BUT LIKELY
STILL REQUIRES DIFFERENT POLY
ANINE TO LYSINE RING.
I'D LIKE TO THANK ALL MEMBERS OF
THE LAB AND OUR COLLABORATORS,
ESPECIALLY MY CO-MENTOR.
I WISH I KNEW WHO WROTE THE
PROGRAM.
AND OUR LAB RATERS AND THOSE WHO
PROVIDED REAGENTS FOR US.
I'D BE HAPPY TO TAKE QUESTIONS.
THANK YOU.
[APPLAUSE]
>> WE'RE DOING EXCELLENT ON
TIME.
ANY QUESTIONS FOR WILLIAM?
>> GREAT TALK.
HAVE YOU LOOKED AND IF SO DO YOU
SEE ANY INDICATION OF REGULARITY
IN MATRIX LAYER IN YOUR
PARTICLES?
>> YEAH, I MEAN, I CAN SEE
MATRIX IN MICROGRAPHS AND I
TYPICALLY DON'T BOX LARGE ENOUGH
TO SEE MATRIX BUT HAVE FOCUSED
IN, YOU CAN SEE ORDERED LAYER OF
MATRIX IN THESE PARTICLES, MAYBE
NOT COMPLETE THROUGHOUT THE
ENTIRE PARTICLE AS YOU'D EXPECT
BASED OFF HETEROGENEITY IN THE
LATTICE BUT I THINK IT IS
DEFINITELY THERE.
>> COOL.
THANK YOU.
>> YEAH, THANK YOU.
>> ANY OTHER QUESTIONS OF
WILLIAM?
IF NOT THEN LET'S GIVE HIM
ANOTHER APPLAUSE.
[APPLAUSE]
AND NOW AVIK BISWAS FROM SALK
INSTITUTE FOR BIOLOGICAL
STUDIES.
GO AHEAD, AVIK.
>> ALL RIGHT.
GOOD AFTERNOON, EVERYONE.
I'M AVIK, POSTDOC AT THE SALK
INSTITUTE FOR BIOLOGICAL STUDIES
IN TODAY I'M GOING TO BE TALKING
ABOUT SOME OF OUR RECENT EFFORTS
LOOKING AT PATHWAYS AND
MECHANISMS OF HOW DRUG
RESISTANCE EVOLVES IN HIV, IN
COLLABORATION WITH RON LEVI'S
GROUP AT TEMPLE, MY Ph.D.
ADVISOR.
THIS WORK BEGAN IN RON'S GROUP.
THE KEY QUESTION WE ASKED HERE
IS WHY DO SOME DRUG RESISTANCE
MUTATIONS ARISE FASTER THAN
OTHERS?
SO IF YOU TAKE THE EXAMPLE FROM
HIV INTEGRASE WE SEE THE
MUTATION AND Q1 48 MUTATION ARE
PRESENT IN DRUG EXPERIENCE
PATIENT SAMPLES IN STANFORD
DATABASE FOR EXAMPLE WITH
ROUGHLY EQUAL FREQUENCES YET N
155H IS REPORTED TO BE FASTER
THAN THE Q148H MUTATION, WE WANT
TO KNOW HOW AND WHY AND MORE
BROADLY WHAT ARE THE KINETIC
PATHWAYS THAT LEAD TO DRUG
RESISTANCE STARTING FROM DRUG
NAIVE PATIENT POPULATION, IF WE
CAN FINDS A BASIS FOR THIS SLOW
OR FAST ACQUISITION OF
RESISTANCE.
SO WITH THIS IN MIND WHAT WE
WANTED TO DO IS START FROM DRUG
NAIVE PATIENT SAMPLES, IN THE
STANFORD DATABASE, EVOLVE THEM
SUCH THAT THEY THEN BECOME DRUG
EXPERIENCE PATIENT SAMPLES IN
STANFORD DATABASE.
SO WE CAME UP WITH A KINETIC
KIEM TO DO THIS, FIRST BUILD A
MODEL OF THE PROTEIN FITNESS
LANDSCAPE BASED ON DRUG
EXPERIENCE IN STANFORD COUPLED
TO THE PROCESS WHERE WE START
FROM THE DRUG NAIVE PATIENT
SAMPLES, SELECT SEQUENCE AND TRY
MUTATE RANDOM, WE CAN ITERATE
AND THAT MAKES DRUG NAIVE
SEQUENCE BECOME THOSE IN THE
STANFORD DATABASE.
WE CAN TRACK HOW THESE MUTATIONS
ARE EVOLVING STARTING WITH DRUG
NAIVE STATE.
WHAT DO WE GET?
FROM THIS PROCESS I'M SHOWING
TWO EXAMPLES, ON THE TOP RIGHT
IS A FAST RISING DRUG RESISTANCE
MUTATION, BOTTOM RIGHT IS SLOW
ARISING DRUG RESISTANCE
MUTATION.
IN EITHER CASE WE SEE THE
MUTATION STARTS FROM CLOSE TO
ZERO FREQUENCY IN THE DRUG-NAIVE
PATIENT SAMPLES, GRADUALLY
ARISES TO EQUAL DRUG EXPERIENCE
FREQUENCY IN THE DRUG
EXPERIENCED PATIENT.
HOWEVER, IT'S ALSO TRUE IN OUR
SIMULATION THE SLOWER MUTATION
TAKES MUCH LONGER THAT EQUAL ITS
DRUG EXPERIENCE FREQUENCIES.
CAN WE USE THIS PROCESS TO
PREDICT THE TIMELINE FOR DRUG
RESISTANCE MUTATIONS?
TAKING THE EXAMPLE FROM HIV
INTEGRASE, HERE I'M SHOWING YOU
THE TIME CONSTANT WHICH COMES
FROM OUR SIMULATIONS, AND THAT
GIVES THE HEIGHT OF THESE BAR
GRAPHS HERE AND THE COLOR OF THE
BAR GRAPHS ARE ACTUALLY THE TIME
THAT WE FIND IN THE CLINICAL
LITERATURE FOR THESE MUTATIONS
TO ARISE IN PATIENTS.
AND FROM THE GRADIENT WE CAN SEE
THAT THERE'S A VERY GOOD
CORRELATION WITH THE TIME
CONSTANTS THAT PREDICTED FROM
OUR SIMULATIONS WITH WHAT'S SEEN
IN THE CLINICAL LITERATURE.
THE CORRELATION COEFFICIENT IS
.9.
THEN WE ASKED THE QUESTION THAT
WHAT ABOUT OTHER FACTORS THAT
CAN PLAY A ROLE?
IS THIS RELATED, FOR EXAMPLE, TO
THE PREVALENCE OF THE MUTATIONS
OR ITS DRUG EXPERIENCE
FREQUENCIES WHICH WE CAN THINK
OF AS A PROXY FOR THE FITNESS OF
THE MUTATION, OR HOW DOES THE
GENETIC CODE SUCH AS TRANSITION
VERSUS TRANSVERSION PLAY, AND
USUALLY AALONE DO NOT CORRELATE
WELL OR WITH PREDICTED TIME
CONSTANTS FROM SIMULATIONS.
SO WHAT IS IT THAT DETERMINES
KINETICS, WHY IS IT SOME
MUTATIONS ARE SLOW, SOME ARE
FAST?
WE FIND THAT THERE ARE GENERALLY
TWO BROAD PROCESSES WHICH CAN
EXPLAIN WHY SOME MUTATIONS ARE
SLOW, SOME MUTATIONS ARE FAST.
SO IN CASE OF THE FAST MUTATION,
OR FAST ARISING MUTATIONS, WHAT
WE SEE IS THERE'S A PREEXISTING
BIAS THAT'S ALREADY PRESENT IN
THE DRUG NAIVE PATIENT
SEQUENCES, TO GET THIS MUTATION.
THAT BECOMES APPARENT WHEN DRUG
PRESSURE IS TURNED ON IN OUR
SIMULATIONS SO IN THIS CASE, FOR
EXAMPLE, AGAIN THIS IS HIV
INTEGRASE, THE SLOW ONE IS Q1
48H, WE SEE BOTH OF THEM START
OFF AT ZERO FREQUENCIES, THAT'S
THE BLACK AND YELLOW LINES OVER
HERE.
HOWEVER, THE FAST MUTATION WHICH
IS IN THE -- HAS MUCH HIGHER
PROPENSITY OR BIAS IN DRUG NAIVE
SAMPLE CLOSE TO FINAL FREQUENCY,
THE GREEN DASHED LINE HERE FOR
N155H MUTATION.
ALSO CONTRIBUTION FROM ACCESSORY
MUTATION, SPECIFIC ACCESSORY
MUTATION THAT IT'S COUPLED TOO.
IT BECOMES CONTINGENT ON THE
OTHER MUTATION BEING PRESENT
BEFORE IT OR AT THE TIME WHEN
IT'S OCCURRING IN THE SEQUENCE
THAT IT'S OCCURRING IN.
SO THESE TWO PROCESSES GENERALLY
EXPLAIN THE KINETICS OF OUR SLOW
VERSUS FAST MUTATIONS.
HOWEVER I'D LIKE TO POINT OUT
IT'S NOT THAT THESE TWO ALONE
CORRELATE WELL BUT IT'S ACTUALLY
THE RELATIVE STRENGTH OF THESE
THAT CORRELATE WITH THE
TIMELINES WE SEE.
SO WITH THAT WE THEN WANTED TO
ASK THE QUESTION WHAT IF WE TAKE
ONE OF THE SLOWEST ARISING
MUTATIONS, ALONG WITH ASSOCIATED
PATTERN OF MUTATIONS, AND CAN WE
PREDICT THE SEQUENCE THAT LET TO
THE PATTERN OF MUTATIONS?
I'M SHOWING TWO EXAMPLES,
STARTING FROM NL SEQUENCE, USING
FITNESS MODEL TO GIVE US A
LIKELIHOOD OF EACH OF THE
MUTATIONS STARTING FROM THIS
PARTICULAR SEQUENCE.
AND THEN ONCE THAT MUTATION HAS
EVOLVED IN THE SEQUENCE WE CAN
ASK WHAT'S THE NEXT MOST LIKELY
MUTATIONAL EVENT BASED ON THIS
EVOLVED SEQUENCE BACKGROUND, AND
THIS WAY WE CAN DERIVE
PROBABILISTIC PATHWAY THAT LEADS
TO THE PATTERN OF MUTATIONS.
E138K, G140S AND Q148H, AS WELL
AS THESE, IN INTEGRASE, IN THESE
TWO CASES THE RESIDUE SITES ARE
THE SAME EXCEPT THAT THE
MUTATIONS HAPPENING AT 140 ARE
DIFFERENT AND THAT SWITCHES
WHICH WOULD BE THE MOST LIKELY
PATHWAY LEADING TO THIS TRIPLE
MUTANT.
SO THEN WE ASKED THE QUESTION
CAN WE THEN RATIONALIZE THESE
PREDICTED TEMPORAL ORDERS OR
PREFERRED PATHWAYS WITH
STRUCTURAL BIOLOGY USING HIGH
RESOLUTION CryoEM STRUCTURES
ALONG THE SPECIFIC PATHWAY,
STARTING FROM WILD TYPE HERE,
NL43 AND I'VE HIGHLIGHTED KEY
RESIDUES WITH THE HISTIDINE,
THAT BECOMES IMPORTANT LATER ON
IN THE STORY.
SO WHEN THE E138K HAPPENS FIRST,
THIS THEN LEADS TO A SLIGHT
SHIFT IN THE DNA OVER HERE.
THAT THEN ALLOWS ROOM HERE AT
148 TO MUTATE TO A LYSINE.
LAST THIS 140 MUTATES TO ALANINE
WHICH CAUSES SHIFT IN
CONFIRMATION OF 148 LYSINE
TOWARDS THE DRUG, LEADS TO DRUG
EXCISION, THIS AGREES WITH THE
PATHWAY WE PREDICTED USING THE
FITNESS MODEL.
HOW IF Q 148K WOULD HAVE BEEN
THE FIRST?
WE SEE IF Q 148K WAS FIRST THIS
COULD HAVE CAUSED SHIFT IN THE
BACKBONE OF THE RESIDUES 139 TO
141 WHICH WOULD HAVE BEEN
UNFAVORABLE.
SIMILARLY THIS HISTIDINE 114 IN
THE SAME CONFIRMATION BEFORE IS
NOW IN A VERY UNFAVORABLE
CONFIRMATION.
SIMILARLY G 140 LEADS TO
UNFAVORABLE CONFIRMATION AT 148,
RATIONALIZING THE PATHWAY USING
THE FITNESS MODEL.
THIS IS STILL A WORK IN PROGRESS
AND WE'RE NOW LOOKING AT OTHER
POSSIBLE PATHWAYS, INCLUDING
THOSE TO OTHER PATTERNS OF
RESISTANCE MUTATIONS INCLUDING
MORE COMPLEX PATTERNS THAT
INVOLVE FIVE OR MORE MUTATIONS.
SO WITH THAT I'D SUMMARIZE BY
SAYING HERE WE'VE USED A MODEL
OF HIV FITNESS LANDSCAPE COUPLED
TO KINETIC MONTE CARLO
SIMULATION WHICH WE SEE CAN GIVE
US TEMPORAL BEHAVIOR OF THESE
RESISTANCE MUTATIONS IN HIV.
AND TO EXPLAIN THE KINETICS OF
THESE FAST VERSUS SLOW MUTATIONS
THERE ARE GENERALLY TWO BROAD
PROCESSES THAT CONTRIBUTE.
ONCE A PREEXISTING BIAS ALREADY
IN THE DRUG NAIVE PATIENT
POPULATION THAT BECOMES APPARENT
WHEN DRUG PRESSURE IS TURNED ON,
WHICH IS ESPECIALLY HIGH FOR THE
FAST ARISING MUTATIONS, AND THE
OTHER IS THE CONTRIBUTION FROM
ACCESSORY MUTATIONS, IN
PARTICULAR VERY STRONGLY
AFFECTING SLOWLY ARISING
RESISTANCE MUTATIONS COUPLED TO
SPECIFIC OTHER MUTATIONS AND
BECOME CONTINGENT ON MUTATIONS
BEING PRESENT WITH THEM.
WE CAN ALSO USE THIS FITNESS
MODEL TO PREDICT SEQUENTIAL
ORDER OF EVENTS THAT LEAD TO
SPECIFIC PATTERNS OF RESISTANCE
MUTATIONS AND THAT THESE
PATTERNS CAN THEN BE
RATIONALIZED USING STRUCTURAL
BIOLOGY.
WITH THAT I'D LIKE TO THANK
EVERYONE INVOLVEDIN THIS WORK,
ESPECIALLY DMITRY AND RON, ALSO
COLLABORATIONS AND FUNDING.
THANK YOU FOR YOUR TIME.
[APPLAUSE]
>> NICE WORK.
YOU MUST TAKE INTO ACCOUNT, I
TRY TO GO THROUGH QUICKLY, YOU
PRESENT EVERYTHING ON THE
PROTEIN SIDE BUT CHANGES TAKE
PLACE AT THE NUCLEIC ACID.
I THINK EVERYTHING YOU THREW AT
US TODAY IS A SINGLE NUCLEOTIDE
CHANGE, YOU MUST THINK ABOUT
THAT.
>> MOST OF THESE RESISTANCE
MUTATIONS, VAST MAJORITY OF
THEM, SINGLE NUCLEOTIDE CHANGES.
THERE ARE A COUPLE DOUBLE, THAT
DOESN'T CORRELATE WELL WITH THE
TIMELINES.
>> WHY DO YOU THINK THAT?
YOU WOULD THINK IF YOU NEED
TWICE AS MANY CHANGES IN THE
TEMPLATE THAT WOULD AFFECT
KINETICS.
>> RIGHT.
I THINK IN TERMS OF THE
CORRELATION THERE'S SO FEW OF
THE DOUBLE NUCLEOTIDE CHANGES IN
TERMS OF THE PRIMARY RESISTANCE
MUTATION, SO WE CAN FIND
EXAMPLES OF A DOUBLE NUCLEOTIDE
CHAIN THAT'S SLOWERTHAN SINGLE
NUCLEOTIDE CHANGE.
>> ANY OTHER QUESTIONS FOR AVIK?
I HAVE A QUESTION.
HOW DO YOU TAKE INTO ACCOUNT
BIOFITNESS PRIMARY ISOLATE, THEY
REPLICATE FASTER THAN OTHER
VIRUSES, CONSTANTLY CHANGES,
PRIMARY ISOLATE, HARD TO PREDICT
IN THE ENVIRONMENT.
>> WHAT WE'RE DOING HERE --
>> MICROPHONE PLEASE.
>> SORRY.
WE'RE ACTUALLY TAKING PATIENT
SAMPLES FROM DATABASES, BECAUSE
THERE'S A DIVERSITY OF PATIENTS,
WE'RE ESSENTIALLY ASSUMING WE'RE
ARRANGING OVER -- SORRY,
IMMUNOLOGIC ASPECTS OF HOW OF
VIRUS IS RESPONDING TO DIFFERENT
PATIENTS.
>> OKAY.
THANK YOU.
>> HI.
A FOLLOW-UP, MOST ARE SINGLE
NUCLEOTIDE CHANGE BUT DIFFERENT
MUTATIONS HAVE DIFFERENT RATES,
MUCH MORE RARE THAN TRANSITIONS
SO HAVE YOU COMPARED RATES OF
DIFFERENT CHANGES THAT MIGHT BE
REQUIRED?
>> YEAH, LOOKED AT TRANSITION
VERSUS TRANSVERSION, DOES THAT
EXPLAIN THE TIMELINES WE
OBSERVED BUT THAT DOESN'T.
>> OKAY.
THANK YOU.
>> OKAY.
IF THERE ARE NO MORE QUESTIONS
FOR AVIK LET'S GIVE HIM ANOTHER
APPLAUSE.
[APPLAUSE]
AND WE HAVE A VERY NICE LAPTOP.
IS IT YOURS?
I WAS JUST ABOUT TO AUCTION IT
OFF.
[LAUGHTER]
IN ANY CASE, THANKS TO ALL THE
PREVIOUS SPEAKERS.
IT WAS A GREAT SESSION.
AND WE'LL TAKE A BREAK NOW UNTIL
4:00 AND IF THE NEXT FOUR
SPEAKERS COULD JUST QUICKLY
CHECK IN WITH ME.
SEE YOU AT 4:00.
>> THIS IS THE SECOND PART OF
THE ABSTRACT SESSION TODAY.
FIRST SPEAKER IS HUNG DO FROM
LOS ALAMOS NATIONAL LABORATORY.
>> I'M AT LOS ALAMOS LABORATORY,
TALKING ABOUT DEVELOPMENT OF THE
WORKFLOW TO ACCELERATE
CONFIRMATION OF SAMPLING OF
BIOMOLECULAR MECHANICS, I'D LIKE
TO RECOGNIZE THE ORIGINAL PERSON
WHO DEVELOPED THE IDEA FOR THE
WORKFLOW, BEFORE I CAME.
FIRST HIV RELEVANT PROCESS
INVOLVED CONFIRMATIONAL CHANGE
OCCUR OVER MICROSECOND, HIV
VIRAL MEMBRANE HIGHLY COMPLEX,
CONSIST OF SEVERAL LIPID TYPE
WHICH WILL BE DISCUSSED IN
POSTER, 78 AND 89, COLORFUL
MEMBRANE.
WE SHOW THE DIFFERENT STATE
IMMUNOGEN IMLID COMPOSITION AND
PROSECUTES ITTY DISCUSSED IN
ABSTRACTS.
CAME UP WITH THE IDEA TO COMBINE
CO-STIMULATION, WHICH ARE FAST,
MANY CONFIRMATIONAL TRANSITION
DUE TO FLAT UNDER SURFACE,
THERE'S NO BARRIER RIGHT HERE SO
CAN SAMPLE ACROSS LANDSCAPE, LOW
BUT PROVIDES DETAIL DUE TO
COMPLEX SURFACE, MANY LOW
ENERGY, MANY HIGH ENERGY
BARRIER, TRANSITION FROM ONE
STATE TO ANOTHER IS VERY SLOW.
THAT'S WHY THE ITERATED
MULTI-SCALE WORKFLOW IS
POWERFUL, IN PARTICULAR FOR
MODELING MEMBRANE PROTEIN CAN
USE CLOSE RANGE, TO GET INTO THE
CORRECT POSITION, AROUND
MEMBRANE PROTEIN.
ONE BIG CHALLENGE REMAINED,
MULTI-SCALE CONSISTENCY, NO
RELATIONSHIP BETWEEN THE
FUNCTIONS HOW HOW CAN WE COMBINE
THE TWO ACCURATELY, ONE QUESTION
THAT REMAINS TO THINK ABOUT.
I WILL TALK ABOUT OUR CURRENT
IMPLEMENTATION OF THE
MULTI-SCALE WORKFLOW IN THE
PACKAGE, ALONG WITH SOME
INTEREST AND CHALLENGE FROM
APPLYING TO SIMILAR.
FOR THE SIMULATION, CYCLOPHILIN,
IF YOU'RE INTERESTED COME TO OUR
POSTER 77 AND WE CAN DISCUSS
MORE TOMORROW.
LET'S TALK ABOUT METHODOLOGY,
YOU DON'T NEED TO SET UP THE
SIMULATION SYSTEM BEFOREHAND.
WORKFLOW SET UP THE SIMULATION
SYSTEM FOR YOU AND RUN THE FIRST
ATOM STIMULATION, CONVERT USING
MARTINIZE, WE CAN RUN THE FINAL
CONFIRMATION OF THE
COARSE-GRAINED, CONVERT BACK
USING M TOOL.
THEN BEFORE AND ATOM CYCLE TO
CONTINUE MULTI-SCALE SIMULATION
WORKFLOW.
WE DEMONSTRATED THE MULTI-SCALE
APPROACH BY SHOWING THAT IT CAN
CAPTURE, TWO MICROSECOND, AND
ONE WITH 15 NANOSECOND COMPARED
TO THE REQUIRED 14 MICROSECOND
TIME WITH PURE CONVENTIONAL.
YOU CAN SEE WE STARTED FROM
FOLDED STATE, TWO ITER RATION
WITH OBTAIN, OBSERVED FROM THE
PLOT RIGHT HERE.
AT THE END OF THE FIRST
SIMULATION PROTEIN IS STUCK,
RIGHT HERE, WITH SOME HELICAL
SECONDARY STRUCTURE ALREADY
FORM, STUCK RIGHT HERE, NO WAY
TO TRANSITION, THAT'S WHY WE GO
BACK, BACK PROTEIN CONFIRMATION,
TO FOLDED STATE, VERY STABLE IN
THE SECOND ITERATION.
WE ALSO OBSERVED MOLECULAR
DIFFUSE THREE TO FIVE TIME
FASTER COMPARED FROM THE
STIMULATION, BASED ON THE
COEFFICIENT, FROM THE
SIMULATION, THE BAR FOR THE
DIFFUSION CO-F OF THE MOLECULAR
TWO TO THREE TIME HIGHER THAN
THE BAR FOR THE SIMULATION SO
SUBMIT THE ORIGINAL DEVELOPMENT
PURPOSE.
BUT ONE PROBLEM, ONE CHALLENGE
REMAIN.
WE OBSERVED SECONDARY STRUCTURE
OF SOLUBLE REGION OF PROTEIN,
WHEN WE PERFORMED SIMULATION,
RIGHT HERE HAD SIP LAYINGS OF
RECEPTOR, WHEN WE START HAVE
CLEAR SECONDARY STRUCTURE,
SECONDARY STRUCTURE OF THE
REGION BECOME COMPLETELY
DISTORTED.
WHEN WE PERFORM SIMULATION AND
RESTRAINED THE POSITION, THE
PROTEIN SOLUBLE DOMAIN STILL
BECAME DISTOWARDED.
RIGHT HERE 400 NANOSECOND, STILL
RETAIN STRUCTURE IN THE
SOLUTION.
WHAT COULD BE THE CHALLENGE
HERE?
THE THIS INVOLVED MINIMIZATION,
HE SOLVED THIS CHALLENGE.
BECAUSE THE BIG PROBLEM WE
ALREADY MENTIONED IN THE FIRST
SLIDE BASICALLY THE FUNCTION OF
THE CLOSE RANGE REPRESENTATION
HAVE NO RELATIONSHIP WHATSOEVER,
THE OPTIMAL FOR THE PROTEIN ATOM
ARE DIFFERENT FROM OPTIMAL FOR
THOSE AT CLOSE RANGE
STIMULATION, VICE VERSA, THE
CURRENT PROTOCOLS BACK MAP, THE
DISTORTION BECOME MINOR BUT IF
YOU RUN THE CYCLE OVER MULTIPLE
TIME THEN THE DISTORTION BECOME
ACCUMULATED AND EVENTUALLY IT
BECOME VERY OBVIOUS.
SO AS YOU CAN SEE FROM THIS
FIGURE RIGHT HERE FOR SIMULATION
OF THE FINAL CONFIRMATION OF THE
FIRST CLOSE RANGE SIMULATION
LOOKS HIGHLY SIMILAR BUT WHEN WE
BACK MAP, WE GO BACK FROM FINAL
CG1 TO AA2 OBSERVE DISPORTION IN
THE BACKBONE, CIRCLE IN RED
RIGHT HERE.
RIGHT NOW WE DON'T HAVE A BACK
MAPPING PROTOCOL, SO ONE
PROPOSED SOLUTION WE BACK MAP
FROM CLOSE RANGE STIMULATION TO
AND COMBINE WITH ORIGINAL
CONFIRMATION BEFORE CONVERSION
TO CLOSE RANGE AND PROCEED TO
ATOMISTIC ITERATION, RETAIN
SECONDARY STRUCTURE OF THE
PROTEIN.
SO WITH THAT, THE CURRENT
MULTI-SCALE IMPLEMENTATION FOR
THE MEMBRANE PROTEIN SIMULATION
WE ONLY USE THE ATOMISTIC TO
SAMPLE PROTEIN INTERACTION AND
USE CLOSE CHANGE TO FACILITATE
LIPID MIXING.
SO WITH THAT I WANT TO -- WE TO
DEVELOP ITERATED MULTI-SCALE
WORK FLOW COMBINING CLOSE RANGE
BUT SIMULATION MUCH FASTER, AND
WE HAD DEMONSTRATED ENHANCE THE
SAMPLING CAPABILITY OF WORKFLOW
ON FACILITATING LIPID DIFFUSION,
METHODOLOGY USED IN UNCOVERING
ANTIGEN-DEPENDENT ACTIVATION
MECHANISM, IN GNANA'S TALK
TOMORROW.
FIRST QUESTION IS MULTI-KALE
CONSISTENCY, HOW CAN WE COMBINE
THESE TWO VERY DIFFERENT
RESOLUTION, WITH NO
RELATIONSHIP, AND BECAUSE OF THE
FIRST CHALLENGE WE HAD SECOND
CHALLENGE BECAUSE OF THE CURRENT
BACK MAPPING PROTOCOL USED SOME
FIRM SO HOW CAN WE BACKMAP
ACCURATELY, MAYBE IN THE FUTURE
WE'LL NEED A BACKMAPPING
PROTOCOL THAT DON'T USE
MINIMIZATION, WITH THAT I WANT
TO THANK OUR FUNDING SOURCE, SO
DUKE CENTER FOR HIV STRUCTURAL
BIOLOGY, NIH, LOS ALAMOS
NATIONAL LABORATORY, DR. KRISHNU
ANDER WON -- AND EVERYONE IN OR
GROUP -- DR. CHRIS NEALE AND
EVERYONE IN OUR GROUP.
[APPLAUSE]
>> THANK YOU.
ANY QUESTIONS FOR HUNG?
DON'T BE SHY.
THEN WE'LL MOVE TO THE NEXT
SPEAKER.
THANK YOU VERY MUCH, HUNG.
LET'S GIVE HIM ANOTHER ROUND OF
APPLAUSE.
[APPLAUSE]
WE'LL MOVE TO THE NEXT SPEAKER,
SHI-JIE CHEN, UNIVERSITY OF
MISSOURI.
>> YOU'LL GET MORE MINUTES IF
YOU SPEAK IN THE MICROPHONE.
[LAUGHTER]
>> I'M GOING TO TALK ABOUT OUR
RECENT WORK IN COLLABORATION
WITH THE LAB ON THE KINETIC
PATHWAYS OF HIV TAR RNA.
I'M FROM UNIVERSITY OF MISSOURI.
THIS SLIDE SHOWS TWO DIMENSIONAL
STRUCTURE OF THE FIVE PRIME UTR,
I DON'T THINK I HAVE TO EXPLAIN
WHAT THIS IS.
I FEEL LIKE IF I TALK TOO MUCH
ABOUT THIS I'M LIKE PREACHING OF
GOD, TODAY I'M GOING TO FOCUS ON
PAR, THIS SHOWS NATIVE
STRUCTURE, RNA FOLDING IS
CO-TRANSCRIPTIONAL.
LET ME START WITH EXPERIMENTAL
RESULTS.
THIS SLIDE, THIS FIGURE SHOWS
THE CLASSICAL EXPERIMENTAL DATA
FROM ROB'S LAB, AND FROM THIS WE
CAN SEE ELONGase, THERE'S A
TRANSCRIPTION PATH HERE, THE
PATH OCCURS.
IN THE POLYMERASE THESE 18
NUCLEOSIDES THEY DON'T
PARTICIPATE IN FOLDING.
WITH THIS 44 NUCLEOTIDES WE HAVE
NATIVE STRUCTURE, THESE APPEARS
IN THE NATIVE FINAL FOUR TAR
STRUCTURE.
LANDIC'S EXPERIMENT, THREE
CONCLUSIONS.
FIRST, YOU HAVE THE PATH HERE,
WE SEE THAT.
SECOND, THE PATH IS INDUCED WE
THE OTHER STRUCTURE, P4
STRUCTURE.
IT'S OUR NOTATIONS, P4
STRUCTURE.
THIS STRUCTURE IS A MISFOLDED
STRUCTURE, MEANING BASE PAIRS
ARE COMPLETELY DIFFERENT FROM
NATIVE PATH.
IT'S MISFOLDED.
ONCE THIS IS FORMED, THEY OCCUR.
70% GETS PASSED, 30% THROUGH.
THAT'S 70 VERSUS 30 THIS RATIO.
ANOTHER NUMBER IS 22 SECONDS,
PAUSE FOR 22 SECONDS.
WE WANT TO UNDERSTAND WHY 70/30,
WHY 22 SECONDS, AND WHAT IS
EXACTLY HAPPENED WHEN THE TAR IS
PASSED FROM STRUCTURE POINT OF
VIEW.
CO-TRANSCRIPTION FOLDING IS
DIFFERENT FROM REFOLDING.
WE HOLDING I MEAN YOU GET A VERY
DIFFERENT FOLDING BECAUSE IT'S A
PROCESS, COMPETITION BETWEEN
THREE RATE PROCESSES, ONE IS
TRANSCRIPTION RATE, ELONGATION
RATE.
SECOND PROCESS IS FOLDING, THIRD
PROCESS IS UNFOLDING, IS IT
COMPETITION, SO LET ME QUICKLY
GO THROUGH THIS SLIDE,
SUPPOSEDLY YOU HAVE TWO HAIRPIN
STRUCTURE, THE HELICES, LOOMS.
THE RNA WILL QUICKLY FORM THIS
LOCAL HAIRPIN STRUCTURE, IT'S
FAST, DOWN HERE.
THIS IS FORM BY BASE PAIRING OF
GREEN STRANDS.
IN ORDER TO FORM THIS STRUCTURE
YOU HAVE TO BREAK DISRUPT,
THAT'S SLOW.
ELONGATION KEEPS GOING.
VERY LIKELY YOU FORM THIS
STRUCTURE INSTEAD OF
THERMODYNAMIC STABLE STRUCTURE,
DIFFERENCE BETWEEN
CO-TRANSCRIPTION OF FOLDING AND
REFOLDING.
IN OUR MODEL, I'LL QUICKLY GO
THROUGH THE KEY POINT OF THE
MODEL.
I WON'T GO THROUGH DETAILS.
IN OUR MODEL WE WANT TO USE
COMPUTATIONAL MODEL TO
UNDERSTAND WHAT'S GOING ON FROM
STRUCTURE POINT OF VIEW, WANT TO
UNDERSTAND THE PATHWAYS.
SO IN OUR MODEL WE MODEL THE
TRANSCRIPTION LIKE MOVIE FRAME,
EACH FRAME RNA MOLECULE
STRUCTURE EVOLVE, KINETIC FROM
INITIAL TO FINAL STRUCTURE,
ELONGATE BY ONE UNIT, TIME
DURATION DETERMINED BY THE
TRANSCRIPTION SPEED.
OUR MODEL CAN MODEL THE
VARIATION OF THE TRANSCRIPTION
SPEED, AND ANOTHER IMPORTANT
POINT CO-TRANSCRIPTION FOLDING
IS DIFFERENT FROM REFOLDING,
SOMETIMES PEOPLE LIKE TO DO
FOLDING AT DIFFERENT THAN MAKE
CONNECTION TOGETHER, YOU THINK
THIS IS PATHWAY BUT THAT'S NOT
ALWAYS THE CASE.
WE HAVE TO BE CAREFUL.
ANOTHER COMPONENT IS FOLDING
MODEL, STRUCTURE PREDICTION
MODEL.
SO IN OUR MOLDING MODEL WE
DEVELOPED A SIMULATION, AND IN
OUR MODEL WE USE COARSE-GRAINED,
TWO DIMENSIONAL COMPARING THE
MODEL SIMULATE MOLECULE, THIS
ONE IS 412 NUCLEOTIDES, CAN GET
THE FINAL STRUCTURE, IT'S NOT
PERFECT, IT'S 17-ANGSTROM BUT I
JUST WANT TO SHOW THE
SIMULATION, WE CAN GET DOWN TO
THE 10-ANGSTROM SO IT WORK.
HERE IS MY LAB, FIRST ONE IS
FROM COMMERCIAL COMPANY,
INTERNATIONAL COMPANY.
SO LET'S COME BACK TO OUR REAL
TOPIC TODAY, THAT'S THE TAR
FOLDING.
SO WE FOUND WITH OUR MODEL WE
FOUND THAT WE HAVE THE TAR FOLDS
AROUND TWO PARALLEL PATHWAYS BUT
THIS SLIDE SHOWS IT'S TOO BUSY,
SO TO HELP THE VISUAL INSPECTION
I DRAW TWO LINES, RED AND BLUE,
TWO PATHWAYS, FIRST PATHWAY IS
BLUE ONE.
BASE PAIRS FOR THE NATIVE BASE
PAIRS, NATIVE STRUCTURES, WE CAN
ALSO GET THREE DIMENSIONAL
STRUCTURES AND THIS MOVIE SHOWS
HOW THE STRUCTURE EVOLVED, AS
TRANSCRIPTION PROCEEDS.
AND LET ME SKIP THIS SLIDE.
I DON'T HAVE TIME TO FINISH ALL
THESE MOVIES.
AND THE OTHER PATHWAY, THE OTHER
PATHWAY WE CALL THE P PATH, P
MEANS PATH.
THAT'S THE CULPRIT TO CAUSE THE
PATH.
THIS STRUCTURE ALONG THIS
PATHWAY CAN ALSO GET A MOVIE FOR
THE THREE DIMENSIONAL STRUCTURES
AND THIS STRUCTURE P4 CAUSES
PATH, NEW PATH, WHAT'S GOING TO
HAPPEN?
SO WE ALSO MODEL WHEN THERE'S NO
PATH, P4 WILL KEEP GOING AS IT
ELONGATES, WILL KEEP GOING, IT
WILL FORM P5, P 6, P7, FINALLY
GO TO TAR.
THE PROCESS IS SUPER SLOW, VERY
SLOW.
WHEN THIS TAR IS FORMED
ELONGATION TRANSCRIPTION GOES TO
PBS OR FURTHER DOWNSTREAM.
WHEN THIS NATIVE TAR FORMED
PROTEIN BONDS THE TAR, TO
ACCELERATE TRANSCRIPTION THAT
MIGHT OCCUR, IF THAT OCCUR, THAT
WILL BE TOO LATE.
TIME IS TOO LATE.
SO THAT WILL LEAD TO THE
DETRIMENTAL CONSEQUENCES BECAUSE
FROM HERE TO DOWNSTREAM TO PBS
SLOW TRANSCRIPTION, YOU GET ALL
SCREWED UPU GET MULTIPLE
SPLICING, FOR EXAMPLE.
AND THE PATH TRIGGERS REROUTING
BECAUSE IT GET STUCK HERE, P4,
NO ELONGATION, IT GOES TO M4, A
NATIVE TAR.
ONCE IT FOLDS TO N4, TAR PROTEIN
BIND, IT GET ACCELERATED REAL
SLOW.
AND SO THE FIRST QUESTION WE
WANT TO ANSWER IS WHY 70-30?
THIS FIGURE SHOWS OUR MODEL
PREDICTED POPULATIONS OF EACH
PATHWAY, YOU CAN SEE AT 44 THE
PATHOCURSE, THE POPULATION
BETWEEN P AND N IS AROUND 80 AND
20.
IT'S VERY CLOSE TO 70 AND 30.
SO HAPPY ABOUT THE RESULT.
AND ANOTHER THING IS THAT WE
WANT TO UNDERSTAND WHY 22
SECONDS?
AS I EXPLAINED WHEN P 4 GOES TO
N 4 THE PATH GET RELEASED SO WE
CALL PAUSE-ESCAPE.
WHY 22 SECONDS?
TO ANSWER THAT QUESTION WE NEED
TO SEE HOW P4 GOES TO N4?
FIRST UNFOLD THIS, UNWIND THE
HELIX HERE.
AND THEN THIS SEQUENCE, THIS
CHAIN, THIS TAIL FOLD BACK TO
HERE TO THE HAIRPIN LOOP, FORM
PSEUDOKNOT.
THEY FORM BASE PAIR TO FORM
ANOTHER, AND UCU, PERFECT.
THEN KEEP GOING.
THIS PSEUDOKNOT PATHWAY IS
EXCITING.
AND DR. SHAW HAINES LAB DID AN
EXPERIMENT TO CONFIRM THESE TWO
BASE PAIRS, THESE TWO SETS OF
BASE PAIRS WHICH INITIATED
PSEUDOKNOT FORMATION ARE LESS
STABLE SHOWN FROM NMR, YOU'RE
EXPERTS SO I WON'T EXPLAIN
DETAILS.
BASED ON THIS PSEUDOKNOT FOLDING
MODEL WE DID CALCULATION, HERE
IS OUR RESULT.
SYMBOLS ARE FROM EXPERIMENT,
THIS LINE FROM OUR CALCULATION.
YOU SEE THAT AGREEMENT IS GOOD.
WE GOT 18 SECONDS, VERY CLOSE.
VERY CLOSE.
SO, WE'RE QUITE HAPPY ABOUT THIS
RESULT.
AND SO FINALLY I WANT TO SHOW
THAT THIS IS PSEUDOKNOT.
WE MIGHT BE ABLE TO FIND A NEW
DRUG TARGET HERE BECAUSE
PSEUDOKNOT MIGHT SERVE AS DRUG
TARGET AND THIS MOVIE SHOWS
FOLDING P4 TO N4.
IF WE DESIGN A DRUG MOLECULE
THAT CAN TARGET THE HAIRPIN, FOR
EXAMPLE, ANYWHERE TO PREVENT THE
FORMATION OF PSEUDONOT THEN
PROBABLY WE CAN STOP THIS
PATHWAY.
SEE, THIS IS FOR ILLUSTRATION,
IT'S NOT A REAL CALCULATION.
AND THEN PSEUDOKNOT CANNOT FORM.
SO THAT'S -- WE'RE VERY EXCITED
THIS WORK WAS JUST -- WAIT,
WHAT --
>> RANDY?
THERE'S A PROBLEM.
YOU'RE GETTING CLOSE TO TIME
ANYWAY.
[LAUGHTER]
>> MAGICALLY.
>> ONE MORE MINUTE.
>> OKAY, YEAH.
I'M DONE.
SO THIS WORK WAS JUST PUBLISHED
A MONTH AGO.
AND WE'RE ALSO WORKING ON TRY TO
USING THIS AS PARADIGM TO STUDY
THE CO-TRANSFER FOLDING OF FIVE
PRIME REGION WITH COLLABORATION
WITH XIAO AND KATHLEEN'S LAB.
LAST SLIDE, I WANT TO THANK MY
GROUP AND STUDENT AND FOUR
HIGHLY TALENTED POSTDOC, XIAO
AND KATHLEEN, I JOINED THE CRNA
THREE MONTH, I ALREADY
ESTABLISHED VERY EXCITING
COLLABORATIONS WITH KWAKU AND
VICKIE AND STEVE AND NATE AND
MANY OTHER PEOPLE.
I THANK NIH FOR THE MONEY.
THANK YOU.
[APPLAUSE]
>> WE HAVE TIME FOR ONE QUICK
QUESTION.
AND ONE MINUTE.
>> HOW DO YOU BACKMAP CLOSE
RANGE REPRESENTATION BACK TO THE
REPRESENTATION?
GOOD QUESTION.
WE FIND MAPPING FROM RANDOM
STRUCTURE TO ALL-ATOM STRUCTURE.
>> ONE MORE ROUND OF APPLAUSE.
[APPLAUSE]
>> NEXT SPEAKER IS FUMIAKI ITO,
UNIVERSITY OF CALIFORNIA LOS
ANGELES.
>> ALL RIGHT.
I'M A POSTDOC, BETWEEN UCLA AND
USC.
WE'VE BEEN STUDYING APOBEC FOR
NINE YEARS, EXCITED TO SHARE
FROM THE COMPLEX TODAY.
DURING HIV INFECTION IN HUMAN T
CELL APOBEC 3 FACTORS ARE
EXPRESSED PACKAGED, THE PROCESS
IS KNOWN TO BE DEPENDENT ON RNA
BINDING WHICH CAN BE FROM EITHER
VIRUS OR HOST.
ALL RIGHT.
UPON HIV REACHING THE PROTEINS
TARGET THE NEGATIVE STRAND
cDNA FOR MUTAGENIC REACTION, AS
A RESULT WE SEE G TO A
HYPERMUTATION ON INTEGRATED
GENOME.
THIS IS ESSENTIALLY HOW APOBEC 3
PROTEIN RESTRICT REPLICATION.
HOWEVER HIV HAS EVOLVED
ACCESSORY PROTEIN SPECIFICALLY
BY APOBECS.
HIJACKS HOST FACTORS SUCH AS CGF
BETA, TO POLY UBIQUINATE
APOBECS, DEGRADED IN
PROTEASOME.
THIS IS CLASSIC EXAMPLE, WE'VE
BEEN INTERESTED IN THIS PROTEIN
COMPLEX ASSEMBLY ESPECIALLY THIS
APOBEC INTERFACE.
IT'S BEEN QUITE CHALLENGING TO
CHARACTERIZE THIS COMPLEX BUT IN
RECENT YEARS THERE'S BEEN
PROGRESS IN THE FIELD AS THE
FIRST APOBEC 3 COMPLEX, BY THE
LAB OVER HERE, MORE RECENTLY
THIS COMPLEX IN THREE
INDEPENDENTY STUDIES BY JOHN'S
LAB AND BY OUR LAB.
THE SURPRISE IN THE INTERACTION
WAS THAT RNA STRAND MEDIATING
THE A3G INTERACTION HERE, IN ALL
THREE STUDIES THIS SINGLE STRAND
RNA IS IN THE SPACE FORMED
BETWEEN.
AT THIS POINT THE COMPLEX
REMAINED UNRESOLVED, WE WERE
WONDERING IF THIS TYPE OF RNA
MEDIATED INTERACTION CAN BE
SEEN.
WITH THAT IN MIND WE
RECONSTITUTED THIS COMPLEX IN
VITRO, PREVIOUS STUDIES HAVE
SHOWN THAT FROM DIFFERENT HIV
STRANDS HAVE DIFFERENT EXTENT OF
ANTAGONISM AGAINST THIS.
FOR EXAMPLE, LAI STRAND DEBATES
A-3H MORE EFFECTIVELY,
DIFFERENCE ATTRIBUTED TO SINGLE
AMINO ACID ACID POLYMORPHISM AT
POSITION 48.
WE CONFIRMED N 48 MUTANT REDUCED
LEVEL OF A3H MORE EFFECTIVELY.
AND FOR A3H THERE WAS A STUDY
DEMONSTRATING HOMOLOG FROM
CHIMPANZEE IS MORE SENSITIVE TO
THIS HIV MEDIATED DEGRADATION.
AND THEY ALSO PINPOINTED THAT
AMINO ACID AT 97 DETERMINED
SENSITIVITY TO HIV IF MEDIATED
WITH DEGRADATION.
AND THIS RESULT WAS HIGHLY
REPRODUCIBLE AS YOU CAN SEE
HERE.
K9 7K MUTANT OF HUMAN A-3H
DEGRADED -- SORRY, THIS WAS MORE
SENSITIVE, MUTANT BECAME MORE
SENSITIVE.
AND A3H NEVER WAS COMPLETELY
DEPLETED WHEN MUTANT WAS
COMBINED.
SO THIS STRAND WAS BASICALLY THE
SAME IN OUR PURIFIED SYSTEM.
AS YOU CAN SEE THE MUTANT K9 7Q
AND N48H, RESULTED IN MOST
PREDOMINANT PEAK SHIFT IN THE
PROFILE.
WE TOOK THIS COMPLEX PEAK FOR
CryoEM.
OKAY.
THIS IS OUR MAIN MAP SHOWING THE
A-3H VIF AND CDF BETA, AS
EXPECTED A3H IS BOUND TO
DOUBLE-STRANDED RNA, AND WE
OBSERVED -- LARGE INTERFACE, THE
INTERACTION IS EXCLUSIVELY
PROTEIN BASED, BASICALLY THERE'S
NO RNA INVOLVED IN THIS
INTERFACE WHICH IS DIFFERENT.
A3H VIF INTERACTION IS MAIN LIP
MEDIATED BY THE ALPHA HELICAL
SIDE OF A3H, AND FIVE-STRANDED
BETA SHEET OF VIF, AND MORE
SPECIFICALLY A3H RESIDUES ON 3
AND 4 ARE INTERACTING WITH THE
RESIDUES AND CONNECTING LOOP.
AND NOTABLY AT 90 ESSENTIAL TO
INTERFACE.
ALL RIGHT.
THEN WE VALIDATED THIS INTERFACE
BY USING THE SAME DEGRADATION
ASSAY.
AND AS YOU CAN SEE POINTING
MUTATION ON ALPHA 3 RESIDUES,
ALPHA 4 RESIDUES INCREASED THE
RESISTANCE TO VIF.
AND INDEED MUTATION ALONG 90
RENDERED FULLY RESISTANT TO VIF
MEDIATED DEGRADATION.
SO OUR STRUCTURE SHOWS THAT THIS
SENSITIVITY MODULATING RESIDUE
AT 97 WOULD BE ACTUALLY
ELECTROSTATICALLY REPULSIVE WITH
THESE.
WE THINK THIS IS PARTIALLY WHY.
NATIVE PAIR IS SUBOPTIMAL IN
FORMING STABLE COMPLEX.
AND PREVIOUSLY VIF VARIANT FROM
LACK ADAPTATION STUDY AND ALSO
HIV ISOLATES FROM THE
PARTICIPANTS SHOWS THAT THERE'S
GAIN OF FUNCTION MUTATION ON THE
VIF WHICH IS K63E, OUR STRUCTURE
SHOWS THAT GLUTAMATE AT THIS
POSITION IS ELECTROSTATICALLY
MORE FAVORED WITH THE WILDTYPE
RESIDUE K97.
BASED ON COLLECTIVE EFFORTS, WE
NOW HAVE MORE COMPREHENSIVE
UNDERSTANDING OF VIF SURFACE
AREA THAT BINDS TO DIFFERENT
APOBECs.
A3H BINDING RESIDES IN
FIVE-STRANDED BETA SHEET, A3G
RESIDES AROUND THE CONNECTING
NERVE AND ALPHA HELIX ONE, THIS
HIGHLIGHTS WHICH USES DISTINCT
SURFACE AREA TO ANTAGONIZE
DIFFERENT APOBECS.
IN SUMMARY A-3H MODULATES THE
SENSITIVITY TO HIV VIF MEDIATED
DEGRADATION, ALSO A3H VIF
INTERACTION IS PROTEIN BASED
WITH NO INTERFACE.
ALPHA HELICAL BINDS IN VIF IS
DISTINCT WITH LIMITED OVERLAP.
WITH THAT, I WOULD LIKE TO THANK
OUR FANTASTIC TEAM FROM BOTH
LABS, AND ALSO COMPUTATIONAL
CENTER, OF COURSE FUNDING
SUPPORT.
THANK YOU.
[APPLAUSE]
>> ANY QUESTIONS FOR FUMIAKI?
I HAVE A QUESTION.
WHY DO YOU THINK IT'S SO
DIFFICULT TO DEVELOP INHIBITORS
OF THE VIF APOBEC PATHWAY,
PEOPLE HAVE TRIED HARD TO
INHIBIT, IT'S BEEN A LITTLE BIT
ABANDONED.
DO YOU HAVE ANY IDEAS?
>> RIGHT.
I THINK FOR A3G BECAUSE
INTERFACE IS NOT JUST PROTEIN
PROTEIN INTERACTION, IT'S RNAs
COMING INTO THE INTERFACE, IT'S
COMPLICATED, AND ACTUAL PROTEIN
PROTEIN INTERFACE FOR A3G IS
LIMITED, THREE OR FOUR AMINO
ACID, ALSO FOR A3H, YEAH, I
DON'T HAVE MUCH INSIGHT INTO A3H
INHIBITOR DEVELOPMENT BUT I
THINK A3H IS PLAYING A BACKUP
ROLE.
>> A MINOR ROLE.
>> PROBABLY MORE MINOR COMPARED
TO A3G.
>> MOST PEOPLE FOCUS ON A3G I
GUESS FOR DRUG DEVELOPMENT.
>> THAT'S RIGHT, YEAH.
>> OKAY, THAT MAKES SENSE.
THANK YOU.
ANY QUESTIONS IN THE MEANTIME?
IF NOT, LET'S GIVE ANOTHER ROUND
OF APPLAUSE TO FUMIAKI.
[APPLAUSE]
AND LAST BUT NOT LEAST SPEAKER
OF THE DAY KATE SKORUPKA FROM
NCI CANCER INNOVATION LABORATORY
AT THE NIH.
>> THANK YOU ALL FOR FREEZING
THIS LONG.
[LAUGHTER]
I'M A POSTDOC HERE AT NCI, AND I
WILL BE CONTINUING THE TOPIC OF
VIF-INDUCED YOUR BIC UBIQUITINT
A BRIEF OVERVIEW OF THE SYSTEM,
THE PROTEIN WE'RE WORKING ON,
THESE, AS FOR APOBEC 3H WE CHOSE
CHIMPANZEE, HERE WE HAVE
SEQUENCE ALIGNMENT, THE MAIN
DIFFERENCE AT POSITION 97, IN
CHIMP GLUTAMINE RESIDUE, THIS
MAKES IT MORE SENSITIVE TO
VIF-INDUCED UBIQUITINATION AND
DEGRADATION.
I'M SHOWING THE CryoEM
STRUCTURE WE SOLVED TO 3.9
RESOLUTION.
IN GREEN WE HAVE A3H, GOLD
STRAND RNA.
THIS IS DIMERIC STRUCTURE BUT WE
SEE A3H IS A DIMER THROUGH
DOUBLE-STRANDED RNA.
RESOLUTION IS ONLY 3.9 SO WE
DECIDED TO IMPOSE 2D SYMMETRY,
AND OUR RESULTING STRUCTURE IS
3.6 RESOLUTION, WITH BETTER
QUALITY, AND DOMAINS ARE COLOR
CODED.
AS I MENTIONED BECAUSE IT IS A
C2 SYMMETRY WE SEE WHO
DIFFERENT -- EXCUSE ME.
WE SEE WHO PROTOMERS, IN EACH
CASE THE PROTOMER IS BUILD FROM
DOUBLE-STRANDED RNA WITH A3H
THE INTERESTING PART OF OUR
STRUCTURE IS IT'S A DIMER
THROUGH VIF-VIF INTERACTION.
AMINO ACID AT THE INTERFACE MOST
ARE BULKY AROMATIC LIKE TRIP
FAN, HISTIDINE.
THESE PARTICIPATE IN BINDING
WITH A3G BUT WE'RE TALKING ABOUT
A3H, IN CONTACT WITH WHO VIF
MOLECULES, FIRST IS EXTENSIVE,
PROTEIN PROTEIN INTERACTION,
SECOND PROTEIN-PROTEIN
INTERACTING SURFACE IS MUCH
SMALLER AS MOLECULES ARE
POSITIONED FURTHER APART.
SO WE HEARD ABOUT THIS
INTERFACE.
WE SEE TRYPTOPHAN 90 AND
INTERACTION BETWEEN TWO PROTEINS
IS FOCUSED ON THIS ALPHA HELIX 3
AND 4 AND RESIDUES ON VIF ARE ON
THE SIDE.
THE SAME RESIDUES IMPORTANT ON
VIF HAVE BEEN DETERMINED
EXPERIMENTAL IN MUTAGENESIS
STUDIES, AND WE ALSO SEE THEM ON
OUR INTERFACE.
THE SECOND INTERFACE IS MUCH
SMALLER.
AND INTERACTION BETWEEN TWO
PROTEINS IS MOSTLY CONFINED TO
LYSINE-RICH LOOP.
WHAT'S INTERESTING IT SEEMS ONE
OF THE DOUBLE-STRANDED RNA
MEDIATED INTERACTION BETWEEN TWO
PROTEINS SO IF WE HAVE CLOSER
LOOK ON DOUBLE-STRANDED RNA HERE
ARE THE MAIN RESIDUES THAT ARE
IN CLOSE PROXIMITY TO ONE OF THE
STRANDS FROM DOUBLE-STRANDED
RNA.
AND I WOULD LIKE TO BRIEFLY TALK
ABOUT THE 116.
THIS IS A BEGINNING OF PPLP
MOTIF RIGHT IN HERE, AND PPLP
MOTIF HAS BEEN IMPLICATED IN
DIMERIZATION AND
OLIGOMERIZATION, ADDITIONALLY IN
PREVIOUS STRUCTURES OF VIF WITH
A3G THERE ARE NUCLEOTIDES THAT
BIND IN THIS PPLP BINDING POCKET
BECAUSE OF HOW WE PREPARE OUR
A3H, OUR DOUBLE-STRANDED RNA IS
EXTENSIVELY CHOPPED AND WE
DIDN'T DETECT ANY NUCLEOTIDES
INSIDE OF THIS POCKET.
AND NOW LET'S TALK ABOUT THE
UBIQUITINATION.
THIS IS PARTLY DONE IN
COLLABORATION WITH THE LAB, WE
PURIFIED THE COMPONENTS OF THE
SYSTEM, A3H HAS 11 RESIDUES THAT
CAN BE UBIQUINATED.
FROM THOSE 11 IN MASS
SPECTROMETRY ANALYSIS, MUTATED
THEM TO ARGININE, ACH IS
PROTECTED FROM THIS MEDIATED
UBIQUITINATION AND DEGRADATION.
WE REVERSE EACH ONE OF THE
MUTATIONS BACK TO THEIR ORIGINAL
LICENSE.
AND WE SEE THAT REVERSAL AT
POSITION 50 AND 51 IS ENOUGH TO
GRANT WILDTYPE LEVEL DEGRADATION
OF A3H.
THIS BRINGS TO WHETHER PROXIMAL
OR DISTAL IS UBIQUINATED.
TO ANSWER THIS QUESTION WE
DESIGNED AN EXPERIMENT WHERE WE
TRANSFECTED CELLS WITH TWO
WILDTYPE PLASMIDS OR MUTANT
PLASMIDS OR MIXTURE OF THE TWO.
IN PRESENCE OF THIS THE
WILDTYPE-WILDTYPE HETERODIMER
EACH ONE OF THE MOLECULES IS
DEGRADED TO SIMILAR LEVEL.
IN CASE OF THE HETERODIMER WITH
MUTANT THE WILDTYPE IS DEGRADED
BUT WE SEE ACCUMULATION OF W 90A
MUTANT INSIDE THE CELLS, THIS
STRONGLY SUGGESTS THAT ONLY THE
WILDTYPE PROTEIN IS UBIQUINATED
AND DEGRADED IN THE DEPENDENT
MANNER.
THIS BRINGS TO US A VERY GENERAL
MODEL WITH THE PRIME TO
UBIQUITIN.
ON THIS SIDE DOUBLE-STRANDED
DNA.
THE SECOND MOLECULE OF A3H FALLS
APART AT SOME LEVEL IN THE
TRANSFER, THAT'S ONE.
AND TWO IS HARD TO SEE,
LYSINE-RICH LOOP, PRIMARILY
ACCEPTORS OF THE UBIQUITIN, THE
TRANSFER IS FAVORABLE, THEY ARE
POSITIONED.
TO SUMMARIZE, IN OUR STRUCTURE
WE SEE THE VIF-VIF NOVEL
INTERACTION NOT SEEN BEFORE,
IDENTIFIED AMINO ACIDS THAT LIE
AT THE INTERACTION BETWEEN A3H
AND IF I HAVE MOLECULES.
WE ADDITIONALLY IDENTIFIED
INTERACTIONS BETWEEN
DOUBLE-STRANDED RNA AND VIF.
WE SHOW K 50 AND K 51 PRIMARY
ACCESSORS OF UBIQUITIN.
PROXIMAL A3H IS DEGRADED.
I WOULD LIKE TO ACKNOWLEDGE
THESE PEOPLE.
THANK YOU.
[APPLAUSE]
>> IF I UNDERSTOOD THE
DIFFERENCE FROM PREVIOUS SPEAKER
YOU'RE SEEING RNA AT THE
SECONDARY INTERACTION INTERFACE,
IS THAT CORRECT?
>> YES.
SO BECAUSE WE HAVE TWO
PROTOMERS, OUR INTERACTION
OCCURS THROUGH THE PROTOMER.
RNA 1 INTERACTS WITH VIF 2.
>> EVERYTHING IS MADE FROM
PURIFIED COMPONENTS INCLUDING
THE RNA, IS THAT RIGHT?
>> SO THE RNA PURIFIES WITH A3H.
WE DO NOT ADD IT AND CANNOT
REMOVE IT.
>> A3H MADE IN BACTERIA?
>> BACTERIA.
E. COLI.
>> OKAY.
>> I'M SORRY IF I MISSED THIS.
DID YOU TRY THIS IN THE CELL, IN
THE PRESENCE OF PROTEASOME
INHIBITOR?
>> WHICH PART?
>> THE UBIQUITINATION, I'M
SORRY.
>> I THINK SO BUT I'M NOT ONE
PERCENT SURE.
>> OKAY, THANK YOU.
>> ANY MORE QUESTIONS?
I ALSO HAVE A FINAL QUESTION.
YOU ARE SEEING RNA-RNA
INTERACTION WITH APOBEC 3H IS
THAT RIGHT OR NO?
BECAUSE FUMIAKI SAID IT WAS
PROTEIN BASED.
DO YOU SEE ANY RNA INTERACTIONS
OR NOT?
>> THAT BRINGS ME TO THIS POINT
WHERE HIS STRUCTURE IS BASICALLY
A MONOMER, SO HE HAS ONE
MOLECULE OF A3H WITH
DOUBLE-STRANDED RNA.
WE ALSO SEE IT IN OUR STRUCTURE.
BUT HE ONLY HAS ONE MOLECULE OF
VIF.
WE HAVE TWO MOLECULE OF VIF,
EVERYTHING A DIMER OF THE
COMPLEX.
THERE'S NO INTERACTION INSIDE
THE SAME PROTOMER, BETWEEN VIF
AND DOUBLE-STRANDED RNA.
WE SEE INTERACTION ACROSS THE
PROTOMERS.
>> OKAY.
>> THIS IS THE UNIQUE FEATURE OF
OUR VERSUS HIS STRUCTURE.
>> THANK YOU FOR THE
CLARIFICATION.
THAT CONCLUDES THE AFTERNOON
SESSION.
[APPLAUSE]
I'LL TAKE OUR FINAL APPLAUSE FOR
ALL THE SPEAKERS, ESPECIALLY FOR
KATE.
[APPLAUSE]
I'LL GIVE THE MICROPHONE TO
DAVID.
>> IF I COULD HAVE THE TALKS FOR
THE NEXT SESSIONS, MORNING TALK,
TWO MORNING TALKS.
IF YOU WANT TO TELL ME IF YOU'RE
GOING TO GO ON A LAPTOP, I DON'T
SEE ALICE.
I HOPE YOU WARM UP WHEN YOU GET
OUTSIDE.
[END OF PROGRAM]