>> GOOD MORNING, EVERYONE.
I'M A COLLEGIATE DAVID McDONALD
AT THE BASIC SCIENCE PROGRAM AND
THANK YOU ALL FOR A WONDERFUL
PRESENTATION YESTERDAY AND IT'S
GOOD TO SEE NEW SCIENCE AND
WE'RE IN THE SECOND DAY AND WE
HAVE THREE MORE CENTERS
PRESENTING TODAY.
I JUST WANTED BEFORE WE GO THERE
SOME FEW HOUSEKEEPING
ANNOUNCEMENT.
YOU SAW A SLIDE A MOMENT EARLIER
THE NEXT MEETING PLEASE MARK
YOUR CALENDAR.
LOOKING FORWARD TO IT NEXT YEAR
AND PEOPLE ARE PRESENTING
ABSTRACTS, PLEASE COME UP BEFORE
LUNCH AND GIVE YOUR PRESENTATION
SO WE COULD BE READY AND COME
BACK AFTER LUNCH IN THE POSTER
SESSION.
WITH THAT LET ME INTRODUCE THE
FIRST CENTER PRESENTER TODAY AND
WE'LL HAVE A PRESENT BY ALICE
TELESNITSKY.
>> WE'RE THE CENTER FOR
STRUCTURAL BIOLOGY OF HIV RNA.
AS THE NAME IMPLIES THE PROJECTS
THAT WE'RE WORKING ON HAVE DO
WITH INTERACTIONS OF MOLECULAR
COMPLEXES AND PROCESSES IN THE
HIV REPLICATION CYCLE THAT
INVOLVE RNA.
WE HAVE A FABULOUS GROUP OF
INVESTIGATORS AND COLLABORATIVE
AWARD WINNERS AND
INTERGENERATIONAL AND
INTER-CENTER INTERACTIONS AND
I'LL SKIP THOSE AND HIGHLIGHT
ONE OF OUR ACTIVITIES WHICH IS
WE HAVE AN UNDER GRADUATE
RESEARCH PROGRAM -- AH, WE CALL
IT THE HIV OPPORTUNITY IN RNA
FOR UNDERGRADUATE SCHOLARS WE
CALL ROAR.
VERY CUTE NAME, HUH.
WE CAN SEE THE SECOND YEAR OF
THE PROGRAM AND TRAINEES ARE
EMBEDDED.
THIS IS MY FAVORITE TRAINING LAB
AND JEREMY LUBAN AND I WERE
TRAINEES AND HE IS HOSTING ONE
OF OUR ROAR STUDENTS THIS YEAR
AND THEY'RE ALREADY EMBEDDED IN
THEIR LABS BUT BECAUSE LIKE
OTHER CENTERS WE'RE REMOTE AND
IT WAS JUST NICE THEY COULD COME
TO THIS MEETING AND INTERACT
WITH YOU SO YOU'LL HAVE SEEN
SOME OF THEM AT THE POSTERS
YESTERDAY AND TODAY.
I WANTED TO CUT STRAIGHT TO THE
SCIENCE BECAUSE WE HAVE EXCITING
THINGS TO TELL YOU ABOUT TODAY
AND FIRST UP IS PAUL BENASH.
-- PAUL BIENIASZ.
I'LL TELL YOU A COLLABORATIVE
LAB ON THE ZINC FINGER COMPLEX
BASED ON A FOUNDATIONAL RESULT
IF YOU TAKE HIV-1 AND TAKE A
SMALL SEGMENT OF ITS GENOME AND
BY SYNONYMOUS SUBSTITUTION
ELEVATE THE CPG CONTENT TO
APPROXIMATELY THE LEVEL YOU'D
SEE IN RANDOM RNA SEQUENCE
IMPOSES A LETHAL DEFECT IMPOSED
BY THE PRESENCE OF THE ZAP
PROTEIN NOT ALLOWING THE VIRUS
TO REPLICATE LIKE WILD TYPE.
AND ZINC PROTEIN ROSE IN AN
ANCESTRAL AQUAPOD AFTER THE
LINEAGE OF TRANSFERASES WHICH
LED TO THE PURGING OF CPG WILL
NUCLEOTIDES FROM THE
TRANSCRIPTOMES OF THAT LINEAGE
CREATING AN OPPORTUNITY FOR
SELF/NON-SELF DISCRIMINATION BY
RECOGNITION OF CPGs CARRIED BY
THE VIRUSES OF WILL WILL INSECTS
AND ARTHROPODS WHICH IS NOT
UNDER GO THIS TRANSFERASE.
BECAUSE THIS EVENT HAPPENED
HUNDREDS OF MILLIONS OF TIMES
PER DAY FOR HUNDREDS OF MILLIONS
OF YEARS MAMMALS HAVE BEEN
BOMBARDED BY CPG RICH VIRUSES
BUT ONLY THE ONES THAT MANAGED
TO PURGE CPG FROM THEIR GENOME
HAVE BEEN SUCCESSFUL IN
COLONIZING MAMMALIAN OF HOSTS.
WE THINK IT'S LARGELY DO YOU TO
THE PROTEIN OF ZAP.
IT OCCURS IN TWO NATURALLY
OCCURRING ISOFORMS.
THE BUSINESS END MUCH THE
PROTEIN IS THIS AMINO TERMINAL
BINDING DOMAIN THAT JANET SMITH
LAB'S SOLVED THE STRUCTURE OF
THE PROTEIN IN COMPLEX WITH AN
RNA TARGET WHICH EXPLAINS IN
ATOMIC DETAIL HOW THE PROTEIN
SPECIFICALLY INTERACTS WITH CPG
DINUCLEOTIDES AND THE SINGLE
EVENT IS NOT ENOUGH FOR THE
ANTIVIRAL ACTIVITY AND LEARNED
FROM GROUPS IMPLICATING A NUMBER
OF PROTEINS BUT MOST
COMPELLINGLY AND THE PROTEINS
I'LL TALK ABOUT TODAY, TRIM 25
AND K9 KHNYN IMPORTANT AND
ESSENTIAL FOR THE ACTIVITY OF
ZAP.
OUR OWN AND I'LL SHOW DATA WHERE
WE DO A SIMPLE CYCLE EXPERIMENT
AND MEASURE WITHIN A DAY OR TWO
HOW MUCH VIRUS COMES ON YOU IN
CELLS KNOCKED OUT FOR ONE OF
THESE PROTEINS THAT MAY OR MAY
NOT BE INVOLVED.
SO HERE'S THE BASELINE
PHENOTYPE.
A VIRUS THAT HAS A SEGMENT OF CP
G ENRICHED CONSEQUENCE.
IT HAS A 50 FOLD SEQUENCE OVER
REPLICATION AND COMPLETELY GONE
WHEN YOU KNOCK OUT ZAP.
IT'S DOWN BUT NOT OUT.
IF YOU KNOCK OUT THIS CO-FACTOR
TRIM 25 IS UNAFFECTED IF YOU
KNOCK OUT THE NUCLEASE KHNYN AND
KNOCK OUT A RELATED FAMILY
MEMBER LITTLE AFFECT BUT IF YOU
KNOCK OUT BOTH THEN THE
PHENOTYPE GOES AWAY.
IT LOOKS LIKE THE PROTEINS ACT
REDUNDANTLY AND WE CAN
RECONSTITUTE THE DOUBLE
KNOCKOUTS WITH ONE OR EITHER OF
THE PROTEINS.
SO ARMED WITH THAT KNOWLEDGE WE
DEVELOPED A RECONSTITUTION
ASSAY.
A SIMPLE TRANSFECTION ASSAY
WHERE WE GENERATED A CELL LINE
KNOCKED OUT FOR ALL THE PLAYERS
WILL INTERESTED AND TRANSFECT
PROVIRAL DNA WITH OR WITHOUT ONE
OF THE PROTEINS IN WILD TYPE OR
MUTANT FORM AND MEASURE
INFECTIOUS VIRUS.
IN THIS PARTICULAR ITERATION WE
HAVE WILD TYPE HIV-1 WITH THE CV
ENRICHED SEGMENT AND YOU CAN SEE
AU YOU NEED ALL THREE TO GET IT
A COUPLE ORDERS OF MAGNITUDE OF
INFECTIOUS VIRUS RELEASE.
INDIVIDUAL PROTEINS ON THEIR OWN
DO NOTHING IN THIS
RECONSTITUTION SYSTEM AND KHNYN
HAS SOME ACTIVITY AND CURIOUSLY
SOMEWHAT OF A LOSS OF
SPECIFICITY AND HAVE WEAK
ACTIVITY AGAINST THE WILD TYPE
VIRUS.
SO I'M GOING TO SHOW YOU A
NUMBER OF EXPERIMENTS USING THAT
RECONSTITUTION SYSTEM.
HERE FIRST WE'RE TAKING THE ZAP
PROTEIN AND DOING SIMPLE
TRUNCATION EXPERIMENTS AND YOU
CAN SHOW AS STEVE GOS' LAB DID
THE AMINO FERM NAL FRAGMENT THE
RNA BINDING DOMAIN IS SUFFICIENT
FOR ACTIVITY.
YOU GET INHIBITION OF THE VIRUS
AND SOME INHIBITION OF THE WILD
TYPE VIRUS.
YOU NEED LONGER FORMS AND
ISOFORMS TO GET THIS
SPECIFICITY.
YOU CAN SEE A HIGH DEGREE OF
SPECIFICITY FOR CG ENRICHED
VIRUS WITH THE FULL LENGTH
PROTEIN COMPARED AMINO TERMINAL
FRAGMENT.
THE OTHER ESSENTIAL COMPONENT IS
AS I SAID A NUCLEASE AND USE THE
KHNYN FOR THESE EXPERIMENTS.
S THIS IS THE PUTATIVE DOMAIN
AND YOU HAVE NUCLEIC ACID DOMAIN
COME TO THAT POINT IN A MINUTE
AND YOU HAVE C TERMINAL DOMAIN
OF UNKNOWN FUNCTION.
YOU NEED ALL THESE DOMAINS TO
GET FULL ACTIVITY OF THE KHNYN
PROTEIN.
ANY OF THESE TRUNCATION OR POINT
MUTATIONS ABOLISH FOR SEVERELY
ABLATE THE ACTIVITY.
THAT C TERMINAL DOMAIN SEEMS TO
BE AN IMPORTANT PART OF
ASSEMBLING THE COMPLEX AND THIS
WAS SOLVED IN JANET SMITH'S LAP
WITH THE RBD DOMAIN AND THAT
SMALL KHNYN C TERMINAL DOMAIN.
YOU CAN SEE THE INTERFACE IF YOU
LOOK IN DETAIL WE CAN IDENTIFY
BASICALLY THREE POINTS OF
CONTACT BETWEEN KHNYN AND ZAP
AND THIS IS NOT THE ONLY ONE
IT'S AN IMPORTANT ONE AND IF YOU
MUTATE THIS PARTICULAR AMINO
ACID AT INTERFACE 1 YOU CAN
COMPROMISE DRAMATICALLY THE
ABILITY.
SORRY, THIS IS A ZAP MUTATION
YOU CAN COMPROMISE THE ANTIVIRAL
ACTIVITY.
IMPORTANTLY THE KHNYN CTD
INTERACTION OCCURS AT A
DIFFERENT PLACE THAN THE RNA
BINDING AND IT'S FULLY
COMPATIBLE WITH ASSEMBLY OF RNA,
ZAP AND KHNYN TOGETHER.
SO I'VE CALLED KHNYN A PUTATIVE
NUCLEASE.
IS IT ACTUALLY A NUCLEASE?
THE ANSWER IS YES.
HERE'S THE FULL LENGTH PROTEIN
WITH APPROXIMATELY 100
NUCLEOTIDE WITH CG DINUCLEOTIDES
OR DOESN'T HAVE THAT HAVE BEEN
REVERSED TO GCs.
YOU CAN SEE THE NUCLEASE DOESN'T
REALLY CARE ABOUT THE PRESENCE
OF CGs IN THE RNA SUBSTRATE.
IT WILL DIGEST COMPLETELY REMOVE
THE RNA IN ABOUT 20 MINUTES.
NOW, IF YOU EXPRESS JUST THE
WILL NIN DOMAIN THAT ALSO IS AN
ACTIVE NUCLEASE AND ALSO DOESN'T
CARE ABOUT THE NUCLEOTIDE
CONTENT OF THE TARGET RNA.
AND HAS LIMITING AMOUNT OF
NUCLEASE AND YOU CAN SEE THE
PRODUCTS BUT IF YOU ASSEMBLE THE
DUPLEX IT'S COMPLETELY
RESISTANT.
SINGLE STRAND RESISTANT
NUCLEASE.
MORE DETAILS HOW THIS COMPLEX
MIGHT INTERACT WITH RNA.
WHERE IN FACT WOULD KHNYN BIND
ON THE FIRST EXPERIMENT THIS IS
A POLARIZATION EXPERIMENT AND
DOESN'T DIGEST THE RNA BUT CAN
AFFECT THE FLOUROPHORS AND THE
POLARIZATION IS INCREASED TO I A
GREATER DEGREE SHOWING THE THREE
PRIME POSITION.
IF YOU DO A LIMITING QUANTITY OF
NUCLEASE DIGESTION ASSAY AND ASK
WHY THE FIRST CUT YOU CAN SEE
THE CUT IS INITIATING THREE
PRIME TO THE CG BINDING SITE.
THAT SUGGESTS A MODEL FOR THE
CORE ESSENTIAL MINIMAL PART OF
THE ANTIVIRAL BINDING DOMAIN
THAT RECRUITS THE KHNYN CTD AND
POSITIONS IT TO CLEAVE THE RNA A
FEW NUCLEOTIDES AND THREE PRIME
TO THE DINUCLEOTIDE.
WE CAN TEST WHETHER THIS IS
FUNCTIONALLY SUFFICIENT TO
RECONSTITUTE IN AN IN VIVO
ASSAY.
WHAT WE'VE DONE IS DONE AWAY
WITH ALL THE PROTEIN DOMAINS
THAT WOULD OTHERWISE BE
ESSENTIAL AND SIMPLY FUSE THE
ZAP RNA BINDING DOMAIN TO THE IT
NYN DOMAIN OF KHNYN.
WHAT YOU GET WHEN YOU DO THAT
RATHER REDUCTION EXPERIMENT IS A
VERY POTENT ANTIVIRAL PROTEIN
AND ENRICH BY TWO ORDERS OF
MAGNITUDE.
YOU DO LOSE SPECIFICITY SO THE
WILD TYPE VIRUS IS INHIBITED
SIGNIFICANTLY BUT IMPORTANTLY
THE ACTIVITY IS DEPENDENT ON THE
NUCLEASE ACTIVITY OF THE KHNYN
PART.
WHAT DO THE OTHER COMPONENTS OF
THE COMPLEX DO?
IS LET'S LOOK AT TRIM 25.
BECAUSE THAT'S OF SOME INTEREST.
SO IN THIS EXPERIMENT WE HAVE
DONE THE RECONSTITUTION ASSAY ON
WILD TYPE OR CG ENRICHED HIV.
THIS TIME WE'RE ADDING FIXED
AMOUNT OF KHNYN AND ADDING IN
TRIM 25 AND SEE THE WILD TYPE
STIMULATES ANTIVIRAL ACTIVITY
NICELY.
THE TERMINUS IS IMPORTANT AND
THE AMINO TERMINUS IS IMPORTANT
FOR THIS ACTIVITY THIS COMPLEX
WAS SOLVED WITH THE UBIQUITIN
AND MAKE MUTATION WERE SHOWN TO
ABOLISH THE UBIQUITIN TO THE
ABILITY TO STIMULATE THE
ACTIVITY AND IF WE MAKE
MUTATIONS THAT ABOLISH THE
DIMERIZATION THEN YOU SEVERELY
COMPROMISE THE ABILITY OF TRIM
25 TO STIMULATE THE ACTIVITY.
CLEARLY THE ABILITY OF TRIM 25
TO WILL CREATE HIGH ORDER MULT
IMERS IS IMPORTANT.
AS FAR AS THE SPRY DOMAIN IS
CONCERN, IT BECOMES COMPLETELY
DISPENSABLE IF YOU SIMPLY GET
RID OF IT AND FUSE INSTEAD THE
ZAP RNA BINDING DOMAIN AND
REDUCE IT TO A TWO IT COMPONENT
SYSTEM AND GET RID OF THE DOMAIN
WHOSE FUNCTION IS RECRUITMENT BY
PUTTING THE RNA BINDING DOMAIN
YOU CAN SEE THAT ACTIVITY HERE.
AGAIN IT'S VERY POTENT AND NOT
AS SPECIFIC AS THE FULL COMPLEX
BUT HAS A HIGH LEVEL OF
ACTIVITY.
LET ME SUMMARIZE WHAT WE KNOW IN
TERMS OF THE BITS THAT COMPRISE
THE STRUCTURE OF THIS PURPORTED
ANTIVIRAL COMPLEX.
I'VE SHOWN THE RBD DOMAIN OF ZAP
INTERACTS WITH KHNYN IN
PARTICULAR THE C TERMINAL
DOMAIN.
THAT FORMS THIS CORE COMPLEX OF
KHNYN AND THE RNA TARGET.
HOWEVER, THAT IS SUFFICIENT FOR
ACTIVITY BUT NOT COMPLETELY
SUFFICIENT FOR SPECIFICITY.
THERE ARE ACCESSORY ACTIVITIES
THAT WE THINK REALLY CONTRIBUTE
TO THAT SPECIFICITY.
THE FIRST OF THESE IS THE C
TERMINAL PORTION OF ZAP.
SO TO SOLVE A STRUCTURE OF PART
OF THAT IS WWE DOMAIN BINDS TO
POLY ADP RIVOS AND THAT COULD BE
AN IMPORTANT PART OF DRIVING THE
MULTIMERIZATION AND JANET SOLVED
THE KH DOMAIN INVOLVED IN
NUCLEIC ACID BINDING THAT IS
CLEARLY NOT THE CASE HERE.
THE KH DOMAIN IS ACIDIC AND DOES
NOT BIND TO RNA IT FORMS PART OF
THE INTERACTION WHICH I HAVEN'T
SHOWN YOU WITH TRIM 25 AND WITH
ZAP.
TRIM 25 ITSELF WOULD BE A DIMER
BUT CAN ASSEMBLE TO HIGHER ORDER
MULTIMERS IMPORTANT FOR THE
ENHANCING ACTIVITY OF ZAP.
SO AND THAT'S ESSENTIALLY WHAT
WE KNOW ABOUT THE PROTEIN
COMPONENTS OF THIS ANTIVIRAL
COMPLEX.
BEFORE I COME TO A MODEL OF HOW
WE ALL THINK THIS FITS TOGETHER
LET ME TALK JUST FOR A FEW
MINUTES ABOUT THE NATURE OF THE
TARGET RNA BECAUSE I THINK
THAT'S QUITE IMPORTANT FOR
CONSIDERATION.
WHAT IS AN OPTIMAL TARGET OF
ZAP?
THE DEFINED STRUCTURE IS A
MONOVALENT OF MOLECULE AND USING
HIV AS A MODEL WE FIGURED OUT
THE RULES FOR WHAT CONSTITUTES
AN OPTIMAL ZAP TARGET AND THE
NUMBER OF AND WHAT'S IMPORTANT
IS THE SPACING BETWEEN THE CPGs
ABOUT 12 TO 30 NUCLEOTIDES AND
THE SINGLE STRANDED CHARACTER IN
WHICH THOSE NUCLEOTIDES ARE
EMBEDDED.
WE KNOW THESE RULES WELL ENOUGH
TO DESIGN VIRUSES AND WE'VE DONE
IT WITH THIS VIRUS THAT BECOMES
ZAP SENSITIVE AND KNOCKOUT CELLS
AND THE VIRUS WE INCREASED THE
SINGLE STRANDED CHARACTER AND IT
BECOMES ZAP SENSITIVE IN CELL
CULTURE IN MICE.
THIS IS A ZAP KNOCKOUT MICE AND
IT WILL REPLICATE WHEN ZAP IS
KNOCKED OUT AND KILLED THOSE
MICE.
SO THE QUESTION ARISES, DOES ZAP
IN ADDITION TO EFFECTING VIRAL
RNAs DOES IT AFFECT HOST RNAs?
USING THESE RULES WHAT WE
DEVELOPED IS PREDICTION
ALGORITHM THAT TRIES TO PREDICT
WHETHER AN INDIVIDUAL TRANSCRIPT
WILL BE SENSITIVE TO IT BASED ON
THESE CRITERIA.
IF YOU COMPARE MAMMALIAN AND
INSECT TRANSCRIPTOMES ON CGs AND
THE DISTANCE BETWEEN THEM AND
COMPOSITION BY ALL THREE
CRITERIA HUMAN TRANSCRIPTS ARE
PREDICTED TO BE FAR LESS
SENSITIVE THAN INSECT
TRANSCRIPTS AND IF YOU PLOT A
ZAP SENSITIVITY SCORE AN AVERAGE
FLY TRANSCRIPT WOULD BE AS
SENSITIVE AS THE MOST SENSITIVE
.1% OF HUMAN TRANSCRIPT.
WE THINK WE CAN PREDICT WHAT
HUMAN GENES WOULD BE SENSITIVE
TO ZAP.
AND VERY VERIFIED THAT USING
KNOCKOUT MICE.
THESE ARE THE HUMAN TRANSCRIPTS
WITH THE ZAP SENSITIVITY SCORES
AND WHEN WE MEASURE THEIR LEVELS
IN THE SPLEENS OF ZAP KNOCKOUT
AND COMPETENT MICE VIRTUALLY ALL
THOSE APPEAR TO BE ELEVATED WHEN
YOU KNOCKOUT ZAP.
THE AVERAGE ZAP SENSITIVITY
SCORE OF UPREGULATE WILL GENES
IS HIGHER AS WELL.
SO ZAP REGULATED GENE IS IT
HIGHER.
THE MAGNITUDE ON HOST GENE
EXPRESSION IS MODEST BUT BECOMES
EXAGGERATED WHEN YOU INDUCE ZAP
WITH POLY I.C.
WE DON'T KNOW WHAT THE BIOLOGY
ASSOCIATED WITH THE GENES ARE
BUT MANY OF THEM ARE INVOLVED IN
DEPOSITION OF EXTRA CELLULAR
MATRIX AND THERE'S SECONDARY
EFFECTS BASED ON TRANSCRIPTION
FACTORS.
THIS IS BASICALLY A MODEL.
SO AN OPTIMAL TARGET RNA HAS CGs
SPACED THAT AMOUNT A PART AND WE
THINK THIS IS WHY THE ABILITY OF
TRIM 25 TO MAKE MULTIMERS AND
THE EXTENDED IT WILL ZAP PROTEIN
IS IMPORTANT BECAUSE IT CONVERTS
WHAT'S A MONOVALENT INTERACTION
TO A MULTIVALENT INTERACTION.
AND OF COURSE CREATES A
NIGHTMARE FOR STRUCTURAL
BIOLOGISTS WHO WANT TO STUDY THE
COMPLEX.
THAT'S ALL I HAVE TO SAY.
THE PEOPLE IN MY GROUP WHO
CONTRIBUTED ARE LISTED THERE BUT
IT'S VERY MUCH A TEAM EFFORT.
AND THERE'S MORE DATA ON A
POSTER SO I ENCOURAGE YOU TO
STOP BY.
JENNIFER AND JANET AND MELANIE
ARE ALREADY CONTRIBUTORS TO
THIS.
THANK YOU FOR LISTENING.
S
>> I'D LIKE TO TELL YOU ABOUT
OUR EFFORTS TO UNDERSTAND THE
RNA DETERMINATES OF PACKAGING
AND IT'S A COLLABORATION WITH
SEVERAL GROUPS WITHIN THE CRNA.
SO THIS WAS JUST TO REMIND YOU
THE SINGLE INTEGRATED PROVIRUS
PRODUCES A FULL LENGTH
TRANSCRIPT AND THAT HAS TWO
FUNCTIONS.
IT HAS THE FUNCTION AS AN mRNA
AND GENOME AND IT'S BEEN THE
PAST SEVERAL YEARS PEOPLE
REALIZED ONE OF THE DETERMINATES
OF THE FUNCTIONAL CONTROL IS
HETEROGENEOUS START SITE USAGE.
FOR MANY YEARS IT WAS UNKNOWN
WHAT THE START SITE IS.
THESE ARE THE THREE GUANOSINES
THAT COULD START AS A SITE BUT
IT'S NOT PEOPLE DIDN'T CARE.
AND THERE'S 1G AND IN 2015 THEY
SUGGESTED ALL THREE ARE MADE AND
3G IS THE PREDOMINANT ONE.
IF YOU GO TO THE NIH COMPENDIUM
ALL THREE ARE REPORTED BUT
THEY'RE SPECULATION ABOUT WHAT
THEY WERE.
WITH ALICE AND SERGEI WHAT WE
SHOWED IN 2016 IS THAT ALL THREE
ARE TRANSCRIBED IN CELLS THAT
THE 1G FORM IS ENRICHED IN
VARIANTS AND WHAT THEY SHOWED
THE IT 3G FORM IS ENRICHED IN
POLYSOMES AND THE 13 AND 3G ARE
PREDOMINANT AND SEEN IN HUMAN
GENES AS WELL AND IT'S BEEN
CALLED TWIN TRANSCRIPTIONAL
START SITE USAGE.
WHAT LEADS TO THIS PARADIGM
WHERE THE 3G CAPPED RNAs AND ONE
AS A GENOME AND NOW THIS HAS
BEEN SPORTED BY A NUMBER OF
ADDITIONAL STUDIES AND TWO THAT
STAND OUT IN MY MIND ARE ALICE'S
GROUP THAT SHOWED THE 3G FORM IS
ENRICHED IN SPLICE TRANSCRIPTS
AND ALSO WILL ANOTHER GROUP
SHOWED THE 3G ONLY VIRUS HAS A
PACKAGING DEFECT WHICH WOULD
MAKE SENSE BASED ON THE MODEL
AND THE 1G VIRUS IS REDUCED IN
GAG EXPRESSION WHICH ALSO MAKES
SENSE WITH THE PARADIGM.
WE'VE USED NMR TO UNDERSTAND
STRUCTURALLY HOW IT WORKS AND
FOR THE IT WILL VERSION OF HIV
WE SHOWED THE FIVE 2G AND 3G
RNAs FORM A STRUCTURE LIKE THIS.
THIS IS JUST A CARTOON BUT
RESIDUES THAT HAD BEEN PREDICTED
TO FORM A HAIRPIN OR POLY A
REGION THIS FORM OF THE RNA
EXPOSES THE SPLICED DONOR SITE
ON THE NUKE LEO CAPSID BINDING
SITES ARE SEQUESTERED AND THIS
FUNCTIONS AS mRNA AND THIS IS
WORK OF JOSH BROWN SHOWED THAT
IF YOU HAVE THE KAPPA 1G FORM
THESE STACK END TO END.
WHAT THAT DOES IS IT SEQUESTERS
THE CAP IN THIS STRUCTURE THE
SPLICE DONOR SITE AND THE GAG
START CODE ARE ALSO SEQUESTERED.
NUKE NUCLEOBINDING SITES ARE
EXPOSED AND THIS IS SEQUESTERED
AND THIS IS THE FORM THAT
FUNCTIONS AS THE GENOME.
SO WE THINK WE UNDERSTAND THE
STRUCTURAL DETERMINATES OF RNA
PACKAGING.
THE QUESTION IS DOES
TRANSCRIPTIONAL START SITE
HETEROGENEITY INFLUENCE
TRANSLATION AND WHAT IS THE ROLE
OF POLY A AND THIS WAS STUDIED.
SO THIS IS INTERESTING BECAUSE
YOU'LL NOTICE IT'S THE POLY A
ELEMENT THAT INTERACTS WITH THE
ADDITIONAL GUANOSINE.
NO OTHER MMR STRUCTURE WE DIDN'T
SEE INTERACTIONS GOING FROM THE
1G TO 3G.
THE POLY A ELEMENT PLAYS AN
IMPORTANT ROLE.
OUR WORK IS BASED ON ACTUAL
OBSERVABLE INTERACTIONS.
THIS IS THE OBSERVED SOLVED OF
NMR AND WHAT IS BOXED ARE
EXPERIMENTALLY DETERMINED NOT
PREDICTED BASED ON FREE ENERGY
CALCULATIONS AN IT'S CLEAR POLY
A IS NOT MAKING LONG RANGE
INTERACTIONS.
I THINK IT'S WORTH POINTING OUT
THERE ARE SEVERAL OTHER MODELS
OF THE MONOMERRICK -- MONOMERIC
AND WE OBSERVED THESE
INTERACTIONS BUT DIDN'T LOOK FOR
OTHER INTERACTIONS.
IN FACT WE BELIEVE NOW THAT
THESE PROBABLY ADOPT THE SAME
STRUCTURE THOUGH THIS STILL A
PREDICTION ON OUR PART.
THERE ARE OTHER STRUCTURES
SOLVED OR PROPOSED ON THE BASIS
OF CHEMICAL PROBING.
THIS IN 2003 BY BERKEHOUT AND
THERE'S INTERACTION WITH THE
DIMER INITIATION SITE AND
SHIFTED A BIT BUT THERE'S DOWN
STREAM INTERACTIONS WE DID NOT
SEE.
SIMILAR TO ANOTHER RECENT MODEL
THAT MIKE AND HIS LAB PROPOSED
BASED ON CHEMICAL PROBING.
I SHOULD ALSO POINT OUT THEY'RE
DIFFERENT SIZES.
THESE ARE RNA'S THROUGH THE GAG
START CODE AND THESE ARE
SOMEWHAT LARGER RNAs.
ONE IS IN THE MIDDLE BY THE
GROUP REPORTED.
THIS IS QUITE A BIT DIFFERENT
FROM ALL THESE OTHER PROPOSED
MODELS.
THE COMMON FEATURE IS THEY ALL
HAVE AN EXTENDED POLY A ELEMENT
THAT IS NOT A HAIRPIN IN THE
MO
MONOMER FORM AND WANTED TO SEE
IF THE STRUCTURE IS CONSERVED.
WE DID AN ANALYSIS OF ALL THE
MAJOR HIV SUB TYPES IN THE LOS
ALOMOS COMPENDIUM AND THERE'S
ENERGY OF THE HAIRPIN FORM POLY
A AND HAVE DIFFERENT LOCATIONS
OF BULGES AND NON-CANONICAL BASE
PAIRS BUT CONSERVED THROUGH THE
SEQUENCES.
WE MADE THE POLY A AND DELETED
THESE AND CALL THIS AN IDEALIZED
HELIX AND CALLED IT AN AU AND
GOT RID OF THE BULGES AND LED TO
A 15 DEGREE STABILIZATION.
WE WOULD CALL THIS A POLY A
HAIRPIN WITH NO BULGES OR
IDEALIZED HAIRPIN.
WE MADE THESE SAME MUTATIONS IN
THE INTACT LEADER IN THE NMR
SHOWED WILL POLY A IS A HAIRPIN
IN THE INTACT LEADER AND THIS
HAS THREE GUANOSINES IN THE FIVE
PRIME END WHERE IT WOULD
NORMALLY NOT BE A HAIRPIN.
THIS FORM BINDS NUCLEOCAPSID
LIKE THE DIMERIC FORM AND THIS
IS A MODEL FOR THE CAP 3G AND
WHEN HAVE YOU MORE Gs THE WILD
TYPE IS MONOMERRICK -- MONOMERIC
IT'S A DIMER LONG AS POLY A IS A
HAIR PIN.
AND THIS FORMS A DIMER AND THAT
BINDS NUKE LEE CAPSID IT STILL
HAS AN EXPOSED CAP.
AND WE HAVE THE HAIRPIN FOR THE
POLY A THOUGH THERE'S AN EXPOSED
CAP AND THOUGH IT DIMERIZES IN
CAPSID.
SO WITH ALICE AND SERGEI THEY
ARE SHOWED IF YOU TAKE AND LOOK
AT THE MAL STRAIN IN CELLS IF
THE TWO RNA'S ARE MADE AT
SIMILAR LEVELS ONLY THE CAP 1G
FORM GETS INTO VIRUSES AND SAME
FOR THE NL43 AND THEY'RE BOTH
MADE INDEPENDENTLY IN CELLS BUT
ONLY THE 1G IS COMPETITIVELY
PACKAGE SOD THE 3G THOUGH IT HAS
ALL THE PROTEIN BINDING SITE IT
DIMERIZES AND NUCLEOCAPSID
BINDING ARE NOT SUFFICIENT TO
OVERCOME THE DOMINANT NEGATIVE
EFFECT OF CAP EXPOSURE.
AND AS CONSISTENT WITH PRIOR
FINDINGS WE PUBLISHED IF WE MADE
THIS CORE SIGNAL THAT GETS
CAPPED IN CELLS NOT PACKAGED BUT
IF WE ADD AN RIBOZYME THIS IS
COMPETITIVELY PACKAGED AND ADD A
FEW RESIDUES ON TO THE DIMERIC
PACKAGING AND WHAT ABOUT
FUNCTIONS THAT COULD BE
INFLUENCED BY HETEROGENOUS START
SITE USAGE?
WE ENTERED INTO A COLLABORATION
WITH THE SHEER LAB AND THEY
PLAYED A HUGE ROLE IN THIS WORK
AND THESE ARE TWO VECTORS THAT
WOULD BE CO-TRAN SPECTED INTO
CELLS AND ENCODE FOR RNAs WITH
DIFFERENT COLORS.
THIS IS THE YFP AND CFP.
THEN WHAT WE CAN DO IS PUT
DIFFERENT TRANSCRIPTION START
SITES BASED ON THE WORK OF ALICE
AND SERGEI.
NOW WE CAN HAVE LET'S SAY A
YELLOW RNA BEGINS WITH 1G AND
CYAN RNA WITH 3G.
HERE'S AN EXAMPLE ON THE LEFT
WITH A CYAN COLORED AND YELLOW
COLORED 1G.
YOU CAN SEE THE CYAN IS GROWING
AND MORE INTENSE.
IT LOOKS A LITTLE BIT BETTER
WHERE WE REVERSE THE COLORS.
AND IT'S FWROEG MORE QUICKLY AND
MORE INTENSE THAN THE CYAN
SHOWING IT'S THE 3G FORM THAT IS
MORE EFFICIENTLY TRANSLATED IN
CELLS IN THIS COMPETITIVE ASSAY
AND PLOT THESE AND PICK REGIONS
WHERE WE CAN DO COMPARISONS.
SO WHAT WE NOW KNOW IN OUR
CONTROLS IF WE COMPARE 1G AND 3G
AND THEN OUR 3G NO BULGE IF THE
TWO CONTROLS HAVE THE SAME FIVE
PRIECHL -- PRIME END AND
TRANSLATED WELL AND LOOK AT 1G
VERSUS 3G THE 3G IS MORE
EFFICIENTLY TRANSLATED AND NOW
BY REVERSING THESE TO SHOW AS A
CONTROL IT'S ALWAYS THE 3G MORE
EFFICIENTLY TRANSLATED.
IF WE LOOK AT THE 3G WITH A NO
BULGE YOU CAN SEE THIS IS NOT AS
EFFICIENTLY TRANSLATED IS MORE
EFFICIENTLY TRANSLATED THAN THE
1G AND THEN IN FACT THE 3G WITH
NO BULGE AND THE 3G ARE
TRANSLATED ABOUT EQUALLY WELL.
WHAT DOES THIS ALL MEAN?
WHAT IT MEANS IS THAT IF YOU
HAVE THE WILD TYPE RNA THAT HAS
A POLY A AND HAIRPIN FORM THE
FIVE PRIME CAP IS SEQUESTERED
AND THIS LIKES THE DIMERIZED.
IT HAS HIGH AFFINITY NUKE LEO
CAPSID BINDING SITE AND
TRANSLATION EFFICIENCY IS LOW.
IF YOU HAVE THE 3G FORM OF THE
RNA, THIS HAS AN EXTENDED POLY A
AND EXPOSED FIVE PRIME CAP AND
THE MONOMER AND THIS HAS ALL THE
PROTEIN BINDING SITES BUT
PACKAGING EFFICIENCY IS LOW
BECAUSE THE CAP IS EXPOSED AND
TRANSLATION EFFICIENCY IS HIGH.
WE DON'T JUST THINK IT'S A POLY
A SWITCH BECAUSE IT'S NOT THE
ONLY REGION THAT UNDER GOES
CHANGES AND WE'RE COMPARING THE
FREE ENERGY CALCULATED FOR THE
WILD TYPE SEQUENCE VERSUS THE
ENERGY OF A MUTATED FORM THAT
WOULD BE AN IDEALIZED HAIRPIN
AND HELIX.
YOU CAN SEE THESE ALL HAVE
HIGHLY SUPPRESSED FREE ENERGIES
OF FOLDING.
SO WE THINK THESE DEPRESSED
FOLDING FREE ENERGIES ARE
IMPORTANT FOR ALLOWING THE RNA
TO ADOPT MULTIPLE CONFIRMATIONS.
THE ENTIRE LEADER IS
ENERGETICALLY TUNED TO ENABLE
POLYMORPHISM.
TO CONCLUDE IN TERMS OF
PACKAGING DIMERIZATION EXPOSES
BINDING SITES AND IT'S A
DOMINANT NEGATIVE DETERMINANT OF
PACKAGING.
IF YOU WANT TO GET RNAs TO
VIRUSES YOU HAVE TO EXPOSE THESE
PACKAGING SIGNALS AND SEQUESTER
THE CAP.
IN TERMS OF TRANSLATION CAP
EXPOSURE IS A POSITIVE EFFECTER
AT EFFICIENT TRANSLATION.
THE WORK IN MY LAB STARTED BY
JOSH BROWN NOW A PHYSICIAN
SCIENTIST AT EMORY BUT ALL THE
WORK I TALKED ABOUT WAS CARRIED
OUT BY A CURRENT Ph.D. STUDENT
IN MY LAB.
THANKS.
>> OKAY.
SO I'M GOING TO TELL YOU A
LITTLE BIT OUR TAKE ON TRYING TO
UNDERSTAND WHAT'S HAPPENING
DURING THE TRANSCRIPTION
INITIATION COMPLEX FORMATION.
ALL THIS WORK HAS BEEN DONE BY
THESE TALENTED INDIVIDUALS AND
COLLABORATION WITH THE LABS AND
ZOOM THROUGH THE WORK I
PRESENTED A FEW YEARS AGO HERE
AND CONCENTRATE ON SOME OF THE
RD STRUCTURES WE'RE SEEING RIGHT
NOW.
SO AS A QUICK INTRODUCTION FOR
PEOPLE WHO DON'T THINK ABOUT
TRAN SKRINGS, AND THERE'S A
SITE -- TRANSCRIPTION AND
THERE'S A SITE WITH A NUCLEOTIDE
COMPLIMENTARITY AND THIS IS WHAT
IT RECOGNIZES AND STARTS MAKING
DNA.
AND WE STARTED SOLVING THE
STRUCTURE OF THE RNA COMPETENCE
AND I WON'T SHOW YOU STRUCTURES.
I'LL DO THIS ALL BY CARTOON.
SO IF YOU LOOK AT THE U5PBS IT
HAS HELIX 2 AND 1 AND AS THE A
LINK THAT WAS HARD TO FIGURE OUT
THE FOLD BUT IN THE NMR IT FOLDS
BEAUTIFULLY INTO THE MODULES AND
THEY'RE CLOSED BY A SINGLE BASE
PAIR.
ONCE THE TRNA COMES IN I'LL
COLOR CODE THIS DIFFERENTLY.
THE TRNA COMES IN AND HELIX 1
OPENS UP AND GET TWO OTHER
MODULE FORMATIONS.
AND THIS IS KIND OF SPECIAL AND
THE M5 OVER HERE IS A PART OF
THE TRN AND THE NUCLEOTIDES.
WHAT WE KNOW ABOUT IT HELPS RNA
STRUCTURES FORM BUT CAN ALSO DO
SOMETHING CALLED LONG RANGE
DOCKING INTERACTIONS.
AND USING BASE PAIR STACKS AND
THAT'S WHAT'S HAPPENING HERE.
THE M1 DOCKS ON TO THE NEWLY
FORMED STEM OF THE RNA AND TO AN
M3 DOCKS AND 18 BASE PAIR PRIMER
AND THE FIRST GA THAT IS GOING
TO BE THE INITIATION SITE.
TO US IT LOOKS LIKE A HIDDEN
COMPLEX REPRESSED AND SOMETHING
THAT NEEDS TO BE OPENED UP TO
RECOGNIZE IT.
I'M GOING TO QUICKLY DO THIS
AGAIN AND THIS IS HAPPENING.
YOU HAVE THIS STRUCTURE IT AND
M2 AND M3 ARE SEQUESTERING THE
NUCLEOTIDE PRIMER AND
SEQUESTERING THE FIRST
INITIATION NUCLEOTIDE.
WE INITIALLY THOUGHT IT WAS
GOING TO BE THE NUCLEIC ACID
PROTEIN THAT RIVALLED THIS BUT
THE R2 BOUND TO THE HELIX AND
THIS IS A HIGH AFFINITY BINDING
AND THE JUNCTION GETS REMODULED
AND THE M2, M3, M4 ALL COME OFF
AND THE SECOND RT BINDS AND YOU
CAN GET INITIATION.
SO WE'RE CALLING THIS THE
REMODELER IT.
SOME MODULES ARE CONSERVED AND
SOME STRAINS ARE INSERTED TWO
OTHER MODULES.
THEY'RE VERY IMPORTANT FOR
INITIATIONS.
EVEN IF YOU MAKE MUTATION DON'T
ALLOW THE ARCHITECTURE THIS IS
WORK FROM ALICE'S LAB AND SEE
THE INITIATION SITE THAT'S
SUPPOSED TO START AND IT GETS
VERY MESSED UP EVEN BY TINY
MANIPULATIONS.
IN VITRO LET'S DID AN EXPERIMENT
WHERE YOU TAKE THE FOUR DOCKING
INTERACTIONS OUT AND DELETE THEM
AND YOU WOULD THINK IT SHOULD BE
ABLE TO BIND HERE AND IT
INITIATES ACCURATELY BUT IT
DOESN'T HAPPEN.
50% OF THE STUFF YOU GET OUT IS
INITIATED IN THE WRONG PLACE.
WHAT THIS TELLS US IS ABOUT
GLOBAL ARCHITECTURE IS IMPORTANT
FOR INITIATION.
SO WHAT REALLY THEN GIVES YOU
THE PERFECT COMPLEX?
SO HERE IF YOU HAVE TWO RTs AND
YOU NEED TWO NUCLEIC ACID
MOLECULES BOUND TO THE LOOPS
THEY'RE ALSO THE HIGH AFFINITY
INTERACTIONS NOT THE CHAPERONE
TAIL INTERACTIONS.
WHEN YOU ASSEMBLE THE COMPLEX
EVERYTHING YOU GET IS ALMOST
RACKET.
-- RACKET.
-- ACCURATE.
WHAT WE STARTED TO GET
INTERESTED IN IS WHAT DOES THE
RD MODEL LOOK LIKE?
WHAT WAS FASCINATING WHEN WE DID
THE FIRST TRITRATIONS INTO THE
NMR IT HAS A COMBINING THAT IS A
TINY AMOUNT OF RT WE EXPECTED
THE BOTTOM TIER AND DOUBLE
STRANDED ASSUMED IT WOULD BE
INTERACTING BUT THAT DOESN'T
HAPPEN.
THE 18 BASE PAIRS DO NOT
INTERACT BUT THE U5 STEM THE
MOTIF ALL SHOW MASSIVE
PERTURBATIONS.
WE KNEW IT WOULD BIND
DIFFERENTLY THAN THE OTHER
STRUCTURES THAT ARE OUT THERE.
SO HERE IS THE DATA THEN THAT
MELANIE COLLECTED.
AND WE HAVE SOME INFORMATION NOT
COMPLETE BUT IT ALLOWS US TO
SEE WHAT IS HAPPENING HERE SO
THIS IS KNOWN TO HAVE TWO
STATES.
ONE IS THE BOUND STATE IN WHICH
THE FINGER AND THUMB ARE
SEPARATED AND THEN WE KNOW OF
THE APOS STATE WHERE THE FINGER
AND THUMB ARE IN CLOSE CONTACT
AND WHEN THE FINGER AND THUMBS
ARE OPEN THE ENZYMATIC CLIMBING
IS AVAILABLE AND NUCLEIC ACIDS
CAN BIND IN AND YOU CAN SEE WITH
THE MOVEMENT YOU GET APO AND
BOUND STATE.
IN THE MIDDLE IS A STRUCTURE FOR
THE REMODELER.
WHAT YOU'LL SEE HERE AGAIN IN
THE LEFT AND RIGHT ALL
STRUCTURES WE KNOW OF RIGHT NOW
ARE WHERE THE NUCLEIC ACID BINDS
IN THE DEEP BINDING TARGET BUT
IF YOU LOOK AT THE MODELER IT'S
ACTUALLY BINDING IN A COMPLETELY
DIFFERENT WAY.
IT'S ACTUALLY IN THE CLOSE STATE
AND THERE IS A BINDING
INTERACTION THAT HAPPENS MOSTLY
BY THE THUMB DOMAIN AND I'LL
SHOW YOU A LITTLE BIT OF THAT.
THERE'S A DIFFERENT WHEEL AND
YOU GET THE ENZYMAIYMATIC CLASPY
FROM THE BINDING SITE AND THE
HELIX IS SITTING THERE.
WHAT WE THEN DID IS TOOK THE
THUMB AND SAID CAN WE JUST
LOOK -- THE RESOLUTION WASN'T
THAT GREAT WE WANTED TO SEE IF
YOU CAN GET MORE DETAILS BY
LOOKING AT NMR AND WITH THE
THUMB DOMAIN WE SAW MANY
BEAUTIFUL STRUCTURES AND I'LL
SHOW YOU WHAT IT LOOKS LIKE.
IN THE BLUE IS BOUND.
IN THE GREEN IS HOW THE THUMB
WOULD BE IN ITS NATURAL FORM AND
THIS LOOP BETWEEN H AND THUMB
OUT OF THE RESTING PLACE AND YOU
GET THE ARGININES AND LYSINE AND
THIS STRUCTURE IS DIFFERENT WE
MADE THE TWO MUTANTS LYSINE AND
ARGININE AND YOU GET ONLY ONE RT
BINDING TO THE HELIX 2 STILL AND
THE REMODELLING DOESN'T HAPPEN
SO THE SECOND COMES IN.
IN THAT SCENARIO YOU GET NOTHING
STARTING CORRECTLY.
IF YOU MAKE THE LYSINE AND
ARGININE MUTATION YOU GET THE
SAME THING.
GET SOME BINDING ON BUT THEN THE
RNA'S NOT REMODELLED AND
INITIATION DOESN'T GO ANYWHERE.
THAT IT DOUBLE MUTANT SEEMS TO
BE PRETTY BAD FOR INFECTIVITY.
AND WE SAW THE STRUCTURE OF THAT
DOUBLE MUTANT AND THAT ONE NOW
BINDS LIKE REGULAR RT IT GOES
DEEP IN THE ENZYMATIC POCKET AND
TO ME IT LOOKS LIKE THE ENDS ARE
NOW COMPLETELY CLEARED FROM THE
RNA H AND IT WILL BE HAPPY AND
THERE WON'T BE REMODELLING AT
THE JUNCTION AND THAT EXPLAINS
WHY THE SECOND RT DOESN'T COME
IN.
SO, OUR CURRENT MODEL IS ALL
BASED ON INTERACTIONS WITH THIS
ABULGE AND THE NATURAL STATE
INTERACTS WITH A-BULGE.
WHEN WHAT THE CRYO IS SAYING THE
A BULGE WILL MAKE INTERACTION
WITH THE CONNECTION DOMAIN.
AT THAT POINT WHAT MY HYPOTHESIS
IS IT WILL CAUSE A HINDRANCE
WITH THE RNA H DOMAIN AND THAT'S
WHAT WE'RE CURRENTLY TESTING.
AND THEN THE NMR DATA SHOWS THE
A BULGE WILL INTERACT WITH THE
THUMB DOMAIN TO GIVE A DIFFERENT
KIND OF STEP 2 AFTER WHICH THE
REMODELLING HAPPENS AND THEN THE
SECOND RT BINDS.
AND THEN FINALLY I WANT TO LEAVE
YOU WITH WHAT HAPPENS ONCE THE
WHOLE SYSTEM HAS ASSEMBLED.
LIKE THE FIRST EXPERIMENT WE DID
THIS TO SEE WHEN THE FIRST
REMODELER WILL BE BUMPED BY THE
ENZYMATIC RT COMING OUT AND WE
CAN SAY IT GOES ALL THE WAY UP
TO THIS GAC STRETCH BEFORE IT IS
KICKED OUT.
BUT DURING THAT PROCESS,
INTERESTINGLY WHAT HAPPENS IS
THE 5 PREM REGION FORMS A MOTIF
AND THIS M5 IS NOW IN CONTACT
WITH THE THUMB DOMAIN AFTER THE
BENDING.
PRESUMABLY WE THINK M6 IS ALSO
GOING TO BE IN CONTACT WITH THE
FIRST.
WHAT THAT SUGGESTS TO ME IS
THROUGHOUT THE INITIATION
PROCESS THE FIRST RT IS NOT ONLY
HELPING THE REMODELLING PROCESS
BUT ALSO MAKING SURE THAT IT IS
ORCHESTRATING ALL THE STRUCTURE
FORMATIONS AND INTERACTION.
FINALLY, THESE THINGS COME WITH
A COST.
FOR EXAMPLE, WHEN YOU FORM THESE
MOTIFS RT THEN HAS A PROBLEM
GOING THROUGH THAT AND YOU CAN
SEE THAT WITH THIS DROP HOWEVER
IF YOU HAVE THE TWO NCs SITTING
ON THE TRNA YOU HAVE THAT.
AND MADE FOR THE RNA TO GO
THROUGH THE WHOLE THING
ACCURATELY YOU NEED ALL OF THIS
IN THE RIGHT PLACE.
SO WITH THAT THE MOST IMPORTANT
FEATURE I WANT TO HIGHLIGHT IS
THAT DISCOVERY OF THIS RT
REMODELER FUNCTION WHICH IS
DIFFERENT THAN BOTH STRUCTURALLY
AND FUNCTIONALLY.
WE THINK WE HIGHLIGHTED A ROLE
FOR RT THAT IS NOT AN ENZYME.
IT'S A REEL STRUCTURAL REMODELER
AND THE MOST IMPORTANT THING IS
WE CAME UP WITH A NEW RNA
BINDING SITE WE DIDN'T KNOW
EXISTED AND NEW MODE OF
INTERACTION.
MY OPINION IS WE NEED TO STUDY
THE ENTIRE INITIATION COMPLEX IN
ITS ENTIRE COMPLEX TO FIGURE OUT
HOW THE WHOLE PROCESS GOES.
WITH THAT I THINK I'M ON TIME.
[APPLAUSE]
>> PLEASE COME ON STAGE.
>> THOROUGHLY AMAZING ALL OF
YOUR TALKS, THANK YOU.
I HAVE QUESTIONS FOR ALL OF YOU
BUT PAUL YOU GAVE A COMPLEX
DESCRIPTION OF ZAP AND ACTIVITY
AND SPECIFICITY AND YOU PROBABLY
THANKFULLY ELIMINATED THE ROLE
OF UBIQUITIN ON THE BINDING.
THERE'S MANY E2s.
HOW WELL DO WE KNOW THAT'S THE
ONLY RELEVANT FOR ZAP AND IS
THERE ORTHOLOGOUS EXPERIMENT
WITH UBIQUITIN SHOWING AS THE
NOT PLAYING A ROLE.
TRIM 25 IS AN E3.
>> SHORT ANSWER IS NO.
NO INTERACTIONS WITH UBIQUITIN.
WE HAVEN'T DONE PROTEOME
INHIBITERS AND THAT SORT OF
THING.
>> FOR MIKE, SOME CELLULAR
PROMOTERS I THINK YOU CALL THEM
TWINED HAVE A PROPENSITY TO HAVE
NEARBY SITES IS IT DUE TO
SEQUESTATION.
IS IT KNOWN?
>> IT'S NOT KNOWN BUT WHAT IS
KNOWN IS THERE ARE CERTAIN GENES
IF YOU EXPRESS IN ONE CELL TYPE
YOU GET 1G AND ANOTHER 3G.
SO THAT RAISES QUESTIONS HOW
THERE COULD BE RESPONSE.
THERE'S AN AREA THAT NEEDS TO BE
STUDY MORE CAREFULLY.
>> SHORT QUESTION FOR VICTORIA.
IT'S GREAT TO SEE YOUR MODEL
CONTINUING TO EVOLVE.
WHAT ABOUT ON THE PLUS STRAND
FROM THE PPT?
DO YOU ENVISION STILL A SINGLE
MOLECULE OR THINK THERE'S
REMODELLING REQUIRED THERE AS
WELL?
>> I DON'T KNOW HOW TO ANSWER
THAT QUESTION.
I KNOW PEOPLE THINK THERE'S A
STRUCTURE THERE AND IT'S
POSSIBLE.
THESE REMODELERS CAN REMODEL
THEM TOO BUT WE HAVEN'T TOUCHED
THAT YET.
>> THANK YOU.
>> FOR PAUL, IS THERE PRESSURE
FOR GCs.
>> MOST ARE NOT BUT A SUBSTRATE
ARE.
CONVERSELY IN INSECTS AND
ARTHROPODS AND THOSE VIRUSES ARE
PREDICTED TO BE ZAP SENSITIVE.
>> ALSO A QUESTION FOR PAUL.
I WAS CURIOUS ABOUT YOUR DATA
SHOWING KHNYN MAY BE REDUNDANT
IN FUNCTION AND DID MAPPING OF
KHNYN BUT ARE THEY TRULY
REDUNDANT DO YOU THINK OR
POSSIBLE CELL TYPE SPECIFIC OR
TAR GET DIFFERENCES?
>> RIGHT.
WE HAVEN'T DONE A CELL SURVEY
CLEARLY IN THE PARTICULAR CELLS
THEY USE THEY'RE SORT OF
REDUNDANT.
IN THE RECONSTITUTION ASSAY IT'S
CLEAR KHNYN IS A BIT MORE POTENT
AND WHY WE FOCUSSED ON THAT
PARTICULAR ONE.
WE DON'T REALLY HAVE ANY
INFORMATION ABOUT BP1.
>> THANKS.
>> I HAVE A QUESTION FOR
MICHAEL.
WE KNOW FROM STUDIES IN
HEPATITIS C VIRUS FROM DUKE THAT
6 MA SUPPORTS FUNCTIONING AND IS
IT DIFFERENT IN THE 1G AND 3G
RNA?
>> I'M NOT SURE.
I JUST STARTED LOOKING INTO THAT
LITERATURE.
I DON'T KNOW WHAT PERCENTAGE OF
THE RNAs ARE METHYLATED.
IS IT A SMALL FRACTION OR ARE
THEY ALL OF THEM METHYLATED?
I WAS UNDER THE IMPRESSION THESE
ARE PRETTY SMALL FRACTIONS OF
THE RNA THAT ARE METHYLATED.
SO I WOULD LOVE TO DO THAT
EXPERIMENT BUT WE HAVE TO HAVE A
WAY OF MAKING THOSE SPECIFIC
ADENOSINES METHYLATED.
THERE MAY BE A WAY OF DOING THAT
WITH TECHNOLOGY THAT VICTORIA
HAS DEVELOPED.
BUT WE HAVEN'T TRIED THAT YET.
IT'S A GOOD IDEA.
THANKS.
>> A QUESTION FOR MIKE.
I WAS SURPRISED TO LEARN THE
MECHANISM IS DETERMINING THE
VIRUS IS THIS EFFECT SEEN IN
PRIMARY CELLS ALSO OR IS THAT
SOMETHING --
>> YES.
>> GOOD TO KNOW.
>> THAT IS SOMETHING THAT ALICE
IS LOOKING AT AS WELL.
>> YEAH.
WE SHOULD TALK MORE ABOUT THAT
AND WE HAVE A PAPER WE'LL SEND
TO YOU TO REVIEW SHORTLY.
>> QUESTION FOR MIKE.
SO FOR VIRUSES THAT HAVE I
PACKAGING BUT LOW TRANSLATION
ARE THEY MORE EFFECTIVE VARIANTS
AT THE END OF THE DAY?
>> THAT'S WHAT WAS SHOWN.
SHE DID THE SAME THING THAT
ALICE AND SERGEI FIGURED OUT HOW
TO DO.
THEY WERE ABLE TO MAKE TRANSFECT
CELLS WITH A VECTOR THAT ONLY
MAKES THE 1G.
AND SO THOSE VIRUSES THAT ARE
MADE ARE NOT HEALTHY.
THEY HAVE A REPLICATION DEFECT.
AND IF THEY TRANSFECT WITH A
VECTOR THAT ONLY MAKES THE 3G
THERE'S ALSO A DEFECT.
NORMALLY THE WILD TYPE VIRUS
MAKES BOTH.
THERE'S A MIXTURE.
IT WOULD BE INTERESTING TO KNOW
IF THE PROPORTIONS CHANGE DURING
THE VIRAL REPLICATION CYCLE BUT
NOBODY HAS BEEN ABLE TO FIGURE
OUT HOW TO DO THAT EXPERIMENT
YET.
>> THANK YOU.
>> QUESTION FOR PAUL.
IN YOUR INITIAL EXPERIMENT THE
CAGE NYN KNOCKOUT DIDN'T REDUCE
VIRUS REPLICATION BUT LATER IN
THE EXPERIMENT IT WAS HAVING AN
EFFECT.
I DON'T KNOW IF I MISSED THAT.
IT SEEMS I REQUIRED SINGLE
STRANDED RNA TO WALK WAS THAT
DIFFERENT THAN THE INITIAL VIRUS
YOU WORKED WITH.
>> IT'S IN A FAMILY OF NUCLEASES
AND AT LEAST TWO ARE EXPRESSED
IN THE CELLS OF THE INITIAL TO
SEE THE EFFECT YOU HAVE TO
KNOCKOUT BOTH.
ONCE YOU KNOCKED OUT BOTH YOU
CAN PUT ONE BACK AND ANALYZE THE
FUNCTION THAT WAY.
>> THANK YOU.
>> WHAT IS THE THOUGHT OF CAP
INDEPENDENT TRANSLATION.
>> IT'S BEEN A CONTROVERSIAL
TOPIC A LONG TIME AND WE HAVEN'T
LOOKED AT IT.
HIV USES IT IN GENERAL AND COULD
USE AN IRIS BUT NOT SOMETHING
I'VE LOOKED AT.
>> FOR TRANSLATION THAT WAY?
WHO KNOWS.
>> NEEDS SOME VIROLOGY, STEVE.
>> THANK YOU.
WE HAVE THE NEXT SESSION FROM
DUKE WE HAVE PRIYAMVADA AND
MUNIR AND GNANA.
YOU
>> ALL RIGHT.
IT'S A PLEASURE TO INTRODUCE THE
DUKE CENTER FOR HIV STRUCTURAL
BIOLOGY.
SO THE DUKE CENTER FOR HIV
STRUCTURAL BIOLOGY IS FOCUSSED
ON THE HH1 ENVELOPE YOU SEE
HERE.
AND COLORED ACCORDING TO SITES
OF VULNERABILITY WHERE
ANTIBODIES CAN BIND AND
NEUTRALIZE.
WE HAVE THREE PROJECT.
THE FIRST LOOKS AT THE ROLE OF
THE ENVELOPE IN HIV-1 ENTRY.
THE SECOND LOOK AT HIV-1
ENVELOPE INTERACTION WITH THE B
CELL RECEPTOR TRIGGERING ANTIGEN
PRODUCTION AND THE THIRD LOOK AT
THE ENVELOPE IN CONTROLLING
HIV-1 REBOUND FROM THE LATENT
RESERVOIR.
SO THE THREE PROJECTS ARE
SUPPORTED BY THREE CORE
STRUCTURAL COMPUTATIONAL AND
IMMUNOBIOLOGY.
THE CENTER IS SUPPORTED BY AN
ADMINISTRATIVE CORE AND RUNS A
DEVELOPMENTAL CORE.
THAT'S THE STRUCTURE OF THE
CENTER.
THE TWO TALKS YOU'LL HEAR TODAY
ARE BY DR. ALAM AND GNANAKARAN
AND THE B CELL RECEPTOR PROJECT
2 AND SEE IT'S CLOSE WITH
STRUCTURAL AND IMMUNOBIOLOGY IN
THE TALKS.
TO GIVE AN IDEA OF WHAT WE ARE
DOING AND BIG PICTURE WE'RE
TRYING TO UNDERSTAND THE COMPLEX
PROCESS AND WE'RE TRYING TO
UNDERSTAND HOW THE BCR SENDS
SIGNAL OVER INCLUDING TO T CELL
HELP.
SO SIGNAL ONE IS THE PART YOU'LL
HEAR ABOUT TODAY.
THE SIGNAL 2 ASPECT IS IN
COLLABORATION WITH THE
UNIVERSITY OF OXFORD.
YOU'LL PROBABLY HEAR ABOUT IT AT
A LATER MEETING.
THERE'S ONLY SO MUCH WE CAN
SQUEEZE INTO ONE.
SO THERE'S THREE STRUCTURES OF
PCRs KNOWN SO FAR AND CRYO EM
STRUCTURES AND THEN LEAVE A LOT
OF QUESTIONS UNANSWERED.
HOW DOES THE DYNAMICS LOOK
BECAUSE YOU HAVE THREE
STRUCTURES, TWO IGM AND ONE IGG
STRUCTURE.
THERE ISN'T MUCH TO COMPARE
WITH.
HOW DO THE DYNAMICS LOOK AND HOW
DOES IT SENSE THE ANTIGEN
BINDING AND THEN SENDS THOSE
CONFIRMATIONAL SIGNALS TO WHERE
IT NEEDS TO SIGNAL.
THOSE ARE THE GAPS IN KNOWLEDGE
WE ARE PLANNING TO FILL WITH
PROJECT 2 OVER THE LONG TERM.
THE PROJECT 2 IS BEING
SPEARHEADED BY MONET RALAM AND
THEY HAD POSTERS YESTERDAY
THEY'RE HERE IF YOU HAVE NOT HAD
A CHANCE TO TALK TO THEM, PLEASE
DO.
THE COMPUTATIONAL ASPECT YOU'LL
HEAR ABOUT FROM GNANA HAS BEEN
LED AND MING XIO AND BETWEEN KEN
HIS POSTER WILL BE WELL TAKEN
CARE OF AND TALK TWO FROM THE
STRUCTURAL BIOLOGY CORE AND
ALBERTO BARTESAGHI WILL TALK
ABOUT WHAT THEY CALL THE MAGICAL
NEXT STEP.
HE'LL TALK ABOUT HIS
COMPUTATIONAL PIPELINE FOR
PROCESSING CRYO ET DATA.
SUPPORTING ALBERTO IN THIS
EFFORT ARE TWO GRADUATE STUDENTS
FROM HIS LAB LAURA AND ABBY.
THEY HAVE POSTERS TODAY SO
PLEASE STOP BY AND LEARN MORE
ABOUT THE MAGICAL NEXT STEP.
SO NOW THESE ARE THE TALKS
YOU'LL HEAR ABOUT TODAY.
I ALSO WANTED TO GIVE YOU A
GLIMPSE OF WHAT ELSE IS HOT AND
HAPPENING IN THE CENTER BEYOND
THESE TALKS WE HAVE TIME TO
INCORPORATE IN TODAY'S MEETING.
PROJECT 1 IS CLOSELY SYNERGIZED
WITH THE CORE AND WE'RE LOOKING
AT MANY THINGS BUT THE CRUX OF
THE PROJECT IS WILL TIME SCALE
DYNAMICS.
WE'RE LOOKING AT THE PROCESSING
IN HIV-1 ENTRY WHICH HAPPEN TOO
FA OF THE FOR CONVENTIONAL
METHODS TO CAPTURE THEM.
TWO LABS ARE LEADING AT THE
FOCUS OF THIS EFFORT AND ONE IS
FROM SWITZERLAND.
YOU SEE HERE HIM ON THE LEFT
WITH TWO STUDENTS AND THEN THE
LAB OF RORY HENDERSON FROM DUKE
UNIVERSITY AND SEE HIS LAB ON
THE RIGHT WITH THEIR LAB
MEMBERS.
AND THEY'RE DOING TEMPERATURE
JUMP CRYO EM AND AT DUKE RORY IS
DOING JUMPS ACROSS THE AXIS AND
SIMULATION TO CAPTURE THESE FAST
INTERMEDIATES.
I WANTED TO GIVE A QUICK
OVERVIEW OF THE CRYO EM AND I'LL
TELL YOU WHY.
FOR AN OVERVIEW YOU FREEZE THE
SAMPLE ON THE GRID AND EXCITE IT
BY PULSING A LASER SO THE ICE
AROUND IT MELTS AND YOU
STIMULATE IT WITH A SMALL
MOLECULE OR WHATEVER.
THE MOLECULE MOVES AND BEFORE IT
HAS A CHANCE TO FIGURE OUT WHAT
JUST HAPPENED YOU FREEZE IT BACK
AND THEN HOPEFULLY CAPTURE AN
INTERMEDIATE.
THAT'S THE CRUX THE TECHNOLOGY.
THEY'VE BEEN ABLE TO ESTABLISH
THE TECHNOLOGY IN MANY SYSTEMS
FOR ENVELOPE WE'RE STILL TRYING
TO FIGURING OUT SOME OF THE
THINGS LIKE HOW TO MINIMIZE
REPARATION AND THESE ARE
ENVELOPE SPECIFIC THINGS AND
LEAD TO TECHNOLOGY DEVELOPMENT
APPLICABLE.
BUT FOR NOW THERE IS THE
TECHNOLOGY THERE.
IT RICK LAURENS STUDIED THE
CAPSID SYSTEM AND I HEARD THE
TALKS GOING ON THE CAPSID HERE
IN THE MEETING.
I FELT LIKE A LOT OF THE STUDIES
THAT I HEARD WOULD BENEFIT FROM
THIS TECHNOLOGY.
SO I'LL PUT IT OUT THERE FOR YOU
ALL.
SO THIS IS BASICALLY FROM HIS
REVIEW RECENTLY WHICH TELLS YOU
WHAT THE TECHNIQUE CAN DO AND
CAPTURE INTERMEDIATES YOU WOULD
NOT SEE OTHERWISE.
THAT WAS THE FOCUS OF
HIGHLIGHTING THIS.
WE ARE WORKING ON IT AND
INVESTING TIME ON MAKING THE
METHOD TO ENVELOPE.
IT CAN BE USED FOR OTHER THINGS.
AND YOU CAN SEE THE STRUCTURAL
AND IMMUNOBIOLOGY CORE THE
PHILOSOPHY IS THROUGH THIS.
THE SCIENTIFIC QUESTION PROJECT
3 ASKING IS THE ROLE OF
ORTHOLOGOUS ANTIBODY REBOUND.
IT'S CLOSELY ENMESHED WITH
MULTIPLE INSTITUTIONS AND CORES.
HOW IS THIS BEING DONE?
THE SAMPLES AND THE BIOLOGY ARE
ALL COMING FROM THE CURE WORLD
ON ORTHOLOGOUS ANTIBODIES AND
THE ISOLATION TECHNOLOGIES WHICH
HAVE BEEN WELL DEVELOPED IN THE
VACCINE WORLD ARE HELPING US
ISOLATE THESE ANTIBODIES AND
STUDY THEM.
THE EPITOPE MAPPING AND THE
STRUCTURAL BIOLOGY TECHNIQUES
WELL ESTABLISHED IN THIS
RELEVANT QUESTION OF HOW
ORTHOLOGOUS ANTIBODIES BLOCK
REBOUND AND WHAT CAN WE DO TO
BLOCK THE REBOUND OF THE VIRUSES
NOT BLOCKED BY THE ORTHOLOGOUS
ANTIBODIES?
SO THEN OUR PROJECT 3 TEAM IS
HERE.
THIS IS THE PANEL LAB MEMBERS
HERE YESTERDAY AND UNFORTUNATELY
I DON'T THINK THEY'RE HERE TODAY
BUT ON ZOOM OR STREAM AND THEY
HAVE POSTERS AND KAYLA WILSON
WILL COVER THE IMMUNOBIOLOGY
ASPECT OF THE WORK FROM MY LAB
ELI ZANG WILL PRESENT A
STRUCTURE OF AN ENVELOPE AND
THIS WAS A NICE PICTURE SO I PUT
IT THERE AND TWO ARE POSTERS.
MARLENE AND LOUISA AND YOU SEE
THEM BONDING AT OUR RECENT
ANNUAL MEETING AND WITH THAT I
WILL ASK FOR HIM TO COME DO HIS
TALK.
>> SO I NEED TO TALK ABOUT SOME
OF OUR RECENT WORKS WHERE WE ARE
LOOKING AT THE STRUCTURE AND
SIGNALLING OF B CELL RECEPTORS
EXPRESSING BROADLY NEUTRALIZING
ANTIBODY.
IT'S A HETTERO METRIC
DIMERIZATION AND IT'S SYMMETRIC
BETWEEN THE IMMUNOGLOBULIN AND
THE BETA AND THIS FUNCTIONS AS A
SIGNALLING COMPONENT OF THE BCR
COMPLEX THROUGH THE I10 MOTIF IN
THE ISOLIC TABLE AND ADOPTS THE
ARCHITECTURE OF THE SOLUBLE
ANTIBODY.
WHEN WE THINK WILL ANTIBODY
SHAPE THE CANONICAL Y SHAPE IS
WHAT WE'RE FAMILIAR WITH AND
THEY ADOPT A CONFIGURATION TO
BIND TO CLOSE THE SPACE GLYCANS
AND WITH STRONGER AFFINITY.
AROUND THE 2G AND HAVE SEEN THE
NON-DOMAIN SWAP FDG ANTIBODIES
AROUND ARE CURRENTLY WORKING TO
GET BCR STRUCTURES FOR THEM AS
WELL.
WE HAVE EARLIER HYPOTHESIZED THE
BCRs OF GLYCAN BINDING
ANTIBODIES ADOPT CONFIGURATIONS
AND HAVE STRUCTURALLY DISTINCT
WILL FORMATIONS AND THE
SIGNALLING WOULD BE DISTINCT.
WE EXPRESSED B CELL RECEPTORS ON
B CELLS AND THESE WERE AFFINITY
PURIFIED AND BY PHYSICAL
ANALYSIS INDICATED THE PURIFIED
BCRs IN MYCELLAR FORMS IS INTACT
WITH THE BETA COMPONENT AND THE
ANALYSIS SHOWED THE SOLUBLE
ANTIBODIES AND AS EXPECTED IT
ADOPTS THE CANONICAL Y STHAP.
-- SHAPE.
AND WANTED TO MAKE SURE IT BINDS
TO THE RELEVANT AND THE BOUND
THE ANTIBODY WITH DIFFERENT
AFFINITIES AND THE IGG AND IGM
PCRs BIND WITH THE SAME
AFFINITY.
THE COMPLEX IS PURIFIED AND
FUNCTIONAL IN RECOGNIZING
RELEVANT HIV TRIMERS.
IN THE NEXT SLIDES I'LL GO OVER
WHAT WE KNOW FROM THE TWO BRR
STRUCTURES RECENTLY REPORTED AND
WHAT WE DO NOT KNOW.
SO THIS IS A MOUSE BGM FOR A
MOLECULE AND WHAT WE LEARNED
FROM THIS STRUCTURE IS THAT
FIRST THE ASYMMETRIC
STOICHIOMETRY DEFINED BY
CHEMICAL STUDIES WAS CONFORMED
IN THE BCR STRUCTURE.
WE LEARNED AND THE INFORMATION
CAME LARGELY FROM THE TRUNCATED
BCR DUE TO THE FLEXIBILITY OF
THE FAB AND THAT WAS REPORTED
HOWEVER, THE BCR DYNAMICS
INFORMATION WAS NOT STUDIED DUE
TO TECHNICAL HURDLES AND THUS WE
HAVE NO INFORMATION ABOUT THE
FLEXIBILITY AND HOW THAT MIGHT
INTACT THE REST OF THE BCR
DOMAINS.
AND THE MECHANISM IS DEFINED.
THE SECOND SET OF STRUCTURE AND
HERE WE HAVE BOTH THE IGM AND
IGG CLASS BCR AND BOTH ADOPT THE
SAME ORGANIZATION THAT WAS
PREVIOUSLY REPORTED FOR THE
MOUSE BCR.
WE GET ATOMIC INTERACTION AT THE
DOMAIN AND THE TRANS MEMBRANE
DOMAIN.
SO THE OVER ALL ORGANIZATION OF
THE BCR WAS SIMILAR FOR BOTH THE
IGG AS WELL AS THE IGM CLASS AND
AGAIN THERE IS NO INFORMATION
ABOUT THE FAB AND THUS WE KNOW
THERE WAS NO DESCRIPTION OF THE
BCR DYNAMICS THAT RESULTS FROM
THE OBSERVED FLEXIBILITY OF THE
FAB.
ONE KEY INFORMATION THAT CAME
FROM THAT STUDY WAS A KEY
DIFFERENCE BETWEEN THE IGG AND
IGM BCR CLASS.
THAT DIFFERENCE COMES FROM HOW
THE FC DOMAIN INTERACTED OR
ASSOCIATED WITH AG ALPHA.
SO IF YOU LOOK HERE IN THE IG
AND BCR AND THIS IS THE IGL IN
BLUE YOU CAN SEE IT'S STACKED
TIGHTLY WITH THE FC DOMAIN
UNLIKE WHAT WE HAVE SEEN THE IGG
BCR STRUCTURE.
WE THINK THIS IS PERHAPS
RELEVANT FOR SIGNALLING BECAUSE
WHEN WE LOOK AT SIGNALLING AND
I'M SHOWING DATA FOR CALCIUM
FLUX RESPONSES FOR 2G12IGG CELLS
AND IGM WE FOUND THE SIGNALLING
WAS DIFFERENT FOR THE SAME PANEL
OF PRITRIMER AND OBSERVED WEAKE
SIGNALLING IN THE IGG EXPRESSING
CELLS COMPARED TO THOSE
EXPRESSING IGM.
SO WE DID SINGLE CRYO EM STUDIES
TO GET THE STRUCTURE OF BOTH THE
I SHAPED 2G12 AS WELL AS THE CD4
BINDING SITE THAT ADOPTS THE
CANONICAL SHAPE CONFIGURATION.
WE WERE ABLE TO LOOK AT THE
STRUCTURE AND THE STRUCTURE CAME
FROM A SMALL SUBSET OF
PARTICLES.
THE MAJORITY SHOWED STRUCTURES
WHERE WE COULD VISUALIZE THE
SIGNALLING COMPONENT AND THE FC
ARE THE FIVE ASSOCIATED WITH THE
FC.
THIS IS TRUE FOR CG12 AND IGM
AND ANOTHER SHOWING OF THE FAB
AND TOOK THE STRATEGY OF
RECONSTRUCTING IT BY USING
INFORMATION THAT CAME FROM THE
MAJORITY OF THE PARTICLE WITH
THE BOTTOM PART OF THE BCRs AND
COMPLETED THE STRUCTURE.
CONSISTENT WITH THE PUBLISHED
BCR STRUCTURE AND HERE BGR12 AND
THE ASYMMETRY OF THE BETA IN ITS
ORIENTATION WITH THE
IMMUNOGLOBULIN.
THAT WAS CONSISTENTLY SEEN IN
BOTH OF OUR STRUCTURES.
UNLIKE WHAT WAS REPORTED IN THE
STRUCK STTURES WE OBSERVED KEY
DIFFERENCES.
I'M SHOWING THE IGG BCR
PUBLISHED STRUCTURE WITH OUR
RECONSTRUCTED STRUCTURE.
HERE YOU CAN SEE THE VARIATION
FROM THE PUBLISHED STRUCTURE ON
THE IG ALPHA DOMAIN AND HERE IS
IT THE LOOP THAT CONNECTS TO THE
TRANS MEMBRANE DOMAIN AND ALSO
WITHIN THE BCR WE OBSERVED MUCH
VARIABILITY WHEN WE COMPARED OR
ALIGNED OUR STRUCTURE TO THOSE
OF THE IGG STRUCTURE THAT WAS
PUBLISHED.
WHEN WE LOOKED AT THE
INTERACTION OF THE C LAMBDA 3
DOMAIN WE FOUND THE
IMMUNOGLOBULIN HAS STRONG
INTERACTIONS WITH THE DOMAIN AND
THIS IS CONSISTENT WITH WHAT WAS
DESCRIBED IN THE IGG PUBLISHED
STRUCTURE.
AND THESE STRONGER INTERACTIONS
WERE NOT OBSERVED WHEN WE LOOKED
AT THE INTERACTIONS BETWEEN THE
SIGNALLING COMPONENT AND THE FC
AND AGAIN THIS WAS AN INDCAT
INDCATION -- INDICATION OR
SUPPORTS THE MALLEABILITY OF THE
STRUCTURE.
WE WERE ABLE TO GET
RECONSTRUCTED MAPS OF THE
STRUCTURE AND THESE COMPOSITE
MAPS VARIED IN THEIR RELATIVE
ORIENTATION OF THE FACT TO THE
FC.
WHEN YOU FIT THEM ALL TOGETHER
YOU CAN SEE THE FAB ARE
POSITIONED WITH A LOT OF
VARIATIONS WITH RESPECT TO HOW
THEY ORIENTED WITH THE FC AND
REST OF THE BCR.
YOU CAN GET A SENSE OF THE
EXTEND MUCH THIS VARIATION IF
YOU LOOK AT THE DISTANCE WHICH
RANGES FROM 132 TO 167 ENSTRON
AND YOU SEE HOW IT'S ORIENTED
RELATIVE TO THE FC HERE WE GOT
GOOD DENSITY OF THE FAB AND WE
SEE FLEXIBILITY IN THE
ORIENTATION OF THE FAB AS
EXPECTED ADOPTS THE CANONICAL Y
CONFIGURATION AND WE COULD SEE
WHERE THE VARIABILITY OF WHERE
IT OCCURS AND THIS IS THE FAB FC
HINGE REGION AND THEN WE CR CAN
CME1 AND 2 AND DISTAL TO IT IN
THE CME 4 WE SEE A LOT OF
FLEXIBILITY.
IT'S NOT ONLY COMING FROM THE
FAB BUT WE ARE SEEING
FLEXIBILITY WITHIN THE FC
DOMAIN.
NOW, IF YOU PUT SIDE BY SIDE TO
WHAT THE PUBLISHED STRUCTURE
LOOKS LIKE AND SO THIS IS NOW
THE IGM THE SAME CLASS AS THE
CH31IGM WE MAPPED AROUND YOU CAN
SEE THE STRUCTURE THAT'S
PUBLISHED IS AN ABRIDGED
STRUCTURE AND STANDS TALL AND
FULLY EXTENDED FORM FOR THE
PLASMA MEMBRANE WHEREAS THE
THREE COMPOSITE MAPS SHOW
WIDESPREAD VARIATION AT THE FAB
FROM THE FAB DOMAIN ALL THE WAY
DOWN EXTENDING TO THE FC.
SO THIS LEADS US TO OUR CURRENT
MODEL WHICH IS DISTINCT FROM THE
PUBLISHED MODEL AND WE ARE
HYPOTHESIZING THE BENDING OF THE
IMMUNOGLOBULIN UNIT ALTERS THE
SIGNALLING COMPONENT AND AN
IMPLICATION OF THIS IS WHEN
ANTIGEN BINDS BASED ON THIS
MODEL WE HYPOTHESIZE THE BINDING
WILL INDUCE SOME CHANGES TO
THIS.
SO THAT'S SOMETHING WE ARE
CURRENTLY WORKING TO UNDERSTAND.
IN CONCLUSION WE ARE IN
AGREEMENT THAT CH31 AND THE BCR
STRUCTURES SHOW ASYMMETRIC
ORGANIZATION.
THE WITH I SHAPED ECTODOMAIN
SHOW CONFIRMATIONAL VARIABILITY
THAT INDICATE HIGHLY DYNAMIC
STRUCTURES.
WE OBSERVED DISTINCT
CONFIRMATIONS OF THE FAB REGIONS
AS WELL AS ADDITIONAL HINGE
REGION WITHIN THE FC THAT
CONTRIBUTES TO FURTHER FAB
DYNAMICS.
SO WE HYPOTHESIZED THAT THE
HINGE POINTS WE'VE IDENTIFIED IN
OUR STRUCTURES ARE KEY FOR BCR
ALLOSTERIC SIGNALLING AND I
ACKNOWLEDGE THE LARGE NUMBER OF
PEOPLE WHO CONTRIBUTED TO THIS
WORK.
THANK YOU.
>> THANKS AGAIN AND I WOULD LIKE
TO THANKS DAVID FOR THE
OPPORTUNITY TO PRESENT RESULTS
FROM OUR COMPUTATIONAL WORK.
IT FOCUSES ON LOOKING AT
ANTIGENS AND WHAT HAPPENS
OUTSIDE AND HOW DO YOU STABILIZE
THEM AND OUR INTEREST WAS TO SEE
HOW THEY GET PRESENTED INSIDE
WHEN GIVEN AS A VACCINE ANTIGEN
FOR EXAMPLE.
AND NORMALLY WHEN YOU HAVE GIVEN
VACCINE ANTIGEN IT'S BEEN TAKEN
UP PART NEF IMMUNE AND THEY CAN
PRESENT TO THE BCR OR THE
ANTIGEN CAN BE PRESENTED TO THE
BCR AS YOU HEARD FROM MUNIR AND
THEN TO THE PLASMA CELL
DIFFERENTIATION.
THE KEY COMPONENTS IS THE CELL
MEDIATED IMMUNITY WHICH IS HOW
YOU'RE GETTING THE T CELL HELP
TOE INDUCE THE BCR IN THE CD8 T
CELLS AND OUR FOCUS IS SEEING
HOW THE ANTIGENS ARE GETTING
RECOGNIZED BY THE B CELL AND T
CELL AS A VACCINE ANTIGEN AND WE
LOOKED AT THE T CELL REPLICATION
A
AND YOU'LL NOTICE FROM WHAT YOU
HEARD FROM MUNIR THEY'RE MODULAR
AND IT BINDS BUT THERE'S A
SIGNALLING THAT IS DIFFERENT AND
THERE'S A SEQUENCE AND TALK
ABOUT THE SIGNALLING.
WHEN WE STARTED THIS WORK I
THINK SOME TIME BACK -- LET ME
GO BACK TO THIS ONE.
WHEN WE HAVE THE DIFFERENT
MEMBRANES WE FOUND WHEN YOU HAVE
A DISORDERED MEMBRANE THEY GET
BURIED INSIDE BUT WHEN YOU GO IN
A DIFFERENT MEMBRANE LIKE A
LIPID MEMBRANE THEY GET EXPOSED.
SO THIS LED US TO PROPOSE A
MODEL WHERE MAYBE WHEN YOU HAVE
THE SIGNALLING MOTIF IN
DIFFERENT REGIONS THEY'RE HIDDEN
AND THEY COME AND MOVE ON TO A
DIFFERENT LOCALIZATION POINT IN
THE MEMBRANE DOMAIN AND THEY
DECIDE TO GET EXPOSED AND LEAD
TO A DOWN STREAM SIGNALLING.
AND I THINK SOME OF THE WORK HAS
BEEN SUPPORTED EXPERIMENTALLY.
SO TYPICALLY WHEN PEOPLE TALK
ABOUT HOW THE CELLULAR BINDING
LEAD TO INTRASIGNALLING
DIFFERENT MODELS HAVE BEEN
PROPOSED SOME CONFIRMATIONAL
CHANGE OR THE EMERGING CONCEPT
SEEMS TO BE THAT THE MEMBRANES
CAN PLAY AN ACTIVE ROLE AND
SOMETHING RELEVANT HAPPENS TO
BCR AND I DON'T NEED TO GO IN
THE DETAILS OF IT.
I THINK AS MUNIR MENTIONED
PEOPLE THOUGHT IT WAS A
SYMMETRIC COMPLEX BUT TURNED OUT
TO BE THE STRUCTURES CONFIRM
WITH ASYMMETRIC.
SO ONE OF THE GOALS OF THE
COMPUTATIONAL WORK IS THE EFFECT
OF HAVING AN ASYMMETRY IN THE
CONTEXT OF MEMBRANE AND THOUGH
EVERYONE HAS KNOWN FOR A LONG
TIME THE BCR IS EFFECTIVE FOR
IMMUNITY WE DON'T KNOW THE
MECHANISMS.
SOME MODELS HAVE BEEN PROPOSED
WHERE THE ANTIGEN BINDS AND
CREATE CROSS LINKING AND LEADS
TO SOME SORT OF SIGNALLING.
AND RECENTLY THERE WAS A
DIFFERENT CALLED DISSOCIATION
ACTIVATION STATE AND IT STAYS
TOGETHER AND SOME ANTIGEN BINDS
AND SOME SHIFTS INTO A MORE OF
AN ACTIVE BOND.
AND WE'LL LOOK AT THE
SIMULATIONS.
SO WE SELECTED THE STIMULATION
WORK ON THE BCR COMPLEX BECAUSE
IT'S A WAY WE CAN CONNECT TO THE
WORK GOING ON AT DUKE.
AND MODEL THE MONOMER AND AND WE
IS ASSEMBLED THE COMPLEX B CELL
LIKE BILAYER.
TODAY I THINK I'LL TALK MAINLY
ABOUT THE OUTCOME OF THE
SIMULATIONS FROM THIS KIND OF A
COMPLEX WHERE THE B CELL
MEMBRANE WAS CAPTURED WITH THE
DIFFERENT LIPID COMPOSITION IN
AN EXPERIMENTAL PAPER.
WE TRIED TO USE THE COMBINATION
OF LIPIDS AND LOOK AT THE EFFECT
OF ANTIGEN BOUND VERSUS NOT
BOUND AND SEE WHETHER WE CAN SEE
ANYTHING MORE IN THE BCR
ACTIVATION.
WE FIND FOUR INTERESTING
DYNAMICAL MOTIONS EVENTS FROM
SIMULATION AND IT'S SOMETHING
I'LL GO THROUGH IN THE NEXT FEW
SLIDES.
ONE OF THE FIRST THINGS WE
NOTICED ANTIGEN INDUCES CHANGES
ACROSS THE WHOLE BCR.
SO HERE WE COMPARE THE
COMPLEXABILITY OF BOUND AND
UNBOUND AND THERE'S A REGION
WHERE THE BOUND REGION GETS
RIGIDIFIED AND THE SECOND THING
WE NOTICED AND THEY COME MORE
TOWARDS ALONG THE Z AXIS IF YOU
LOOK AT THE CASE HERE THE ANGLE
REDUCES IN ALL THE TRANS
MEMBRANE HELLISMSS -- HELICES
AND THE IGL BETA.
THEN THE THIRD PART WE OBSERVED
IS IF YOU FOCUS ONLY ON THE
SIGNALLING MOTIF THE ANTIGEN
BINDING SEEMS TO REDUCE SOME
INTERACTIONS MAINLY IN THE
REGIONS OF THIS NEAR THE
MEMBRANE REGION AND INCREASED
CONTEXT IN THE REGIONS WHICH ARE
LIKE THE EXTRA CELLULAR AND WHAT
CAPTURED IN THIS CONTACT
FREQUENCY MAP YOU SEE.
AND THERE'S ONE OTHER ASPECT WE
WANTED TO SEE BECAUSE WE KNOW
REGARDLESS OF WHATEVER THE MODEL
A CROSS LINK O
OOR DISASSOCI
OR DISASSOCIATION/ACTIVATION
THEY NEED TO COME TOGETHER.
THIS COULD HAPPEN THROUGH MANY
WAYS.
MAYBE THREES RE-ORIENTATION OF
THE TRANS MEMBRANE REGION WHERE
THINGS BECOME MORE STICKY AND
PUT OTHER BCRs TOGETHER OR
ALTERNATIVELY YOU COULD HAVE A
CASE WHERE SOMETHING CHANGES
BECAUSE THE TRANS MEMBRANE
REGION CHANGES SOMEWHAT AND
CHANGES TO LIPID COMPOSITION
AROUND IT AND COULD LEAD TO A
DIFFERENT LOCALIZATION IN THE
MEMBRANE.
AGAIN JUST TO FIND OUT THESE ARE
SOME PAPERS OF QUESTIONS PEOPLE
HAVE ASKED BEFORE.
AND WE WERE CURIOUS THAT I
MENTIONED THE THIRD THING WHEN
THE ANTIGEN BINDS IT BECOMES
MORE STRAIGHT.
AND IN A MEMBRANE-BASED SYSTEM
THAT CAUSES SOME HYDROTROPHIC
MISMATCH AND THE TRENDS IS
THEY'LL GO TO A MEMBRANE DOMAIN
WHICH IS THICKER.
SO I THINK YOU HEARD FROM HUANG
YESTERDAY WE HAVE DEVELOPED THE
CROSS LINK AND THE LIPID MIXING
WHEN YOU HAVE A COMPLEX LIPID IT
TAKES A LONG TIME TO MAKE SO WE
TOOK THIS AND RAN EXTENSIVE
SIMULATIONS.
AND WHAT WE ARE SHOWING HERE THE
COARSE-GRAINED SIMULATIONS AND
WE'RE LOOKING AND ASKING THE
QUESTION WITHIN THE SHORT REGION
ARE THE LIPIDS CHANGING WITH THE
ANTIGENS BOUND VERSUS NOT BOUND.
WE SEE SOME CHANGES WHEN ANTIGEN
IS BOUND VERSUS NOT BOUND.
WE SEE INCREASED IN CHOLESTEROL
AND LIPIDS SOISHTED WITH MORE
ORDER DOMAIN OR A THICKER
DOMAIN.
THIS RAISES WHETHER THERE'S AN
EFFECT WHERE THIS COULD LEAD TO
SOME SORT OF LOCALIZATION AND IN
SUMMARY WHAT WE ARE FINDING OUT
IS THAT WHEN THE ANTIGEN BINDS
THERE SEEMS TO BE A DIFFERENTIAL
MOTION WITH ALLOSTERIC
INTERACTIONS AND THIRD WE SEE
THE ALTERATIONS ARE IN THE
SIGNALLING MOTIF AND THE IDEA IS
THE SIGNALLING MOTIF MIGHT
IMPACT THE ONE MOTIF EXPOSED.
AND AGAIN I THINK MOST THE WORK
WAS DONE BY HUANG DO AND NATURAL
WAS INVOLVED IN THE
COMPUTATIONAL BIOLOGY CENTER HIV
CENTER GROUP AT DUKE.
AND THAT'S ALL I HAVE.
THANK YOU.
>> I'LL MENTION WE ARE IN THE
CORE OF STRUCTURAL BIOLOGY AND
ONE MANDATE IS TO PROVIDE
SUPPORT IN TERMS OF MUTANT
KNOWLEDGE THAT CAN BE APPLIED TO
THE BIOLOGY AND ONE WE'RE
EXCITED ABOUT IS SINGLE
CRYOELECTRON TOMOGRAPHY AND IN A
NUTSHELL WHAT THE TECHNOLOGY
ALLOWS YOU TO DO IS TO LOOK AT
LOOK AT STRUCTURAL BIOLOGY SO
YOU CAN LOOK AT THE CELL.
IT'S TOO THICK TO GET THROUGH
THE MICROSCOPE AND NEED TO THIN
IT THROUGH THE PROCESS.
ONCE YOU HAVE IT THIN ENOUGH YOU
CAN TAKE PROJEBBICTIONS AND LOO
FOR REPEATING FEATURES AND THAT
COULD BE LIKE THE ENVELOPE OR
ANY OTHER PROTEIN YOU HAVE IN
THE CELL IN THIS CASE AND
COMBINE THOSE VOLUMES BY USING
TOMOGRAPHY TO GET INFORMATION
FROM YOUR DATA.
TO GIVE A SENSE WHERE THE FIELD
IS.
IT'S BEEN AROUND A LONG TIME BUT
RECENTLY HAS COME A LONG WAY IN
TERMS OF TECHNOLOGY DEVELOPMENT.
THIS IS THE LAY OF WHAT CAN BE
ACCOMPLISHED THESE DAYS.
SO IN THIS GRAPH YOU HAVE ON THE
X AXIS THE COMPLEXITY OF THE
DIFFERENT SAMPLES.
AT THE BEGINNING ARE THE
PURIFIED SAMPLE WHAT YOU
TYPICALLY DO WITH CRYO EM AND
YOU SEE IN VITRO PROTEIN LIKE
HIV GAG AND THEN YOU MOVE NEXT
STEP BACTERIAL SAMPLES AND TO
THE RIGHT IS THE LARGER
EUKARYOTIC CELLS TO THIN THE
SAMPLE.
AND THE STRUCTURES ON THE GRAPH
THE HIGHER THEY WILL THE HIGHER
THE RESOLUTION.
FOR THE SAMPLES LIKE PURIFIED
PROTEIN WE CAN GET PURIFIED
RESOLUTION USING CRYO ET AND
THEN AS YOU GO TO THE RIGHT TO
THE MORE COMPLEX SAMPLES THE
RESOLUTION SUFFERS A LITTLE BIT
BUT WE'RE TRYING TO DEVELOP
METHODS TO TRY TO IMPROVE THE
FEATURES WE CAN SEE EVEN IN THE
MORE COMPLEX SAMPLES.
ONE THING THAT IS IMPORTANT TO
NOTE HERE IS THAT ONE PARAMETER
THAT IS IMPORTANT IS THE NUMBER
OF COPIES WE HAVE ACCESS TO AND
OBVIOUSLY IN THE MORE COMPLEX
SAMPLES AND HAD PHYSIOLOGICAL
CONCENTRATIONS AND YOU CAN SEE
THESE START TO GO DOWN AS YOU
MOVE TO THE RIGHT.
HOPEFULLY IT GIVES AN OVERVIEW
OF WHERE THE FIELD IS ON THINGS
MOVING FORWARD IT'S VERY
EXCITING.
ONE OF THE THINGS THAT HAS
DRIVEN IMPROVEMENTS IS
PREPARATION AND ALSO IMAGING AND
WHAT MY LAB HAS BEEN FOCUSSING
ON IS ONCE YOU HAVE YOUR DATA
HOW YOU IMPROVE THE DATA
ANALYSIS TO GET THE MOST OUT OF
YOUR SERIES.
AND YOU CAN SEE HERE THAT
ACTUALLY THE COMBINATION OF
DIFFERENT IMPROVEMENTS AND
METHODS CAN TAKE YOU FROM WHAT
IS ON THE LEFT THROUGH START
SEEING SIDE CHAINS AND MODELLING
BECOMES A LOT EASIER.
SO JUST TO GIVE AN OVERVIEW.
IF YOU'RE FAMILIAR WITH CRYO EM
CRYO ET HAS A FEW MORE STEPS AND
TRYING TO DESCRIBE THE
COMPLEXITY OF THE PROCESS AND
THERE'S MULTIPLE STEPS.
RIGHT NOW IT'S QUITE INVOLVED
AND YOU NEED TO KNOW A LOT OF
THE DIFFERENT TOOLS AND PIECE
THEM TOGETHER IN THE RIGHT WAY
TO GET A HIGH RESOLUTION
STRUCTURE AT THE END.
SO WE RECENTLY WROTE A REVIEW
WITH ONE OF MY STUDENTS WHO WILL
HAVE A POSTER IN THE AFTERNOON,
JAVI, AND WE REVIEWED THE ENTIRE
LAY OF THE LAND IN SINGLE CRYO
TOMOGRAPHY AND HOPEFULLY IT WILL
BE A GOOD RESOURCE FOR THE
COMMUNITY.
AND IN ADDRESSING THE COMPLEXITY
WE WERE TRYING TO BUILD A LAYER
ON TOP TO MAKE THE TECHNOLOGY
AVAILABLE TO PEOPLE USING THIS
STRUCTURES AND THE NAME OF THE
PLATFORM WE DEVELOPED AND WE
RELEASED LAST YEAR AND IT'S
WEB-BASED AND INTUITIVE AND
ALLOWS YOU ONCE YOU GET YOUR
HANDS ON YOUR DATA YOU CAN PUSH
IT THROUGH THIS PIPELINE AND
ANALYZE AND SEE THE INTERMEDIATE
RESULTS AND HOPE IT WILL MAKE A
BIG DIFFERENCE FOR THE FIELD AND
THIS IS OUR LAB THAT WAS
FACILITATED TREMENDOUSLY AND THE
AMOUNT OF DATA WE CAN PROCESS
AND THE TRAINING YOU NEED TO
HAVE TO CONVERT RAW DATA TO HIGH
RESOLUTION STRUCTURES.
WE'RE EXCITED ABOUT THIS TOOL.
WE ARE HOPING IT WILL BE USEFUL
IN A LOT OF THE PROJECTS WITHIN
THE PROJECTS UP OUR CENTER BUT
ALSO MORE BROADLY IN OTHER
CASES.
SO ONE OF THE THINGS WE DO AND
ONE EXAMPLE ON VIRUSES IS WE CAN
ANALYZE THE VULNERABILITY IN
SITU AND WE LOOKED AT ONE TEST
DATA SET ON THE RIBOSOME ON
BACTERIAL CELLS AND CAN T CELL
THE DIFFERENT FACTORS ATTACHED
TO THE RIBOSOMES.
YOU CAN DO ALL THESE KINDS OF
THINGS IN A MORE ROUTINE WAY
SIMILAR TO WHAT YOU CAN DO WITH
SINGLE CRYO.
AND THIS LEVERAGES THE POWER IN
THE TOOLS AND THIS IS ON A
STRUCTURE WE SAW RECENTLY IN
COLLABORATION WITH THE IMMATURE
CAPSID PROTEIN IN MISS CASE THEY
IMAGED THIS AND YOU CAN SEE HERE
IN THE UPPER LEFT YOU CAN SEE
THE PROTEIN INSIDE THE BLPs AND
ESSENTIALLY ONCE YOU TAKE THE
TOMMO -- TOMOGRAM AND CUT OUT
THE POSITIONS WHERE YOU HAVE THE
GUT PROTEIN.
ALL THAT INVOLVES DOING A LOT OF
PREPARATIONS IN 3-D.
YOU NEED TO DO DETECTION,
SEGMENTATION AND FIND ALL THE
EXAMPLES AND WE HAVE THE SUB
VOLUMES THAT CAPTURE A SMALL
PIECE OF THE HEXAMERIC LATTICE
AND YOU NEED THINGS FOR THE HIGH
RESOLUTION AND ONCE YOU HAVE
THOSE YOU HAVE TO ALIGN THEM IN
3-D AND GET A 3-D STRUCTURE AND
THIS SHOWING DIFFERENT STEPS IN
STAGES IN ORDER TO INCREASE
RESOLUTION AND EVENTUALLY WE
ENDED UP WITH A RESOLUTION
RECONSTRUCTION YOU CAN SEE WITH
THE CYAN PART HIGHLIGHTED.
YOU CAN SEE IN THE STRUCTURES
YOU CAN SEE SIDE CHAINS MAKING
IT EASIER TO PRODUCE ATOMIC
MODELS FOR THE DENSITY.
THAT WAS GETTING TO HIGH
RESOLUTION AND BECAUSE IT WAS IN
SITU YOU CAN MAP BACK AND SEE
HOW UNIFORM THE DISTRIBUTION IS
OR WHETHER THERE'S VARIATIONS
FROM THE STRUCTURE.
ALL OF THAT THIS IS ONE EXAMPLE
OF THE TOOLS.
AND THE BIGGEST CHALLENGE IS HOW
TO FIND THE FEATURE THAT YOU
WANT TO WILL USE TO GET HIGH
RESOLUTION.
AND THESE TOOLS HELP YOU SPEED
UP THE PROCESS OF MINING THE
DATA AND LOOKING FOR THINGS THAT
ARE INTERESTING.
WITHOUT GETTING INTO THE DETAILS
AND YOU GET THESE THAT REPEAT
WITHIN YOUR VOLUMES AND CAN
CHOOSE THERE'S SOMETHING I
RECOGNIZE I WANT TO GO AFTER
THAT AND PUSH IT FORWARD.
THESE TAKE A MINUTE TO
UNDERSTAND HOW THEY LOOK BUT
THEY'RE USEFUL.
WE HAVE THE SARS COV2 VIRUS AND
AND YOU ISOLATE WHAT IS ATTACHED
TO THE MEMBRANE AND IF YOU ZERO
IN ON THAT YOU CAN PICK YOUR
PARTICLES FROM THE DATA SET AND
START PRODUCING STRUCTURES USING
THE AVERAGING AND IN THIS CASE
WE DID CLASSIFICATION AND THAT
GIVES US THE STATE OF THE SARS
COV2 SPIKE BUT THIS IS AGAIN A
GENERAL PURPOSE TOOL YOU CAN USE
FOR MINING CELLULAR TOMOGRAMS.
I WANTED TO ACKNOWLEDGE PEOPLE
IN MY LAB AND THE TWO MAIN
FORCES BEHIND DEVELOPING THIS
AND ACKNOWLEDGE COLLABORATORS
AND THE SUPPORT THAT MAKES ALL
THE WORK POSSIBLE.
THANK YOU.
PLEASE
>> PLEASE COME UP TO THE STAGE.
>> HELLO.
QUESTION FOR MUNIR AND GNANA
COOL WORK WITH THE B CELL
RECEPTORS.
HOW SPECIFIC ON EPITOPE AND
PERITOPE AND IT SEEMS IT WOULD
AFFECT THE REGION IN SIGNALLING.
DO YOU THINK DIFFERENT EPITOPES
MAY HAVE DIFFERENT SIGNALLING
AFFECTS AND IS THAT A BASIS FOR
IMMUNODOMINANCE?
>> I THINK THAT'S WHAT WE LIKE
TO UNDERSTAND RIGHT NOW.
WHEN WE LOOKED AT THE PURIFIED
BCR WHEN WE LOOKED AT HOW THEY
BIND THEY LOOK AT THE AFFINITY
AND THE RATES ON AND OFF RATES
AREN'T VERY DIFFERENT FROM WHAT
WE OBSERVED FROM THE SOLUBLE
ANTIBODIES AND FRAGMENT.
THE SPECIFICITY WAS DEFINED BY
THE SEQUENCES.
NOW, WHEN YOU BRING IN THE OTHER
COMPONENTS OF THE B CELL
RECEPTOR AND THAT MIGHT CHANGE
BECAUSE WHAT WE THE MODEL BASED
ON OUR STRUCTURE AND THE
TRANSMISSION AND WE SHOWED
THERE'S A LOT OF COMPLEXABILITY
AND VARIABILITY AND THE ANTIGEN
BINDING WILL AFFECT THAT
FLEXIBILITY.
AND THAT IS WHAT WOULD BE
TRANSMITTED TO EACH OF THE
DOMAINS OF THE BCR AND
SUBSEQUENTLY TO THE SIGNALLING
MOLECULE BUT I THINK WE ARE NOW
WORKING TO LOOK AT ANTIGEN BOUND
COMPLEX AND THAT WILL GIVE US
THE CLUE TO ANSWER YOU'RE
LOOKING FOR.
>> DO YOU THINK IT MATTERS IF
ONE FAB ISN'T BOUND OR BOTH?
>> THAT'S AN INTERESTING
QUESTION BECAUSE WE THINK BOTH
FAB NEEDS TO BE ENGAGED AND WE
HAVE A LOT OF DATA LOOKING AT
THE SIGNALLING AND IF YOU TOOK A
MONOMERIC ANTIGEN IN VITRO AS
WELL AS EX VIVO B CELLS FROM
MOUSE MODELS WE DON'T SEE AC
ACTIVATION TO ONE FAB TO ONE
ANTIGEN INTERACTION WE THINK
BOTH FAB NEED TO BE ENGAGED.
>> COOL, THANK YOU.
6:
>> HI.
I HAVE A QUESTION FOR ALBERTO.
FOR YOUR MOLECULAR PATTERN
MEANING HAVE YOU CHECKED HOW IT
FAIRS ON CELLULAR TOMOGRAMS THAT
ARE A LITTLE BIT MORE COMPLEX?
>> HERE I SHOWED THE EXAMPLE
THAT WAS MORE RELEVANT TO THINGS
HAPPENING IN THE CORE.
BUT YES, WE CAN COMBINE
INTERIORS AND WE HAVE SEVERAL
CASES WE VALIDATED THESE AND
HOPEFULLY AN UPCOMING
PUBLICATION WILL HAVE THE
DETAILS BUT THE ANSWER IS YES.
>> IT'S A FASCINATING TOOL.
I'M REALLY EXCITED.
>> FOR US IT'S BEEN A GAME
CHANGER BAYS NOW WE DON'T NEED
TO SPEND TIME IN THE DATA.
>> THIS MAY BE A QUESTION
PRINCIPALLY FOR RICK IT'S BEEN
GREAT TO HAVE ENVELOPE PEOPLE IN
THE MEETING.
I WAS TAKEN BY THE FACT YOU
THOUGHT THE TILT OF THE TRANS
MEMBRANE DOMAIN CHANGED ON
BINDING AND THAT WOULD
PROBABLY -- IT'S ALWAYS
INTERESTING TO THINK OF CAUSE
AND EFFECT BUT YOU POINTED OUT
IT WOULD CHANGE THE LIPID
FLUIDITY AND WONDER IF YOU CAN
DRIVE IT IN THE OPPOSITE
DIRECTION.
CAN YOU CHANGE THE AFFINITY FOR
LIGAND BY CHANGING THE LIPID
COMPOSITION OF THE MEMBRANES?
>> ONE QUESTION WE ASKED IS THE
BCR WORK WAS DONE WITH
DETERGENTS.
WE WANTED TO SEE WHAT HAPPENS
WHEN HAVE YOU THE SIMPLE PRPC
VERSUS LIKE A B CELL MEMBRANE
AND WE DO SEE A DIFFERENCE.
I THINK THE MEMBRANE MIGHT
COMPOSITION YOU'RE LOOKING AT
THE BCRs MAY AFFECT THE WAY
THISE THE INFORMATION GETS BOUND
TO THE TRANS MEMBRANE REGIONS.
SO I THINK ONE OF THE MAIN THING
WE SEE IS WHENEVER YOU HAVE THE
MAME BRAIN THE MOTIONS ARE
LARGER.
AND SO WE ARE STILL TRYING TO
HAVE ASSIMILATION AND IT'S NOT
BY CHANCE YOU'RE SEEING
DIFFERENCES.
WE'RE ELEVATING THAT PART RIGHT
NOW.
>> THANK YOU.
[APPLAUSE]
IT'S A GREAT PLEASURE FOR ME TO
INTRODUCE OUR CENTER THAT IS
DIRECTED BY TATIANA POLENOVA AND
MYSELF AND I'LL GO VERY BRIEFLY
THROUGH A BIT OF THE BACKGROUND
OF THE CENTER.
WE HAVE P.I.s ALL OVER THE WORLD
AND WHAT IS IMPORTANT WE HAVE
WORKED ON NUCLEAR INTERPMPORT A
INTEGRATION AND WE HAVE CORES
AND COLLABORATORS AND THE
COMPUTATIONAL CORE AND CRYO EM
CORE AND VIROLOGY CORE.
IN THE DEVELOPMENT CORE AND KWAE
HAVE A MENTORED SCIENTIST
PROGRAM AND STARTED A RESEARCH
EMBEDDING PROGRAM WHICH IS
UNFORTUNATELY SORT OF THROWING A
WRENCH THAT THAT AND HAVE
SEVERAL EXAMPLES WHERE PEOPLE
FROM ONE P.I.'S LAB GO TO
ANOTHER P.I.s LAB AND STAY IN
THAT LAB FOR WEEKS OR MONTHS AND
LEARN TECHNOLOGIES AND THEN
BRING THAT BACK TO THEIR HOME
LAB AND THIS IS SOMETHING THAT
WE STRONGLY FEEL IS A BIG
ADVANCEMENT IN TRYING TO TRAIN
OUR NEXT GENERATION SCIENTISTS.
OUR CENTER ALWAYS HAD A STRONG
EMPHASIS ON TECHNOLOGY
DEVELOPMENT AND ALL OF OUR CORES
CONTRIBUTE TO ALL THE PROJECT
BUT WE ALSO REALLY THINK ABOUT
WHERE WE COULD PUSH THE
TECHNOLOGIES.
AND UP PARTICULAR THE MOLECULE
CORRELATED MICROSCOPY CORE
CONTRIBUTES TO THE DIFFERENT
PROJECTS IN THE LAB AND THE
COMPUTATIONAL CORE.
WE HAVE PUBLISHED OVER THE LAST
YEAR AND SEVERAL OF THE
PUBLICATIONS ARE PUBLICATIONS
BETWEEN DIFFERENT CENTERS
UNFORTUNATELY MOST OUR POSTERS
WERE YESTERDAY AND HOPEFULLY YOU
CAN SEE THE POST DOCS LISTED
HERE AND I'M SURE THEY'LL TALK
TO YOU AGAIN AND THERE ARE FOUR
POSTERS WE HAVE TODAY.
THE TALKS TODAY ARE FROM THREE
PEOPLE TATYANA WILL TALK ABOUT
THE MATURATION INHIBITOR BINDING
AND SHE WILL HIGHLIGHT THE
TECHNOLOGY OF SPINNING SOLID
STATE AND MEL IS LA KANE WILL
TALK ABOUT THE RANBP2 IN HIV-1
INFECTION AND YONG WILL TALK
ABOUT THE BIOLOGY OF HOST VIRUS
INTERACTIONS.
WITH THIS I HAND IT OVER TO
TATIANA WHO WILL TALK ABOUT THE
SCIENCE THAT'S GOING ON.
>> GOOD MORNING, EVERYBODY.
IT'S ALWAYS GREAT TO BE HERE AND
THANK YOU FOR BRINGING US TO THE
SCIENCE CELEBRATION.
AND I KNOW THAT WE ARE
INTEGRATING THAT WITH THE
TEMPERATURE IN THE ROOM SO I'LL
TRY TO BE ENERGETIC TO GIVE YOU
AN OVERVIEW OF WHAT WE DO AND
IT'S SOMETHING THAT HAS NOT YET
BEEN DISCUSSED OVER THE PAST DAY
AND A HALF, MATURATION WITH THE
VIRAL HIV LIFE CYCLE TARGETED BY
SMALL MOLECULES.
WHAT I WILL TELL YOU TODAY ABOUT
IS THE WORK TRULY AN INTEGRATION
FROM SEVERAL CENTERS AND THE
LONG STANDING COLLABORATION
BETWEEN THE CORE AND MYSELF AND
THE POSTDOC CENTER LABS DONE BY
TALENTED PEOPLE AT THE
UNIVERSITY OF DELAWARE AND IS WE
HAVE AN NMR CORE CALLED SPINNING
NMR TO LOOK AT STRUCTURAL
BIOLOGY.
THEY TALKED ABOUT THE DIFFERENT
TYPE OF NMR AND THIS IS MAGIC
ANGLE SPINNING NMR.
WHAT'S SO MAGIC?
AS FAR AS THE APPLICATIONS GO WE
CAN LOOK AT THINGS THAT ARE
PRETTY LARGE AND INTACT CELLS
AND ORGANELLES AND THERE'S CRYO
ELECTRIC MICROSCOPY AND ISOLATE
YOUR ASSEMBLIES OR OR PARTICLES
AND FOR MAGIC ANGLE SPINNING AND
MORE WE USE A SPECIAL SAMPLE
CONTAINER CALLED ROTORS.
WE HAVE TO ROTATE THE SAMPLES
VERY FAST UP TO 110,000
REVOLUTIONS PER SECOND AT TIME
110 KILO-HERTZ AND THIS IS
IMPORTANT FOR THE TYPE OF
APPLICATIONS I WILL BE
DISCUSSING.
WE HAVE MULTIDIMENSIONAL
EXPERIMENTS AND WE CAN DETERMINE
STATIC STRUCTURES AND WE
DEMONSTRATED OVER THE YEARS AND
ALSO WHAT'S IMPORTANT IS WE GET
INFORMATION ABOUT DYNAMICS FROM
THE MEASUREMENTS.
AND WHEN WE COUPLE OUR
EXPERIMENTS WITH POWERFUL
MOLECULAR DYNAMIC SIMULATIONS
AND MECHANICAL STIMULATIONS WE
CAN LOOK AT DYNAMIC STRUCTURES.
LOOKING AT THE ARCHITECTURE AND
MOTION.
THE ADVANTAGE IS QUITE A FEW.
AND IN SOLUTION NMR WE CAN SEE
THE QUOTE, UNQUOTE, IN VISIBLE
STRUCTURES AND I'LL SHOW SOME
EXAMPLES TODAY AND WE CAN LOOK
AT SMALL MOLECULES WITH DETAIL
AND THERE'S NO SYMMETRY REQUIRED
AND AS THE ONLY EXPERIMENTAL
TECHNIQUE THAT YIELDS DYNAMIC
INFORMATION IN ASSEMBLIES WITH
RESOLUTION ON A BROAD
TEMPERATURE RANGE FROM
PHYSIOLOGICAL TO CRYO GENIC.
OF COURSE THERE ARE SOME
COMPLICATIONS BUT TODAY I WANT
TO TALK ABOUT AND HIGHLIGHT THE
POWER OF LOOKING AT HIV
STRUCTURAL VIRAL GENE.
WE'RE INTERESTED IN A NUMBER OF
STEPS IN THE VIRAL LIFE CYCLE.
AND WE HAVE LOOKED AT THE GAPS
IN ARCHITECTURE AND LOOKING AT
THE INTERACTIONS WITH
MICROTUBULES AND THERE'S A
POSTER AND WE HAVE LOOKED AT
INTERACTIONS WITH THE FACTOR AND
AS I TOLD YOU WE'LL FOCUS ON THE
MATURATION PART OF THE VIRAL
LIFE CYCLE.
AND THIS IS A PROCESS TIGHTLY
REGULATED SPATIALLY AND THE LAST
PART IS THE PEPTIDE AND THE
MATURE VARIANTS WITH THE
CANONICAL CAPSIDS AND THE LAST
STEP IS ACCOMPANIED BY A LARGE
MORPHOLOGICAL CHANGE FROM
IMMATURE TO MATURE LATTICE AND
THE ORGANIZATIONAL WILL DCA IS
SUCH THE CR TERMINAL DOMAIN HAS
THE SHAPE OF A GOBLET AND SO
MATURATION HAS BECOME AN
ATTRACTION FOR THE INHIBITORS
AND OTHER COLLEAGUES HAVE WORKED
OVER THE YEARS ON DESIGNING
POTENT INHIBIT.
THE FIRST IS THE MOLECULE WHICH
IS WILL AN ACID AND THERE'S ALSO
A UNIQUE INHIBITOR DESIGN BY
PFIZER.
WHAT HAPPENS WHEN THE MATURATION
INHIBITORS ARE ADDED TO THE
VIRUS THEIR VIRIONS ARE FORMED
AND STABILIZE THE BUNDLE.
THE PROCESS HAS BEEN BEAUTIFULLY
STUDIED BY MANY PEOPLE IN THE
AUDIENCE EXPERIMENTS IN
CRYSTALLOGRAPHY AND
COMPUTATIONALLY AND IT MICRO-ID
THERE'S BEAUTIFUL INSIGHTS AND
DECIDED TO LOOK INTO THE
COLLABORATION A FEW YEARS AGO
BECAUSE ALL OF THESE STUDIES AT
THE TIME HAD DETAILS OF THE
BINDING WERE NOT CLEAR.
SO WE CONTRIBUTED TO THE
PLETHORA OF EXPERIMENTS AND
CALCULATIONS BY SOLVING THE
STRUCTURAL DVM PUBLISHED LAST
YEAR AND QUICKLY WALK YOU
THROUGH THAT BEFORE I TALK SOME
OF THE RECENT RESULTS BECAUSE
I'M NOT SURE I DISCUSSED IT.
WE HAVE THE PROTONS AN
DETERMINED THE PROTEIN STRUCTURE
AND USED PROTONS AND LOOKED AT
INHIBITOR INTERACTIONS AND ALSO
AS MANY KNOW FROM THE SAMPLING
OF THE VIRUS THIS WAS REQUIRED
AND IT'S EXPLOIT THIS PROPERTY
TO IN VITRO ASSEMBLIES AND
LOOKING AT THE IP6 DYNAMICS BY
USING PHOSPHORUS NMR SO THIS IS
OUR TOOL KIT.
LET'S LOOK AT HOW WE LOOKED AT
THE ASSEMBLY.
AND THE RESOLUTION IS SO HIGH WE
CAN PULL OUT DISTANCE RESTRAINTS
AND THEY'RE SOME OF THE BEST
DEFINED STRUCTURE AND WE CAN SEE
DYNAMICALLY DISORDERED PARTS OF
THE PROTEIN NOT VISIBLE IN THE
CRYO EM AND DETERMINED BY THE
RESIDUES AS WELL AS THE FIRST OF
THE CTD.
HOW DO WE LOOK AT THE BINDING OF
BVM?
WE HAVE SPECIAL CORRELATIONS
BETWEEN THE PROTEINS AND THE
SMALL MOLECULES AND YOU CAN SEE
HERE THE OVERLAYS OF THE ASIMPLY
ASSEMBLY AND CONCLUDED IT'S
BOUND WITH IP6 ALSO WHAT OTHERS
DISCOVERED AND IMPORTANTLY WE
CAN UNAMBIGUOUSLY DETERMINE THE
MOLECULE AND IT POINTS TO THE 6
AND POINTS TO THIS REGION AN
I'LL TELL YOU BASED ON MANY
EXPERIMENTS AND THE CALCULATION
OF THE STRUCTURE WE FOUND BVM
BINDS SIMULTANEOUS LY WE SEE IT
BINDS TO THREE CHAINS AND THAT'S
WHY YOU KNOW IT DIDN'T DEVELOP
TO CLINICAL TRIALS AND WE
CHARACTERIZED THE DETAILS IN THE
TWO THAT ARE INTERESTING AND
IMPORTANT THE MUTATION THE
VARIANTS WE HAVE STUDIED AND
BASED ON EXPERIMENTAL DATA AND
MORE MICROSCOPY BASED WE
CONCLUDED SEVERAL INTERESTING
THINGS.
FIRST IF WE LOOK AT THE MUTANT
WHAT HAPPENS IS BVM SIMPLY DOES
NOT BYPRODUCT THERE AND THE
VARIANT IS MORE DYNAMIC.
WE CAN GET THIS INFORMATION
DIRECTLY FROM THE NMR SPECTRA
AND THIS DOES BIND BVM BUT MORE
VARIANT THAN THE WILD TYPE AND
IT'S MORE DYNAMIC THAN THE WILD
TYPE.
THIS IS A VERY UNIQUE WAY THE
RESISTANCE IS CONFERRED.
ANOTHER WAY TO LOOK AT WILD TYPE
AND RESISTANCE VARIANTS IS TO
LOOK AT THE PHOSPHORUS BY
PROBING IP6 ITSELF AND
ORIENTATION AND DYNAMICS AND
WALK YOU THROUGH THAT QUICKLY
BECAUSE IT'S INTERESTING.
IF WE DO THE EXPERIMENTS WE'RE
LOOKING AT THE SIGNALS AND ONE
PROBES BOTH DYNAMIC MOLECULES
AND HERE WILD TYPE AND THEN A1V
VARIANTS WITH OR WITHOUT BVM OR
THE SIGNAL AND THAT'S A GREAT
CONTROL EXPERIMENT.
NOW WE HAVE CROSS
PHOSPHORYLIZATION AND THERE IS
SOMETHING INTERESTING HERE.
WHEN WE HAVE NO B VM THERE IS NO
SIGNAL.
THIS TELLS US IP6 IS DYNAMIC
INSIDE THE CORE ON THE TIME
SCALES OF MILLISECONDS.
IT MEANS WE KNOW FROM ALL THE
EXPERIMENTS THIS ROTATION INSIDE
THE CORE.
SO NOW WHAT HAPPENS WHEN WE ADD
BVM?
IN THE CASE OF THE WILD TYPE WE
GET SIGNAL BUT YOU CAN SEE THERE
ARE DIFFERENT TYPES OF SIGNALS.
SO A1V THERE IS NO SIGNAL WHICH
ARE IS NOT SURPRISING BECAUSE
A1V DOES NOT BIND BVM AND IT'S
DYNAMIC.
THE EXPERIMENTS ARE QUICK.
THIS COULD BE A NICE SCREENING.
WE CAN DO CORRELATION
EXPERIMENTS IN TWO DIMENSIONAL
WAYS AND THESE ARE CORRELATIONS
BETWEEN PHOSPHORUS AND PROTON
ATOMS OF THE PROTEIN AND BASED
ON THE EXPERIMENT AND YOU CAN
SEE THE WILD TYPE IN BLACK AND A
VARIANT IN PURPLE.
VERY DIFFERENT CORRELATIONS.
THIS TELLS US IP6 ADOPTS A
DIFFERENT ORIENTATION AND YOU
CAN SEE IN THE WILD TYPE IT'S
TILTED IN THIS WAY AND IT ADOPTS
A MORE PARALLEL ORIENTATION.
THESE ARE THE EXPERIMENTS WE
APPLY WHEN WE LOOK AT THE
MOLECULE.
THIS IS THE PFIZER COMPOUND WITH
A PROPELLER SHAPE AND THREE
CHEMICAL GROUPS AND BENZENE AND
TRI FLUORO ANTIMER AND WE SOLVED
THE SOLVED OF THE CRYSTAL AND
THEN LOOKED AT THE BINDING.
THE FIRST THING DISCOVERED WAS
INTERESTING.
BF BINDS ISO METRICALLY AND THIS
IS PRETTY OBVIOUS.
WE LOOKED AT THE INTERACTION AND
THE RESIDUES PARTICIPATING HERE
ARE HIGHLIGHTED.
BASED ON THESE CORRELATIONS THEY
WERE ABILITY TO DETERMINE THE
BINDING MOLD AND YOU CAN SEE THE
SMALLER MOLECULE ACTING ON THE
CHAINS AND THE IP6 AND LED TO
DIFFERENT ORIENTATION.
ONE THING UNIQUE AND UNEXPECTED
IN OUR STUDY WAS THAT WE WERE
ABLE TO ASSIGN BIASED ON THE
SPECTRA FOR THE RAS ARE
DIFFERENT.
THIS COULD SERVE AS A QUICK
PROBE OF ENAN TIMER STRUCTURE
AND IT'S AN INHIBITOR AND
STARTED WITH ERIC AND COLLEAGUES
LOOKING AT THE SECOND GENERATION
MATURATION OF INHIBITERS AND
ANALOGS OF BVM AND THEY HAVE
DIFFERENT GROUPS AND SLIGHTLY
LONGER.
AND THERE WAS THE NUCLEIC ACID
DOMAIN AND THERE'S A
STABILIZATION OF THE SP 1 TAIL
AND THE NC RESIDUES.
WE'RE WORKING TOWARDS TO THE
STRUCTURE AND HAVE CLUES WHAT
HAPPENS AS FAR AS THE BINDING
AND HOPEFULLY TELL YOU IN THE
NEXT YEAR OR TWO.
AND WE CAN GET ATOMIC RESOLUTION
STRUCTURES OF PROTEINS
ASSEMBLIES AND COMPLEXES WITH
SMALL MOLECULES AND WE DON'T
NEED TO DO ASYMMETRY AND WE'RE
WORKING TOWARDS UNDERSTANDING
HOW THE UNIQUE SECOND GENERATION
IN WHY INHIBITORS TO THE HELIX
BUNDLE AND I WANT TO ONCE AGAIN
THANK THE PEOPLE WHO HAVE BEEN
INVOLVED IN THE WORK.
I CAN'T STRESS ENOUGH HOW
IMPORTANT THE CENTERS ARE TO
PROMOTE THESE KINDS OF
INTEGRATIVE COLLABORATIVE
STUDIES AND THANK YOU VERY MUCH
FOR LISTENING.
>> SO I'LL TALK ABOUT RANBP2 AND
IT LOCALIZES TO THE NUCLEAR CORE
COMPLEX AT THE END TERMINUS.
AND IT INHIBITS THE INTEGRATION
COMPLEX OF HIV AND OTHER PRIMATE
LENTI VIRUSES BECAUSE OF THIS
THE VIRAL CAPSID IS THE
DETERMINATE FOR SENSITIVITY AND
CAPSID INTERACTION A MAJOR DE
TERMINUS AND THIS IS SENSITIVE
TO MX2 BUT IF WE HAVE A CAPSID
MUTANT THAT DOESN'T BIND OR
KNOCKOUT CELLS OR CYCLOSPORINE
IT'S SOMETIME ENHANCED IN THESE
SITUATIONS.
BUT THIS IS ONLY TRUE FOR HIV-1
AND NOT HIV-2.
CONTROL CELLS BOTH VIRUS
SENSITIVE TO MX2 BUT IT REMAINS
SENSITIVE EVEN WHEN NOT BOUND TO
PSYCYCLOPHILIN AND ONE OF THE
QUESTIONS WE'RE INTERESTED IS
WHAT ARE INVOLVED IN THE PORE OF
AND THE ABILITY TO INHIBIT
INFECTION.
STUDIES HAVE DEMONSTRATED
INTERACTIONS BETWEEN THE CAPSID
BUT THEY MAY NOT HAVE FUNCTIONAL
CONSEQUENCES AND WHEN WE KNOCKED
DOWN CERTAIN METHODS THERE WAS
AN INDICATION THEY'RE PLAYING A
ROLE BUT WILL THERE COULD BE
CONFOUNDING RESULTS.
HOW CAN WE DETERMINE WHAT THEY
ARE?
SRNA COULD MESS UP THE PROBLEM
AND IF YOU DO MORE PRECISE
ENGINEERING USING A CRISPR 9 AD
APPROACH YOU CAN SEPARATE THEM
FROM IT THEIR DOMAIN DELETED.
I WANT TO HIGHLIGHT THE WORK HAS
DIDN'T DONE BY TWO OUTSTANDING
TECHNICIANS IN MY LAB HAILEY AND
ANANYA AND WE'RE FOCUSSED ON THE
PEN
PENTIM
PENTIMERS THIS BINDS THE VIRAL
CAPSID AND WHEN WE KNOCK IT DOWN
YOU SEE THIS NICE RING AROUND
THE NUCLEUS BUT WHEN YOU KNOCK
IT DOWN IT'S MISLOCALIZED AND NO
LONGER INHIBITING INFECTION.
BECAUSE OF THESE THE EXONS AND
WHAT THEY LOOK LIKE AND A
WESTERN SHOWING WE GET THE SMALL
REDUCTION IN THE SIZE OF THE
PROTEIN.
WHEN WE LOOK AT THE CELLS YOU
CAN SEE BOTH PROTEINS ARE AT THE
NUCLEAR ENVELOPE AND IT'S ONE OF
THE FIRST NUCLEAR CORE TO GO
THERE AT THEN OF MITOMITOSIS.
AND THEY BIND AS THE WILL GD9V
AND ALL THREE CAPSIDS ALSO STILL
BIND WHEN LACKING THE
CYCLOPHILIN HOMOLOGY AND SIT
INDEPENDENT BETWEEN RANBP2 AND
THE CAPSID AND WHEN WE LOOK AT
THE INFECTIVITY MX2 WITH AND
WITHOUT WE SEE INTERESTING
THING.
A SMALL BUT REPRODUCIBLE IN
HIV-1 AND LACK OF REPRODUCTION
IN THE CELLS IN CONTRAST TO
HIV-2 MORE SENSITIVE THAN HIV-1
BUT REMAINS SENSITIVE TO MX2 IN
THE CELLS.
WE CAN TAKE THE CONCLUSION OF
THE SENSITIVITY ONE STEP FURTHER
AND THE DOMAIN IS REQUIRED FOR
HIV-1 ONE TO BE SENSITIVE BUT
NOT HIV-2.
WE NEXT ASKED IF THE DELETION OF
THE DOMAIN HAD SIMILAR EFFECTS
WE SEE AN INCREASE IN
INTEGRATION IN LAMIN ASSOCIATED
DOMAINS AND INCREASE IN GENE
DENSE REGIONS.
AS FAR AS INTEGRATION SITE
SELECTION THEY APPEAR TO PLAY A
SIMILAR ROLE.
AND WE TOOK YOU'RE CONTROL AND
TRANSDUCED THEM WITH OUR MX2 AND
TRANS FECTED THEM WITH AND ADDED
THE EXPRESSION AND INFECTED WITH
THE PURO VIRUS AND QUANTIFIED
INFECTIVITY AND ON THE FAR RIGHT
ARE THE CONTROL CELLS WITH AND
WITHOUT MX2 AND SOME REQUIRE
VIRAL INFECTION AND ACTIVITY.
AND THESE ARE IN CONTROL CELLS
AND SEE A DIFFERENT PICTURE
LOOKING AT THE DEL SIP CELLS.
THESE MEMBERS ARE OF THE
COMPLEX.
THE VIRUS BECOMES MORE SENSITIVE
TO DEPLETION OF THIS WHEN IT'S
GONE AND WE ALSO SEE INTERESTING
THINGS WITH THE SIGHT J JO --
CYTOPLASMIC MOUSE AND THIS IS
WHAT WE SEE WITH GV9V INFECTION
IN OUR CONTROL CELLS.
WE SEE REDUCTION UPON DEPLETION
OF THE KNOBS RESCUED BY MX2
EXPRESSION AND SEE A SIMILAR
EFFECT OF THE 155 DEPLETION AND
THINK THE CAPSID INTERACTIONS
ARE AFFECTING THE INFECTION AND
SENSITIVITY BUT THIS IS A LITTLE
BIT AT THE END SO WHAT ARE THE
OTHER DOMAINS DOING FOR VIRAL
INFECTION?
AND THIS IS THE DOMAIN
ARCHITECTURE AND WE SEE AN
PICTURE LOOKING AT HIV INFECTION
IN CELLS.
WE SAW A SMALLER EFFECT ON
INFECTIVITY AND THIS IS THE DE
MAIN ARCHITECTURE AND NOW WE SEE
WE HAVE NO EFFECT ON INFECTIVITY
OR SENSITIVITY OF THE VIRUS IN
THE CELLS.
SO THIS IS TELLING US THERE ARE
COMPLEX INTERACTS BETWEEN THE C
TERMINAL DOMAIN THAT AFFECT
HIV-1 INFECTION AND MX2
SENSITIVITY.
WE SEE SMALL EFFECT BUT MX2
ACTIVITY IS ABROGATED AND WHILE
IT'S SENSITIVE TO THE IT
CYCLOPHILIN HOMOLOGY IT'S A NO
THE WHEN YOU GET RID OF THIS AND
SENSITIVE TO MX2 AND LOOK AT
LOCALIZATION OF THE CELLS AROUND
RANBP2 STILL GOES TO THE NUCLEAR
ENVELOPE BUT MX2 IS LOCALIZED
AND THINK THE RAMIFICATIONS ARE
AFFECTING THROUGH DIRECT
INTERACTION THE VIRAL CAPSID AND
INDIRECTLY THROUGH THE MX2
LOCALIZATION.
IN CONTROL CELLS HIV-1 AND HIV-2
ARE SENSITIVE TO MX2 AND HIV-1
IS RESISTANT AND HIV-2 IS
NEGATIVE AND WHEN WE REMOVE BOTH
BOTH ARE SENSITIVE.
AGAIN THE INTERACTIONS ARE
COMPLEX BETWEEN THE CAPSID AND
MX2 SENSITIVITY AND THINK THE
FOURTH BINDING DOMAIN PLAYS AN
INDIRECT ROLE AND INSPENCEABLE
FOR HIV INFECTION.
AND WE INDICATE IT'S INTERACTING
WITH THE CAPSID AND FG
INDEPENDENT MANNER BUT THE
EXPRESSION OF THE TRUNCATED
RANBP2 IS REQUIRED AND MX2 IS
REQUIRED FOR NUCLEAR ENVELOPE
LOCALIZING AND ANTIVIRAL
ACTIVITY.
AND WITH THAT I WANT TO THANK
EVERYBODY IN MY LAB AND ANANYA
IS GOING TO MADISON FOR GRAD
SCHOOL THIS FALL AND WILL BE
MISSED AND THANK YOU FOR THE
FUNDING AND IT'S BEEN GREAT TO
BE PART OF THIS GROUP.
>> OKAY.
SO I'M NOW STANDING BETWEEN YOU
AND MEALS.
I'LL TRY TO SPEAK AS FAST AS
MELISSA.
SO WHAT I'M TRYING TO STUDY HERE
IS APPLICATION OF A SINGLE
PARTICLE CRYO EM AND NOT CRYO-ED
FOR IN SITU STUDIES IN A NATIVE
SELL ENVIRONMENT.
FOR THE FIRST PART OF MY TALK
I'LL TALK BRIEFLY ON VIRUS WORK
BUT THIS IS THE TREND IN THE
FIELD AS YOU HEARD ABOUT
YESTERDAY AND TALK ABOUT
STRUCTURAL BIOLOGY AND MY LAB IS
ALSO LOOKING AT THE SINGLE
PARTICLE CRY SCRO -- CRYO EM
FROM MAMMALIAL CELLS AND GET A
RANGE AND THIS WORK IS
SPEARHEADED AND CO-LED BY MY LAB
WITH HELP WITH RACHEL AND FOR
THE INTEREST OF TIME I WILL ONLY
PRESENT ONE STRUCTURE TO YOU
TODAY OF THIS CAREFUL VIRUS.
SO WHY THE TREATMENT?
WHAT IS PFO.
IT'S BACTERIAL TOXINS ON THE
LIPID MEMBRANE.
THIS IS A FIRST APPLIED TO THE
MATURE VIRAL VLPs AND LABS USE
THIS FOR CRYO EM WORK AND THIS
IS MATURE CAPSIDS AND CAN WE
APPLY THIS BECAUSE IT'S CLOSE TO
THE MEMBRANE.
DOES IT WORK FOR TREATMENT?
AND YOU CAN COMPARE FROM A
COMPARISON OF THE BEFORE AND
AFTER TREATMENT YOU CAN CLEARLY
SEE THE PORES FORM AS INDICATED
BY ARROWS AND WE'RE VERY HAPPY
TO SEE IT WORKS ON THE IMMATURE
VLP AND IT APPEARS TO BE INTACT.
IT'S NOW DISRUPTING THE LATTICE.
IT POKE HOLE TO HELP INFLUX OF
DRUG OR INHIBITOR PRECURSORS SO
WHETHER THEY'RE OPTIMIZED WILL
HELP WITH PERMEABILITY.
WE USE THIS TO STUDY A FEW
DIFFERENT DRUG COMPOUNDS WHEREAS
I SAID I'LL ONLY TALK TO YOU
ABOUT A DOUBLE TREATED VLP.
SO IT WORKED WELL AND GOT HIGH
RESOLUTION OF 2.1 IN RESOLUTION
AND LOOKS AT THE BVM AND YOU SEE
NICE IP6 ON TOP OF THE BUNDLE OF
CSP1 AND FROM THE SIDE VIEW YOU
SEE THE IG6 AND BVM IS WELL
DEFINED DENSITY.
WE KNOW PRECISELY THE LOCATION
AND CAN FIT A NICE MODEL INTO
IT.
HOW ABOUT LENACAPAVIR AND IF I
TAKE THIS AND TURN IT 90 DEGREE
VIEW AND IV6 THE LENACAPAVIR
BINDS TO NTD ONLY IN THE
IMMATURE VIRION AND WE HAVE GOOD
DENSITY TO DEFINE THE FUNCTIONAL
GROUPS AND IT DOES ACTUALLY HAVE
A DIFFERENT POST COMPARED TO THE
MATURE LENACAPAVIR BINDING TO
THE MATURE CAPSID.
AND THE BINDING ACTUALLY CAUSES
A CONFIRMATIONAL CHANGE PART OF
THE LAG.
IT'S A RELATIVE ORIENTATION OF
THE NTCA RELATIVE TO THE CTD.
AS A CONSEQUENCE WE SEE A
MORPHING MOTION OF THE LATTICE
TYPE AND THE IMMATURE LATTICE IS
MALLEABLE AND CAN CHANGE.
AS A QUICK SUMMARY OF THIS PART,
PFO TREATMENT WORKS FOR INHI
INHIBITOR STUDIES AND ALLOWED US
TO OBTAIN A HIGH RESOLUTION
STRUCTURE OF LENACAPAVIR AND
IDENTIFY A NEW LOCATION AND POST
OF BINDING AND AT THE SAME TIME
WITH GOOD DEFINITION OF BBM
INDICATING THEY CAN WORK
TOGETHER.
AND ALSO WE IDENTIFIED AN
IMMATURE CAPSID MAY HAVE
SIGNIFICANT INDICATIONS IN THE
PATHWAY OF THE CAPSID.
SO THE STRUCTURAL BIOLOGY YOU
HAVE SINGLE BIOLOGY CRYO EM WORK
AND WHAT WORKS WELL FOR VIRUSES
HAS GREAT OUTCOMES AND I WANT TO
SPEND MORE TIME WITH YOU ON THE
HIGH RESOLUTION RESOLUTION IN
CELLS BASICALLY FROM TEST TUBE
TO CELLS.
THIS IS REALLY A DREAM FOR MANY
OF US FOR DECADES AND ALSO
ACCELERATED BY THE MAJOR
DEVELOPMENT IN THE FIELD
BASICALLY THE THINNING THE CELL
SAMPLE BY CRYO ET AS ALBERTO
NICELY ILLUSTRATED IN THE
MORNING.
AS NICE AS IT IS DUE TO
INTRINSIC LIMITATIONS AND THE
HIGH LEVEL OF EXPERTISE NEEDED
TO DO IT WELL.
AND YOU CAN TAKE A SINGLE
PARTICLE TYPE OF WORK WITHOUT
ANY TILT CAN WE DO HIGH
RESOLUTION STRUCTURES.
THIS IS SPEARHEADED BY OUR
POSTDOC IN THE LAB.
I'LL WALK YOU THROUGH THE
PIPELINE.
ON THE LEFT ARE FROZEN CELLS
POSITIONED TO BE MARK AND FOR
THE TALK THE WORK I'LL PRESENT
TODAY I HAVE TWO DATA SETS ON
NATIVE UNTREATED CELL AND THE
INHIBITOR TREATED CELL.
THEN WE ACTUAL LY ACTUALLY HAD
BOTTLENECK ON THE WORK SO WE
NEED BETTER EQUIPMENT AND THE
TRAINING AND EXPERIMENTING TO
GET THERE TAKES ABOUT 100 DAYS.
THIS WOULD HAVE BEEN IMPOSSIBLE
WITHOUT THE GREAT SUPPORT OF OUR
COLLEAGUES AT YALE ESPECIALLY
JUN LU WHO REALLY TOLERATED US
WHO TRAINED US TO DO IT WELL.
IF WE GET OVER THIS HURDLE THE
REST IS SMOOTH AND ENJOYABLE.
WE TOGETHER WITH OUR COLLEAGUES
WE DEVELOPED A QUITE EFFICIENT
SINGLE PARTICLE PIPELINE LIKE
YOU COLLECT PURIFIED SAMPLE IN
THE DATA SET.
YOU CAN DEFINE A FOCUS REGION TO
DO DATA COLLECTION AND MOVE TO
DIFFERENT LOCATIONS TO DO THE
SAME.
SO WITH THIS SCHEME WE CAN GET
ABOUT 4,000 TO 5,000 MICROGRAPHS
PER DAY WHICH IS NOT THE SAME AS
SINGLE PARTICLES VERY CLOSE TO
THE THROUGHPUT PURIFIED SAMPLE.
THESE ARE THE TYPICAL
MICROGRAPHS WE LOOK AT THESE
DAYS.
THE PROOF OF PRINCIPLE STUDIES
AND FOCUS ON THE BLACK DOTS.
AND IT TURNED OUT PAR BETTER
THAN WE EXPECT.
THIS IS A WILL E-MAIL TO THE
TEAM THE SECOND DAY AFTER THE
INTERACTION AND 3.6 RESOLUTION
ENTIRELY USING THE PIPELINE
USING CRYO SPARK NO SPECIAL
TREATMENT AND ASSEMBLED A LARGE
TEAM AND TOOK ADVANTAGE OF THE
ALGORITHM DEVELOPED THAT WORKED
BETTER.
A MONTH LATER WE HAD 2.5
ASSOCIATION AND THESE ARE THE
SEN
SENSITY WE SAW AND --
SENSITIVITY AND THIS IS THE
DENSITY WE HAVE NOW.
IT'S NICE TO SEE THE AROMATICS
AND SMALL SIDE CHAINS AND GOOD
DEFINITION WITH THE RNA POST
TRANSLATIONAL GROUP ON THE BASE
AND WE USE THE INHIBITOR AND WE
SEE GOOD DENSITY IN THE CELL.
HIGH RESOLUTION IS GOOD.
WE ALSO SEE NICE INTERACTION
BETWEEN TRNA AND CODEINE
INTERACTION AND IF I DON'T SPEAK
THIS FAST IT GIVES TIME TO THINK
AND YOU MAY THINK WAIT A MINUTE
AND THIS IS A HIGHLY COMPLEX AND
DYNAMIC PROCESS.
CLOSE TO 60TRNAs BRING ACIDS
PACKING THROUGH MANY SITES WITH
CONFIRMATIONAL CHANGES.
BASICALLY WE HAVE EVERYTHING
EVERYWHERE ALL AT ONCE.
THIS A CHALLENGE BUT ALSO
BLESSING.
IF WE CAN FIGURE OUT THE
DIFFERENT STATES WE LEARN MORE.
THAT'S WE -- THAT'S WHAT WE DID
AND WE USED THE SINGLE PARTICLES
AND SOME CALL IT PURIFICATION IN
SILICO AND HERE'S A CLASS OF
HIBERNATING RIBOSOME AND HERE'S
ANOTHER PTRNA AND BOUND TO THE
SIDE AND MORE AND MORE AND MORE
AND MORE.
AND THIS IS A Z tRNA IN THE
CELL.
AT THE END WE HAVE AN EXTENSIVE
TRANSLATION CYCLE WITH 21
DISTINCT STATES AND ALL BUT ONE
IS DETERMINATED IN THE
RESOLUTION AND THIS IS 3.3 NOT
TOO SHABBY.
AND ALSO BECAUSE WE HAVE OVER A
MILLION HIGH QUALITY PARTICLES
WE CAN DO STATISTICS TO FIGURE
OUT THE STATE DISTRIBUTION AND
THE TRANSLATION LANDSCAPE.
YOU HAVE TIME TO TILL ABOUT ONE
STORY AND CRBP1 AND THIS CENTRAL
REGION OF THIS LONG NOODLY
MOLECULE HAS BEEN OBSERVED
OCCUPYING THE mRNA CHANNEL AND
PUT IT INTO A NON-TRANSLATING OR
HIBERNATING STATE AND WE SEE THE
END TERMINUS AND THIS INDICATES
IT'S A POTENT SO TRANSLATE TO
HIBERNATION AND MORE STRIKINGLY
WE SEE THIS ALWAYS ON THE UNIT
WHICH IS A TANTALIZING MODEL
THAT MAYBE PERHAPS IN THE CELL
EVERYTHING IS COMING AS TETHERED
PAIRS.
THIS IS HIGHLY SPECULATIVE.
WE DON'T HAVE DATA TO SUPPORT
THIS.
TO MORE SLIDES TO SHOW.
WE HAVE AN INHIBITOR TREATED
CELL AND SEEING INTERACTION WE
SEE DO TRANSLATION LANDSCAPE
ANALYSIS TO SEE HOW THEY CHANGE
THE TRANSLATION STATES
DISTRIBUTION.
WE SEE OTHER COFACTORS SUCH AS
POLY AMINES AND THIS IS ENGAGED
IN HEXAMIDE IS LIKELY FROM
PURIFIED SAMPLE STUDY AND IT'S
NOT INCLUDED.
OF COURSE THEY DON'T SEE IT.
THIS EMPHASIZES TO DO THE WORK
IN THE CORRECT ENVIRONMENT AND
CELL IS THE BEST YOU CAN GET.
IN SUMMARY IT HOLDS GREAT
POTENTIAL FOR STUDIES BUT THIS
IS JUST THE BEGINNING.
SO MUCH NEEDS TO BE OPTIMIZED
AND SO MUCH CAN BE DONE.
WE CAN DO RIBOSOME RELATED
BIOLOGY IN CELLS AND SOME
PHARMACOLOGY BUT COMING BACK TO
VIROLOGY IS WHERE WE DEVELOPED
THE SYSTEM.
WE CAN STUDY THE VIRAL
ALTERATION A COMMON PATHWAY WITH
THE RIBOSOMES SHIFTING THE
EFFECT ON TRANSLATION EFFICIENCY
AND ALTERNATIVE INITIATION
VIRUSES.
AND WE CAN ALSO STUDY HIV
MATURATION TETHERED TO CELL
SURFACE.
WE'RE WELL UNDERWAY AND HAVE
DATA FOR THE CELLS AND
COLLABORATING WITH YALE WE HAVE
A VIRUS ON PRIMARY CD4 T CELLS
AND TAKING ADVANTAGE OF THE
RESTRICTION TETHERING WORKING TO
HELP LOOK AT THE TETHERED
PARTICLES TO ENRICH TO THE CELL
SURFACE.
WITH THIS I WILL ACKNOWLEDGE
PEOPLE WHO DID THE WORK AND
COLLABORATORS AND THANKS FOR THE
GENEROUS SUPPORT FROM NIAID AND
THE IN SITU WORK IS NOT FUNDED
YET.
THANK YOU FOR YOUR ATTENTION.
>> THANK YOU.
WHERE WE GO FOR THE QUESTIONS I
WANT TO REMIND PEOPLE WE HAVE A
PRESENTATION THIS AFTERNOON
PLEASE BRING THE PRESENTATIONS
NOW AS SOON AS THE QUESTION
SESSION IS OVER WE CAN DO THAT.
>> GREAT WORK.
DO YOU HAVE ANY IDEA HOW THE B
FACTORS FOR THE RECONSTRUCTION
IS COMPARED BETWEEN THE IN SITU
AND EX SITU SAMPLES AND HOW MUCH
MORE DATA DO YOU NEED IN THE
CELL FOR THE RESOLUTIONS AND
NOISE?
>> IT'S PROBABLY AT THE LAW
LAWYER -- LOWER END OF THE
SIGNAL TO NOISE.
THE 3.6 IS FROM 20 TO 30K
PARTICLES GETTING TO HIGH
RESOLUTION WE NEED OVER A
MILLION PARTICLES.
>> THANK YOU.
SOONER OR LATER PEOPLE WORK ON
THE RIBOSOME.
PRESUMABLY THERE'S NO PREFERRED
ORIENTATIONS AND GET ISOTROPIC
SETS OF IMAGES.
IS THAT TRUE?
>> I THINK SO BECAUSE IN THE
CELL THERE'S NOTHING AND YOU SEE
IT INSIGHT THE CELL.
-- INSIDE THE CELL.
>> A QUESTION FOR MELISSA.
>> HI, HARRY.
>> HEY.
I WAS WONDERING IF MX2 IS
INHIBITED BY CELL CYCLE STAGE.
IN SOME IT CAUSES CELL CYCLE
ARREST PHENOTYPE AND HAVEN'T
BEEN ABLE TO DISENTANGLE THE
CELL CYCLE IN YOUR SYSTEMS.
>> DISENTANGLES NO BUT I CAN
TELL YOU MX2 ACTIVITY AGAINST
HIV IS ENHANCED WHEN YOU BLOCK
CELL DIVISION SO WHICH MAKES
SENSE THAT IT'S BETTER ABLE TO
INHIBIT THE VIRUS WHEN THERE'S
NO NEW MITOSIS HAPPENING OR
PORES BEING GENERATED.
IT DOES HAVE ENHANCED ACTIVITY.
HOW THE CELL CYCLE IS PLAYING A
ROLE WE DON'T KNOW BUT SHORT
ANSWER, YES.
THANKS.
>> ANYMORE QUESTIONS?
IF NOT I'LL THANK THE SPEAKERS
FOR THE MORNING'S PRESENTATIONS.
MAY I CALL ON THE SPEAKERS WHO HAVE THEIR PRESENTATIONS THIS
AFTERNOON TO COME UP AND LOAD UP THE SLIDES.
MODERATING THIS AFTERNOON.
THANK YOU FOR JOINING US FOR
THIS LAST SESSION.
OUR FIRST SPEAKER IS ANDREW
DELAITSCH FROM CALIFORNIA
INSTITUTE OF TECHNOLOGY.
>> GREAT.
HELLO, EVERYONE.
I'D LIKE TO THANK THE ORGANIZERS
FOR THE OPPORTUNITY TO SHARE MY
WORK TODAY.
I'M ANDY DELAITSCH, FIFTH YEAR
GRADUATE STUDENT, TODAY TALKING
ABOUT NANOBODY WITH A COOL NAME,
007.
AND SO, THIS ANTIBODY CAME FROM
A COLLABORATION BETWEEN OUR LAB
AND KLEIN LAB IN COLOGNE,
GERMANY.
PEOPLE LIVING WITH HIV, THEY
SCREENED SERUM FROM 2,000 PEOPLE
LIVING WITH HIV, AND SCREENED
THE SERUM FOR ITS ABILITY TO
NEUTRALIZE DIVERSE VIRUSES, TAKE
THE TOP, ELITE NEUTRALIZERS,
ISOLATE SINGLE B CELLS FROM
THESE PEOPLE.
AND SO TO DO THIS THEY LOOK FOR
IgG POSITIVE BG 505 SINGLE B
CELL CLONES, FROM THE TOP ELITE
NEUTRALIZERS, ISOLATED HUNDREDS
OF MONOCLONAL ANTIBODIES,
SCREENED THESE FIRST ON A
SCREENING PANEL AND THEN ON
LARGER PANELS.
SO THE ANTIBODY THAT I'M GOING
TO TALK ABOUT TODAY CAME FROM
THIS TOP ELITE NEUTRALIZER, IT
LANDED IN WELL D 07 MISREAD AT
007, THE NAME STUCK.
IT APPEARS TO BE BEST IN CLASS,
HERE SHOWN AGAINST 119 STRAINS.
SO THEIR LAB TURNED TO OUR LAB
TO A SINGLE PARTICLE CryoEM
STRUCTURE OF THE ANTIBODY, I'M
SHOWING HERE THE TYPICAL WORK
FLOW THAT OUR LAB USES, AND SO
THIS INVOLVES MAKING FAB WITH
PRIMER, WE USE EXCESS OF FAB,
3.3 MOLES PER EVERY MOLE OF
TRIMER.
AND THEN PURIFIED SAMPLE BY SIZE
EXCLUSION CHROMATOGRAPHY, INDEED
WE SEE EXCESS UNBOUND FAB IN THE
SEC PROFILE.
WE TAKE THE COMPLEX PEAK, FREEZE
IT, AND THEN IMAGE ON ELECTRON
MICROSCOPE.
FROM THOUSANDS OF IMAGES LIKE
THIS WE'RE ABLE TO EXTRACT
PARTICLES AND DO DATA PROCESSING
AND ONE OF THE BEAUTIFUL THINGS
ABOUT E.M., IT DEALS WITH
HETEROGENEITY QUITE NICELY SO
FROM A SINGLE DATASET WE GET NOT
ONE STRUCTURE BUT FOUR
STRUCTURES, HERE YOU CAN SEE
UNBOUND, ONE BOUND, TWO BOUND,
THREE BOUND STRUCTURES.
SO WHILE THIS UNUSUAL
STOICHIOMETRY IS THE FOCUS, I
WANT TO HIGHLIGHT SOME FEATURES
OF THE EPITOPE OF THIS ANTIBODY,
LOOKING SPECIFICALLY AT THE ONE
BOUND STRUCTURE.
AND SO AS I MENTIONED IT'S A V3
ANTIBODY AT THE APEX, THEY BIND
WITH DIFFERENT ANGLES OF
APPROACHES.
AS YOU CAN SEE ON THE LEFT 007
SEEMS TO BIND TO ONE SIDE
RELATIVE TO OTHER HIV V3
bNAbS, IT DOES SO, HOISTED UP
IN BETWEEN THESE N 156 AND M 301
GLYCANS, DOES NOT COMBER ACT
WITH N332, AND HAS 22 AMINO
ACID-LONG CDRH 3 WHICH MAKES
CONTACT WITH COULD BE SERVED
GDIR MOTIF ON V3.
THE MOST HAVING THING TO ME WE
OBSERVED BIND BUYING ELECTRON
MICROSCOPY, AND SO THIS IS
REALLY UNUSUAL BECAUSE THIS IS A
VERY POTENT ANTIBODY, IT WAS
POTENT AGAINST 505, WE THINK OF
NEUTRALIZATION POTENCY AS
CORRELATED WITH BINDING, AND SO
BY EM LOOKS LIKE THIS ANTIBODY
IS NOT BINDING WELL.
YOU CAN SEE THE DISTRIBUTION OF
PARTICLES HERE ON THE RIGHT.
AND SO WHAT IS GOING ON?
THIS WAS THE QUESTION.
AND WE REALLY MADE NO PROGRESS
FOR WELL OVER A YEAR UNTIL ONE
DAY I HAD SORT OF A CRAZY
HYPOTHESIS, WHICH IS THAT
PERHAPS 007 IgG IS BINDING
BIVALENTLY TO A SINGLE OM.
IN THE TOP RIGHT YOU CAN SEE THE
FAB USED FOR THE STRUCTURAL
DETERMINATION BUT THE IgG,
BIOLOGICALLY RELEVANT FORM, WAS
USED FOR NEUTRALIZATION ASSAY ON
THE RIGHT.
THIS IS A CRAZY HYPOTHESIS, FOR
TWO REASONS.
ONE IS THAT TO MY KNOWLEDGE THIS
HAS NEVER BEEN SHOWN FOR ANY HIV
ANTIBODIES BEFORE, SECONDLY THE
WAY PEOPLE INFERRED IS LOOKING
AT HEAVY CHAINS.
C TERM NIGH ARE THE SHOULDERS,
THEY HAVE TO BE WITHIN A CERTAIN
DISTANCE, SHOULDER WIDTH APART
FOR ANTIBODY, ABOUT 50 OR 60
ANGSTROMS.
IN MY TWO BOUND STRUCTURE YOU
CAN SEE THE C TERMINI ARE TOO
FAR APART.
THIS IS A DYNAMIC MACHINE AND AS
MANY PEOPLE HERE KNOW, THERE
HAVE BEEN OPEN STATES OF OHMs
SOLVED, HERE OCCLUDED OPEN OHMS,
WHEN I TOOK MY STRUCTURE, A
CLOSED OMM, TOOK THE FAB
RELATIVE TO THE GP120 AND DOCKED
TO VARIOUS OPEN FORMS C TERMINI
COME IN CLOSE PROXIMITY, COOL
BUT STILL A MODEL, NO EVIDENCE
FOR THIS.
BUT WHAT IT DID DO IS PROMPTED
US TO RUN ANOTHER EXPERIMENT,
WHICH IS SOAP -- SHOWN HERE,
NEUTRALIZATION ASSAY, COMPARING
HOW POTENT IS FAB RELATIVE TO
IgG.
THE IgG IS ABOUT THREE TIMES
THE SIZE OF THE FAB, TO CONTROL
FOR THE STERICS, ONE ARM IS
NON-HIV BINDING FAB, AND SO
THAT'S A BETTER CONTROLLED
COMPARISON THIS IS SHOWN FOR ONE
EXAMPLE PSEUDOVIRUS, MOVING FROM
THE FAB TO BI-SPECIFIC WE GET A
20-FOLD INCREASE IN POTENCY,
INDICATING THERE'S SOME STERIC
COMPONENT, AND FROM BI-SPECIFIC
TO NATIVE IgG WE GET
ADDITIONAL 250-FOLD INCREASE IN
POTENCY FOR THIS SPECIFIC
STRAIN.
SO, TOGETHER THAT MEANS THAT THE
IgG IS ABOUT 5,000 TIMES MORE
POTENT THAN FAB FOR THIS STRAIN.
AND SO THAT WAS PRETTY
UNEXPECTED AND COOL.
AND SO WE THOUGHT THIS WAS GOING
TO BE SOMEWHAT STRAIGHTFORWARD.
LET'S DO A SINGLE PARTICLE EM
STRUCTURE AND SEE IF WE SEE THAT
STRUCTURE THAT WE EXPECTED.
AND SO FOR THIS I SIMPLIFIED THE
WORKFLOW, MIXED EQUI MOLAR 007
WITH THE 505 SOSIP, WE SEE
SOMETHING THAT LOOKS LIKE THIS,
TO OUR SURPRISE.
THE EQUILIBRIUM IS SHIFTED TO
THE WEIRD TRIMER-DIMER
STRUCTURE, FOR THOSE FAMILIAR
WITH EM THIS IS A GOOD REMINDER
THAT THINGS THAT ARE FLEXIBLE IN
EM SUCH AS UNBOUND FABS OR SD
ARE NOT OBSERVED BECAUSE THIS IS
AT ITS CORE AN AVERAGING
TECHNIQUE, STRUCTURES CAN BE
INTERPRETED MORE LIKE THAT,
IgG AROUND THE FABs, BUT ON
THE RIGHT THE STRUCTURE IS
REALLY WEIRD.
AND SO WE ASKED THE QUESTION CAN
THIS STRUCTURE ACTUALLY EXPLAIN
THIS INCREASED NEUTRALIZATION
POTENCY OF THE IgG?
THE WAY WE THINK OF THIS THE
POTENTIAL MECHANISM WOULD BE
THAT YOU HAVE ONE ON ONE VIRUS,
ANOTHER ON A DIFFERENT VIRUS,
AND YOU WOULD HAVE VIRAL
AGGREGATION AS A MECHANISM OF
NEUTRALIZATION, FORMER THIS
TRIMER-DIMER STRUCTURE IN
BETWEEN VIRIONS.
THERE'S ALSO OUR ORIGINAL
HYPOTHESIS OF INTRASPIKE
CROSS-LINKING TO SINGLE OPEN OM
OR BINDING TO ADJACENT TO SPIKES
ON A SINGLE VIRUS.
AND SO WE WANTED TO NAIL THIS
HOME, AND FIGURE OUT WHAT WAS
GOING ON WITHIN OUR PSEUDOVIRUS
ASSAY, AND SO WHAT WE WOULD LIKE
TO DO IS IMAGE THE 007 IgG IN
COMPLEX WITH OTHER VIRUS OR
RATHER ENVELOPE VIRUS-LIKE
PARTICLES.
OKAY.
AND SO TO DO THIS WE UTILIZED
TECHNOLOGY THAT WAS PREVIOUSLY
DEVELOPED IN OUR LAB, EABR
TECHNOLOGY, THE MAIN PURPOSE AT
THE TIME WAS AS A VACCINE
PLATFORM, BUT I WANT TO MAKE THE
CASE IT'S USEFUL FOR STRUCTURAL
BIOLOGY AND SO THE IDEA HERE WE
HAVE A GENE SHOWN IN TOP LEFT,
FULL LENGTH HIV OMs, SWITCH TO
ENDOSIGH THOSE IS PREVENTING
MOTIF FOLLOWED BY EABR SEQUENCE.
THAT LEADS TO THE FORMATION OF
THESE VIRUS-LIKE PARTICLES.
WITH MEMBRANE PRESENT.
THIS IS A REALLY ADVANTAGEOUS
METHOD, BECAUSE RELATIVE TO
WORKING WITH VIRUSES, SAFE AND
EASY TO WORK WITH, VERSATILE,
ANYTHING YOU CAN CLONE YOU CAN
TEST TO SEE WHETHER YOU GET
EXPRESSION, NOT LIMITED TO A FEW
VIRAL STRAINS AND RELATIVE TO
SOSIPs, STRESSING FULL LENGTH
OMS WITH A MEMBRANE.
THE WORKFLOW IS PRETTY
STRAIGHTFORWARD.
I'M NOT GOING INTO DETAIL BUT
WE'VE PURIFIED ON A SUCROSE
GRADIENT, DIALIZED, LOOKED ON
MICROSCOPE.
THESE THINGS LOOK REALLY
BEAUTIFUL.
RELATIVE TO ACTUAL VIRUSES
SPARSELY COATED IN OMS, YOU CAN
SEE A LOT PRESENT.
WE'RE GETTING A NUMBER OF OMS,
RELATIVE POSITIONING AND TILT
RELATIVE TO MEMBRANE.
ONE THING NOT IMMEDIATELY
OBVIOUS YOU CAN PERFORM SINGLE
PARTICLE ON SIDE VIEW OMS ON
PERIMETER OF THE VIRUSES SO WE
DID THIS, AND WE'VE GOTTEN
ROUGHLY 7 OR 8-ANGSTROM
STRUCTURE OF THIS.
THAT'S WHERE WE'RE AT NOW.
SO I'LL LEAVE YOU WITH A
CLIFFHANGER OF THE NEXT THING WE
HAVE TO DO, WE WANT -- NOW THAT
WE'VE ESTABLISHED WE CAN USE
EVLPs, WE WANT TO COMPLEX THEM
WITH ANTIBODY OF INTEREST.
FUTURE DIRECTIONS TO DO THIS,
THIS IMAGE OF THE STABILIZED
CONSTRUCT, WE WANT TO SEE IF WE
CAN ALSO GET A NATIVE
NON-STABILIZED CONSTRUCT.
FINALLY I DIDN'T GO INTO IT BUT
AVIDITY IS A CONSEQUENCE, WE
WANT TO LOOK FURTHER INTO
BINDING KINETICS OF 007 WITH
DIFFERENT OMS.
I A TON OF PEOPLE TO THANK,
PAMELA, ADVISER, PEOPLE IN THE
LAB, WONDERFUL COLLABORATORS,
AND POSTDOC IN THE LAB, AND THEN
VARIOUS PEOPLE WHO HELPED
IMMEDIATE WITH EM, FUNDING
SOURCES, HAPPY TO TAKE
QUESTIONS.
[APPLAUSE]
>> THANK YOU, ANDREW.
ANY QUESTIONS?
>> I WAS ABOUT TO ASK ABOUT THE
KINETIC RATES UNTIL I SAW THE
LAST SLIDE.
>> YEAH, A REALLY INTERESTING
THING WHEN YOU LOOK AT THESE
NEUTRALIZATION CURVES, HERE
STERIC COMPONENT, WE ALWAYS SEE
THIS, BUT THE AVIDITY COMPONENT
VARIOUS FROM A FEW TO FEW
HUNDRED.
>> DO YOU KNOW WHAT THE SPACING
DISTANCE IS BETWEEN THE OMS?
>> YOU CAN JUST SEE VISUALLY A
LOT OF THE EVLPs ARE DENSELY
COATED, SPIKES ALL OVER.
YEAH, I'D LIKE TO DO TOMOGRAPHY
TO FURTHER LOOK INTO THAT.
>> THANK YOU.
>> HI.
CAN YOU JUST EXPAND ON WHICH
ESCORTS SEEM TO DO A BETTER JOB
AND WHY YOU THINK THAT MIGHT BE
HAPPENING?
>> ON WHICH ESCORT?
EXCUSE ME?
YEAH, SO I DIDN'T REALLY TEST
ANY DIFFERENT LIKE ESCORT
MACHINERY.
WE USED THIS EABR SEQUENCE
PREVIOUSLY PUBLISHED, WENT
FORWARD AND IT WORKED, SO WE
HAVEN'T DONE ANY TROUBLESHOOTING
SO I DON'T HAVE MUCH.
>> OKAY, THANKS.
>> THANK YOU AGAIN, ANDY.
>> THANK YOU.
[APPLAUSE]
>> NEXT SPEAKER IS SHARIDAN
BROWN FROM SCRIPPS RESEARCH.
WE'LL GIVE HER A SECOND.
>> I'M SHARIDAN, GRADUATE
STUDENT IN ANDREW WARD'S LAB AT
SCRIPPS, TALKING ABOUT MY WORK
ON ANTI-IMMUNE COMPLEX
ANTIBODIES ELICITED AGAINST HIV
OMS.
FIRST I WANT TO TOUCH ON SOME OF
THE CORE GOALS OF RATIONAL
VACCINE DESIGN.
PRETTY MUCH WE WANT TO ANALYZE
ANTIBODY RESPONSES, AND
CHARACTERIZE DIFFERENT
NEUTRALIZATION CAPABILITIES OF
ANTIBODIES TO GO BACK INTO OUR
IMMUNOGENS, PRODUCING GERMLINE
TARGETING IMMUNEEGENS, I'LL TAKE
YOU THROUGH THE PIPELINE.
WE'RE ABLE TO TAKE IMMUNIZED
SERA, ISOLATE IgG, DIGEST INTO
FAB WHICH WE CAN RECOMPLEX WITH
ANTIGEN, PUT THAT PUT THAT ON
CryoEM OR NEGATIVE STAINING
GRIDS.
THROUGH SERIES OF CLASSIFICATION
STEPS WE CAN ISOLATE HIGH
RESOLUTION MODELS OF OUR ANTIGEN
BOUND TO POLYCLONAL ANTIBODIES.
THIS COMPLEMENTS RATIONAL
VACCINE DESIGN TECHNIQUES.
WE'VE USED IT TO HELP PRODUCE
STABLE AND SOLUBLE
GLYCOPROTEINS, THAT COULD BE
USED AS IMMUNOGENS.
I WANT TO HIGHLIGHT WORK BY
OTHER PEOPLE IN OUR LAB BECAUSE
I THINK IT SHOWCASES EXACTLY HOW
POWERFUL EMPEM.
SHE WAS ABLE TO PRODUCE A
DATASET CONTAINING 20 UNIQUE
POLYCLONAL ANTIBODIES, RANGING
FROM 3 TO 3 1/2 ANGSTROMS,
TARGET SIX THREE ON THE ENVELOPE
SEES BEYOND INCLUDING MALARIA,
GLYCOPROTEIN, INFLUENZA VIRUS,
CORONAVIRUS SPIKE, AND HEPATITIS
C VIRUS.
MY WORK, I WANT TO FIRST
INTRODUCE THE CONCEPT OF
ANTI-IDIOTIPPIC ANTIBODIES, THAT
TARGET AND BIND TO PRIMARY
ANTIBODIES, IN THREE CLASSES,
AB2 BIND TO PRIMARY ANTIBODIES.
AB2 BETA BIND INTO THE CDR
REGION OF THE PRIMARY ANTIBODY.
AND THIS BLOCKS ANTIGEN BINDING.
AND THEN AB2 GAMMA ANTIBODIES
BIND ADJACENT TO CDR BINDING
SITE BLOCK ANTIGEN BINDING BUT
ARE NOT INTERNAL IMAGE
ANTIBODIES.
WE CAME ACROSS A SERIES OF
ANTIBODIES, ANTI-IMMUNE COMPLEX
ANTIBODIES, IN FOUR CLASSES.
I'LL WALK YOU THROUGH EACH OF
THOSE IN DETAIL.
OUR FIRST EXAMPLE IS CLASS 1
ANTI-IMMUNE COMPLEX ANTIBODIES,
AND THESE ARE ANTIBODIES THAT
HAVE DISTINCT ANTIGEN EPITOPES
BUT MAKE MINOR NON-SPECIFIC
CONTACTS ALONG THE FRAMEWORK
REGIONS.
IN THIS EXAMPLE WE HAVE A BASE
ANTIBODY, N625 FAB MAKING MINOR
CONTACTS.
SAME ANIMAL, SAME BASE EPITOPE,
BUT WE ALSO HAVE AN ADDITIONAL N
66 ANTIBODY BINDING, WE THINK
ONE BINDS, HELPS STABILIZE
EPITOPE, ALLOWS OTHER ANTIBODY
TO COME IN AND BIND.
THESE CLASS 2 ANTI-IMMUNE
COMPLEX ARE EPITOPE OF ANTIGEN
AND ANTIBODY, SO IN THIS CASE
THE ANTI-IMMUNE COMPLEX ANTIBODY
IN GREEN IS BINDING TO A GLYCAN
ON OUR SOSIP ANTIGEN, ALSO
BINDING TO THE FRAMEWORK REGIONS
OF THIS N 241 FAB.
ANOTHER EXAMPLE BETWEEN THE BLUE
AND B1B 3 FAB CONTACTING
FRAMEWORK REGIONS BUT ALSO THIS
138 GLYCAN.
CLASS III ARE YOUR
QUINTESSENTIAL AB2 ALPHA
IDIOTYPIC ANTIBODIES, COMPOSED
OF ANTIBODY, DOES NOT MAKE
CONTACT WITH THE ANTIGEN,
THERE'S EXTENSIVE CONTACTS WITH
FRAMEWORK REGION 1 AND FRAMEWORK
REGION 3 OF THIS V2 FAB.
PERHAPS CLASS 4 ANTIBODY HAS
EPITOPE OF TWO DISTINCT
ANTIBODIES, THIS IS BINDING TO
BOTH THIS V2 FAB IN BLUE AND GP1
INTERFACE IN RED.
ONE KEY QUESTION, WHEN DURING
THE IMMUNE RESPONSE ARE THESE
ANTIBODIES BEING ELICITED?
WE PERFORM LONGITUDINAL NEGATIVE
STAIN TO ANSWER, THE SLIDE IS
BUSY.
FIRST WITH THE RABBIT
IMMUNIZATION, SO THE FIRST
IMMUNIZATION IN GREEN, THERE'S
LITTLE TO NO ANTIBODY RESPONSES
WHICH IS EXPECTED.
BY THE SECOND IMMUNIZATION WHICH
IS IN BLUE, WE SEE ANTIBODIES TO
IMMUNODOMINANT EPITOPES ARISE,
SO TYPICALLY THE BASE ANTIBODY
WHICH IS PURPLE HERE AS WELL AS
SOME GLYCANS, GLYCAN HOLES, AND
THEN BY THE THIRD IMMUNIZATION
WE SEE WIDE VARIETY OF EPITOPES
ELICITED AS WELL AS ANTI-IMMUNE
COMPLEX ANTIBODIES.
FOR THE MACAQUE IMMUNIZATION,
THIS TREND IS BORNE OUT BUT TOOK
LONGER.
THIRD IMMUNIZATION V 5 AND
INTERFACE FAB APPEAR, BY FOURTH
YOU SEE THAT CLASS IV
ANTI-IMMUNE COMPLEX ANTIBODY
BRIDGING.
UNFORTUNATELY THIS IS POLYCLONAL
SO IT'S HARD TO STUDY THESE
ANTIBODIES IN DETAIL USING OTHER
ASSAYS.
BUT PEOPLE IN THE WARD LAB,
MYSELF INCLUDED, HAVE BEEN
WORKING ON DIFFERENT STRATEGIES
TO DETERMINE SEQUENCE OF
POLYCLONAL ANTIBODIES ONE USING
SOFTWARE MODELANGELO.
WE CAN TAKE OUR POLYCLONAL MAP
AND B CELL RECEPTOR REPERTOIRE,
AND COMBINE WITH MODELANGELO,
WILL SPIT OUT A SEQUENCE BASED
ON OUR FEATURES, AND USE THAT
INFORMATION TO SEARCH OUR
REPERTOIRE.
I WAS ABLE TO FIND DECENT ENOUGH
MATCHES.
SO I JUST ORDERED THE
CONSTRUCTS, WE'LL SEE IF THEY
EXPRESS AND IF THEY BIND
APPROPRIATELY.
SO, I GUESS THE BIG QUESTION IS
HOW ARE THESE ANTI-IMMUNE
COMPLEX ANTIBODIES ELICITED?
AND SO I'LL WALK YOU THROUGH OUR
CURRENT HYPOTHESIS.
WE BELIEVE THAT AFTER THE FIRST
IMMUNIZATION ACTIVATED B CELLS
ENTER THE GERMINAL CENTER,
UNDERGO SOMATIC HYPERMUTATION
AND SELECTION.
THESE BECOME MEMORY B CELLS PLAS
MOW BLASTS, TARGETING EPITOPES,
AND AFTER THE SECONDS
IMMUNIZATION AID IN FORMATION OF
IMMUNE COMPLEXES, AND THERE'S
LITTLE SURFACE AREA LEFT DUE TO
THIS GLYCOSYLATION AND THESE
PRIMARY ANTIBODIES, THAT'S
CALLED EPITOPE MASKING, IN THE
GERMINAL CENTER THEY ARE
PRESENTED TO FOLLICULAR
DENDRITIC CELLS, UNDERGO SOMATIC
HYPERMUTATION TO BECOME PLAS MOW
BLASTS AND MEMORY CELLS, CALLED
AND FORM ANTI-IMMUNE COMPLEXES.
IMAGING COMPLEXES USING EMPEM
ALLOWED YOU HAD TO CHARACTERIZE
THEM, IN FOUR CLASSESES, ARISE
AFTER THE THIRD AND FOURTH
IMMUNIZATION, MODELANGEL CAN
HELP US DETERMINE SEQUENCES OF
POLYCLONAL ANTIBODIES AND MOST
IMPORTANTLY THESE ANTIBODIES
PROBABLY HAVE IMPORTANT
IMPLICATIONS FOR CURRENT
APPROACH TO HIV VACCINE DESIGN.
AND WITH THAT I WANT TO THANK
THE PEOPLE IN MY LAB WHO
SUPPORTED THIS WORK, SHOUT OUT
ALEX, MY POSTDOCTORAL MENTOR,
NOW RUNS HIS OWN LAB, AIDED IN
THE COLLECTION OF A LOT OF THIS
DATA.
SO THANK YOU.
[APPLAUSE]
>> VERY INTERESTING STORY.
DO YOU KNOW ANYTHING ABOUT THE
FUNCTION OF THESE ANTI-IMMUNE
COMPLEX ANTIBODIES, AND
ESPECIALLY DO THEY INFLUENCE
NEUTRALIZATION?
>> GREAT QUESTION.
SO NONE OF THESE ANIMALS SHOWED
ANY REMARKABLE NEUTRALIZATION,
THEY WERE VERY STANDARD FOR
THEIR COHORTS SO I CAN'T SAY
THEY WERE BETTER NEUTRALIZERS OR
WORSE.
UNFORTUNATELY WE DON'T KNOW A
LOT ABOUT THEIR FUNCTION BECAUSE
WE ONLY HAVE POLYCLONAL
INFORMATION AT THE MOMENT BUT
WE'RE EXCITED TO HOPEFULLY
PRODUCE AND START LOOKING AT FC
RECEPTOR FUNCTION.
>> ANY OTHER QUESTIONS?
IF NOT LET'S GIVE SHARIDAN A
LAST ROUND OF APPLAUSE.
THANK YOU.
[APPLAUSE]
>> NEXT WILL BE ZHUANROMROM
YALE UNIVERSITY.
>> I WANT TO THANK THE
ORGANIZERS FOR THE OPPORTUNITY
TO HEAR OUR WORK.
I'M ASSOCIATED WITH SCIENTISTS
IN THE LAB, I'M GOING TO TALK
ABOUT THE DYNAMIC OF THE
MEMBRANE-PROXIMAL EXTERNAL
REGION.
PRIMARY GOAL TO INTRODUCE
BROADLY NEUTRALIZING ANTIBODIES
THAT PROTECT AGAINST HIV
STRAINS.
THIS BROADLY NEUTRALIZING
ANTIBODY, ENVELOPE GLYCOPROTEIN,
INCLUDING THE MEMBRANE
PROXIMAL-EXTERNAL REGION, MPER.
AMONG THOSE ANTIBODIES THE
ANTI-MPER ANTIBODY STAND OUT FOR
POTENCY.
TWO REASON STUDY SHOW MPER
EPITOPE CAN BE USED IN VACCINE
DEVELOPMENT.
AND THOSE TWO STUDIES
SUCCESSFULLY INDUCED BROADLY
NEUTRALIZING ANTIBODY.
IN PREVIOUS STUDIES THE ANTIBODY
FAB BINDS TO ENVELOPE HAS BEEN
VISUALIZED USING CryoEM SINGLE
PARTICLE ANALYSIS.
TWO STUDIES OF THE DIFFERENT
STATES OF ONE, TWO, AND THREE
MPER ANTIBODY 28 FAB AND 4010
FAB, SHOW DIFFERENT -- SHOWING
DIFFERENT DISTANCE OF ENVELOPE
FROM THE MEMBRANE.
THEY FOUND ONE FAB COMBINES TO
THE ENVELOPE AND THREE FABS
COMBINE TO ENVELOPE FROM THE
MEMBRANE.
THE STUDY ALSO SHOWS THAT
DIFFERENT MEMBRANES SOMETIMES
HAVE DIFFERENT CUMULATIVE WORK,
ROTATIONAL POSITION AND
COMPOSITIONAL PROPERTY,
ADDITIONALLY THE MPER ANTIBODY
SHARE -- THEY ALIGN MPER EPITOPE
FROM CRITICAL INFRASTRUCTURE
,
BINDS TO ANTIBODY IN A SIMILAR
WAY.
THOSE STUDIES USE PURIFIED
ENVELOPE.
WE WANT TO LOOK AT THE ENVELOPE
FROM MULTIPLE VIRUS AND STUDY
PROPERTY.
IN OUR STUDY WE USE THE MPER
ANTIBODY FAB, TH 511.
WE FOUND IN ABSENCE OF 34 YOU
CAN ALREADY SEE THE DH 511
BINDING FROM THE TOMOGRAM.
THEN WE DID IMAGING ON THE
CLASSIFICATION, WE FOUND HALF OF
THE ENVELOPES DON'T HAVE DH 511,
AND ABOUT A HALF HAS THREE DL511
BIND SITE.
WE DID NOT OBSERVE ANY ONE OR
TWO BINDING.
SO THEN WE THOUGHT AVERAGE
STRUCTURE, TO AVERAGE MAP, WE
FOUND THE MEMBRANE
REORGANIZATION IN THE CENTRAL
SLICE, WE ALREADY KNOW THAT THE
MPER REGION PLAY A ROLE IN THE
MEMBRANE FUSION AND SAW HOW THIS
AMPLIFIED FAB BINDING CAUSE THE
THIS MEMBRANE.
ALL RIGHT.
THIS IS THE CRYSTAL STRUCTURE OF
THE DH511 FAB BINDS TO THE AMPER
AND PLACE THIS STRUCTURE INTO
OUR ELECTRON DENSITY, THIS IS
THE 3D RENDERING.
WE USE THE CRYSTAL STRUCTURE
FROM DH511 FAB AND CLOSE THE
ENVELOPE STRUCTURE.
YOU CAN SEE THE MEMBRANE
DISTORTION IS RIGHT IN CENTER,
UNDERNEATH THE ENVELOPE.
AND TO FACILITATE THIS BINDING,
THIS EPITOPE NEED TO BE PLACED
AT ONE SIDE OF THE ENVELOPE.
HOWEVER, IN THE LAMAR STRUCTURE,
THE REGION FORMS SYMMETRIC ABOUT
THE TRANSMEMBRANE DOMAIN,
DIFFERENT, THE MPER EPITOPE IN
THE STRUCTURE IS DIFFERENT FROM
EPITOPE IN OUR STRUCTURE.
MPER NEED TO BE ON ONE SIDE OF
THE ENVELOPE.
SO WE'RE USING THIS TO STUDY,
THE REASON WE USE STIMULATION
ALTHOUGH PART 69 EPITOPE IS
STABILIZED, THE OTHER PART THAT
CONNECTING TO THE ENVELOPE IS
STILL FLEXIBLE SO WHEN I ALIGN
THE TOP ENVELOPE PART FROM THE
GROUND YOU CAN SEE THE DH511 FAB
IS SHIFTING, WHEN ALIGN DH 511
FAB AT THE BOTTOM OF THE
SUBTOMOGRAM YOU CAN SEE THE
EPITOPE SHIFTS AND ROTATES.
NEXT I ANALYZE THE FAB BIND
ENVELOPE AND FOUND THE FREQUENCY
PEAK AT ABOUT 30-DEGREE.
COME DOWN TO THE UNBOUND
ENVELOPE, FREQUENCY PEAK AT 12
DEGREE.
NOW WE HAVE THE QUESTION ARE THE
ENVELOPE ALREADY INHIBITED?
TO ANSWER THIS QUESTION WE
PREPARED ANOTHER SAMPLE IN WHICH
WE ADDED DH511 FIRST, CD4 AND
17B.
WE FOUND ONE-THIRD -- WE FOUND A
HALF -- WE FOUND A HALF OF THE
ENVELOPE HAS -- ALL OF THE 17B
BOUND AND HALF OF THE DH511 FAB
BINDS FROM ONE SIDE.
AND ABOUT A THIRD HAS THE DH 511
FAB BINDS SYMMETRICALLY.
SO THE CD4 AND 17B TO STILL BIND
TO THE ENVELOPE SUGGESTING THAT
THE ENVELOPE STILL UNDERGOES CD4
DEPENDENT ACTIVATION.
ALSO FROM THE NUMBERS YOU CAN
SEE THE CDs 4 BINDING INCREASE
DH511 BINDING, ALREADY REPORTED
IN LITERATURE.
HOWEVER THIS ENVELOPE STILL
RETAIN FLEXIBILITY, SO FROM THE
ANALYSIS WE CAN SEE THE CD4 AND
THE 17B BINDS ENVELOPE WITHOUT
FAB LARGER RAIN OF TRT AND THE
DH511 BINDING OF FAB ENVELOPE,
START BINDING ENVELOPE,
CONJOINED SYMMETRICALLY IN
ENVELOPE.
IN SUMMARY, WE HAVE SHOWN THAT
THE DH 511 COULD BIND TO OPEN
AND CLOSED ENVELOPE.
NEED TO BE SHIFTED AT ONE SIDE
OF THE ENVELOPE, SO INDICATING
IF EXCEPTIONAL CONFIRMATION OF
FLEXIBILITY OF THE MPER REGION.
THE ENVELOPE CAN UNDERGO
CD4-DEPENDENT ACTIVATION.
HOWEVER, LOOK AT ALL THOSE
STRUCTURES WE STILL HAVE MANY
QUESTIONS.
SO FIRST THAT'S THE MPER
ANTIBODY INDUCED ENVELOPE
CHANGING, JUST SIMPLY BINDS TO
TRT ENVELOPE.
ADDITIONAL DH511 CD4, CAN EITHER
BE FRONT OF TRT ENVELOPE.
GIVE THIS STRUCTURE.
AND THE LAST BUT NOT THE LEAST
CAN THIS TWO STRUCTURE
TRANSITIONAL CONFIRMATIONAL
EQUILIBRIUM.
I WANT TO THANK ALL THE LAB
MEMBERS AND OUR COLLABORATORS.
THANK YOU.
[APPLAUSE]
>> GREAT WORK.
I HAVE A QUESTION ABOUT THE
DIFFERENCE BETWEEN THE STRUCTURE
AND STRUCTURE WITH THE
ANTIBODIES, DO YOU THINK THE
ANTIBODY BINDING IS CHANGING
THAT MPER OR IS JUST THAT SINCE
YOUR SYSTEM IS A LITTLE BIT MORE
NATIVE THAT IS ACTUALLY THE
STRUCTURAL WITH THE MPER, THEN
MY SECOND YES IF IT'S THE
ANTIBODIES THAT ARE CAUSING THAT
CHANGE HOW DOES THAT TRANSLATE
TO THE TRANSMEMBRANE REGION AND
THE CYTOPLASMIC TAIL OF THE
ENVELOPE, BASICALLY CAN MPER
BINDING CREATE PROPAGATING
CHANGES THAT CHANGE THE
CYTOPLASMIC TAIL OF THE ENVELOPE
AND MAYBE THAT'S WHY MPER
ANTIBODIES ARE SO GOOD?
>> THAT'S A VERY GOOD QUESTION.
WE DON'T KNOW THAT.
>> OKAY, THANK YOU.
>> GO AHEAD.
>> VERY NICE.
DO YOU SEE ANY NEAREST NEIGHBOR
EFFECT, LIKE 50% BOUND, 50%
UNBOUND, ARE BOUND ONES
CLUSTERED TOGETHER OR IS IT
PRETTY RANDOM?
>> FROM THE -- YEAH, SOMEONE
ELSE ASK ME THIS QUESTION.
FROM THE TOMOGRAM YOU SEE THEY
ARE CLUSTERING.
YOU'LL SEE THEM CLUSTERING.
>> THEY ARE CLUSTERED TOGETHER?
>> YEAH, BY THE WAY WE NEED LIKE
ANALYZE THE DATA THAT YOU GIVE A
CONCLUSION.
>> THANK YOU.
>> THANK YOU AGAIN, ZHUAN.
[APPLAUSE]
NEXT IS JAMIN WILLOUGHBY,
UNIVERSITY OF UTAH.
>> TECHNICAL DIFFICULTIES.
>> SWEET, OKAY.
I'M A SECOND YEAR GRADUATE
STUDENT IN THE STAR LABS,
UNIVERSITY OF UTAH.
I'D LIKE TO THANK THE ORGANIZERS
FOR INVITING ME TO GIVE A LITTLE
TALK.
I'D LIKE TO TELL YOU A BIT OF A
STORY HOW I STARTED TO MAP
MUTATIONAL LANDSCAPE OF BROADLY
NEUTRALIZING ANTIBODIES,
bNAbS ARISE FROM THE ARMS
RACE, TRYING TO EVOLVE TO COMBAT
A POOL OF HIV IN THE HOST, AND
SO THEREFORE THEY DEVELOP THE
ABILITY TO NEUTRALIZE
HETEROLOGOUS HIV ISOLATES FROM
OTHER INDIVIDUALS THROUGHOUT THE
WORLD.
AND THE ANTIBODIES THAT TARGET
CD4 BINDING SITE ARE BROAD
BECAUSE IT'S HIGHLY CONSERVED
VITAL FOR CELL ENTRY AND MIMIC
CD4 RECEPTOR.
HERE IN SORT OF A STRUCTURAL
COMPARISON BETWEEN CD4 AND HEAVY
CHAIN IMMUNIZING IMMUNOGLOBULIN
FOLD THAT'S TYPICAL OF THE CD4
RECEPTOR.
SO THEREFORE A LOT OF ANTIBODIES
ARE ABLE TO NEUTRALIZE OVER 90%
OF GLOBAL HIV STRAINS.
AND OVER THE YEARS OF MORE SORT
OF -- AS PEOPLE BEGAN TO ISOLATE
ANTIBODIES WITH MORE BREADTH AND
POTENCY BEGAN TO SEE MANY
SIMILAR bNAbS AROSE IN
DIFFERENT DONORS, SUGGESTING A
CONVERGENT EVOLUTIONARY THING
THAT WE MIGHT BE ABLE TO DIRECT,
SHOWN HERE WITH A STRUCTURAL
OVERLAP OF FIVE DIFFERENT CD4
BINDING SITE bNAbS FROM FIVE
DONORS TARGETING GP120.
THE COMMON BINDING MODE THAT
DICTATES INTERACTION IS
STRUCTURALLY CONSERVED AMONGST
THESE ANTIBODIES.
FURTHERMORE THIS IS ENCODED
COMMONLY BY THE VH1-2 GENE WHICH
MOST INDIVIDUALS IN THE WORLD
HAVE.
IT HIGHLIGHTS THAT WE MIGHT
LEVERAGE FOR EFFECTIVE VACCINE
TO INDUCE CD4 BINDING SITES.
TRYING TO ELICIT THESE
ANTIBODIES, THEY HAVE BEEN USING
SORT OF THE MATURE ANTIBODY
PROTOTYPES THEY HAVE ISOLATED AS
BENCH MARKS FOR SUCCESS.
ALL THESE IMMUNOGENS HAVE SO FAR
FAILED TO ELICIT IN A BROAD AND
PROTECTIVE MANNER HIGHLIGHTING
CRE FEATURES THAT WE HAVE TO
TAKE INTO ACCOUNT AND HAVE BEEN
YET TO BE OVERCOME DEPENDABLY.
THERE WE GO.
SO, FIRST AND FOREMOST THESE
ANTIBODIES ARE EXTENSIVELY
MUTATED FROM NAIVE PRECURSORS,
HARDER TO CAPITULATE IN A
SHORTER TIME SPAN.
THERE'S PRESENCE OF ISOLATION AT
276, THIS GLYCOSYLATION IS
PRESENT IN 95%.
DURING THE EVOLUTION OF A LOT OF
THESE ANTIBODIES THEY KIND OF
TRIED TO ALEVE BY TRUNCATING
LOOPS, YET TO BE DEPENDABLY
ELICITED IN MOST VACCINE
RESPONSES.
SO THIS HIGHLIGHTS TWO THINGS
THAT WE NEED TO KNOW HOW ALL THE
POSSIBLE WAY THE 2376 GLYCAN.
A MORE HOLISTIC ANALYSIS THAT
THEY CAN PICK UP ALONG THE WAY
WOULD BE ABLE TO INFORM HOW NEXT
GEN IMMUNOGENS COULD ELICIT
PATHWAYS WE WANT.
WE WOULD LIKE TO TRY AND ELICIT
BROAD AND POTENT ANTIBODIES WITH
MINIMAL MUTATIONS NECESSARY TO
BE BROAD, POTENT, PROTECTIVE.
THIS LEADS ME TO THE IOMA CLASS,
AND SO THIS CLASS IS
CHARACTERIZED IN 2016, AND
CONTRARY TO MOST COMMON CD4
BINDING SITE bNAbS, THE
CLASS OF ANTIBODIES THAT HAS
BEEN GIVEN MUCH EFFORT INTO
VACCINE ELICITATION, THEY HAVE
ACQUIRED THE IOMA -- THE CLASS
ACQUIRED MORE, AND HAS A NORMAL
LENGTH CDR L1 THAT CAN
ACCOMMODATE THE GLYCAN THROUGH A
DIFFERENT MECHANISM, SHOWN HERE
IN THIS FIGURE WHERE IOMA HAS A
10% SEQUENCE DIVERSITY FROM
NAIVE ANTIBODY PRECURSOR
COMPARES TO 25%.
WE'VE ONLY BEEN ABLE TO
NEUTRALIZE 25%, IT HAS LESS
NECESSARY MUTATIONS ALONG THE
WAY.
AND THE BJORKMAN GROUP HAS SHOWN
THIS TO SUCCESS A COUPLE YEARS
AGO DEVELOPING TWO IGT 1 AND 2
IMMUNOGENS, IN THE ANTIBODIES
THEY ELICITED IN THEIR STUDY
THEY DID HAVE SOME POTENT
NEUTRALIZING ACTIVITY.
AND SO I'VE BEEN COLLABORATING
WITH THE BJORKMAN GROUP TO
COMPLEMENT RESULTS AND
VACCINATION STUDIES USING MORE
UNBIASED MUTATIONAL SCANS TO
HOLISTICALLY DETERMINE HOW ALL
THE POSSIBLE MUTATIONS IN THIS
IOMA ANTIBODY CAN AFFECT THE
POTENCY OF RECOGNITION, TRY AND
ENGINEER IOMA TO BE MORE BROAD
AND POTENT, IT CAN ALSO AID THE
NEXT GENERATION OF IMMUNOGENS TO
TARGET IOMA CLASS PAINTING A
BETTER TARGET OF WHAT WE MIGHT
WANT TO SHOOT FOR.
THAT LEADS ME TO WHAT I WANT TO
PROPOSE, WHERE I'D LIKE TO USE
DEEP MUTATIONAL SCANNING TO
DETERMINE HOW THE POSSIBLE
MUTATIONS IOMA CAN ACCRUE, WHAT
THEIR EFFECT WOULD BE ON BINDING
AFFINITY.
DEEP MUTATIONAL SCANNING ALLOWS
US TO SAMPLE MUTATIONS AND
DELETIONS IN A PROTEIN OF
INTEREST AND BE APPLIED TO THE
HEAVY AND LIGHT CHAINS OF
ANTIBODIES.
TO DO THIS, I CAN TRANSFORM A
SINGLE MUTANT PROTEIN LIBRARY OF
THE IOMA INTO YEAST WHERE THEY
CAN DISPLAY THAT PROTEIN ON
THEIR CELL SURFACE, IN LIKE A
SINGLE OPEN READING FRAME BY
EXPRESSING ANTIBODIES IN A
SINGLE CHAIN VARIABLE FRAGMENT
FORMAT WHERE WE TAKE THE HEAVY
CHAIN AND LIGHT CHAINS OF THE
ANTIBODIES AND TETHER TOGETHER
WITH GLYCINE SERINE LINKER
RESULTING IN EACH YEAST CELL
DISPLAYING A DIFFERENT MUTANT,
WE CAN DETERMINE HOW THEY CAN
BIND TO HIV OMS BY LABELING THE
CELL SURFACE AND OMS LIGAND OF
CHOICE, THIS ALLOWS US TO
LEVERAGE FLUORESCENCE ACTIVATED
ACTIVATED
CELL SORTING, WE CAN RUN THIS
YEAST CELL THROUGH A CYTOMETER
AND SORT INTO BINS BASED ON THE
BINDING FLUORESCENCE INTENSITY.
IF WE'RE TAKING A POOL OF YEAST
CELLS EXPRESSING A DIFFERENT
VARIANT ON CELL SURFACE WITH
DIFFERENT BINDING AFFINITIES AS
YOU TITRATE UP YOU'LL SEE THE
ONES WITH HIGHER BINDING
AFFINITY WILL TITRATE INTO BINS
3 AND 4 AT MUCH QUICKER RATE.
ONCE WE DO THE ASSAY WE CAN
EXACT THE DNA, DEEP SEQUENCE
VARIANTS, AND FROM THERE WE'LL
BE ABLE TO QUANTIFY AT PER
VARIANT LEVEL BINDING AT EACH
LIGAND CONCENTRATION WHICH AFTER
RUNNING COMPUTATIONAL ANALYSIS
CAN GET US TO VARIANT-SPECIFIC
TITRATION BINDING CURVES.
SO TO THIS EFFECT I'VE RUN THE
IOMA SINGLE MUTANT SCAN AND THIS
ASSAY WITH IOMA ANTIBODY, WITH
TWO LIGANDS.
I'VE DONE THE 4, 2, 6, A NATIVE
PROTEIN AND IgG 2 IMMUNOAGAIN
FROM 4, 2, 6C, THE READOUT WE
MIGHT EXPECT LOOKING AT A HEAT
MAP, CHANGE IN BINDING AFFINIT,
AT THE HEAVY A AND LIGHT RAIN,
ON THE Y-AXIS WE'RE LOOKING AT
ALL MUTATIONS WE CAN MUTATE AT
THAT SITE, WHERE QUITE SQUARES
WITH Xs WITH WILDTYPE RESIDUE.
IF WE LOOK AT THE HEAVY CHAIN,
THESE POSITIONS WERE CYSTEINE IS
THE WILD TYPE RESIDUE ANYTHING
ELSE YOU MUTATE THAT RESIDUE TO
WILL TANK BINDING AFFINITY,
INFERRING WE MIGHT BE DISRUPTING
A DISULFIDE BOND, FOR INSTANCE,
SEEING A LOWER BINDING AFFINITY.
WHAT WE'RE LOOKING AT HERE IS
TOP-DOWN VIEW OF THE BINDING
INTERFACE THAT IOMA USES TO
CONTACT OMS, THIS IS WHAT YOU
WOULD SEE FLYING AT YOU, WHERE
I'VE OUTLINED THE KEY RESIDUES
THAT CONTACT THE GP120 IN BLACK.
AND AS YOU MIGHT GLEAN FROM JUST
LOOKING AT THE HEAT MAP MOST OF
THESE MUTATIONS ARE NEGATIVE.
IF WE'RE LOOKING -- OR ON
AVERAGE, NOT NEGATIVE.
IF WE LOOK AT MAXIMUM BENEFICIAL
EFFECTS OF MUTATIONS WE START TO
SEE INTERESTING THINGS.
AROUND THE PERIPHERY OF THAT
CORE BINDING INTERFACE WE'RE
STARTING TO SEE COMPLEMENTARY
DETERMINING LOOPS WITH MUTATIOS
WHICH BIND AFFINITY, EXCITING TO
SEE.
I WANT TO HIGHLIGHT EACH OF
THESE TILES IN A HEAT MAP IS A
TITRATION SERIES WITH PROTEIN
MUTANT, HIGHEST AFFINITY
ENHANCING MUTATION SERINE 100
WHEN WE MUTATE WE CAN INCREASE
BY ABOUT TEN-FOLD OR SO.
WHEN I LOOK AT THE STRUCTURE
WITH MODELING I CAN SEE I'M NOT
ABLE TO SEE ANY SIDE CHAINS TO
THAT MIGHT ACCOUNT FOR INCREASED
BINDING AFFINITY I'M SEEING A
STABILIZING EFFECT WITH LYSINE
28 MAY BE FORMING A SALT BRIDGE
BUT I HAVE TO RUN MORE ASSAYS TO
CHASE THAT DOWN AND CONFIRM.
IT GOES TO SHOW YOU CAN GO FROM
LARGE AERIAL VIEW OF THE HEAT
MAP LEVEL TO MORE OF A DISTINCT
MOLECULAR CONTACT LEVEL.
I'VE ALSO RUN THIS WITH THE
IgG 2 MUTAGEN, WE'RE SEEING
SIMILAR EFFECTS, BUT THE KEY
IDEA IS THERE'S A LOT OF
DIFFERENCES AS WELL AND YOU
WOULD BE ABSOLUTELY CORRECT.
I DECIDED TO HONE IN ON ONE
DIFFERENTIAL MUTATION THAT
LYSINE 58 HERE IN THE CDRH 2
LOOP.
WHEN WE MAP THE DIFFERENCE IN
BINDING AFFINITY WE CAN SEE
THAT'S MOSTLY A 1:1 EFFECT BUT
THERE ARE QUADRANTS WHERE WE'RE
SEEING DIFFERENTIAL MUTATIONAL
EFFECTS, AND THAT, AGAIN, LYSINE
58 AND HEAVY CHAIN IS SITTING IN
THE AREA WITH INCREASED
AFFINITY.
THAT MUTATION IS JAM PACKED INTO
WHERE ALL THE MUTATIONS FROM
426C WERE MADE.
THAT'S EPRESIDENT-ELECTED IN THE
BINDING CURVES THAT CORRELATE.
MY SUMMARY, TAKEHOME MESSAGE
I'VE STARTED TO BUILD A SYSTEM
THAT CAN PROBE THESE FUNCTIONAL
FLEXIBILITIES AND CONSTRAINTS IN
A HOLISTIC MANNER AND I HOPE
I'VE CONVINCED YOU MUTATIONS
SHOW DIFFERING EFFECT, WHEN YOU
LOOK AT THIS MUTATIONAL PATHWAYS
CAN BE INDUCED.
NEXT STEPS, I WANT TO EXPAND TO
VRC-01 AND OTHER ANTIBODIES, THE
LIGAND PANEL THAT I CAN WORK
WITH SO I CAN SEE HOW MUTATIONS
AFFECT RECOGNITION ON A GLOBAL
HIV DIVERSITY LEVEL AND SOME
OTHER IMMUNOGENS AND AUTOLOGOUS
PROTEINS TO FLESH OUT SURFACE
GLYCOSYLATION CAN BE OVERCOME
WITH DIFFERENT MUTATIONS.
I'D LIKE TO ACKNOWLEDGE MY LAB,
HELPFUL GETTING THIS OFF THE
GROUND, ESPECIALLY THE BJORKMAN
GROUP WHO PROVIDED REAGENTS AND
SCIENTIFIC EXPERTISE AS WELL AS
CHEETAH GROUP FOR ALLOWING ME TO
COME AND TELL MY STORY.
THANK YOU.
>> ANY QUESTIONS FOR JAMIN?
NOBODY?
>> ALL RIGHT.
THANK YOU.
>> NICE JOB, JAMIN.
THANK YOU.
[APPLAUSE]
NEXT SPEAKER WILL BE RUTH
PARSONS, DUKE UNIVERSITY.
>> HEY, EVERYBODY.
I'M RUTH PARSONS, A FOURTH YEAR
GRADUATE STUDENT, AT DUKE
UNIVERSITY.
TODAY I'M GOING TO TELL YOU
ABOUT CONFIRMATIONAL VARIATION.
CD4 BINDING IS IMPORTANT AT THE
BEGINNING OF THE TRANSITION OF
THE ENVELOPE FROM PRE-FUSION TO
POST-FUSION.
THERE'S A PRIMARY BINDING SITE
AND SECONDARY BINDING SITE FOR
CD4.
SHOWN IN BLUE IS PRIMARY BINDING
SITE.
ON ON THE ADJACENT GP120 THERE'S
A SECONDARY BINDING SITE.
THIS IS IMPORTANT FOR INITIAL
CONTACT OF CD4.
THERE ARE MANY bNAbS THAT
BIND TO CD4 AS WE'VE BEEN
DISCUSSING.
AND THERE ARE A FEW THAT BIND TO
BOTH THE PRIMARY BINDING SITE
AND THE SECONDARY BINDING SITE.
AND THAT'S WHAT IS OF INTEREST
IN MY TALK TODAY.
THERE ARE OBSTACLES FOR BROADLY
NEUTRALIZING ANTIBODIES BEING
ABLE TO BIND TO THE CD4 BINDING
SITE ONE AND TWO.
THERE'S VERY -- IT'S A VERY
VARIED SITE BETWEEN TWO
PROTOMERS, LONG LOOPS, GLYCANS
SHIELDING THE REGION, AND
THERE'S ALSO FLEXIBILITY BETWEEN
THE TWO ENV MONOMERS.
THAT BINDING SITE IS BETWEEN TWO
MONOMERS, FLEXIBILITY IN
MOVEMENT BETWEEN THEM.
OUR GOAL IS TO UNDERSTAND HOW
THE CD4 BINDING SITE bNAbS
ACCESS THE CD 4 BINDING SITE AND
CD4 BINDING SITE 2.
OUR GROUP STUDIED ENV
FLEXIBILITY, THAT IS BETWEEN TWO
PROTOMERS.
WE'VE FOUND DIFFERENCES IN
FLEXIBILITY BETWEEN DIFFERENT
SEQUENCES AND ENVELOPES.
SO HERE SHOWN A BG 505 SOSIP,
TIGHTLY CLOSED ENVELOPE, AND
THEN 1059 SOSIP, COMPLETELY
OPEN.
IT'S ALSO MORE FLEXIBLE.
AND A CONSEQUENCE OF THIS WAS
FOUND TO BE DECREASED BINDING TO
THE CD4 BINDING SITE ANTIBODIES.
THEY BIND FINE TO JUST THE GP120
BUT IN THE TRIMERIC FORM THEY
DON'T BIND AS WELL.
AND SO WE BELIEVE THAT'S DUE TO
THE FLEXIBILITY.
SO, THERE ARE DIFFERENT WAYS
THAT DIFFERENT bNAbS HAVE
FIGURED OUT HOW TO GET TO THE
SECONDARY BINDING SITE.
LIKE ONE EXAMPLE, VRC 03 HAS
EXTENDED FR 3 REGION HERE, THAT
ALLOWS US TO REACH TO THE
SECONDARY BINDING SITE.
VRC CH 31 AS EXTENDED REGION
WHICH ALLOWS IT TO EXTEND TO
THAT REGION.
AND THEN ANOTHER ANTIBODY THAT
WE'RE INTERESTED IN CR 103
DOESN'T HAVE EXTENDED LOOPS.
DIFFERENT ANGLE OF APPROACH,
20-DEGREE DIFFERENT IN ANGLE,
THAT ALLOWS THIS FRAMEWORK
REGION 3 LOOP TO GET CLOSE
ENOUGH TO THE SECONDARY BINDING
SITE TO INTERACT.
SO WE'RE INTERESTED IN THIS CH
103 bNAb AND WANT TO
UNDERSTAND HOW THE RESIDUES
EVOLVED TO BIND TO THIS SITE.
WE'RE INTERESTED IN TWO RESIDUES
IN THE FRAMEWORK REGION 3 THAT
EARLY ON IN THE ANTIBODY LINEAGE
DEVELOPMENT THEY MUTATED TO
BECOME MORE AN-IONIC, MAINTAINED
THROUGH THE LINEAGE AND MATURE
CH 103.
AS I MENTIONED THEY ARE SITUATED
IN REGION 3 WHICH IS WHAT
INTERACTS WITH THE CD4 BINDING
SITE 2, CDRH 3, INTERACTING WITH
BINDING SITE 1.
AND SO THE UNMUTATED COME AN
ANCESTOR, IF YOU MUTATE THESE
TWO RESIDUES BACK TO THE
UNMUTATED COMMON ANCESTOR AT
POSITION 75 AND 76, THAT
DECREASES NEUTRALIZATION BY
THESE ANTIBODIES AS WELL AS
BINDING TO THE THE ENV.
THE UNMUTATED COMMON ANCESTOR
REVERTED FORM ALL REFER TO THAT
AS THE KN MUTANT.
SO WE COLLECTED DATASETS ON BOTH
OF THESE AND GOT MULTIPLE
STRUCTURES WITH DIFFERENT
NUMBERS OF ENVs BOUND.
MANY OF YOU ARE FAMILIAR WITH
THE CryoEM DATA PROCESSING
WORKFLOW, COLLECTING
MICROGRAPHS, TAKING PARTICLES,
DOING 2D CLASS AVERAGES, WE GET
RID OF BAD PARTICLES AND
CLASSES, THEN WE CAN GET
MULTIPLE STRUCTURES OUT OF ONE
DATASET.
SO FOR MY TWO DATASETS I'M
TALKING ABOUT NOW, WILD TYPE AND
REVERTED, I HAVE STRUCTURES OF 1
FAB BOUND, 2 BOUND, THEN NO FAB
FOR THE KN MUTANT.
NOW ZOOMING INTO THE BINDING
SITE TO SEE WHAT'S GOING ON, AT
THE POSITION 75, THIS INTERACTS
WITH THIS ARGININE HERE SO
THERE'S AN EXTENSIVE LIKE
HYDROGEN BONDING NETWORK THAT'S
CREATED HERE SO THESE RESIDUES
ARE SITUATED CLOSE TO THE
ARGININE.
THEN IN CONTRAST THE LYSINE THAT
THIS 75 HAS BEEN MUTATED TO IS
NOW SITUATED TOO FAR TO BE
HYDROGEN BONDING.
POSITION 76, IN THE WILD TYPE,
IT IS SITUATED CLOSE ENOUGH TO
HAVE ELECTROSTATIC INTERACTIONS
WITH LYSINE, IN THE KN MUTANT
THIS LYSINE IS NOW SITUATED TOO
FAR AWAY TO BE INTERACTING.
AND THERE'S ALSO NOT VERY GOOD
DENSITY INDICATING THIS IS NOT
CREATING A STABLE INTERACTION AT
THIS PORTION OF THE CD4 BINDING
SITE, THIS LOWER PORTION OF THE
CD4 BINDING SITE 2 IS CREATING
MORE OF THE DIFFERENCE IN THE
BINDING BETWEEN THOSE TWO
MUTANTS.
AND I ALSO TOOK A LOOK AT THE
PRIMARY BINDING SITE TO SEE IF
THE DIFFERENCES IN THE SECONDARY
BINDING SITE WOULD CAUSE ANY
CHANGES IN THE PRIMARY BINDING
SITE.
AND I DIDN'T SEE ANY DIFFERENCES
SO IT SEEMS TO BE BINDING THE
WILD TYPE AND KN SEEM TO BE
BINDING WITH EACH OTHER TO THE
PRIMARY BINDING SITE SO ALL
THESE INTERACTIONS I'M SHOWING
ARE JUST THE SAME BETWEEN THE
TWO.
SO, TO FURTHER LOOK AT THE
FLEXIBILITY IN THESE STRUCTURES,
I USED 3D VARIABILITY ANALYSIS
SO THIS TAKES INDIVIDUAL
PARTICLE IMAGES AND DOES
PRINCIPAL COMPONENTS ANALYSIS TO
GIVE A CONTINUOUS FAMILY OF 3D
STRUCTURES, AND THAT'S WHAT I
WAS SHOWING, THAT'S WHAT WE DID
IN THOSE STRUCTURES OF ENVELOPES
TO SHOW DIFFERENT ENVELOPES HAVE
DIFFERENT FLEXIBILITY.
I DID THAT WITH THESE STRUCTURES
AS WELL.
AND THESE ARE THE MOVIES THAT
RESULTED FROM DOING 3D ANALYSIS,
IN GENERAL THE WILD TYPE FORM
SEEM TO BE LESS FLEXIBLE,
ESPECIALLY AT THIS PROTOMER
SITUATED NEXT TO THE FAB.
AND THEN IN THE BINDING SITE YOU
CAN CLEARLY SEE IN ALL OF THE
FRAMES THERE IS DENSITY AROUND
THIS LYSINE, THAT I MENTIONED
WAS INTERACTING EXTENSIVELY BUT
THAT IS NOT REALLY PRESENT IN
THE KN MUTANT.
AND ALSO YOU CAN SEE THIS
PROTOMER IS JUST MOVING MUCH
MORE AND NOT CREATING A STABLE
INTERACTION AT THIS PORTION OF
THE INTERACTION.
OF THE BINDING SITE.
IN CONCLUSION, HIV-1 ENVELOPE
FOR BINDING SITE BETWEEN TWO
PROTOMERS PROTECTED BY
FLEXIBILITY, SOME bNAbS
INTERACT WITH PROTOMERS AT CD4
BINDING SITE USING MULTIPLE
MECHANISMS, AND CD103 LINEAGE
ACQUIRES, TWO MUTATIONS EARLY.
I WANT TO THANK MY LAB AND THE
FUNDING AND THE ORGANIZERS OF
THE THIS EVENT.
THANK YOU ALL VERY MUCH FOR
LISTENING.
[APPLAUSE]
>> ANY QUESTIONS FOR RUTH?
QUIET GROUP THIS AFTERNOON.
ALL RIGHT.
WELL, FOR AN EXCELLENT
PRESENTATION.
[APPLAUSE]
NEXT AND FINAL SPEAKER IS MAMTA
SINGH FROM NATIONAL INSTITUTE OF
ALLERGY AND INFECTIOUS DISEASES
>> GOOD AFTERNOON, EVERYONE.
I'M A POSTDOC FELLOW AT NIAID.
I'LL BE TALKING ABOUT THE
OPTIMIZATION OF VLP-FORMING
mRNA VACCINE BY
PROTEASE-MEDIATED GAG
PROCESSING.
SO, THE DEVELOPMENT OF THE
PROPHYLACTIC VACCINE REMAINS ONE
OF THE HIGHEST PRIORITIES IN THE
GLOBAL EFFORTS TO END THE
HIV/AIDS PANDEMIC.
AND THERE HAVE BEEN A WIDE
CONSENSUS THAT THE ELICITATION
OF BROADLY NEUTRALIZING
ANTIBODIES WHICH I TARGETED TO
THE ENVELOPE GLYCOPROTEIN IS THE
PRIMARY GOAL OF THE PROTECTIVE
VACCINE.
HOWEVER, THERE ARE SEVERAL KEY
REQUIREMENTS FOR THE ELICITATION
OF THESE bNAbS WHICH
BASICALLY REQUIRE THE ENVELOPE
GLYCOPROTEIN TO BE PRESENTED IN
ITS NATIVE CONFIGURATION.
THE ENVELOPE NEEDS TO BE
PRESENTED IN ITS NATIVE
CONFIGURATION TO RETAIN CORRECT
ANTI--GENICITY, TO STIMULATE
GERMINAL CENTER REACTION, WITH
EFFECTIVE ENGAGEMENT OF THE
PRECURSOR B CELLS FILED BY
MATURATION TOWARDS THE bNAb
DEVELOPMENT.
SO, TO ACHIEVE THESE GOALS IN
OUR LABORATORY WE DEVELOPED ON
THE GAG-BASED mRNA VACCINE
WHICH UTILIZES HOST
TRANSLATIONAL MACHINERY TO
EXPRESS VIRUS-LIKE PARTICLES AND
THE VACCINE WAS ADMINISTERED
USING UNIQUE IMMUNIZATION
REGIMEN, THAT INVOLVES PRIMING
WITH GERMLINE ENGAGER ENVELOPE
FOLLOWED BY INTENSIVE BOOSTING
WITH THE HETEROLOGOUS ENVELOPE
TO DRIVE THE AFFINITY MATURATION
TOWARDS bNAb DEVELOPMENT.
SO THE RESULTS OF THE STUDY WERE
REPORTED IN NATURE MEDICINE IN
2021.
IMPORTANTLY, ALL OF THE
VACCINATED MACAQUES INTRODUCED
TITERS AGAINST 11 OF THE 12
ISOLATES PART OF THE SO-CALLED
SMALL GLOBAL PANEL.
EVEN THOUGH THE TITERS WERE LOW,
THE VACCINE INDUCED REMARKABLE
PROTECTION AGAINST REPEATED
MUCOSAL CHALLENGES WITH
HETEROLOGOUS NEUTRALIZED TITER
TO STRAIN THAT RADIATE WITH
CALCULATED RISK REDUCTION OF
79%.
THIS DATA SUGGESTED THE ENVELOPE
GAG VLP-FORMING VACCINE IS A
PROMISING APPROACH IN VACCINE
DEVELOPMENT, ROOM FOR FURTHER
IMPROVEMENT.
WITH THIS AIM IN THE STUDY WE
EXPLORED BENEFITS OF PRODUCING
MATURE RATHER THAN IMMATURE
VLPs TO DELIVER ENVELOPE
GLYCOPROTEIN.
THIS SHOWED RELATIVE TO GAG
LATTICE, IN CASE OF IMMATURE
PARTICLES IT HAS BEEN SEEN THAT
THE MATRIX DOMAIN OF THE GAG
INTERACTS WITH CYTOPLASTIC TAIL
OF THE ENVELOPE, THERE WERE
MOBILITY ACROSS THE ENTER
INTERFACE.
IT IMPARTS MOBILITY TO THE
INCURRED GLYCOPROTEIN SO WE
SPECULATE THIS ENHANCED MOBILITY
OF ENVELOPE ON THE SURFACE OF
VIRUS-LIKE PARTICLES MAY IMPART
POTENTIAL IMMUNOLOGIC BENEFITS
IN TERMS OF ANTIGEN UPTAKE OR
PRESENTATION, MATURE VLPs MAY
ACT AS EFFECTIVE IMMUNOGEN
COMPARED TO IMMATURE
COUNTERPARTS.
WE INTRODUCED WILD PROTEASE OF A
THIRD COME COMPLEMENT.
WE HYPOTHESIZED THIS WILL
ENHANCE MOBILITY OF ENVELOPE,
PROMOTE CLUSTERING ON THE
SURFACE OF MATURE VLP,
SUBSEQUENTLY LEADING TO
EFFICIENT B CELL STIMULATION BY
CROSS-LINKING, AND TO EXPRESS
THE VIRUS PROTEASE WE DECIDED TO
MIMIC PHYSIOLOGIC MECHANISM
DURING THE COURSE OF HIV
INFECTION, TRANSCRIBED AS GAG
TRANSCRIPT FOLLOWED BY
TRANSLATION IN A DIFFERENT
READING FRAME BY RIBOSOMAL SHIFT
MECHANISM, DESIGNED TO
IDENTIFIER THE MOST SUITABLE ONE
TO INDUCE APPROPRIATELY DOSED,
TIMELY EXPRESSION, FORMATION OF
MATURE VLPs.
THREE OF FIVE HAD THE FRAME
SHIFT SEQUENCES, AND THEY WERE
OPTIMIZES, CONSTRUCT NUMBER 4
WAS THE FULL LENGTH CONSTRUCT
DIVIDE OF THE FRAME SHIFT
SEQUENCES, FIFTH HAD SOME
ADDITIONAL TWO-RUN CASE IN PAUSE
GENE.
WHEN WE LOOKED AT FOR EFFICIENCY
BY MEASURING P16 CAPTURABLE B
2711, HIGHEST LEVEL WITH
CONSTRUCT NUMBER 4, AGAIN A
FRAME SHIFT SEQUENCE 3, FULL
LENGTH GAG POL GENE.
WE SELECTED THIS CONSTRUCT FOR
OUR FURTHER STUDIES, NEXT
DEFINED OPTIMAL AMOUNT FOR
PRODUCTION OF THE MATURE VERSION
OF THE VLPs, AND WE
CO-TRANSFECTED THE ENVELOPE
EXPRESSING CELL LINE WITH FIXED
AMOUNT OF GAG mRNA AND
DECREASING AMOUNT OF GAGPOL,
EFFICIENCY OF VLP PRODUCTION
SHOWED DISTRIBUTION, PEAK
VALUES, THIS PARTICULAR DATA
SUGGESTED CO-TRANSFECTION OF
CELL LINES WITH GAG mRNA AND
GAGPOL N RNA DEVOID OF FRAME
SHIFT SEQUENCES AT RATIO OF 1 IN
210 PROVIDE OPTIMAL CONDITION
FOR PRODUCTION OF MATURE
VIRUS-LIKE PARTICLES.
SO, WE WENT AHEAD WITH THE IN
VITRO EXPRESSION OF IMMATURE AND
MATURE VLPs FOR
CHARACTERIZATION, AND THE
STRUCTURAL CHARACTERISTICS OF
IMMATURE AND MATURE HIV VLPs
REVEALED BY NEGATIVE STAINING
ELECTRON MICROSCOPY, SHOWING
ARRAY OF ENVELOPE SPIKES ON
SURFACE, IN DEPTH STRUCTURE
CHARACTERIZATION OF IMMATURE
VLPs WAS PERFORMED BY CryoEM
ANALYSIS, AND THE PARTICLES
SHOWED A WELL-ORGANIZED GAG
LAYER, DECORATED WITH THE
TRIMERS, HERE I'M SHOWING VIDEO
OF THE TOMOGRAPHIC SLICES.
INTERESTINGLY FOR MATURE VLPs
CryoEM IMAGES SHOWED NO
VISIBLE GAG LAYER, AND WE
COULDN'T SEE THE CONICAL CODE
WHICH MAY SUGGEST THE PROCESS IS
INEFFICIENT, ALTHOUGH SOME
EVIDENCE OF ENVELOPE CLUSTERING
WAS OBSERVED.
SO NEXT TO ASSESS POTENTIAL OF
ADDITION OF THIS GAGPOL TO
VACCINE PLATFORM, WE IMMUNIZED
MICE WITH THE mRNA
FORMULATION, HAVING ENVELOPE
ALONE OR IN COMBINATION WITH SIV
GAG IN THE PRESENCE AND ABSENCE
OF GAGPOL IN DIFFERENT RATIOS,
IMMUNIZED WITH .
AS WE CAN SEE THE TRIMER BINDING
ANTIBODIES AGAINST AUTOLOGOUS
ENVELOPE WERE SIGNIFICANTLY AND
CONSISTENTLY HIGHER FOR THOSE
ENVs THAT RECEIVED VLP-FORMING
VACCINE AS COMPARED TO THE ONE
THAT RECEIVED THE SUBUNIT BASE
VACCINE, WHICH BASICALLY
SUGGESTS SUPERIORITY OF THE
PARTICLE OF VACCINES OVER
SUBUNIT VACCINES, AND ALSO WE
OBSERVED THAT ALL THOSE ANIMALS
THAT RECEIVED PROTEASE
SUPPLEMENTED VACCINE ELICITED
SUBSTANTIALLY HIGHER TITERS OF
ANTIBODIES AS COMPARED TO ENV 2,
GAG ALONE, DEMONSTRATING THE
IMPROVED IMMUNOGENICITY OF
PROTEASE SUPPLEMENTATION TO THE
VACCINE PLATFORM.
SO NEXT WE CHECKED
NEUTRALIZATION ACTIVITY IN THE
SERUM ANTIBODIES AGAINST
DEGLYCOSYLATED VERSION OF
PSEUDOVIRUSES.
ALL OF THOSE ANIMALS THAT
RECEIVED PROTEASE SUPPLEMENTED
REGIMENS ELICITED HIGHER
NEUTRALIZING ANTIBODIES AS
COMPARED TO ENV 2, WITH ONE
HAVING THE LOWEST TITERS.
NEXT WE AGAIN TESTED THE MOUSE
SERA FOR ABILITY TO NEUTRALIZE
THE PARTIALLY CLOSED FORM, A
COMPETITIVELY MORE CHALLENGING
TARGET.
ALTHOUGH WE OBSERVED LIKE
MARKEDLY LOWER TITERS FOR ALL OF
THE TIME POINTS, EXCEPT FOR ARM
3 WHICH RECEIVED THE RATIO OF 1
IN TWO FIVE SUGGESTING THE
PLATFORM WHEN SUPPLEMENTED WITH
PROTEASE mRNA IMPROVED
EFFICACY OF VACCINE BY INDUCING
NEUTRALIZING ANTIBODIES.
THE INCLUSION FOR PROCESSING GAG
RESULTED IN MATURE FORM OF
VLPs ELICITING HIGHER TITERS
AS COMPARED TO VACCINE THAT
PRODUCES IMMATURE VLPs,ODOD AND
THEREFORE IS ONE OF THE IMPROVED
AND PROMISINGS PLATFORMS FOR HIV
VACCINE DEVELOPMENT.
HERE ARE MY ACKNOWLEDGMENTS, MY
ACKNOWLEDGMENT TO MY LAB MEMBERS
AND SPECIALISTS, MY POSTDOC
MENTOR FOR HIS SCHOLARLY INPUT
AND GUIDANCE, PROVIDING mRNA
FORMULATION, SPECIAL THANKS FOR
COLLABORATION, AND WE THANK
UNIVERSITY OF WASHINGTON FOR
HELPING US WITH THE CryoEM
ANALYSIS AND SENDING BEAUTIFUL
IMAGES OF THE VIRUS-LIKE
PARTICLES.
I WOULD ALSO LIKE TO ACKNOWLEDGE
GATES FOUNDATION, NIAID, OFFICE
OF AIDS RESEARCH FOR THE
FINANCIAL ASSISTANCE.
THANK YOU ALL FOR YOUR KIND
ATTENTION AND I'LL BE HAPPY TO
TAKE QUESTIONS IF ANY.
[APPLAUSE]
>> DID YOU LOOK AT GAG
PROCESSING FOR YOUR GAGPOL
VLPs?
>> YEAH, WE DID BLOOD ANALYSIS
TO BASICALLY LOOK AT GAG
PROCESSING FOR DIFFERENT AMOUNTS
BECAUSE OF THE TIME CONSTRAINTS,
I DIDN'T SHOW THE ENTIRE DATA
BUT WE DID TRY DIFFERENT AMOUNTS
OF THE mRNA TO EVALUATE
OPTIMALITY CONDITION.
>> WAS THERE EFFICIENT GAG
PROCESSING?
BECAUSE THE PARTICLES DID NOT
LOOK MATURE IN TERMS OF LACKING
CONICAL CORE.
>> YEAH, EXACTLY.
THAT'S ONE OF THE THINGS WE HAVE
BEEN WORKING ON BECAUSE
ENHANCING THE AMOUNT OF GAGPOL
WAS INDUCING SOME AMOUNT OF
CELLULAR CYTOTOXICITY, THAT'S
WHY WE CHOSE A SAFER RATIO BUT,
YES, WE HAVE TO FURTHER OPTIMIZE
THE CONDITIONS TO GET THE FULLY
MATURE FORM.
THANK YOU.
>> THIS IS A FOLLOW-UP TO ERIC'S
QUESTION, EDDIE ARNOLD, RUTGERS
UNIVERSITY.
QUESTION IS, HOW DO YOU USE THE
PROTEASE OF GAGPOL ADDING TO THE
EQUATION, DO YOU HAVE ANY
INFORMATION ABOUT WHAT REVERSE
TRANSCRIPT TASTE MIGHT BE DOING
BECAUSE PROTEASE IS A SMALL PART
OF POL.
>> THAT IS TRUE BUT LIKE WE TRY
TO TRUNCATE THE GAGPOL AND DON'T
SEE LIKE MUCH DIFFERENCE BUT WE
WE TRY TO KEEP IT AS MINIMAL AS
POSSIBLE.
>> JUST WONDERING IF YOU'RE
LOOKING INTO THAT ASPECT.
THANK YOU.
GREAT TALK.
>> ONE MORE QUESTION.
>> QUICK QUESTION ABOUT THE
mRNA FOR GAG AND ENV.
COVID mRNA VACCINE HAS A RNA
MODIFICATION TO ENHANCE PROTEIN,
ALSO AVOID INNATE IMMUNE
RESPONSE, WONDERED DO YOU HAVE
MODIFICATION.
>> THIS IS A MODIFIED VERSION
OF mRNA BY MODERNA,
EXPRESSING --
>> BOTH GAG AND --
>> YEAH.
>> THANK YOU.
>> IF THERE'S NO OTHER QUESTIONS
WE'LL GIVE MAMTA AND THE OTHER
SPEAKERS A BIG ROUND OF
APPLAUSE.
THANK YOU.
[APPLAUSE]
>> THANK YOU, EVERYONE, FOR
COMING.
THIS HAS BEEN, AS ALWAYS, A
GREAT MEETING.
I ENJOY THIS EVERY YEAR.
AND I REALLY APPRECIATE ALL YOUR
PARTICIPATION, ESPECIALLY THE
POSTERS, HARD TO GET GET YOU AY
FROM THE POSTERS, ALWAYS A GOOD
SIGN.
I DON'T HAVE ANYTHING TO SAY
OTHER THAN SEE YOU NEXT YEAR,
JUNE 23, 2025.
THIS MEETING IS ALWAYS THE LAST
MONDAY AND TUESDAY OF THE MONTH,
IF YOU CAN'T REMEMBER THE DATES.
AND I HOPE I SEE Y'ALL OUT HERE
AGAIN, ESPECIALLY SEE IF KEY CAN
MAKE A RECORD NUMBER OF POSTERS
AGAIN NEXT YEAR.
AND GET OUT THERE AND DO SOME
GOOD SCIENCE.
[APPLAUSE]
[END OF PROGRAM]