GOOD AFTERNOON.
I'M STEVE HUGHES AND I WANT TO
WELCOME ALL OF YOU TO THE DAVE
DERSE MEMORIAL LECTURE.
DAVE HAS NOW BEEN GONE FOR 15
YEARS, AND THAT MEANS,
UNFORTUNATELY, THAT MANY OF YOU
DID NOT HAVE A CHANCE TO GET TO
KNOW DAVE PERSONALLY.
SO THAT BRINGS ME TO THE
QUESTION OF WHO DAVE DERSE WAS
AND WHY WE WANT TO BOTH REMEMBER
AND HONOR HIM.
DAVE WAS A SCHOLAR AND HE LOVED
THE INTELLECTUAL CHALLENGES THAT
SCIENCE PROVIDES.
FOR THAT REASON, IT IS ENTIRELY
FITTING THAT WE HONOR HIS MEMORY
IN A FORM THAT DAVE WOULD
PARTICULARLY HAVE LIKE, A
LECTURE FROM CAROL CARTER.
THIS IS DAVE AND HIS COLLEAGUES
IN, OH, MAYBE 20 YEARS AGO.
DAVE WAS ALSO NOT ONLY A
VALUABLE MENTOR, WAS DEEPLY
DEDICATED TO PEOPLE WHO WORK
WITH HIM, HE WAS A COLLEAGUE AND
THOSE OF US WHO WORKED WITH HIM
GREATLY MISS HIM.
HERE HE'S SHOWN WITH ANOTHER
MEMBER OF THE DRP IN THE
AUDIENCE TODAY, WHO I'M SURE
MISSING HIM JUST AS MUCH AS THE
REST OF US, BUT MOST OF ALL,
DAVE WAS A FRIEND.
WE SHOULD REMEMBER DAVE IN THE
LARGER SENSE.
WE WANT TO REMEMBER HIM, OF
COURSE, AS A SCIENTIST, BUT WE
WANT TO REMEMBER HIM NOT ONLY AS
A SCIENTIST, BUT ALSO AS A
PERSON.
DAVE WAS REMARKABLY KIND AND
AGAIN TELL.
HE HAD A -- GENTLE.
HE HAD A MARVELOUS CLARITY OF
SPIRIT.
I CAN SAY HONESTLY THAT I NEVER
HEARD HIM SAY A HARSH WORD ABOUT
ANYONE.
BECAUSE WE SHARED A LOT OF THE
OUTDOORS AND FISHING, I HAD THE
PRIVILEGE OF SPENDING TIME WITH
HIM AWAY FROM THE LAB.
HERE'S DAVE WITH A REALLY NICE
STRIPER CAUGHT OUT IN CHESAPEAKE
BAY, AGAIN ABOUT 20 YEARS AGO.
IT REALLY WASN'T THE SIZE OF THE
FISH THAT MATTERS.
HERE'S DAVE WITH A LITTLE BROWN
HE CAUGHT ON COTTON CREEK AND
HIS SMILE WAS EVERY BIT AS BIG
AS IT WAS WHEN HE HAD THE BIG
STR
STRIPER.
AND THOSE OF US WHO KNEW DAVE
WELL HAVE MANY FOND MEMORIES OF
HIM.
I CAN GO BACK TO CHESAPEAKE OR
TO JOHN CREEK AND SOME OF THE
OTHER PLACES WE FISH AND BE
REMINDED OF WHAT A WONDERFUL
FRIEND AND COLLEAGUE HE WAS.
AND IT'S PARTICULARLY IMPORTANT,
I THINK, THAT WE SHOULD HONOR
HIS KINDNESS AND HIS GENTLENESS
AS WE APPROACH AN ELECTION THAT
IS REMARKABLY BITTER.
IT'S FILLED WITH HARSH WORDS AND
THE THREAT OF EVEN HARSHER
DEEDS.
SO I WOULD HOPE THAT DAVE IS
WITH US TODAY IN SPIRIT AND HIS
GENTLE APPROACH TO LIFE CAN HELP
US NAVIGATE THESE TROUBLED
TIMES.
WELCOME TO THE LECTURE THANK
YOU.
[ APPLAUSE ]
>> THANK YOU VERY MUCH FOR THAT
STEVE.
WE CERTAINLY DO MISS DAVE VERY
MUCH.
I'D LIKE TO JOIN STEVE AND ALL
THE FACULTY OF THE HIV DRP IN
WELCOMING YOU TO THE 13th ANNUAL
DAVID DERSE MEMORIAL LECTURE AND
AWARD.
AS STEVE ALLUDED TO, MANY IN
THIS AUDIENCE DID NOT KNOW DAVE
PERSONALLY.
TO LEARN MORE ABOUT HIS LIFE AND
HIS CAREER, I WOULD DIRECT YOU
TO THIS COMMENTARY WRITTEN BY
ONE OF HIS TRAINEES, MAUREEN
SHUH, PUBLISHED IN 2009, SHORTLY
AFTER HIS UNTIMELY PASSING.
I'D LIKE TO SHARE ONE EXCERPT
FROM THIS COMMENTARY.
DAVE WAS VERY PASSIONATE ABOUT
SCIENCE AND HIS ENTHUSIASM FOR
WORK WAS CONTAGIOUS TO MEMBERS
OF THE LABORATORY.
DAVE MADE SEVERAL IMPORTANT
CONTRIBUTIONS TO THE FIELD, BUT
HE NEVER SOUGHT ACCOLADES FOR
HIMSELF.
DAVE REMAINS THE MOST GENUINE
AND KIND INDIVIDUAL I KNOW.
I THINK ALL OF US WHO KNEW DAVE
FELT THIS WAY ABOUT HIM.
SO I RECENTLY HAD THE
OPPORTUNITY TO COMB THROUGH THE
COLD SPRING HARBOR PHOTO
ARCHIVES AND FOUND A FEW OLD
PHOTOS OF DAVE THAT I
PARTICULARLY LIKED.
ONE HERE ON THE LEFT TAKEN IN
1992, AND TWO IN 1999, INCLUDING
THE ONE ON THE RIGHT OF DAVE AND
KATE, WHO LATER BECAME KATE
DERSE AND REMAINS ONE OF THE
MAJOR SPONSORS OF THIS EVENT.
DAVE CONTRIBUTED IN SUBSTANTIAL
WAYS IN MANY ASPECTS OF
VIROLOGY.
EARLY IN HIS CAREER, I STUDIED
HSV POLYMERASE BEFORE SWITCHING
TO RETROVIRUSES, GENE EXPRESSION
AND RNA SURPRISING.
HE DEVELOPED A NUMBER OF
RETROVIRAL VECTORS AND CLONES
STILL USED TODAY.
HE STUDIED RETROVIRAL ASSEMBLY
AND TRANSMISSION AND RESTRICTION
OF RETROVIRAL INFECTIVITY BY
APOBEC.
THE DERSE MEMORIAL LECTURE AND
AWARD WERE ESTABLISHED IN 2012
AND FEATURED A NUMBER OF
PROMINENT SCIENTISTS, INCLUDING
PAT GREEN, JOE GIAM, WALTER
MOTHES, QUENTIN SATTEN TAU,
CHARLES, BEATRICE, WE WENT
VIRTUAL DURING COVID IN 2020 AND
2021.
AND THEN WE RESUMED IN-PERSON
MEETINGS IN 2022 WITH DARIA AND
LAST YEAR WITH LOU MANSKY.
SO THAT BRINGS US TO THIS YEAR'S
LECTURE AND AWARDEE, CAROL
CARTER.
CAROL RECEIVED HER BACHELORS IN
DEGREE IN BIOLOGY AND CHEMISTRY
FROM 1967 FROM CITY COLLEGE OF
NEW YORK.
WENT ON TO GET HER PhD IN
MICROBIOLOGY FROM YALE.
DID A TWO-YEAR POST DOC AT ROCHE
AND WAS RESKRUTED AS ASSISTANT
PROFESSOR TO STONY BOOK
UNIVERSITY WHERE SHE'S REMAINED
EVER SINCE, CLIMBING THROUGH THE
RANKS, BECOMING FULL PROFESSOR
IN 1994 AND IN 2022, THE STATE
UNIVERSITY OF NEW YORK
DISTINGUISHED PROFESSOR AT STONY
BROOK.
CAROL HAS RECEIVED A NUMBER OF
HONORS AND RECOGNITION AND IS
WELL-KNOWN FOR HER SERVICE TO
THE COMMUNITY.
IN 2013, SHE RECEIVED THE STONY
BROOK UNIVERSITY PRESIDENTIAL
AWARD FOR PROMOTING DIVERSITY
AND ACADEMIC EXCELLENCE.
TWICE, SHE LEAFED THE STONY
BROOK EXCEPTIONAL SERVICE TO
UNDERGRADUATE EDUCATION AWARD.
HAS SERVED ON NUMEROUS EDITORIAL
BOARDS AND STUDY SECTIONS.
IN 2022, WAS ELECTED TO THE
AMERICAN ACADEMY OF MICROBIOLOGY
AND EARLIER THIS YEAR TO THE
NATIONAL ACADEMY OF SCIENCES.
CONGRATULATIONS TO CAROL FOR
THAT WELL-DESERVED HONOR.
[ APPLAUSE ]
CAROL'S REACH HAS SPANNED A WIDE
SWATH OF RETROVIROLOGY,
ACTIVATION OF THE HIV-1 PROCEED
TEEATION, THE STRUCTURE OF
RETROVIRAL PROTEINS, THE ROLE OF
ESCRT MACHINERY, THE ROLE OF
CALCIUM AND IS FOR TOINNOSITIDE
SIGNALING.
SO CAROL IS NOT ONLY A
WELL-KNOWN AND DISTINGUISH
SCIENTIST, BUT SHE'S ALSO A
CARING MENTOR AND ROLE MODEL FOR
YOUNGER PEOPLE IN THE COMMUNITY.
I THINK THIS IS CAPTURED TO SOME
EXTENT IN THESE PHOTOS TAKEN
WITH CAROL WITH MEMBERS OF MY
LAB AT COLD SPRING HARBOR BACK
IN MAY.
YOU CAN SEE THEY'RE HAVING A
GOOD TIME HERE.
[ LAUGHTER ]
SO WITHOUT ANY FURTHER DELAY, I
WOULD LIKE TO WELCOME CAROL TO
THE PODIUM FOR HER LECTURE
ENTITLED HOST-TARGETED
THERAPEUTICS, PRAZOLES TARGET
TSG101 UBIQUITIN E2 VARIANT
MIMICRY, AND TO RECEIVE HER
DAVID DERSE MEMORIAL LECTURE
AWARD.
[ APPLAUSE ]
IT'S ALL YOUR FAULT.
[ LAUGHTER ]
>> THANK YOU.
READY.
1, 2, 3.
PERFECT.
A COUPLE LIKE THIS.
1, 2, 3.
AND 1, 2, 3.
[ APPLAUSE ]
>> SO ONCE AGAIN, THANK YOU,
THANK YOU, THANK YOU.
THIS IS A VERY SPECIAL HONOR FOR
ME BECAUSE I KNOW SO MANY
FABULOUS SCIENTISTS WHO ARE HERE
AND DIDN'T ACTUALLY EVER MEET
DAVID DURING HIS LIFE, BUT
CERTAINLY ACQUAINTED WITH THE
WORK THAT HE DID.
SO I'M GOING TO START OFF.
STEVE HUGHES JUST SAID TO ME, HE
SAID KNOCK THEM DEAD.
WELL, I'M GOING TO START OFF BY
SIMPLIFYING MY TITLE.
SO HOST-TARGETED THERAPEUTICS,
PRAZOLES TARGET EXIT, AND THAT
IS WHAT WE'RE GOING TO SORT OF
DO TO TALK TO YOU ABOUT TODAY
AND ONE OF THE THINGS THAT HAS
ALWAYS FASCINATED ME.
SO EVERYBODY WHO IS IN THIS ROOM
OR VIRTUAL IS ACQUAINTED, I
THINK, FOR THE MOST PART WITH
HIV, ITS LIFECYCLE, AND THE
INCREDIBLE AND IMPRESSIVE
COMPLEXITY THAT THIS VIRUS
PRESENTS IN DOING WHAT IT
NATURALLY DOES FOR ITSELF,
REPLICATE.
AND ONE OF THE THINGS THAT'S
ALWAYS FASCINATED ME IS THAT
EVERY STEP OF ITS REPLICATION
CYCLE IS IMPORTANT, BUT IN
PARTICULAR, THE LAST STEP, THE
EGRESS STEP IS NOT ONLY WHAT
IMPORTANT IN ITS OWN RIGHT, BUT
IT'S CRITICALLY IMPORTANT
BECAUSE IT SETS THE VIRUS UP FOR
WHAT IT'S GOT TO DO IN THE NEXT
STAGE, WHICH IS TO GET INTO
ANOTHER CELL.
SO I TOOK A QUOTE FROM
SHAKESPEARE'S MAC BETH, NOTHING
IN THE VIRUS' LIFECYCLE BECOMES
IT LIKE THE LEAVING OF IT.
IN OTHER WORDS, IT'S GOT TO DO
IT RIGHT OR ELSE IT'S DONE.
AND SO WE -- THE FOCUS OF THE
TALK TODAY IS REALLY GOING TO BE
WHAT'S INVOLVED IN GETTING OUT
OF AN INFECTED CELL.
IT'S NOT AS EASY AS IT MIGHT
SEEM TO BE.
SO BACK, I GUESS, A COUPLE OF
DECADES AGO NOW, THE FIELD WAS
VERY ANXIOUS TO TRY TO IDENTIFY
SOME ANTIVIRALS THAT WOULD
TARGET HIV REPLICATION, AND IT
WAS SORT OF, IN MY OPINION, WAS
SORT OF A STOPGAP MEASURE
BECAUSE, AFTER ALL, WE'RE GOING
TO HAVE A VACCINE ANY DAY, SO WE
CAN JUST BUSY OURSELVES WITH
GETTING SOME ANTIVIRALS GOING
THAT WOULD SORT OF HOLD US UNTIL
THE VACCINE, YOU KNOW, WAS
AVAILABLE.
AND AT THE TIME, THE FIELD AND
IN PARTICULAR WITH
COLLABORATIONS FROM SEVERAL
COMPANIES, DID WHAT WAS
SUPPOSABLE STRATEGY, WHICH WAS
TO TARGET THE GENES THAT ARE
ENCODED BY THE VIRUS ITSELF AND
TO TARGET THEM FOR DEVELOPMENT
OF AGENTS THAT WOULD INTERRUPT
THEIR FUNCTION.
NOW, AT THE TIME, IF THE IDEA OF
TARGETING NOT JUST THE VIRAL
ENCODED GENES, BUT ALSO THE HOST
GENES THAT ACTUALLY HELP THE
VIRUS TO DO WHAT IT DOES, THAT
IS TO SAY, THE VIRAL GENES THAT
ENABLE THE VIRUS TO EXPLOIT HOST
MACHINERY, WE WOULD HAVE
PROBABLY BEEN LAUGHED OUT OF THE
ROOM, AND IT WASN'T UNTIL
PROBABLY AROUND, I THINK, THE
MID TO LATE '90s, WHEN IT BECAME
APPARENT THAT THIS PROBLEM OF
DRUG-RESISTANCE THAT FOLLOWED
THE TARGETING OF ANY COMPOUND,
WAS NOT GOING TO GO AWAY.
AND SO WE ENTERTAINED THE IDEA
THAT MAYBE -- AND WE STILL DON'T
KNOW, BUT MAYBE WE TARGETED HOST
GENE PRODUCTS THAT WERE HELPING
THE VIRUS, THEY WOULD BE NOT SO
EASY TO ACCRUE RESISTANCE TO.
SO WE STARTED TO LOOK FOR THEM.
WE WERE VERY FORTUNATE THAT THE
PERSON OF THE INVESTOR OF THESE
TWO ASSAY HYBRIDS, WAS AN
ASSISTANT PROFESSOR DOWN ON THE
FIRST FLOOR IN MY BUILDING, AND
WAS VERY ANXIOUS FOR PEOPLE TO
USE HIS ASSAY, AND SO WHAT
ESSENTIALLY WE DID WAS TO SEARCH
A B CELL LIBRARY FOR PROTEINS
THAT WOULD INTERACT WITH THE
STRUCTURAL PROTEIN ENCODED BY
HIV, THE PROTEIN THAT'S NOT ONLY
RESPONSIBLE FOR MAKING VIRAL
PARTICLES, BUT ALSO FORTUNATE,
TO MAKE SOMETHING THAT AT LEAST
LOOKS LIKE AN IMMATURE VIRUS
PARTICLE, WE USED B CELL LIBRARY
TO FISH OUT PARTNERS OF THAT --.
NOW, IN THE INITIAL FORAYS, WE
PULLED OUT SEVERAL HOST GENE
PRODUCTS AND THE GRADUATE
STUDENT WHO WAS WORKING ON THIS
PROJECT, SHE PULLED OUT ACTUALLY
CYCLOCILLIN, AND AT THE TIME,
THEY WERE WORKING ON CYCLOCILLIN
AND BETH SAID, I'M NOT GOING
TO -- YOU'RE NOT GOING TO TALK
ME INTO DOING MY THESIS ON A
PROTEIN THAT NOBODY HEARD OF AND
IS NOT IN THE DATABASE.
I'M GOING TO WRITE A THESIS ON
CYCLOCILLIN.
BUT FORTUNATELY, AS GRADUATE
STUDENTS GO, WE WERE ABLE TO
TALK THE NEXT GRADUATE STUDENT
WHO JOINED THE LAB INTO WORKING
ON THIS PROTEIN THAT BETH HAD
PULLED THE SEQUENCE OUT OF THE
DATABASE, WAS LOOKING LIKE -- IT
WAS -- I'M SORRY, SHE PULLED OUT
OF THE HYBRID ANALYSIS, THAT THE
SEQUENCE WAS NOT IN PROTEIN
DATABASE.
SO WHAT WE STARTED WAS -- WE
WANTED TO UNDERSTAND WHETHER HIV
CARED BIT AND IN WHAT WAY MIGHT
IT BE UTILIZING IT SUCH THAT WE
SAW INTERACTION WITH GAG.
SO AS WE WERE WORKING ON THIS,
WE KEPT EVERY MONTH OR SO WHERE
WE GO TO THE PROTEIN DATABASE
AND LOOK TO SEE IF THE SEQUENCE
OF TSG101 HAD IN FACT BEEN
ENTERED MACE IN THE DATABASE BY
ANYBODY, AND ONE DAY, IT
APPEARED.
IT HAD BEEN PLACED THERE BY A
CANCER GENETICIC STAN COHEN, WHO
WAS AT STANFORD.
WHEN I SAW THE SEQUENCE IN THE
DATABASE, I CALLED HIM AND HE
EXPLAINED TO ME HOW HE HAD
HAPPENED TO COME ACROSS TSG101.
ESSENTIALLY, HE HAD MADE A
STRAIN OF OLIGO KNEW THEO TIDES.
HE HAD INJECTED THOSE INTO NICE
AND HE LOOKED TO SEE WHICH MICE
DEVELOPED TUMORS BECAUSE THE
OLIGONUCLEOTIDES WERE ACTING AS
AN ANTIPHASE.
INSURE ENOUGH, HE PULLED OUT,
THE MOUSE HAD A TUMOR AND THE
CARBON SEQUENCE WAS TSG101.
SO THIS WAS FASCINATING BECAUSE
HERE HE WAS KNOCKING OUT A GENE
THAT CAUSED A CELL TO OVERGROW,
ESSENTIALLY.
HERE WE WERE WORKING WITH THIS
SAME GENE PRODUCT AND AT THAT
POINT, WE KNEW THAT HIV ACTUALLY
NEEDED TSG101 TO REPLICATE.
SO WE WERE SAYING, WELL, MAYBE
THEN, THIS IS A SITUATION WHERE
THE VIRUS IS ACTUALLY EXPLOITING
THE PROTEIN IN A WAY THAT IS A
NICHE.
BY A NICHE, I MEAN SOME WAY IN
WHICH IT'S EXPLOITING THE
PROTEIN, IT'S A CELLULAR
PROTEIN, AN IMPORTANT CELLULAR
PROTEIN, BUT YET THE VIRUS IS
USING IT IN SOME WAY THAT WE
MIGHT THEN BE ABLE TO ULTIMATELY
TARGET IT BECAUSE IT IS ONE THAT
THE VIRUS USES IN A WAY THAT'S
NOT QUITE IDENTICAL TO THE WAY
THE HOST IS USING IT.
SO THAT REALLY BECAME THE SORT
OF DRIVING HYPOTHESIS IN THE LAB
THAT WE THEN FOLLOWED FOR A
COUPLE OF DECADES.
NOW, JUST ABOUT THE SAME TIME,
BECAUSE WE ALWAYS LIKED TO DO
COMPARATIVE VIROLOGY, WE
ALWAYS -- IF SOMETHING IS GOING
ON WITH HIV, WE LIKE TO KNOW WAS
IT ALSO HAPPENING WITH THE
CHICKEN VIRUS, THE MOUSE VIRUS,
THE MONKEY VIRUS, THE HORSE
VIRUS, AND WE THEN, AT SOME
POINT, WE WENT TO THE CULTURE
MEETING.
I -- COLD SPRING HARBOR MEETING.
I REMEMBER BEING A LITTLE THE
COLD SPRING HARBOR, ANOTHER
RETROVIRAL GIST SAID CAROL, THIS
IS REALLY INTERESTING, WHEN I GO
BACK HOME, I'M GOING TO WORK ON
IT WITH -- WE SAID DON'T BOTHER,
JOHN, WE ALREADY KNOW.
WE DECIDED TO COLLABORATE AND IN
COLLABORATING, WE DID SOMETHING
THAT WAS VERY, VERY INFORMATIVE
FOR US.
SO THE AVIAN RETROVIRUS IS A
RETROVIRUS THAT CALLS ESCRT
MACHINERY BY A -- I'LL TALK MORE
ABOUT IN A BIT, WHAT'S CALLED
THE PR MOTIF.
HIV DOES NOT HAVE ONE OF THOSE.
SO IT SEEMED AS THOUGH MAYBE
EVEN THOUGH THEY BOTH COULD CALL
ESCRT MACHINERY, WERE THEY DOING
IT BY SOME DIFFERENT KIND OF
MECHANISM OR MIGHT THESE BE
MECHANISMS THAT ARE GOING TO
OVERLAP?
THAT WAS A SORT OF A QUESTION
THAT WE WANTED TO TRY TO
UNDERSTAND, BUT AT THE SAME
TIME, AS PEOPLE STUDIED IN MANY
LABS INVOLVED IN THIS, AS PEOPLE
STUDIED RETROVIRUSES, INCLUDING
HIV, IN A VERY, VERY BROAD WAY,
WHAT WE CAME TO SORT OF
APPRECIATE IS THE FACT THAT IN
THE CASE OF HIV, IT HAS WHAT'S
CALLED A PRIMARY LATE DOMAIN.
LATE DOMAIN IS A TERM THAT HAD
BEEN ATTRIBUTED TO THE
ESSENTIALLY MOTIFS, DOCKING
SITES, IF YOU WILL, THAT PERMIT
RETROVIRUSES AND OTHER VIRUSES
TO RECRUIT MACHINERY THAT WILL
GET THEM OUT OF THE CELL.
HIV HAD A PRIMARY LATE TO PAIN.
IT WAS TO THIS -- LATE DOMAIN.
IT WAS TO THIS LATE DOMAIN THAT
WE FOUND TSG101 WAS TARGETED.
THIS LATE DOMAIN IN
PARTICULAR -- HERE IT IS OVER
HERE -- IT'S CHARACTERIZED BY
FOLDING, SOMETIMES PROLINE, WHAT
WE CALL A P/SAP MOTIF.
ANYBODY WHO WORKS IN HIV KNOWS
IF YOU WERE TO MAKE A MUTATION
THAT WAS TO DISABLE THE P/SAP
MOTIF, WELL, DEPENDING ON THE
CELL TYPE YOU'RE IN, THE VIRUS
IS DEFINITELY IMPAIRED WITH
RESPECT TO MAKING VIRAL
PARTICLES THAT CAN GET OUT OF
THE CELL, BUT IT CAN STILL MAKE
VIRAL PARTICLES THAT CAN GET OUT
OF THE CELL.
AND THAT'S BECAUSE IT HAS WHAT
WE CALL A SECONDARY LATE DOMAIN.
NOW, THIS SECONDARY LATE DOMAIN,
L DOMAIN 2, THIS ONE CAN TAKE
MANY FLAVORS.
IN THE CASE OF HIV, IT'S
ESSENTIALLY A LEUCINE YXXL
AGAIN, RIGHT?
SPECIFICALLY, IN THE CASE OF
HIV, IT'S AS YOU SEE IT THERE ON
THE BOARD, A LEUCINE TYROSINE
PROLINE, A LEUCINE SO ON AND SO.
IT CAN ALSO TAKE THE FORM OF
YXXL.
SO IF YOU KNOCKED OUT THE
PRIMARY LATE DOMAIN, VIRUS CAN
STILL GET OUT.
AND THAT'S BECAUSE IT'S
DEPENDING ON ITS SECONDARY LATE
DOMAIN.
SO IF YOU NOW KNOCK OUT THE
SECONDARY LATE DOMAIN AND THE
PRIMARY LATE DOMAIN, YES, THE
VIRUS IS IMPAIRED WITH THE
EFFICIENCY WITH WHICH IT CAN GET
VIRAL PARTICLES OUT, BUT IT ASK
STILL GET VIRAL PARTICLES OUT
AND THAT'S BECAUSE OF ITS
TERTIARY LATE DOMAIN.
IT'S A LATE DOMAIN WHOSE
STRUCTURE IS NOT SO CLEAR, BUT
IT IS ONE THAT FUNCTIONS WHEN,
IN FACT, THE OTHER TWO ARE
DISABLED.
NOW, IN THIS KIND OF
RELENTLESSNESS WITH RESPECT TO
REDUNDANCY OF MECHANISM FOR
GETTING OUT, ONE OF THE
QUESTIONS I ASKED MYSELF WAS,
WELL, YES, IT IS A REDUNDANT
MECHANISM TO GET VIRUS OUT OF
CELLS AND, YES, IT MAKES SENSE
THAT A VIRUS MIGHT WANT TO DO
THAT, BUT IN POINT OF FACT,
THERE WAS NOT AT THE TIME AND
STILL TODAY THERE'S NOT -- I DO
NOT KNOW OF ANY HIV VARIANT THAT
HAD BEEN ISOLATED GLOBALLY WHERE
THERE'S A MUTATION IN THE
PRIMARY LATE DOMAIN.
IN OTHER WORDS, THE VIRUS SAYS,
I'M NOT DOING THAT, I'M NOT
MESSING WITH THAT, ALL RIGHT?
BUT IN THE LABORATORY, YOU
CERTAINLY CAN DO IT, AND SO
OBVIOUSLY IT ALWAYS WAS A
NAGGING THOUGHT TO US THAT THIS
CAN'T SIMPLY BE FOR -- HAVING
THESE LATE DOMAINS CAN'T SIMPLY
BE FOR THE QUESTION OF
REDUNDANCY BECAUSE WE HAVEN'T
GOTTEN A VARIANT IN A NATURAL
VARIANT YET.
SO IF THERE'S A REASON THAT THE
VIRUS SHOULD BOTHER TO GO AND
MAKE THIS SYSTEM TO REDUNDANT.
ONCE AGAIN, ANOTHER THOUGHT
LURKING IN THE BACK OF THE HEAD
WAS THAT IN THE CASE OF HIV, IT
RECRUITS TSG101.
IN THE CASE OF THE AVIAN
RETROVIRUS, IT RECRUITS THE --
THROUGH THE PY MOTIF PROTEINS
THAT BELONG TO A FAMILY OF
UBIQUITIN E3 LIGASES, NEDD4 AND
OTHER MEMBERS OF THE FAMILY AND
IN THE CASE OF TSG101, IT HAS
BOTH A P/SAP MOTIF AND WE'LL
CALL IT A SECONDARY LATE DOMAIN
MOTIF AND IT ACTUALLY
UTILIZING -- I'M SORRY, IT MIGHT
HAVE ALL THREE, BUT IT
DEFINITELY HAS THE P/SAP AND THE
PY AND IN THE FACE OF TSG101, IT
ACTUALLY UTILIZES MOST.
SO THIS STIMULATED THIS THOUGHT
PROCESS TO ASK ABOUT THE
POSSIBILITY THAT TSG AND NEDD4
MIGHT ACTUALLY INTERACT WITH
EACH OTHER SO THAT NO MATTER
WHAT DOCKING SITE THE VIRUS
MIGHT USE, WHETHER IT'S HIV OR
SOME OTHER VIRUS, THAT EVEN
THOUGH THEY MAY RECRUIT DIRECTLY
TSG OR THEY MIGHT RECRUIT
DIRECTLY NEDD4, IF TSG AND NEDD4
INTERACT, THAT COMPLEX WAS GOING
TO BE CALLED.
BACK TO THAT -- MEANWHILE,
BECAUSE IT TURNS OUT, WHETHER
HIV OR ANY OTHER VIRUS RECRUITS
TSG101 DIRECTLY, TSG101 AND
NEDD4 INTERACT IN SOLUTION AND
SO THE COMPLEX IS BEING BROUGHT
TO BEAR.
SO THE KEY PLAYERS THAT WE'RE
GOING TO TALK ABOUT TODAY ARE
THE TSG101 PROTEIN.
IT HAS A -- WHAT'S CALLED A
TERMINAL UBIQUITIN E2 VARIANT OR
UEV DOMAIN.
IT'S CALLED UBIQUITIN E2 VARIANT
BECAUSE IT LOOKS LIKE IT'S A
STRUCTURAL COMMON WITH THE
ENZYMES.
THESE ARE UBIQUITIN ENZYMES AND
THEN IT HAS THE DOWNSTREAM
DOMAINS THAT ARE VERY IMPORTANT
FOR IT TO BE ABLE TO READ
DOWNSTREAM FACTORS THAT ARE IN
ESCRT-II AND ESCRT-III TO WHERE
THE TSG101 IS.
AND ONE OF THE THINGS THAT I
THINK YOU KNOW OR SHOULD
APPRECIATE IS THAT ESCRT-III
HOUSES MACHINERY THAT'S CRITICAL
FOR REMODELING MEMBRANE AND
CYTOSKEL
CYTOSKELETON, AND SO WHEN TSG101
RECRUITS THAT BY VIRTUE OF ITS
ABILITY TO INTERACT WITH
ESCRT-II AND IN TURN ESCRT-III,
IT COMPLETES MACHINERY TO THE
VIRAL ASSEMBLY SITE AND PROMOTE
BUDDING.
SO TSG IS A MAJOR PLAYER WITH
RESPECT TO EXIT.
THEN, WE LOOK AT LATE DOMAIN
NUMBER 1.
THE LATE DOMAIN NUMBER 1, ITS
FAVORITE PARTNER IS ACTUALLY A
MEMBER OF THE NEDD4 FAMILY
CALLED NEDD4L FOR LONG.
IT CAN SOMETIMES BE CALLED
NEDD4-2.
THE LATE DOMAIN 2 WILL RECRUIT
AND ADOPT A PROTEIN, LE
CHÂTELIER'S.
THIS LE CHÂTELIER'S PROTEIN WILL
RECRUIT ANOTHER MEMBER OF THE
NEDD4 FAMILY AND THAT IS
NEDD4-1, AND THEN THE LATE
DOMAIN 3 WILL RECRUIT ANOTHER
ADAPTOR PROTEIN AND THAT ADAPTOR
PROTEIN CAN THEN BIND NEDD4-L.
SO THE VIRUS HAS THE ABILITY TO
BRING TO ITS VICINITY,
INDIRECTLY THROUGH TSG, NEDD4,
THIS ISOFORM LONG THROUGH ALI
LICLIX,
NEDD4-L AND ANOTHER ISOFORM OF
NEDD4 ABLE TO FIND DIRECTLY.
SO ONCE AGAIN, RELENTLESS AND
REDUND
REDUNDANT.
NOW -- OOPS, I HAD A FREEZE.
I'M NOT GETTING THIS TO MOVE
FORWARD.
SO I NEED A LITTLE -- OH, GOOD,
THANK YOU.
ALL RIGHT.
SO AS I SAID, THE NEDD4 OF
UBIQUITIN E3 LIGASES IS PRETTY
LARGE.
THERE'S SEVERAL, SEVERAL
MEMBERS.
AND TO JUST REVIEW FOR YOU, THIS
FAMILY, WHAT THIS FAMILY DOES IS
ACTUALLY MODIFY PROTEINS BY
ADDING ON TO THEM UBIQUITIN.
AND THERE ARE MANY DIFFERENT
WAYS THAT IT CAN ADD UBIQUITIN.
IT CAN ADD JUST ONE UBIQUITIN,
TYPICALLY, TO A LYCINE.
IT CAN ADD A CHAIN, COULD ADD
UBIQUITIN AS A CHAIN.
IT COULD ADD UBIQUITIN AS A
DIMER.
IT COULD ADD UBIQUITIN AS A
BRANCHED COMPLEX AND COULD ADD
UBIQUITIN TO MORE THAN ONE SITE
OF THE PROTEIN.
ALL OF THESE DIFFERENT
VARIATIONS COMPRISE UBIQUITIN
SIGNALING, AND THAT SIGNAL IS
READ BY CELLS TO MANY CERTAIN
THINGS.
SOME OF THEM MEAN TAKE THIS KARG
FOR TO THE GARBAGE PAIL.
IF THERE'S A UBIQUITIN MACHINE,
IT SAYS SEND ME TO THE LYSOSOME
OR THE PROTEO SOME FOR
DEGRADATION.
IF IT HAS -- IT SAYS TAKE ME
SOMEWHERE IN THE CELL.
SORT MESO THAT I END UP IN SOME
LOCATION.
AND THE PROCESS BY WHICH THE
UBIQUITIN INTERACTS WITH ANOTHER
UBIQUITIN MOLECULE CAN ALSO VARY
AND THAT ALSO IS BEARING A
SIGNAL TO TREAT THE CARGO IN A
CERTAIN WAY.
SO WHAT HAPPENS IS, THESE
ENZYMES SEE UBIQUITIN, THEY WORK
WITH WHAT'S CALLED E1 ENZYMES.
E1 ENZYMES ACTIVATE THE
UBIQUITIN.
THAT ENABLES THE UBIQUITIN TO BE
TRANSFERRED TO WHAT'S CALLED N
E2 ENZYME AND THE E2 ENZYME IS A
UBIQUITIN CONJUGATING ENZYME.
THE UBIQUITIN CONJUGATING ENZYME
IS WHAT I REFERRED TO BEFORE AS
BEING STRUCTURAL HOMOLOGOUS TO
THE TSG101 TO MAIM.
THESE WERE PASS THE WAITING
UBIQUITIN TO AN E3 ENZYME AND
WHEN IT DOES THAT, IT TRANSFERS
THE UBIQUITIN WAITING TO ASSIST
IN THE ACTIVE SITE OF THE ENZYME
AND THEN THAT IS ABLE TO PICK A
SUBSTRATE AND IT PICKS A
SUBSTRATE AND IT THEN WILL
MODIFY THAT SUBSTRATE WITH
UBIQUITIN.
THE WAY IT PICKS THE SUBSTRATE
IS BY VIRTUE OF A DOMAIN WITHIN
THE E3 LIGASE THAT IS SPECIALLY
DESIGNED FOR SUBSTRATE
RECOGNITION.
TYPICALLY SUBSTRATES WILL HAVE A
PY MOTIF.
NOW, EACH VIRUS HAS ITS
FAVORITE.
IN THE CASE OF HIV, I'VE
MENTIONED THREE THAT ARE ITS
FAVORITE, BUT IF YOU WERE
DEALING WITH, SAY FOR EXAMPLE,
HCLV-1, IT WOULDN'T BE NEDD4-1
OR 2 OR LONG.
IT WOULD BE WWP1.
THAT'S A FAVORITE.
OR IT WOULD BE SMURF, ANOTHER
FAVORITE.
THE REASON FOR THAT IS REALLY
INTERESTING TO ME BECAUSE HIV
DOESN'T LIKE THOSE.
SO EACH VIRUS HAS ITS FAVORITE
AND EACH CASE, YOU'RE CALLING
DIFFERENT CONNECTORS TO THE
ASSEMBLIAGE THROUGH THIS
PARTICULAR PARTNER.
NOW, SO I SAID IT BEFORE.
LATE DOMAINS, THEY FACILITY
CELL-FREE VIRUS EXIT.
AND SO IF YOU'RE DEALING WITH,
SAY, NEDD4L, THEN THAT ENZYME BY
VIRTUE OF ITS WW DOMAIN CAN BIND
THE OTHER PARTNERS IN THE CELL.
AND THIS IS JUST A COLLECTION OF
SOME OF THE PROTEINS THAT ARE
ABLE TO INTERACT WITH NEDD4L,
AND YOU CAN SEE THAT AMOT IS ONE
OF THEM.
AMOT WAS THE ADAPTOR PROTEIN
THAT LEADS NEDD4 TO THE LATE
DOMAIN 3.
IF YOU WERE TALKING ABOUT, SAY,
NEDD4-1, ITS WW DOMAINS ARE
DIFFERENT AND THE COLLECTION OF
PROTEINS THAT IT CALLS ARE
DIFFERENT, AND HERE ACTUALLY,
ONE OF THESE PROTEINS WOULD THEN
BE AN ADAPTOR THAT WOULD PERMIT
ALIX TO CONNECT TO NEDD4-1 AND
SO ON.
SO YOU CAN SEE BY HAVING THIS
KIND OF RELATIONSHIP, THE VIRUS
IS ABLE TO CALL PROTEINS THAT
ARE NOT DIRECTLY ASSOCIATED WITH
THE GAG PER SE TO THE ASSEMBLAGE
FOR VARIOUS PURPOSES, WHICH
WE'LL ALLUDE IN A MINUTE.
OKAY.
SO IRRESPECTIVE OF GAG, I SAID
BEFORE THAT THE TSG101 UEV
DOMAIN AND THE NEDD4 HECT DOMAIN
ARE ABLE TO INTERACT AND FOR
NMR, WE CAN IDENTIFY THOSE
REGIONS IN BOTH THE UEV DOMAIN
AND THE HECT DOMAIN WHERE THIS
INTERACTION OCCURS.
I CAN TELL YOU THAT THIS
INTERACTION IS WEAK.
IT'S LIKE THE INTERACTION OF
UBIQUITIN.
UBIQUITIN'S INTERACTIONS TEND TO
NOT BE HIGH AFFINITY BECAUSE
THEY WANT TO BE MOBILE.
THEY WANT TO BE ABLE TO BE ON
AND OFF IN TERMS OF CONTROLLING
SIGNALING.
IN THE CASE OF THE TSG UEV, WE
KNOW THAT ONE REGION IN NEDD4
THAT INTERACTS WITH IS THIS
REGION HERE CALLED ALPHA
HELIX-1.
WE KNOW THAT THERE'S ANOTHER
REGION OF NEDD4, SO NEDD4 HAS --
AS PART OF ITS ENZYMATIC DOMAIN,
IT'S HECT DOMAIN, IT HAS AN
N-LOBBY AND A C-LOBE.
THE C-LOBE IS WHERE THE PROTEIN
THAT WILL RECEIVE THE UBIQUITIN
FROM THE E2 ENZYME WOULD SIT,
AND THIS N-LOBE IS ACTUALLY
CONSIDERED LIKE A WAITING
STATION, MEANING THAT UBIQUITIN
MOLECULES THAT ARE DESTINED TO
BE, SAY, ADDED TO A UBIQUITIN
CHAIN, THEY WILL SIT IN THIS
WAITING ROOM OVER IN THE N-LOBE.
SO THAT AS YOU HAVE AN E2
TRANSFERRING A UBIQUITIN TO AN
E3 AND THEN E3 TRANSFERRING IT
TO A SUBSTRATE, UBIQUITIN IN THE
WAITING ROOM WILL THEN JOIN TO
THE UBIQUITIN THAT'S ON THE
SUBSTRATE AND MAKE SOME KIND OF
UBIQUITIN CASCADE THAT IS THE
SIGNAL.
ALL RIGHT.
SO ONE OF THE THINGS THAT WE DID
WAS, WE LOOKED AT THE RESIDUES
THAT WERE IN UEV PROXIMITY, AND
WE MUTATED THOSE RESIDUES, AND
WHAT WE FOUND IS THAT IN A
CERTAIN REGION OF THE HECT
ENZYMATIC DOMAIN IN NEDD4,
MUTATIONS IN THIS ALPHA HELIX-1
REGION, WHICH IS IN UEV
PROXIMITY, THAT INTERFERED,
MINIMIZED, DIMINISHED THE
ABILITY OF NEDD4, THE WILD-TYPE
NEDD4 TO RESCUE.
SO YOU'RE DEALING WITH A VIRUS
WHERE YOU'VE ACTUALLY KNOCKED
OUT LATE DOMAIN 1 AND LATE
DOMAIN 2 SO THAT NOW IT IS
TOTALLY DEPENDENT ON LATE DOMAIN
3 TO BUD OUT OF THE CELL.
AND YOU CAN DEMONSTRATE THAT,
BECAUSE IF YOU TAKE THAT VIRUS
WHICH IS IN ONE SELECTIVE
ANALYSIS AND THE MATERIAL IN THE
SUPERNATANT HAS BEEN SEMI
PURIFIED, SO WE'RE LOOKING AT
VIRAL-LIKE PROTEINS, AND THIS IS
HOW MUCH YOU ACTUALLY GET OUT IF
YOU DIDN'T ADD ANYTHING, BUT NOW
IF YOU ADD WILD-TYPE NEDD4L, YOU
CAN RESCUE THE VIRUS.
IF YOU MAKE MUTATION NONTHE
REGION OF ALPHA HELIX-1 THAT ARE
IN CLOSE PROXIMITY TO THE UEV
DOMAIN IN TSG, NOW YOU REPAIR
THE ABILITY OF THE NEDD4 TO
RESCUE.
NOW, YOU COULD DO THE SAME THING
IN OTHER REGIONS OF NEDD4.
THIS IS ANOTHER PHASE OF THE
HECT DOMAIN IN NEDD4 WHERE WE
ENDED UP MAKING MUTATIONS ALL
OVER THIS DOMAIN, ALL RIGHT.
IT'S GOT THE REGION WHICH IS
WHERE THE ACTIVE SITE C SISTINE
WOULD ACTUALLY SIT.
IT'S GOT A REGION WHERE AN E2
WOULD ACTUALLY SIT WHEN IT'S
TRANSFERRING THAT UBIQUITIN TO
THAT CYSTINE.
IT HAS THAT WAITING STATION THAT
I MENTIONED TO YOU BEFORE WHERE
UBIQUITINS JUST SIT LYING IN
WAIT.
BUT WHEN YOU MAKE MUTATIONS,
THESE SITES, THE ONLY SITES THAT
ACTUALLY ENDED UP IMPAIRING THE
ABILITY TO GET INFECTIOUS VIRUS
MADE WERE THE SITES I'VE
HIGHLIGHTED HERE IN RED.
HERE, HERE, AND HERE.
AND WHAT THIS IS, ACTUALLY, IT'S
A REGION THAT'S CALLED THE HINGE
REGION.
IT'S A HINGE REGION BETWEEN THAT
C-LOBE AND N-LOBE AND THE WHOLE
ISSUE OF HOW E3 LIGASES
CATALYZE, DO WHAT THEY DO,
UBIQUITIN TRANSFER, IS NOT
KNOWN, BUT THE CONSENSUS IS THAT
THE HINGE REGION ENABLES
COMMUNICATION BETWEEN THE N-LOBE
AND THE C-LOBE, AND BY OUR
FINDING MUTATIONS IN THAT
REGION, BEING THE ONES THAT
IMPAIR THE ABILITY TO GET
INFECTIOUS VIRUS OUT, IT SAYS --
IT SUGGESTS THAT THIS WHOLE
ACTIVITY OF NEDD4 IS PLAYING AN
IMPORTANT ROLE IN BEING ABLE TO
GET INFECTIOUS VIRUS OUT.
NOW, A KEY THING, INFECTIOUS
VIRUS.
THE REASON I KEEP SAYING
INFECTIOUS VIRUS IS NONE OF
THESE MUTATIONS HAD ANY EFFECT
ON BUDDING.
IN OTHER WORDS, YOU COULD MAKE
MUTATIONS IN THIS REGION, VIRUS
COULD GET OUT JUST FINE AS AN
IMMATURE PARTICLE, BUT IT COULD
NOT MATURE TO BE INFECTIOUS.
WHAT THAT ESSENTIALLY SAYS IS
THAT THE VIRUS USES SEVERAL
PARTS IN RELATIONSHIP WITH NEDD4
TO CONTROL BOTH THE BUDDING AND
THE MATURATION OF THE VIRUS.
NOW, ONCE AGAIN, THE WHOLE THING
HAS TO DO WITH THE FACT THAT WE
MADE MUTATIONS IN THE REGIONS OF
THE HECT DOMAIN THAT WERE IN
CLOSE PROXIMITY BY NMR OF THE
UEV DOMAIN IN TSG.
IF YOU MAKE MUTATIONS OUTSIDE,
SOME REGION THAT IS NOT IN UEV
HECT PROXIMITY, VIRUS COULDN'T
CARE LESS.
ALL RIGHT.
SO WE SAID, ALL RIGHT, WE KNOW
THAT ENZYMES LIKE NEDD4, THE
CELL DOESN'T WANT IT TO BE
ACTIVE, SO THEY ARE TYPICALLY IN
CELLS IN AUTO INHIBITED STATE,
AND IN THAT AUTO-INHIBITED
STATE, TYPICALLY YOU HAVE THE C2
DOMAIN AND THIS IS A DOMAIN THAT
TYPICALLY HELPS TO DECIDE WHERE
NEDD4 CAN DOCK IN THE CELL.
TYPICALLY IN THE AUTO-INHIBITED
STATE, THE C2 DOMAIN IS ACTUALLY
IN CONTACT WITH THE HECT DOMAIN.
AND BECAUSE IT'S IN CONTACT WITH
THE HECT DOMAIN, THE HECT DOMAIN
IS NOT IN A POSITION NOW TO BE
ACTIVE.
ITS ACTIVE SITE IS HIDDEN.
SO WE REASONED THAT MAYBE TSG'S
ROLE WAS TO ACTUALLY INTERFERE
WITH THAT C2 HECT INTERACTION
AND TO TEST THIS, WE TOOK
ADVANTAGE OF THE FACT THAT
THERE'S A NEDD4 ISOMER THAT CAN
BIND DIRECTLY THAT HAD BEEN
IDENTIFIED EARLIER BY THE SUN
QUIST AND DOPLINGER LABS.
THIS IS AN ISOMER THAT HAS A BIT
OF THE C2 DOMAIN, NOT ENOUGH TO
MAKE THE CONTACT WITH THE HECT.
WE ASKED WHETHER IF IN FACT YOU
NEEDED THOSE RESIDUES IN THE
HECT DOMAIN FOR STABILIZING THE
INTERACTION WITH THE UEV DOMAIN,
AND IF IN FACT YOU ACTUALLY USE
AN ENZYME, A NEDD4 THAT DOESN'T
HAVE THAT C2 DOMAIN, THAT SHOULD
ALWAYS BE ACTIVE AND SO MAYBE
THEN YOU DON'T NEED THE TSG AND
WE TESTED THAT IDEA AND THAT'S
WHAT'S HERE ON THE SLIDE.
IN OTHER WORDS, EVEN THOUGH
TYPICALLY WITH A REGULAR NEDD4
MUTANT, IT WOULD NOT BE ABLE TO
RESCUE BUDDING IF IN FACT YOU
RESCUE WITH THE ISOFORM THAT'S
LACKING THE C2 DOMAIN, THEY ARE
ABLE TO RESCUE.
AND SO THAT THEN GAVE US THE
NOTION THAT THE TSG UEV IS
ACTUALLY HOLDING THE NEDD4 IN AN
ACTIVATED STATE.
ALL RIGHT.
NOW, ONE OF THE MOST, I THINK,
INTERESTING STUDIES THAT I HAD
AN OPPORTUNITY TO DO TOOK PLACE
WHEN I WAS COLLABORATING WITH
THE WISE, THE WOMEN'S
INTERAGENCY HIV STUDY GROUP.
AND THE REASON THAT THAT WAS
PARTICULARLY FASCINATING TO ME
IS BECAUSE AS BASIC RESEARCHERS,
WE FORMED SOME HYPOTHESES AND WE
THINK OF EXPERIMENTS TO TEST THE
HYPOTHESIS.
BUT IN THIS PARTICULAR CASE, THE
WIHS WHEN I JOINED THIS
PARTICULAR GROUP WAS 20
PHYSICIANS, CLINICIANS, AND WHAT
THEY DO IS THEY INTERACT WITH
PEOPLE LIVING WITH HIV AND THEIR
PROBLEMS AND THEY TRY TO
UNDERSTAND WHAT'S THE NATURE OF
THE PROBLEM SO THEY CAN, YOU
KNOW, WORK TO SOLVE IT.
SO AT THE POINT THAT I JOINED
IT, THERE WAS THIS SUBSET OF THE
PARTICIPANTS FOR WHOM, AT THE
TIME, VERY FABULOUS ARCH WAS NOT
WORKING.
THESE PEOPLE WERE ON THE
PROTEASE INHIBITORS WHICH AT
THAT TIME WERE THE SAVIOR OF THE
ART WHEN -- IN THOSE DAYS, AND
IT HAD BEEN REALLY WORKING
BEAUTIFULLY, BUT FOR THEM, THEY
WERE NONRESPONSIVE.
AND, OF COURSE, THEY KNEW THAT
THERE WERE NO RESISTANT
MUTATIONS THAT WERE IN THE
TARGET OF THE ART.
THAT'S THE FIRST THING THEY
CHECKED FOR.
SO THEY WERE AT A LOSS OF WHAT
COULD BE GOING ON.
SO THEY PERMITTED US TO ACTUALLY
ISOLATE VARIANTS FROM THESE
PARTICIPANTS, LIST OUT THE
REGION THAT WE WERE INTERESTED
IN, WHICH WAS GAG THROUGH THE
END OF THE PROTEASE CHAIN, AND
TO SEQUENCE IT.
AND WHEN WE DID, WE FOUND THAT
THERE WERE MUTATIONS IN WHAT WAS
IN FACT THE BINDING SITE OF
ALIX.
THAT IS TO SAY, LATE DOMAIN 2,
AND THESE MUTATIONS IN AND OF
THEMSELVES WERE VERY
INTERESTING.
FIRST OF ALL, WITHIN THE GAG
FRAME, THEY WERE NOT IN A VERY
CRITICAL TYROSINE RESIDUE, WHICH
IS THE MAKER OR BREAKER OF ALIX
BINDING.
THEY WERE ACTUALLY IN A HIGHLY
CONSERVED SPARING RESIDUE.
HERE.
AND THE MUTATIONS CHANGED THIS
TO NOT JUST ANYTHING, BUT TO
EITHER ALAMINE OR PHETYLALMA
MEAN.
THIS IS A REGION IN THE GAG
PRECURSOR, WHICH IS WHAT'S
CALLED THE TRANSFRAME REGION.
IT'S IN THE REGION OF OVERLAP
BETWEEN GAG AND POL AND IT'S
REALLY RIGHT UPSTREAM OF THE
PROTEASE.
THAT'S WHY IT'S CALLED
TRANSFRAME REGION.
NOW, ONE SET OF MUTANTS, THE
ONES, THE VARYING SET HAD
SUBSTITUTED F, AND THEY IN FACT
HAD NO CHANGE IN THE POL DOMAIN.
SO THAT WAS REALLY PERPLEXING
BECAUSE THERE WAS NO OBVIOUS
REASON WHY, THEN -- THERE SHOULD
HAVE BEEN MORE RESPONSE TO ART.
THE OTHER ONE GAVE US A REASON
BECAUSE IF IN FACT THE MUTATION
WAS ALANINE, THEN IN THE
OVERLAPPING FRAME, IT ACTUALLY
CHANGED THE RESIDUE TO A CYST
TEEN AND THAT WAS HAPPENING
RIGHT AT THE CLEAVAGE SITE AT
THE END TERMINUS OF THE
PROTEASE.
SO WHAT WAS HAPPENING WAS THOSE
VARIANTS WERE NOT MAKING THE 99
AMINO ACID PROTEASE.
THEY WERE MAKING PROTEASE
EXTENSIONS, SO YOU CAN
IMMEDIATELY SEE WHY THEY MIGHT
NOT BE RESPONSIVE TO A DRUG THAT
IS HONED TO FIT INTO IN THE
SUBSTRATE BINDING SITE IN
PROTEASE BECAUSE IT'S GOT AN
EXTENSION AT ITS END TERMINUS.
SO WE WERE PARTICULARLY
FASCINATED WITH THE VARIANTS,
SUBSTITUTED F IN THE GAG FRAME
AND THERE WAS NO CHANGE IN THE
FOL FRAME.
AND THERE -- IN THE POL FRAME.
SO THERE WERE SEVERAL THINGS
THAT WERE VERY CURIOUS.
NUMBER ONE, THE MUTATION, THESE
VARIANTS WERE TEN TIMES MORE
PREVALENT IN WIHS VARIANTS THAN
IN THE LASS ALLAH -- LOSS ALLAH
MONTHS DATABASE AND AT THE TIME,
I WAS TOLD THAT THE LOS ALAMOS
DATABASE WAS ACTUALLY ARCHIVING
MAINLY VARIANTS THAT HAD BEEN
ISOLATED FROM HIV-INFECTED
MALES, ALL RIGHT?
SO THAT WAS SORT OF INTRIGUING.
AND THEN, THE SECOND THING THAT
WAS UNSUSPECTED -- SO I WAS
COLLABORATING WITH ONE SITE.
THERE WERE MAYBE ABOUT A DOZEN
SITES OF WIHS AROUND THE COUNTRY
AND BECAUSE I WAS COLLABORATING
WITH ONE SITE IN THE BRONX, THE
THOUGHT WAS, WELL, MAYBE THESE
PARTICIPANTS ARE SOMEHOW, SOME
WAY, IN CONTACT AND THEY SPREAD
THE VIRUS FROM ONE TO THE OTHER.
SO THE LAS ALAMOS COLLABORATOR
PREPARED THIS TREE AND SHOWED US
THAT, NO, THESE VARIANTS
APPARENTLY AROSE INDEPENDENTLY
IN THE PARTICIPANTS AND THEY
WERE NOT RELATED.
SO THAT WAS FASCINATING.
WE COULD RECAPITULATE THAT THE
MUTATIONS CONFERRED INDON'T
INAVIR RESISTANCE TO THE CONTEXT
OF NL4-3 LABORATORY STRAIN, AND
YOU HAVE TO REMEMBER THAT THE
INDINAVIR, THE REASON WE CHOSE
IT WAS PART OF THE ART, AND SO
WE COULD RECAPITULATE IN THAT IN
THE CONTEXT OF NL4-3.
THE MUTANTS WERE INDEED
SENSITIVE.
SO IF THERE'S NO DRUG, THEN ONE
OF THE MUTANTS, THE S40A WAS AS
FIT AS THE WILD TYPE.
THE OTHER ONE WAS LESS FIT.
SO IT CASS CARRYING THIS
MUTATION AT A COST, BUT IN THE
PRESENCE OF INDINAVIR, THEN, IN
FACT, THE S40A, HERE'S THE WILD
TYPE DOWN HERE, IT WAS MORE FIT
THAN THE WILD TYPE AND THE S40F
AT LEAST BECAME AS FIT AS THE
WILD TYPE.
SO IN BOTH CASES, THE MUTANTS
WERE DOING BETTER RECAPITULATING
WHAT WAS HAPPENING IN VIVO.
I'M LOOKING FOR TIME.
ALL RIGHT.
NOW, ONCE AGAIN, WE BUILT THESE
MUTATIONS INTO THE CONTEXT OF
NL4-3, AND WE THEN ASKED WHAT
HAPPENED.
AND SO OBVIOUSLY ON THE LEFT
PANEL HERE, YOU CAN SEE IT.
IF IT'S WILD-TIME NL4-3, IT
MAKES VIRAL PARTICLES THAT LOOK
JUST LIKE NORMAL VIRAL PARTICLES
DO.
IF IN FACT THE MUTATION WAS
S40A, IT COULD RELEASE VIRAL
PARTICLES AS EFFICIENTLY AS THE
WILD-TYPE, BUT THEY WERE NOT
MATURE.
IF IN FACT IT WAS THE S40F
VARIANT, THEN IT DIDN'T MAKE
MATURE PARTICLES.
IT MADE IMMATURE PARTICLES, BUT
THEY WERE ACTUALLY NOT RELEASED.
THEY WERE IN THE STRUCTURES
WHICH RESEMBLES FILOPODIA.
SO THEN WE CAME TO UNDERSTAND
THAT THAT COULD PROVIDE AN
EXPLANATION FOR WHY THEY WOULD
ESCAPE IF THEY'RE IN FILOPODIA,
THEN THEY'RE NOT GOING TO BE
SUBJECT TO DRUGS OR EVEN
ANTIBODIES THAT MIGHT CLEAR.
AND ONCE AGAIN, GOING BACK TO
THIS ISSUE OF WHETHER YOU HAVE
LATE DOMAIN 1, 2, AND 3, FOR
REASONS OF REDUNDANCY, WELL,
YES, BUT I THINK THAT HERE'S
ANOTHER REASON WHY YOU HAVE LATE
DOMAIN REDUNDANCY BECAUSE IT
PERMITS THE VIRUS TO HIDE UNDER
CIRCUMSTANCES WHERE THAT MIGHT
BE PRACTICAL.
OKAY.
SO ONE QUESTION THAT WE ASKED
AND WE'RE STILL ASKING NOW IS
WHY WAS S40F TEN TIMES MORE
PREVALENT IN THE WIHS VARIANTS
THAN IN THE LAS ALAMOS DATABASE,
AND SO WHAT WE DID WAS IN
COLLABORATION WITH TOM HOGUE, WE
GOT A NRMENT FOR MY GRANT FROM
THE OFFICE OF WOMEN'S STUDIES AT
NIH, AND WHAT WE HAVE BEEN
TRYING TO DO IS TO LOOK AT HOW
THE MUTANTS FAIRE IN DIFFERENT
KINDS OF TISSUES WHEN THEY ARE
DERIVED FROM MALES OR FEMALES.
SO THE STORY IS NOT YET DONE,
AND SO THAT'S WHY I'M NOT
SHARING IT WITH YOU TODAY.
BUT I THINK IT WOULD BE REALLY A
SOURCE WHERE I KNOW OF WHERE
THERE'S SOME BIAS RELATING TO
THE KIND OF VARIANTS THAT WE
SAW.
ALL RIGHT, BACK TO THE TSG.
SO AT CERTAIN POINTS, WE DECIDED
THAT WE SHOULD TRY TO USE WHAT
WE KNEW ABOUT TSG101 AND NEDD4
TO SEE IF WE COULD TRANSLATE ANY
OF THIS INFORMATION INTO
SOMETHING THAT MIGHT BE A
POTENTIAL ANTIVIRAL.
AND THAT WAS WHEN, ONCE AGAIN,
WE WENT TO A LIBRARY AGAIN.
THIS TIME IT WAS A PROPRIETARY
LIBRARY OF SMALL MOLECULES, AND
WE FED IT THE RECOMBINANT UEV
DOMAIN, AND FED IT THE
RECOMBINANT NEDD4, AND OUT OF
THAT, WE PULLED OUT ABOUT A HALF
A DOZEN HITS.
THIS WAS ABOUT 80,000 COMPOUNDS
THAT WERE SCREENED AND WE PULLED
OUT ABOUT A HALF DOZEN HITS, AND
THEN WENT TO WORK TRYING TO
VALIDATE THOSE HITS.
AND OUT OF THAT VALIDATION CAME
THE PRAZOLES.
NOW, PRAZOLES, MANY OF YOU MAY
KNOW, THEY ARE FDA-APPROVED
COMPOUNDS FOR GASTROINTESTINAL
DISEASE, AND WHAT THEY DO IN
PARTICULAR IS THEY BIND TO AN
ATP-ASE, WHICH IS IN THE SMALL
INTESTINE AREA, AND THEY
INTERFERE WITH ITS ABILITY TO
PUMP ACID -- I'M SORRY -- YEAH,
I'M SORRY, THEY INTERFERE WITH
ITS ABILITY TO PUMP ACID SO
THAT, IN FACT, ACID DOESN'T
BUILD UP IN THE INTESTINE.
AND SO THE THING IS THAT IN
USING IT AS AN ANTIVIRAL, IT
DOESN'T ACTUALLY WORK THE SAME
WAY.
THAT IS TO SAY, IT HAS TO BE
TAKEN UP INTO THE CELL.
IF YOU PREVENT THEM FROM GOING
INTO THE CELL, THEY CAN FUNCTION
THE WAY THEY DO IN THE BODY, BUT
THEY WON'T INHIBIT THE VIRUS.
SO IT TURNS OUT THEN THAT THEIR
USE IN THIS WAY IS A -- WHAT
THEY CALL A COUNTER INDICATION
OR ANOTHER INDICATION OF THIS
COMPOUND.
AND SO WE BEGAN TO STUDY THEM
AND WHAT THEY MIGHT ACTUALLY
REVEAL TO US ABOUT WHAT'S GOING
ON IN TERMS OF TSG101, NEDD4,
HIV ASSEMBLY AND BUDDING.
SO OUR COLLABORATORS WERE ABLE
TO IDENTIFY THE SITE IN THE UEV
DOMAIN OF TSG WHERE THE PRAZOLES
DOCK AND IT TURNS OUT THAT THEY
DOCK ON ONE OF THE
UBIQUITIN-BINDING REGIONS IN THE
TSG MOLECULE.
AND WHEN THEY DOCK, THEY PREVENT
UB FROM BINDING.
YOU CAN SEE IN FAMILIAR A HERE,
I HAVE UBIQUITIN DOCKED IN TO
THE UEV.
IT CONTACTS UEV IN A NUMBER OF
POINTS, BUT IF THE PRAZOLES
BINDING THERE, IT DISPLACES THE
UEV AND PREVENTS THAT.
THE PRAZOLES, ONE OF THE THINGS
I MENTIONED TO YOU BEFORE IS
THAT TSG BY INTERACTING WITH UEV
AND INTERFERING WITH HOW IT
HANDLES UBIQUITIN, IT THEN IS
ABLE TO STABILIZE NEDD4 SO IT
DOESN'T TURN OVER AS RAPIDLY,
AND THAT'S WHAT YOU SEE HERE,
THAT IF IN FACT YOU HAVE GOT AN
INCREASING AMOUNT OF A PRAZOLE,
THEN YOU WILL, INSTEAD OF
PROTECTING NEDD4, THE NEDD4 IS
NO LONGER PROTECTED BY TSG101,
YOU LOSE IT.
IF IN FACT YOU USE A TSG101
MUTANT THAT CAN'T BIND THE
PRAZOLES, THEN YOU CAN'T
MAINTAIN PROTECTION OF NEDD4
THROUGH THAT.
SO THAT MEANS THAT THE PRAZOLE,
BY BLOCKING TSG'S ABILITY TO
BIND UB, ARE ACTUALLY ABLE TO
DISABLE NEDD4 IN ADDITION.
AND YOU CAN INTERFERE WITH
EITHER SIDE.
YOU CAN INTERFERE WITH THE TSG
SIDE WITH A SMALL MOLECULE OR
YOU CAN INTERFERE WITH THE NEDD4
SIDE WITH A SMALL MOLECULE, AND
YOU'LL GET THE SAME RESULT,
WHICH IS TWO ARREST BUDDING AT
AN EARLY STAGE, EARLIER THAN THE
STAGE WHERE, FOR EXAMPLE, YOU
GET THE TETHERED NET, WHICH IS
THE CLASSICAL FORM OF BUDDING
ARREST.
HERE, THE WHOLE PARTICLE IS
MADE.
HERE, IT'S ARRESTED EARLIER.
WE'RE FINDING -- SO AT THIS
POINT, WE'VE MADE LOTS OF
PRAZOLE DERIVATIVES IN
COLLABORATION WITH OUR MEDICINAL
CHEMIST FRIEND, AND JUST LIKE
THE COMMERCIAL PRAZOLES, WHICH
AS YOU KNOW OR MAY KNOW COMES IN
ALL KINDS OF FLAVORS, SO YOU CAN
TAKE A PRAZOLE FOR IMMEDIATE
ACTION.
YOU CAN TAKE IT FOR OVERNIGHT
ACTION.
YOU CAN TAKE IT FOR SIX-HOUR
ACTION.
JUST LIKE THE COMMERCIAL
PRAZOLES, WHEN WE LOOK AT OUR
PRAZOLES, THEN THEY MAKE
DISTINCT FOOTPRINTS ON THE TSG
UEV MOLECULE.
THEY ARE ALL PRO DRUGS.
THEY ALL ATTACK A CYSTINE IN THE
TSG MOLECULE, BUT THEY DON'T
NECESSARILY, JUST BECAUSE OF
THEIR STRUCTURE, STICK IN THE
SAME WAY.
THIS GAVE US THE IDEA THAT MAYBE
YOU COULD HAVE PRAZOLE VIRUS
SPECIFIC TARGETING, AND WE'RE
ENCOURAGED IN THAT THOUGHT BY
LOOKING AT PRAZOLES DERIVATIVE
EFFICACY WITH SARS.
SO IF YOU COMPARE HIV-1 AND
SARS, WHAT YOU SEE IS THAT HERE
IS A GUY WHO IS NOT -- FIRST OF
ALL, JUST TAKE A COMMERCIAL
PRAZOLE.
COMMERCIAL PRAZOLE, IT'S ONE OF
THEM IS NOT PARTICULARLY
EFFICACIOUS WITH EITHER BUD.
HERE'S ONE THAT IS NOT
PARTICULARLY SELECTIVE AS FAR AS
HIV IS CONCERNED, BUT WITH SARS,
YOU KNOW, FOR US AT OUR STAGE OF
INVESTIGATION, WE'RE GETTING
PRETTY GOOD SELECTIVITY WITH
SARS.
ON THE OTHER HAND, TAKE THE GUY
WHO IS NOT PARTICULAR SELECTIVE
WITH RESPECT TO SARS, AND HE'S
NOT BAD WITH RESPECT TO HIV.
SO THAT IS GIVING US THE
IMPRESSION THAT MAYBE THE --
THESE COULD BE VERY FORMATIVE IN
TERMS OF HOW THEY -- INFORMATIVE
IN TERMS OF HOW THEY TELL US
WHAT'S HAPPENING IN TRAFFIC WITH
THESE VARIOUS VIRUSES.
I DIDN'T INCLUDE A SLIDE TO SHOW
YOU HERE, BUT WE'VE ALSO DONE
THIS SORT OF EXPERIMENT USING
DIFFERENT CELL MACHINERIES WHERE
TSG AND ESCRT ARE INVOLVED, AND
AGAIN, WE SEE SELECTIVITY.
THERE ARE CERTAIN MACHINE RIS
THAT ARE PARTICULARLY
SUSCEPTIBLE TO PRAZOLES, BUT
THERE ARE ONES THAT SEEM TO
IGNORE IT ENTIRELY.
EVEN THOUGH THEY'RE ALL -- THEY
ALL HAVE TSG INVOLVEMENT.
ALL RIGHT.
WELL, THERE'S A MODEL TO LEAVE
YOU WITH.
ESSENTIALLY, WE'RE THINKING THAT
THE TSG101 UEV DOMAIN, ITS JOB
IS TO ACTIVATE NEDD4 BY TAKING
IT OUT OF ITS AUTO-INHIBITION
STATE AT LEAST FOR THE PERIOD
THAT THE VIRUS NEEDS IT, AND
DOES SO BY ESSENTIALLY
INTERFERING WITH THIS NATURAL C2
HECT INHIBITION THAT THE PROTEIN
WOULD BE FOUND UNDER IN THE
CELL.
AND ONCE THAT IS THE CASE, THEN
THE ENZYME CAN, YOU KNOW, BRING
THE COHORTS THAT IT BRINGS.
IF IT'S IN THE CASE OF THE
COHORT THAT CAN ENABLE US TO
TAKE ADVANTAGE OF HIDING IN
FILOPO DI-A, IT CAN BRING THAT,
TOO, AND THEN YOU GET A
SITUATION WHERE THE VIRUS CAN
FIND SOME OTHER WAY OF
MECHANISM.
SO WHOO HAVE I TOLD YOU?
I TOLD YOU VIRAL L DOMAINS ARE
DOCKING SITES FOR THE
RECRUITMENT OF CELLULAR FACTORS
TSG101 AND NEDD4.
THE TSG101 UEV DOMAIN ACTIVATES
NEDD4 BY INTERFERING WITH THE
AUTO-INHIBITION MECHANISM BY
WHICH THE CELL KEEPS THE ENZYME
SILENT.
THE TSG REGULATES, THIS WE
BELIEVE BY SUPPRESSING THE
AUTO-INHIBITION THAT TYPICALLY
IS THE WAY -- IS THE CELL'S WAY
OF SORT OF TEMPORALLY
CONTROLLING THE ENZYME'S
ACTIVITY.
THE NEDD4 ACTIVATION PROMOTES UB
MODIFICATION EVENTS THAT
FACILITATE THE BUDDING OF
CELL-FREE VIRUS AND THE VIRUS AT
LEAST AS FAR AS PEOPLE LIVING
WITH HIV TELL US, THE VIRUS CAN
GET ITS WAY AROUND THAT TOO BY
COURTING THIS LATE DOMAIN
MECHANISM.
INTERFERENCE WITH THIS KIND OF
SIGNALING IS, WE BELIEVE, AN
UNAPPRECIATED MECHANISM OF ART
EVASION IN PEOPLE LIVING WITH
HIV.
AND WE THINK THAT THESE PRAZOLES
ARE FDA-APPROVED AS SMALL
MOLECULES AND WE KNOW THAT THEY
INTERFERE WITH TSG-NEDD4
INTERACTION, THEY ARE PROBABLY
GOOD MEANS FOR TRYING TO FIND A
WAY TO MAKE THEM SPECIFIC.
OBVIOUSLY NOTHING THAT I SHOWED
YOU IS WHAT OUR SHORT-TERM GOAL
IS, THAT IS TO SAY, GET
SOMETHING IN THE SUBNANO MOLAR
AFFINITY, BUT WE'RE THINKING IF
ONE CAN ACHIEVE THAT, YOU HAVE A
SMALL MOLECULE THAT'S TARGETING
HOST EXPLOITATION.
NOW, I HAVE TO THANK A WHOLE
BUNCH OF PEOPLE.
I HAVE WONDERFUL -- I'VE ALWAYS
HAD WONDERFUL, WONDERFUL
COLLABORATORS AT STONY BROOK.
THESE ARE PEOPLE WHO WERE MOSTLY
INVOLVED IN THE WORK I SHOWED
YOU TODAY, AND THEN AT MT.
SINAI, BOTH VIVIANA AND ALSO BEN
CHEN, WITH WHOM WE'RE DOING
CELL-CELL TRANSFER KINDS OF
EXPERIMENTS, TOM AND THEN MY
STRUCTURAL BIOLOGY COLLABORATORS
AT NIH AND MEDICINAL CHEMISTRY
AS WELL AS OUR FRIENDS AT
MOREHOUSE.
AND THEN ALSO OUR COMPUTATIONAL
BIOLOGIST FRIENDS AT STONY BROOK
AND ALSO C CHOPEING CHEN.
SO THANK YOU VERY, VERY MUCH.
[ APPLAUSE ]
>> WE HAVE TIME FOR QUESTIONS.
>> IN DESCRIBING FIRST OF ALL,
WHAT -- WHAT A DISCUSSION.
IN THE SLIDE WHERE YOU SHOWED
THAT YOU TREAT INCREASING
AMOUNTS OF THE PRAZOLES, I
FORGOT WHAT IT WAS CALLED, AND
YOU'RE ALSO ADDING NEDD4-2, I
GUESS I'M NOT SURE, SINCE YOU'RE
ADDING NEDD4-2, THAT'S WHY YOU
CAN SEE IT, BUT WOULD THAT SAME
HAVE A SIMILAR EFFECT ON THE
OTHER FORM OF NEDD4?
>> SO THAT'S VERY INTERESTING.
SO I THINK THAT -- SO THE
QUESTION WAS IN THE EXPERIMENT
WHERE WE WANTED TO ASK WHETHER
THE PRAZOLES, BECAUSE THEY'RE
INTERACTING WITH TSG, WOULD THEY
IN FACT IMPACT A NEDD4
DETERMINED FUNCTION, RIGHT?
SO THE THING IS THAT WHAT'S VERY
IMPORTANT FOR THE NEDD4
DETERMINED FUNCTION IS THE
UBIQUITIN MOLECULES THAT TSG IS
HOLDING.
AND SO THE REASON THAT THE
PRAZOLES WOULD BE EFFECTIVE IS
BECAUSE, AS LONG AS IT'S
INTERACTING WITH A NEDD4 THAT
NEEDS THOSE UBs, THEN THE ANSWER
IS YES.
NOW, THE REASON THAT THE UBs ARE
SO IMPORTANT, IF YOU LOOK AT
THE -- HOW WE MODEL THE
STRUCTURE, I SAID BEFORE THAT
THE TSG'S UEV DOMAIN, IT'S A
UBIQUITIN E2 VARIANT.
IF YOU TAKE THE CLOSEST RELATED
E2 AND YOU COMPARE HOW IT
INTERACTS WITH UBIQUITIN TO HOW
TSG INTERACTS WITH UBIQUITIN, IT
IS NOT THE SAME.
AND SO THE TSG UEV IS HOLDING
THE UB IN A MANNER THAT WOULD
APPEAR TO BE MODULATING HOW THE
NEDD4 IS ABLE TO TRANSFER THE UB
TO SUBSTRATE AND TO ITSELF.
AND SO WE THINK THAT THAT'S AT
THE CRUX OF HOW IT'S ABLE TO
CONTROL WHAT NEDD4 DOES, AND AS
LONG AS THE PRAZOLES ARE
INTERFERING WITH THAT TSG
FUNCTION, IT CAN HIT NEDD4-2.
>> GREAT TALK.
SO HIV IS PRIMARY -- FIV, THEY
RADIATED TO T CELLS FOR
MACROPHAGE TROPISM.
WHEN ONE LOOKS AT LATE DOMAIN,
ARE THERE LESS LATE DOMAINS IN,
LIKE, THIS -- AND THE REDUNDANCY
THAT ONE SEES IN HIV WITH
GREATER CELL PURPOSE?
>> LET ME SEE IF I REPEAT THE
QUESTION CORRECTLY, WHICH WAS
THAT IF YOU TAKE SOME OF THE
OTHER MEMBERS OF THE FAMILY,
LET'S SAY WITHIN THE LATENCIES,
NOT NECESSARILY OUTSIDE, BUT
WITHIN, CAN YOU SEE A
CORRELATION BETWEEN HOW
EFFICIENTLY THEY ARE TRANSMITTED
AND LATE DOMAIN ACTIVITY.
>> DOES HIV, AS FAR AS YOU KNOW,
DO THEY HAVE REDUNDANCY?
>> SO THEY DEFINITELY -- THE
REASON I DON'T KNOW, BUT UEV WAS
A FAVORITE TO STUDY BECAUSE, AS
I MENTIONED TO YOU, THERE'S
DIFFERENT KINDS OF ALIX-BINDING
MOT
MOTIFS, AND STRUCTURALLY THERE'S
SOME SIMILARITY, BUT IN TERMS OF
THE MOTIF ITSELF, THE SEQUENCE,
THEY'RE NOT IDENTICAL.
SO EIV WOULD, AS FAR AS WE CAN
SEE, BE VERY, VERY SIMILAR IN
HOW IT INTERACTS WITH ALIX TO --
AS HIV.
WHAT WE DO NOT KNOW IS WHETHER
OR NOT IN FACT IN THE CASE OF
HIV-1, YOU CALL NEDD4-1.
IN THE CASE OF EIV, I DON'T KNOW
THAT YOU NECESSARILY CALL IT
BECAUSE ALIX IS ITSELF ABLE TO
HAVE MANY ADAPTOR FRIENDS THAT
CAN CALL E3 LIGASE NROOP WHAT'S
YOUR EXPLANATION FOR WHY THERE'S
SO MANY DOMAINS THEN?
>> I'M GOING TO TELL YOU WHAT
ALLEN SAID TO ME WHEN I WAS
DEALING WITH HIM EARLIER TODAY.
HE IS SAID THAT WHENEVER ANYONE
ASKS A QUESTION LIKE THAT, HE
SAYS, WHY -- I'LL TAKE A STAB AT
ALLEN'S ANSWER.
>> IT'S RELATED SEQUENCES WITHIN
THE TWO.
I WONDER WHAT COHORT IS ABLE THE
PATIENTS WHO ARE FEMALE.
AND MALE INDEX HOW THE
FREQUENCE --
>> SO THE PERSON WHO
COLLABORATED IN THIS WITH US,
THE ANSWER IS -- THE QUESTION
WAS IN TERMS OF THE LASALAMOS
DATABASE, WHICH I WAS TOLD WAS
PREDOMINANTLY MEN, I CHECKED IT
FOR ACTUALLY 2000 -- WHATEVER
WAS THE LATEST, '23 OR '24, AND
NOW I THINK THEY REPORT
SOMETHING LIKE 20 OR 30%
VARIANTS FROM WOMEN.
BUT AT THE TIME THAT THIS WAS
DONE WITH THE WIHS, I WAS TOLD
THAT IT WAS, LIKE, IN THE 90s
MEN, AND VERY, VERY FEW WOMEN.
SO THE FACT THAT IT WAS TEN
TIMES HIGHER IN THE WIHS SEEMS
SIGNIFICANT OR AT LEAST
NOTEWORTHY.
>> BUT IN THE LAS ALAMOS, THEY
SHOULD HAVE THE SEX -- FEMALE
AND MALE INSIDE THAT --
>> SO YOU MEAN RIGHT NOW?
I HAVEN'T GONE BACK TO DO THAT,
ACTUALLY.
THAT WOULD BE REALLY INTERESTING
TO DO, YEAH.
>> AND THEN A SECOND QUESTION
IS, THE MUTATION THAT YOU FOUND
S40, YOU THINK IF YOU CHANGE THE
BINDING, THEY PROJECT --
>> ABSOLUTELY.
THAT'S EXACTLY WHAT IT DOES.
SO IT'S -- I WON'T SAY
COMPLICATED, BUT IT'S
MULTI-STEPPED, BUT THAT'S
EXACTLY WHAT IT DOES.
THANKS.
>> DO YOU KNOW WITHIN THE
PATIENT WHETHER THERE'S A LOT OF
GENETIC VARIATION AT THAT POINT?
>> SO THAT -- THE REASON THAT
THAT'S STRIKING AS A SEQUENCE IS
BECAUSE THERE'S DEFINITELY --
AND THE STRUCTURE WOULD DO A
GOOD JOB OF EXPLAINING WHY, BUT
THERE'S DEFINITELY SEVERAL
RESIDUES WITHIN THE ALIX-BINDING
MOTIF THAT ARE NOT CONSERVED,
BUT THE S40, THE STEERING AT THE
40 POSITION IS HIGHLY CONSERVED.
IT'S MOST CONSERVED OTHER THAN
THE Y IN THERE, YEAH.
IF YOU MUTATE IT BY ITSELF, YOU
WON'T LOSE ALIX BINDING ABILITY.
>> UNLESS WE HAVE A LOT OF GAG
SEQUENCES GENERATING A GOOD BIT
OF [ INDISCERNIBLE ].
>> YES, YES.
I THINK IT'S FASCINATING,
ACTUALLY.
>> IT'S ONE OF THE TOOLS THAT --
SO ALIX SHOWS IT WILL REDUCE
VIRUS REPRODUCTION AND PRAZOLES
WILL DO THAT.
DO YOU KNOW IF THERE'S ANY
SYNERGY BETWEEN THOSE TWO OR
ADDITIVE EFFECTS?
>> THE QUESTION IS, IF I HEARD
IT RIGHT, DID YOU SAY STATS,
STATINS AND PRAZOLES WILL
BOTH -- I MISSED THE VERB.
>> IS IT AN INDEPENDENT
MECHANISM, BOTH THROUGH TSG101,
WILL THEY -- IS THERE A SYNERGY
BETWEEN THOSE TWO?
>> SO WE DON'T KNOW.
I DON'T KNOW, YEAH.
WE SHOULD ASK THAT.
>> WE HAVE PATIENTS ON SOME, BUT
BOTH, SO THIS EARLY LATE DOMAIN
DEFECT THAT YOU SEE WITH THE
PRAZOLES, MECHANISTICALLY, WHAT
DO YOU THINK IS HAPPENING THERE?
IT SEEMS NOT TO BE A CLASSIC
TSG101 IMPAIRMENT SORT OF LATE
DOMAIN, BUT MORE OF AN ASSEMBLY
LATTICE CONSTRUCTION EFFECT.
WHAT DO YOU THINK
MECHANISTICALLY IT'S HAD?
>> THAT IS ACTUALLY A QUESTION
THAT IS, TO ME -- THE QUESTION
WAS, WHAT DO I THINK IS ACTUALLY
HAPPENING BECAUSE THE PRAZOLES
SORT OF ARREST AT AN EARLY
STAGE, EARLIER THAN WHEN YOU
WOULD CALL ESCRT TO COME FINISH
THE JOB OFF AND SNIP THE NET.
AND SO IT HAS -- WHAT IT SAYS TO
US IS THAT VERY EARLY ON, WHAT
YOU NEED IS A UBIQUITIN NASCENT
EVENT, THAT UBIQUITIN EVENT --
ON TUESDAYS AND THURSDAYS, WE
THINK THE UBIQUITIN NATION EVENT
IS FOR SCAFFOLD.
ON MONDAYS AND WEDNESDAYS, WE
THINK, NO, NO, NO, IT HAS TO DO
WITH WHICH ESCRT MIGHT IN FACT
BE, YOU KNOW, SORT OF LIKE --
YOU GOT A BRILLIANT EARLY TO
ACTIVATE IT, BUT THE OTHER
ASPECT IS THAT THE -- EVEN WITH
RESPECT TO ALIX THAT WE WERE
JUST TALKING ABOUT, IT HAS GOT
TO BE UBIQUITINATED TO FUNCTION
AS WELL.
AND SO IT COULD BE A THIRD
MECHANISM WOULD BE THAT EVEN IN
YOUR ADAPTOR MACHINERY, THE
UBIQUITINATION NEEDS TO BE THERE
EITHER BECAUSE THAT WHOLE --
SOME INTERACTION IN PLACE FOR
SOME TIME OR NOT.
SO DEPENDING ON THE DAY OF THE
WEEK, WE'VE DIFFERENT IDEAS
ABOUT, YOU KNOW, WHY IT MIGHT BE
GOING ON.
>> WE HAVE AN ONLINE QUESTION.
WONDERFUL TALK, THANK YOU.
WHY DO YOU THINK THERE'S SUCH A
DRAMATIC DIFFERENCE IN HOW THE
140A/F MUTATIONS AFFECT VIRAL
FITN DEPENDING SPECIFICALLY ON
WHETHER THERE IS OR NOT DRUG
PRESSURE APPLIED?
>> YES, SO WHAT I'M WONDERING
ABOUT IS THIS AND IN A WAY, IT'S
THE DRIVING FORCE FOR THE
EXPERIMENTS THAT WE'RE TRYING TO
FINISH UP RIGHT NOW.
SO ONE OF THE THINS THAT WE KNOW
IS, IF YOU PUT THE MUTATION IN T
CELLS, THEN IT'S VERY POOR, AS
THE PERSON JUST SAID.
IF IN FACT YOU PUT IT IN JERCA,
LOVES IT.
THAT'S THE REASON WHY WE'RE
EXPLORING ALL THESE DIFFERENT
CELL ENVIRONMENTS.
SO I'M WONDERING WHETHER, AND
THIS MAY GET US NORTHERN, BUT
WHAT WE'RE -- GET US NOWHERE,
BUT WHAT WE'RE TRYING TO DO IS
IDENTIFY WHAT MAYBE THE PLAYERS
IN THE PHENOTYPE THAT IT
MANIFESTS AND TO ASK WHETHER OR
NOT THERE'S DIFFERENTIAL
EXPRESSION OF FORMS OF THOSE IN
THESE VARIOUS CELL ENVIRONMENTS.
IT COULD BE AS, QUOTE-UNQUOTE,
SIMPLE AS THAT.
AT LEAST THAT'S WHERE WE
STARTING.
>> DO YOU FIND THAT CLEAVAGE
WITH A PROTEASE INHIBITOR
RESISTANT PROTEASE AT THAT SITE,
THAT IT'S AFFECTED BY THAT
MUTATION?
WE'RE SEEING THAT WITH A
[ INDISCERNIBLE ].
>> YEAH.
SO NOT AS FAR AS WE CAN TELL.
SO IT WAS ON THE SLIDE, I THINK,
BUT THERE WAS A -- THERE WAS --
WHAT WE DID WAS WE TOOK FROM THE
ONE DATABASE MUTATIONS THAT CAME
UP IN OTHER INDIVIDUALS AS
CONFERRING SOME DEGREE OF
PROTEASE RESISTANCE.
AND WE ASKED WHETHER IN FACT
THAT WAS INFLUENTIAL.
AND WE DIDN'T SEE ANYTHING THAT
WE COULD DETECT.
>> ANYBODY ELSE?
>> I JUST WONDER IS THERE A WAY
TO TEST ON THE SAMPLES
[ INDISCERNIBLE ] THE EXTRA
RECEPTOR ON THE CELLS THAT WE
CANNOT NOW COUNT.
>> THAT'S AN INTERESTING ONE.
WOULD THAT BE SUFFICIENT?
>> I SAW SOME STUDY LOOKING FOR
THE ROLE OF ESTROGEN ON HIV AND
THAT'S WHAT THEY DID.
I'M NOT SURE ABOUT THE
[ INDISCERNIBLE ] SOME POTENTIAL
SEX-RELATED.
>> SO THAT'S SORT OF LIKE
TEASING -- OF COURSE, IF YOU'RE
GOING TO KNOCK DOWN, THEN THE
RECEPTOR, IF YOU JUST PLUG IT
INTO THE RECEPTOR, PERIOD, WELL,
ONE OF THE JOBS OF ESCRT
MACHINERY IS TO DOWN-REGULATE
RECEPTORS.
I DON'T KNOW IF THAT'S ONE OF
THEM, BUT IT WOULD JUST TIE IN
WHAT ESCRTs, SO IF IN FACT THERE
WASN'T A GENDER PLAY, I DON'T
KNOW, BUT THAT'S WHAT SORT OF
LIKE STRIKES ME COULD BE AN
INTERESTING CONNECTION WITH THAT
IDEA ANYWAY.
>> DO YOU HAVE A SENSE OF TSG101
IS DIFFERENTIALLY EXPRESSED
BETWEEN DIFFERENT AGE GROUPS?
>> DIFFERENT AGE GROUPS, YOU
MEAN PEOPLE AGE GROUPS?
HMM.
I DON'T KNOW THAT WE WOULD HAVE
A WAY OF UNDERSTANDING THAT.
THE QUESTION WAS, WOULD TSG101
BE -- RESTATE THE QUESTION.
>> DO YOU KNOW IF THERE IS
DIFFERENTIAL EXPRESSION OF
TSG101 AMONG DIFFERENT HUMAN AGE
GROUPS?
>> I WOULD ASSUME THAT THE
ANSWER IS NO.
IT'S UBIQUITOUSLY EXPRESSES.
I MEAN, PLANTS EVEN HAVE IT.
IT'S EMBRYOTICALLY LETHAL, SO
EVERYBODY NEEDS IT, BUT I HAVE
NO INDICATION -- SO IN THE
THESIS OF GRADUATE STUDENTS WHO
WORKED ON THIS INITIALLY IS A
BUNCH OF DIFFERENT TISSUES,
CERTAINLY, BUT IN TERMS OF AGE,
WE NEVER LOOKED.
I DON'T SEE A REASON WHY IT
SHOULD.
BUT WHO KNOWS?
>> AND ONE MORE QUESTION IF
THERE'S TIME.
DO WE HAVE TIME?
YES?
OKAY.
>> SURE.
>> DO YOU THINK, ARE YOU OR
ANYONE ELSE LOOKING AT THE
EFFECT THAT PRAZOLES MIGHT HAVE
ON OTHER UBIQUITIN LIGASES, LIKE
THE ONES THAT ACTUALLY HAVE THE
ABILITY TO TRANSFER?
>> YES.
SO THAT'S SOMETHING WE'RE DOING
RIGHT NOW.
IT TURNS OUT THAT TSG IS ABLE TO
PROTECT A WHOLE BUNCH OF
DIFFERENT ONES.
NOT EVERYBODY, ALL RIGHT, BUT A
WHOLE BUNCH OF DIFFERENT ONES,
AND THE PRAZOLES WOULD APPEAR TO
DO SO.
I'LL PUT AN ASTERISK THERE FOR
TWO REASONS.
NUMBER ONE, BECAUSE WE WERE
DOING THE EXPERIMENT IN THE HOPE
OF GETTING A HANDLE ON HOW IT
ACTUALLY IS MANIPULATING THE
UBIQUITINATION EVENT.
BECAUSE -- AND SO THE REASON I
PUT AN ASTERISK IS WE'VE ONLY
DONE THE EXPERIMENT WITH A
LIMITED NUMBER OF THEM AND SO WE
HAVEN'T TAKEN ONES LIKE -- YOU
KNOW, ACROSS THE BOARD,
DIFFERENT BY OTHER INDICATIONS
AND TRIED IT.
>> ALL RIGHT.
WELL, THANK YOU VERY MUCH.