GOOD MORNING.
IT'S FANTASTIC TO SEE SO MANY
PEOPLE IN PERSON.
I DON'T THINK THIS ROOM HAS BEEN
THIS FULL IN PROBABLY SINCE
PRE-COVID.
SO FOR THOSE WHO DON'T KNOW ME
OR MIGHT NOT RECOGNIZE ME IN THE
FANCY CLOTHES, I'M ERIC FREED,
DIRECTOR OF THE DYNAMICS AND
REPLICATION PROGRAM, ON BEHALF
OF THE STAFF AND FACULTY OF THE
CRP I'D LIKE TO WELCOME YOU TO
THE CELEBRATION OF ALAN REIN'S
CAREER.
HIGHLIGHTS TO GET US STARTED,
ALAN GRADUATED FROM REID COLLEGE
IN 1961, Ph.D. RESEARCH WITH
HARRY RUBIN AT UC-BERKELEY
BEFORE DOING POSTDOCTORAL
RESEARCH AT M.I.T., WITH SHELDON
PENMAN.
IN THE 70s HE WORKED AT LITTON
BIOMEDICS THAT DID
GOVERNMENT-FUNDED CONTRACT
RESEARCH.
LATE 70s WORKED IN TUMOR VIRUS
GENETICS AT NCI BETHESDA.
IN 1979, ALAN MOVED TO FREDERICK
TO SERVE AS A SENIOR RESEARCH
SCIENTIST, MICROBIOLOGY OF
RETROVIRUS SECTION, IN THE
CANCER RESEARCH FACILITY.
BETWEEN 1984 AND 1999 ALAN WAS
HEAD OF THE RETROVIRAL GENETICS
SECTION AT ABL IN FREDERICK.
AND SINCE 1999, ALAN HAS BEEN
HEAD OF THE RETROVIRUS ASSEMBLY
SECTION IN THE HIV DRP.
HE HAS MANY HONORS AND
ACCOMPLISHMENTS OVER THE YEARS,
SERVED ON EDITORIAL BOARDS OF
SEVERAL JOURNALS INCLUDING JBC,
VIROLOGY, IN 2012 RECEIVED THE
NIH DIRECTOR'S AWARD, 2014
ELECTED FELLOW AMERICAN ACADEMY
OF MICROBIOLOGY.
I WOULD LIKE TO SAY A FEW WORDS
ABOUT HIS GROUNDBREAKING
CONTRIBUTIONS TO THE FIELD OF
RETROVIROLOGY.
I COULD SPEND HALF THE DAY DOING
THIS.
CONDENSING THINGS DOWN -- HELLO?
PLEASE MUTE YOURSELF.
>> WAS THAT YOU TALKING?
>> NO, THE CHAIR OF THE MEETING.
>> PLEASE MUTE YOURSELF.
EARLY TO MID-1970s, ALAN DID
GROUNDBREAKING RESEARCH ON Fv1
REVICES OF MLV INFECTION,
JONATHAN STOYE WILL SAY MORE
LATER.
EARLY INDICATIONS MEMBRANE
BINDING OF GAG IS ESSENTIAL FOR
VIRUS ASSEMBLY SUBSEQUENTLY
SHOWN FOR HIV AND MANY OTHER
RETROVIRUSES.
IN 1988, ALAN SHOWED POINT
MUTANTS FAILED TO PACKAGE VIRAL
RNA, PROVIDING EVIDENCE FOR
SPECIFIC RNA RECOGNITION BY THE
ZINC FINGER-LIKE DOMAIN, NC ZINC
FINGER DOMAIN WE'RE FAMILIAR
WITH.
1994 SHOWED THE MLB
TRANSMEMBRANE GLYCOPROTEIN IS
CLEAVED TO ACTIVATE FUSION
ACTIVITY, THE FIRST INDICATION
OF A CONNECTION BETWEEN
ACTIVATION AND VIRUS MATURATION.
IN 2001, ALAN SHOWED RNA IS A
STRUCTURAL ELEMENT IN RETROVIRUS
PARTICLES, IN 2001 SHOWED THE
MODULATION OF HIV-LIKE PARTICLE
ASSEMBLY IN IN VITRO PREDATED BY
NEARLY TWO DECADES THE CURRENT
APPRECIATION FOR THE ROLE OF IP6
IN RETROVIRAL ASSEMBLY AND
MATURATION.
TOGETHER WITH NCI FREDERICK
CONTRIBUTED TO SOLVING THE
STRUCTURE OF THE HIV REN
RESPONSE ELEMENT AND DISSECTED
SPECIFIC BINDING OF HIV-1 GAG TO
PACKAGING SIGNAL IN VIRAL RNA,
GAG BINDING SPECIFICALLY TO
VIRAL RNA IS TRIGGER FOR VIRUS
ASSEMBLY EXPLAINING SPECIFICITY
OF RNA PACKAGING.
SO, IN ADDITION TO BEING
ENORMOUS CONTRIBUTOR TO THE
FIELD OF RETROVIROLOGY, ALAN WAS
THEN ATTENDING FOR MANY YEARS,
MANY CONFERENCES IN THE FIELD,
INCLUDING THE COLD SPRING HARBOR
RETROVIRUS MEETING.
HERE IS ALAN IN 1989.
AND WITH JACKIE BENTLEY, HERE
WITH US TODAY, IN 1991.
HERE IS ALAN IN 1998 WITH JOHN,
IN THE FRONT ROW, JUST LIKE AT
COLD SPRING HARBOR.
ALAN AND DELPHINE IN 1999.
THEY ACTUALLY LOOK EXACTLY THE
SAME NOW AS THEY DID THEN.
IT'S REALLY REMARKABLE.
IN 2001 WITH STEVE.
SO I'LL PROVIDE A FEW QUICK
HIGHLIGHTS OF THE PROGRAM WE
HAVE A LINEUP OF DISTINGUISHED
SPEAKERS TODAY.
BEGINNING WITH FRANCOIS
BARRE-SINOUSSI FROM INSTITUT
PASTEUR, JONATHAN STOYE.
AFTER THE BREAK DELPHINE MURIAUX
AND MAURICIO COMAS-GARCIA, AND
WE'LL HEAR ON TRANSCRIPTION
START HETEROGENEITY; AND THE TWO
FORMERS DIRECTORS, VIRTUAL
PRESENTATION FROM PETER AND
FINALLY ALAN REIN WILL PROVIDE
CLOSING REMARKS.
THERE WILL BE AN AFTER PARTY
CELEBRATION AT THE END OF THE
DAY AT STEINHARDT BREWERY,
DOWNTOWN FREDERICK.
FINALLY, ALAN, I CONGRATULATE
YOU ON YOUR GROUNDBREAKING
CONTRIBUTIONS TO THE FIELD OF
RETROVIROLOGY, AND WISH YOU THE
BEST FOR A LONG AND HAPPY
RETIREMENT.
ALAN WILL CONVERT TO EMERITUS
STATUS AT THE END OF THE YEAR SO
WE HOPE YOU'LL STAY ENGAGED IN
DRP ACTIVITIES.
HOWEVER, WE HOPE HE WILL ALSO
SPEND TIME WITH FRIENDS AND
FAMILY, OLD AND NEW HOBBIES AND
INTERESTS.
YOU KNOW HE'S AN AVID BIRD
WATCHER, FOR EXAMPLE.
SO WITHOUT FURTHER DELAY I WILL
WELCOME ALAN TO THE PODIUM TO
KICK OFF TODAY'S PROGRAM.
>> THANK YOU, ERIC.
THAT WAS MORE THAN I COULD WISH
FOR.
SO, I ASKED TO INTRODUCE EACH
SPEAKER MYSELF, AND JUST SAY A
FEW WORDS, NOT ABOUT THE USUAL
THEY HAVE SO MANY AWARDS, BLAH,
BLAH, BLAH, BUT WHAT THEY ARE
DOING HERE, WHAT IS THE
CONNECTION SUCH THAT THEY ARE
PARTICIPATING IN THIS EVENT.
SO, FIRST SPEAKER REALLY NEEDS
NO INTRODUCTION, FRANCOISSE IS
THE PERSON WHO ISOLATED HIV, AND
BEFORE SHE ISOLATED HIV, IN FACT
BEFORE HIV APPEARED, SHE STUDIED
MURINE VIRUSES IN THE LAB OF BOB
BASSEN, IN BETHESDA, AS DID I.
AND SO WE WORKED SIDE BY SIDE IN
THE 19, UM, '70s, MID-'70s,
LATE 70s.
WE'VE BEEN FRIENDS EVER SINCE.
AND SO IT'S A GREAT PLEASURE TO
SAY HELLO AGAIN TO MY OLD FRIEND
FRANCOISE.
>> THANK YOU VERY MUCH.
GOOD MORNING, EVERYONE.
TODAY WE GATHER TO HONOR OUR
COLLEAGUE AND DEAR FRIEND ALAN,
PROFESSIONAL LIFE FOR ADVANCES
IN SCIENCE ON RETROVIRUSES.
I'M FILLED WITH BOTH JOY AND
NOSTALGIA, WITH GREAT MEMORIES
COMING BACK IN MY MIND.
AS IT WAS SAID BY ERIC, Ph.D.
IN BERKELEY, M.I.T., ALAN IS
ENJOYING THE NATIONAL CANCER
INSTITUTE IN THE '70s, FIRST
IN BETHESDA, AND THEN IN
FREDERICK.
THAT IS WHEN OUR PATHS FIRST
CROSSED IN THE NCI LAB AT
BIONETTICS IN BETHESDA.
AT THAT TIME, I WAS THERE TO
SUCCEED SYLVIE, WHO
UNFORTUNATELY CANNOT BE WITH US
TODAY.
I WAS THERE FOR PART OF
RESTRICTION OF MURINE VIRUS IN
THE LABORATORY OF BOB BASSEN, AS
WAS MENTIONED, UNFORTUNATELY BOB
PASSED AWAY A YEAR AGO.
I STILL VIVIDLY REMEMBER THAT
PERIOD WITH YOU, ALAN, BOB,
BRENDA, AND OTHERS.
I WAS YOUNG, EAGER, VERY EXCITED
TO LEARN ABOUT RETROVIRUSES FROM
YOU AND PERHAPS A LITTLE NERVOUS
ABOUT BEING OUT OF THE COUNTRY
WITH MY POOR ENGLISH.
BUT I VERY RAPIDLY SENSED THE
INTELLECT ENVIRONMENT, I NOT
ONLY FOUND FANTASTIC MENTORS
WITH BOB AND YOU, BUT ALSO
LIFELONG FRIENDS.
BELIEVE IT OR NOT, ALAN, WE HAVE
KNOWN EACH OTHER FOR NEARLY HALF
A CENTURY.
ALMOST 15 YEARS AGO ALREADY YOU
WERE ATTENDING IN PERSON WITH
BOB AND DAN, ANOTHER LIFELONG
NCI FRIEND, AT MEETING ORGANIZED
FOR MY RETIREMENT AT PASTEUR
INSTITUTE IN OCTOBER 2015.
SO OBVIOUSLY I WANTED TO BE
THERE TODAY FOR YOU, AT LEAST AT
DISTANCE.
LET ME TAKE A MOMENT TO BRIEFLY
REFLECT ON NOT ONLY YOUR
SCIENTIFIC ACHIEVEMENTS, THAT
WAS ALREADY -- THEY WERE ALREADY
MENTIONED, BY ERIC, BUT ALSO THE
INCREDIBLE PERSON YOU ARE.
AS YOU ALL KNOW HERE, ALAN'S
PASSION AND COMMITMENT TO
SCIENCE IS NOTHING SHORT OF
INSPIRING.
HE HAS MADE SIGNIFICANT ADVANCES
THAT WERE MENTIONED TO OUR
UNDERSTANDING OF SEVERAL ASPECTS
OF REPLICATION OF MURINE AND
HUMAN RETROVIRUSES INCLUDING
HIV.
ACCORDING TO THE AGENDA OF THE
MEETING TODAY, THE NEXT SPEAKER
WILL CERTAINLY MENTION ALAN'S
CONTRIBUTION TO OUR KNOWLEDGE OF
MOLECULAR, STRUCTURING, AND
FUNCTIONAL MECHANISMS INVOLVED
IN VIRAL ACTIVITY AND
MATURATION, REGULATION OF
INFECTION.
BUT PIONEERING WORK ON Fv1, AS
WAS JUST MENTIONED, AND ON THE
INTERACTION OF HIV-1 GAG WITH
NUCLEIC ACID LEADING TO
CONFIRMATIONAL CHANGES REQUIRED
FOR VIRAL ASSEMBLY.
ALAN'S RESEARCH HAS NOT ONLY
ADVANCED OUR KNOWLEDGE BUT IT
HAS ALSO PAVED THE WAY FOR
FUTURE DISCOVERIES IN THE FIELD.
BUT BEYOND HIS IMPRESSIVE
SCIENTIFIC CONTRIBUTIONS WHAT
SETS ALAN APART IS HIS
PERSONALITY.
HE'S A MAN OF GENEROSITY, ALWAYS
STANDING BY HIS PRINCIPLES AND
HUMAN VALUES.
IN A WORLD THAT OFTEN
PRIORITIZES ACCOLADES, ALAN HAS
ALWAYS REMAINED REFRESHINGLY
MODEST.
HE HAS NEVER SOUGHT THE
SPOTLIGHT BUT RATHER SHINING IT
ON OTHERS, MENTORING COUNTLESS
STUDENTS AND COLLEAGUES ALONG
THE WAY.
HIS LOYALTY AND FRIENDSHIP HAVE
BEEN UNWAVERING.
THROUGH THE HIGHS AND LOWS OF
OUR CAREERS, ALAN WAS ALWAYS
THERE, OFFERING OR LISTENING
HERE AND FRIENDLY SUPPORT.
HE HAS ALWAYS REMINDED US THAT
SCIENCE IS NOT JUST ABOUT DATA,
REPLICATION, BUT ABOUT THE
PEOPLE WE WORK AND FOR AND ABOUT
THE RELATIONSHIP WE BUILD, ABOUT
COLLABORATION, ABOUT SHARING
GOALS.
I HAVE WONDERFUL MEMORIES OF
TIME WE SPENT TOGETHER IN THE
LAB, AT HOME, OR SOMEWHERE ELSE
EXCHANGING EXPERIENCE, SHARING
OUR YEARS, AND DISCUSSING NOT
ONLY SCIENCE BUT ALSO LIFE,
DREAMS, AND ASPIRATIONS.
ALAN, YOU BELIEVED IN ME WHEN I
SOMETIMES DID NOT BELIEVE IN
MYSELF, OFFERING YOUR GUIDANCE,
ENCOURAGING ME TO PURSUE MY
JOURNEY.
YOU CERTAINLY DID IT FOR MANY
OTHERS AROUND YOU.
YOUR ABILITY TO MENTOR IS A GIFT
THAT HAS BEEN SO MUCH
APPRECIATED.
YOUR HUMAN, YOUR KINDNESS, YOUR
INSIGHTFUL SUPPORT HAVE ENRICHED
OUR LIVES IN WAYS I CANNOT FULLY
EXPRESS.
NOW AS YOU STEP INTO THIS NEW
CHAPTER OF LIFE, I'M EXCITED FOR
YOU.
ALAN, YOU WILL SEE, RETIREMENT
IS NOT AN END.
IT IS A BEGINNING.
IT IS A TIME TO EXPLORE NEW
PASSIONS, NEW HOBBIES, TO
TRAVEL, TO SPEND TIME WITH
FRIENDS, FAMILY, ENJOY NATURE
INCLUDING BIRD WATCHING AND ALL
THE BEAUTY.
I KNOW WHATEVER YOU DECIDE TO
PURSUE YOU WILL APPROACH IT WITH
THE SAME DEDICATION THAT YOU
BROUGHT TO YOUR SCIENTIFIC
CAREER.
ALAN, THANKS FOR BEING NOT ONLY
A SCIENTIST AND MENTOR, ALSO A
TRUE AND LOYAL FRIEND.
YOUR LEGACY WILL LIVE IN THE
HEART OF THOSE YOU HAVE
INSPIRED, AND IN THE WORK THAT
THEY WILL CONTINUE TO DO IN YOUR
HONOR.
WHILE YOU MAY BE RETIRING FROM
PROFESSIONAL LIFE, YOU WILL
ALWAYS BE PART OF OUR SCIENTIFIC
COMMUNITY AND WE LOOK FORWARD TO
SEEING WHERE THIS NEW JOURNEY
TAKES YOU.
MAY THE NEXT CHAPTER OF YOUR
LIFE BE FILLED WITH JOY,
ADVENTURE, AND ALL THE HAPPINESS
YOU SO RICHLY DESERVE, ALAN.
ENJOY, CHEERS TO YOU.
THANK YOU.
>> THAT'S A HARD ACT TO FOLLOW.
I THANK YOU, FRANCOISE.
AGAIN, I COULDN'T WISH FOR
ANYTHING MORE.
OUR NEXT SPEAKER IS JONATHAN
STOYE, AND JUST A FEW WORDS.
SO, AS FRANCOISE INDICATED, I
CAME INTO MURINE LEUKEMIA VIRUS
WORK BY WAY OF BOB BASSEN.
I INHERITED BOB BASSEN AS A
BOSS, WHEN I INHERITED BOB HE
WAS ACTUALLY ON SABBATICAL IN
PARIS.
AND HE CAME BACK AND SAID, OKAY
NOW, THERE'S THIS NEW THING
Fv1 RESTRICTION, BRAND NEW AND
REALLY INTERESTING.
AND SO WE'RE GOING TO WORK ON
IT.
AND SO THIS -- WE NOW SEE Fv1
AS A MEMBER OF THE CLASS OF
RESTRICTION FACTORS, BUT AT THE
TIME NOBODY HAD EVER HEARD OF A
RESTRICTION FACTOR, Fv1 WAS
UNIQUE, SINGULAR FOR MANY YEARS.
I DID SOME -- IN BOB'S LAB I DID
SOME KIND OF ELEMENTARY GENETICS
CHARACTERIZING SOME OF THE
PARTIES OF ITS INTERACTION WITH
MURINE LEUKEMIA VIRUSES WHICH
ARE FASCINATING, STILL NOT FULLY
UNDERSTOOD.
BUT THEN IT GOT HARD.
WHEN IT GOT HARD I WENT WITH
OTHER THINGS TO DO.
AND THAT'S WHERE JONATHAN COMES
IN.
JONATHAN DID NOT DROP Fv1 WHEN
IT GOT HARD.
HE STUCK WITH IT YEAR AFTER YEAR
AFTER YEAR, AND BROKE IT OPEN.
HE CLONED Fv1 AND HAS
CONTINUED TO CHARACTERIZE Fv1
AND NOW OTHER RESTRICT FACTORS
AS WELL.
AND HAS LEARNED A LOT OF AMAZING
THINGS ABOUT Fv1.
SO, JONATHAN, THANK YOU FOR
COMING.
I'M DELIGHTED YOU CAN BE HERE.
>> IT'S A GREAT APPRECIATE TO BE
IN FREDERICK.
IT WAS A PLACE THAT I REMEMBER
WITH PLEASURE.
WE FIRST MET AT COLD SPRING
HARBOR 45 YEARS AGO.
CAN YOU FIND ALAN LOOKING SOBER
CAN YOU FIND ME PARTYING?
[LAUGHTER]
IS THERE ANYONE ELSE IN THE ROOM
WHO CAN FIND THEMSELVES IN THESE
PICTURES?
IT'S INTERESTING.
SIX OF THE SPEAKERS TODAY WERE
PRESENT AT THAT MEETING.
AT LEAST SIX, BY MY COUNT SIX.
I WANT TO TALK ABOUT Fv1.
AND SHOW HOW STARTING WITH THE
BIOLOGY THAT ALAN AND OTHERS
WORKED ON IN THE '70s WE
LEARNED A LOT ABOUT
RETROVIRUSES, HOW THEY WORK.
SO, PEOPLE THOUGHT RETRO CAUSES
CAUSED CANCER, THERE WAS
INTEREST IN GENES IN MICE THAT
WOULD PROTECT AGAINST RETROVIRUS
INFECTION.
AND FORTUNATELY APPROXIMATELY
INDEPENDENTLY THESE TWO
GENTLEMEN, FRANK LILLY AND WALLY
ROWE, DISCOVERED THE GENE CALLED
Fv1.
BASIC PROPERTIES ARE SUMMARIZED
HERE, IN MICE THERE ARE TWO
MAJOR ALLELES, A LITTLE N AND
LITTLE B, THEY RESTRICT B AND N
TROPIC VIRUSES RESPECTIVELY.
THE SUSCEPTIBILITY DETERMINED IN
MLV MAPS TO CAPSID, SPECIFICALLY
AMINO ACID 110 IN THE CAPSID
MOLECULE.
AS WE'LL SEE, THERE ARE OTHER
PARTS OF CAPSID THAT ARE
INVOLVED, CAN INFLUENCE THIS
INTERACTION.
SO, WORK Fv1 IN THE '70s,
THESE ARE SOME PAPERS THAT CAME
FROM HIM AND FROM BOB BASSEN.
I'LL DRAW YOUR ATTENTION TO THE
BOTTOM RIGHT-HAND CORNER, WHICH
FOR ME WAS -- BECAME KEY, THAT'S
THAT YOU CAN OVERCOME Fv1
RESTRICTION IF YOU INFECT WITH
THE RIGHT VIRUS BUT THE CELL
MAKING -- IF THE CELL IS MAKING
THAT VIRUS IT STILL SHOWS Fv1
RESTRICTION, I'LL RETURN TO THAT
BRIEFLY LATER.
SO, THIS SLIDE SUMMARIZES WHAT
WE KNOW ABOUT OR WHERE Fv1
WORKS.
WORK FROM ALAN AND MANY OTHER
PEOPLE, SOME IN THIS ROOM TODAY,
DESCRIBING WHERE Fv1 ACTS.
Fv1 SEEMS TO TARGET VIRUS AT A
STAGE IN THE REPLICATION CYCLE
UNCOATINGS OCCUR, SEEMS TO BLOCK
MURINE IMPORT.
IT DOES NOT INTERACT -- Fv1
DOES NOT INTERACT WITH CAPSID ON
THE WAY OUT.
SO, WE WERE INTERESTED IN Fv1
IN THE '70s.
I WISH I COULD FIND THE
ANNOUNCEMENT, THE PIECE OF PAPER
ON WHICH IT WAS WRITTEN I WAS
GOING TO TALK ABOUT A PAPER ON
Fv1 BUT COULDN'T FIND IT.
THE FACT THAT THERE SEEMS TO BE
A MOUSE GENE, WORKS ON MOUSE
VIRUSES, PEOPLE SORT OF LOST
INTEREST IN THE SUBJECT.
BUT I ALWAYS BELIEVED STRONGLY
IN COMPARATIVE APPROACH,
STUDYING HUMAN RETROVIRUSES BY
STUDYING NON-HUMAN RETROVIRUSES
IN NON-HUMAN SYSTEM.
SO WHEN I HAD THE TUNED TO SET
UP MY OWN LAB, I DECIDED TO TRY
AND USE A POSITIONAL APPROACH,
GENETIC APPROACH THAT RATHER
APPEALED TO ME.
WHEN I WAS A POSTDOC WITH JOHN
COFFIN, TOGETHER WITH WAYNE
FRANKEL, WE IDENTIFIED FOUR
ENDOGENOUS RETROVIRUSES THAT
WERE LINKED TO Fv1
GENETICALLY.
AND I DECIDED TO USE THOSE AS
STARTING POINTS IN THE SEARCH
FOR Fv1.
FIRST GENERATING A BETTER
GENETIC MAP, THEN MAKING YAC
CLONES, LARGE CLONES FOR THE
REGION AND INTRODUCE INTO CELLS
AND SHOW WHICH ONES HAVE Fv1,
AND FOLLOW THE ACTIVITY.
IT WAS A STRUGGLE.
THERE WERE A NUMBER OF
DIFFICULTIES, LIKE A MASS
HEPATITIS VIRUS INFECTION OF THE
ANIMAL COLONY, WE HAD TO DO LESS
OF A SURVEY OF THE GENETIC
CROSS.
AND THERE WERE INSTABILITY REACT
CLONES, AND OTHER THINGS OF THE
EVENTUALLY WE CLONED THE GENE.
GREAT.
THIS WAS MY MOMENT IN THE
SPOTLIGHT.
DID I HAVE A MOMENT IN THE
SPOTLIGHT.
I RATHER ENJOYED IT.
[LAUGHTER]
IT TURNED OUT THAT Fv1 WAS
DERIVED FROM ONE OF THESE
ENDOGENOUS RETROVIRUSES.
I WAS TRYING TO GET AWAY FROM
ENDOGENOUS RETROVIRUS, SPENT SO
MUCH TIME WORKING ON THEM, AND
NOW ANOTHER ENDOGENOUS
RETROVIRUS.
IT IS A VIRUS BELONGING TO THE
ERV-L, RISEN BY COLLEAGUES,
Fv1 CAN BE RECOGNIZED AS OPEN
READING FRAME OF 1400
NUCLEOTIDES THAT ENCODES THE
ABILITY TO RESTRICT VIRUSES.
WE STILL DON'T KNOW WHERE --
WHAT THE ORIGINAL ERV-L WAS.
WE CAN FIND OTHERS IN GENOMES OF
RODENTS, BUT 50% SIMILARITY.
IT'S ONLY ABOUT 10% SIMILARITY,
IF ONE IS INVOKING SOME KIND OF
LIKE WITH LIKE MECHANISM, IT'S
GOT TO BE SEEING SOMETHING, A
MORE COMPLICATED PRINCIPLE THAN
THE AMINO ACID SEQUENCE.
SO, WE ALSO NOTED IN THE
ORIGINAL CLONING THAT Fv1 DID
NOT SEEM TO BE PRESENT IN RAT
DNA, AND SO WE CONCLUDED THAT
THE GENE HAD -- THIS Fv1 HAD
ARISEN LESS THAN 10 MILLION
YEARS AGO, SEPARATION BETWEEN
RAT AND MOUSE.
HOWEVER, A MORE EXTENSIVE STUDY
OF LOTS OF DIFFERENT -- DNA FROM
LOTS OF CREATURES SUGGESTED THAT
IN FACT THE Fv1 INSERTION THAT
GAVE RISE TO Fv1 OCCURRED 45
MILLION YEARS AGO.
IT WAS LOST BY DELETION OF THE
FAIRLY HIGH FREQUENCY OR BY
POINT MUTATION.
NEVERTHELESS IT'S QUITE
REMARKABLE THIS OPEN READING
FRAME HAS PERSISTED FOR THIS
PERIOD OF TIME.
SO, THE CALCULATION SAYS THAT IN
45 MILLION YEARS AGO THERE'S AN
ENORMOUSLY HIGH PROBABILITY THE
GENE WILL BE LOST OVER TIME
WHICH SUGGESTS IN ORDER TO
MAINTAIN THE GENE YOU HAVE TO
HAVE A SELECTIVE FORCE.
THAT SAYS IT'S A GOOD THING.
NOW, THIS RAISES A PROBLEM
BECAUSE MLVs ONLY AROSE, LET'S
SAY ABOUT 5 MILLION YEARS AGO.
SO WHAT WAS HAPPENING IN THE
PREVIOUS 40 MILLION YEARS?
IS IT POSSIBLE THERE ARE OTHER
VIRUSES AROUND -- THAT Fv1
COULD RESTRICT?
AND THAT'S A QUESTION WE SPENT
TIME WORKING ON, TRYING IDENTIFY
OTHER VIRUSES THAT MIGHT BE
RESTRICTED BY Fv1.
AND WE'VE NOW TESTED THE
RESTRICTION PROPERTIES OF 39
Fv1 DERIVATIVES AGAINST 22
VIRUSES.
WITH RESTRICTION OF GAMMA
RETROVIRUS IS MOST COMMON, WE'VE
ALSO IDENTIFIED EXAMPLES OF
ACTIVITY AGAINST MULTIPLE OTHER
KINDS OF RETROVIRUSES INCLUDING
ALPHAS, BETAS, DELTA, LENTI, AND
FOAMY VIRUSES.
I DON'T HAVE TIME TO GO INTO
FOAMY VIRUSES BUT IT'S HAVING
GIVEN WHAT WE KNOW ABOUT THE
STRUCTURE OF FOAMY VIRUSES.
THIS JUST SHOWS YOU A TREE OF
Fv1 IN LOTS OF RODENTS, AND
THE VIRUSES THAT CAN BE
RESTRICTED BY THESE.
SO, THERE ARE MULTIPLE EXAMPLES
OF RESTRICTION.
IT'S RARE TO SEE RESTRICTION OF
ALL THE DIFFERENT VIRUSES BY A
SINGLE Fv1 MOLECULE.
BY COMBINING THEM WE CAN LOSE
BOTH ACTIVITIES, TAKE THE AMINO
ACID OF ONE, TRY TO MAKE
SOMETHING THAT SEE LOTS OF
VIRUSES, WE DON'T GET IT.
WHICH SAYS THIS THING CAN
RECOGNIZE LOTS OF DIFFERENT
VIRUSES, BUT THERE ARE CHANGES
THAT OCCUR, ASSOCIATED WITH THE
DIFFERENT RESTRICTION
SPECIFICITIES.
AROSE 45 MILLION YEARS AGO, CAN
SEE LOTS OF THINGS.
SOMETIMES THESE SIGNAL AMINO
ACID CHANGE IN Fv1 IS
SUFFICIENT TO ALLOW RECOGNITION
OF A DIFFERENT VIRUS.
AND YOU HAVE TO REMEMBER THAT
THIS IS A SINGLE AMINO ACID IN A
PROTEIN THAT DIFFERS FROM ITS
TARGET BY 50 TO 90%.
SO, IT IS VERY -- Fv1 IS
DIFFERENT FROM ITS TARGET WITH A
SINGLE AMINO ACID MAKING A
DIFFERENCE BETWEEN RESTRICTING
AND NOT RESTRICTING.
AND SOME BUT NOT ALL CASES WE
CAN COMBINE MULTIPLE
SPECIFICITIES, THERE'S AT LEAST
ONE CASE WHERE YOU AFEAR HAVE
DUPLICATED, YOU HAVE TWO, ONE
CAN RESTRICT ONE VIRUS, ANOTHER
CAN RESTRICT ANOTHER KIND OF
VIRUS, SEEMS TO BE AN EXAMPLE OF
THINGS Fv1 IS CAPABLE OFF.
I WOULD ARGUE THIS GENE SURVIVED
FOR ALL THIS TIME, IMPLIES A
SERIES OF INFECTIONS BY
DIFFERENT VIRUSES, ALL THROUGH
OUR HISTORY.
AND I GUESS THAT TO A
RETROVIROLOGIST THAT'S NOT A
SURPRISED BUT SOME PEOPLE THINK
ABOUT VIRUSES APPEARING ONCE,
AND CAUSING AIDS, AND, YOU KNOW,
THEY DON'T THINK ABOUT THE PAST
OR THE FUTURE IN THIS REGARD.
SO HOW DOES Fv1 RESTRICT?
UNFORTUNATELY WE STILL DON'T
KNOW.
HOWEVER MUCH WE WORK, WE DON'T
KNOW HOW IT WORKS.
WE CAN SHOW SPECIFIC RESPONDING
OF Fv1 TO ASSEMBLE CAPSID.
YOU CAN ASSEMBLE CAPSIDS ON THE
TUBES IN THE LEFT-HAND CORNER,
FORMS A HEXAGONAL ARRAY IN A WAY
THAT'S SIMILAR TO THAT SEEN IN
MATURE VIROMES.
WE CAN SHOW SPECIFICITY, SHOW
SPECIFIC BINDING OF Fv1 TO
B-MLV BUT NOT N-MLV.
YOU DON'T GET BINDING THIS WAY
IF YOU STICK THE CAPSID MOLECULE
ON TO THESE AND TRY TO PULL DOWN
THIS COMPLEXES WITH THESE TUBES.
SO WE THINK IT'S RECOGNIZING
SOMETHING VIRUS-LIKE.
AND THIS SEEMS TO SHOW A CERTAIN
SIMILARITY TO THAT OF THE
MORE -- PERHAPS THE MORE FAMOUS
RESTRICTION FACTOR WHERE PEOPLE
HAVE BEEN ABLE TO SHOW THAT
Fv1 BINDS TO THE CAUSE FOR
VIRUSES BY MICROSCOPY.
IT BINDS TO ASSEMBLIES.
THAT WAS ONE PIECE OF
INFORMATION.
WE ALSO REALIZED FROM STUDIES OF
DIFFERENT MLVs, SOME OF WHICH
AROSE BY ESCAPE FROM Fv1, BUT
THE BINDING DOMAINS ARE SPREAD
ACROSS THE ENTIRE SURFACE OF THE
MOLECULE, SO -- OR RATHER AMINO
ACID DIFFERENCES THAT CAN
INFLUENCE INTERACTION ARE SPREAD
ALL OVER THE TOP OF THE
MOLECULE.
AGAIN, ARGUING AGAINST THE
SINGLE POINT OF CONTACT BUT A
WIDE POINT OF CONTACT WITH
Fv1, ITS TARGET.
WE'VE LOOKED IN SOME DETAIL AT
THE SEQUENCES OF THESE DIFFERENT
Fv1s WE'VE ISOLATED, THIS
INITIAL -- THE INITIAL ANALYSIS
WAS DONE WITH 19 SPECIES OF
Fv1.
WE'VE NOW LOOKED AT AT LEAST 39
OR SO.
WHAT WE SEE IS A SEQUENCE TENDS
TO BE WELL CONSERVED, WITH
DIFFERENCES CLUSTERING IN FOUR
REGIONS, OF WHICH THREE, WHAT WE
CALL VB AND VC AND VD,
ASSOCIATED WITH THE DIFFERENCES
BETWEEN THE N AND ALLELES OF
Fv1.
SO, YOU'VE GOT A CONSERVED
SEQUENCE IMPLYING CONSERVED
FUNCTION BUT CLUSTERS OF
DIFFERENCES, POSSIBLY IMPLYING
THESE ARE IMPORTANT FOR THE
BINDING INTERACTION.
AND THIS JUST SHOWS ONE EXAMPLE
OF CHANGES YOU CAN SEE IN ONE OF
THESE REGIONS.
WE HAVE ONE AMINO ACID CHANGE
THAT'S IMPORTANT FOR THE
DIFFERENCE BETWEEN Fv1 N AND
B, A DIFFERENT ONE, SLIGHTLY
DIFFERENT POSITION, WHICH IS
IMPORTANT FOR THIRD SPECIFICITY
OF Fv1.
AND OTHER CHANGES THAT ARE
IMPORTANT FOR RECOGNITION OF
STILL MORE VIRUSES.
THE DETAILS ARE PERHAPS NOT
IMPORTANT BUT THE IDEA THAT YOU
CAN HAVE CHANGES OF RECOGNITION
PROPERTIES ASSOCIATED WITH
SIGNAL AMINO ACIDS IS IMPORTANT.
SO, ONE OF THE PROBLEMS IN OUR
STUDIES Fv1 IS FLIPPING
IMPOSSIBLE TO WORK WITH.
IF YOU TRY EXPRESS IT IN
BACTERIA, IT ALWAYS AGGREGATED.
AND THIS MAY BE A REFLECTION,
ITS INHERENT ABILITY TO
AGGREGATE WITH VIRUSES, IT CAN
ALSO AGGREGATE WITH ITSELF.
SO, WE -- 25 YEARS WE'VE SPENT
TRYING TO EXPRESS THIS THING SO
WE COULD DO STRUCTURAL STUDIES.
WHEN ALPHA CAME AROUND, WE
RELUCTANTLY TRIED PUTTING OUR
MOLECULE INTO ALPHAFOLD.
THIS SHOWS THE STRUCTURE OF THE
C-TERMINAL REGION OF Fv1.
THE N-TERMINAL REGION IS STATE
-- IS
STRAIGHTFORWARD.
THIS IS WELL SEPARATED FROM THE
DIMER.
WHAT YOU CAN SEE IF WE MAP IN
THESE -- IF WE INDICATE THE
POSITION OF THESE VARIABLE
REGIONS, AVB, VC, AGAIN
APPROACHING THE SURFACE OF ONE
SIDE, IMPLYING TWO DINNER PLATES
STICKING TO ONE ANOTHER.
PERHAPS THIS IS IMPORTANT FOR
RESTRICTION.
WE TOOK THIS A BIT FURTHER VERY
RECENTLY.
I'M SORRY, THIS ISN'T A
SUBMARINE OR SOMETHING BUT IT'S
A MODEL OF THE CORE HEXAMER
BASED ON WORK FROM ALAN AND
COLLABORATORS, AND WE'VE TRIED
TO ADD THIS Fv1 BINDING DOMAIN
A TOP OF IT.
WHAT WE CAN SEE WHEN WE DID
THAT, THIS IS STILL VERY
PRELIMINARY, AND WE WOULD LIKE
TO HAVE MORE INFORMATION BY
CROSS-LINKING OR MASS SPEC TYPE
DATA APPROACHES.
WHAT WE SEEM TO SEE IS E349 AND
K358, TWO AMINO ACIDS I
MENTIONED ARE BINDING TO OR
ASSOCIATED WITH RESIDUES IN TWO
DIFFERENT PARTS OF THE HEXAMER
CONSIST END Fv1 IS SOMEHOW
CROSS-LINKING DIFFERENT
MOLECULES OF ITS TARGET.
BUT IT IS VERY EARLY DAYS,
MODELING IS STILL VERY
PRELIMINARY, IT'S DIFFICULT, BUT
WE WILL CONTINUE TO PURSUE THAT.
SO, Fv1 IS DERIVED FROM A
RETROVIRUS, DOESN'T APPEAR TO
INTERACT WITH SINGLE CA
MOLECULES, WE'VE DONE THAT IN
CELLS.
WE KNOW THAT Fv1 CAN FORM
PUNCTATE ASSEMBLIES AND THAT IF
WE PREVENT THAT WE CAN PREVENT
RESTRICTION.
SO THE IDEA THERE'S SOME HIGHER
CAPSID-LIKE CAPSID INTERACTION
TAKING PLACE SEEMS REASONABLE.
AND WE WOULD POSTULATE IT ACTED
BY PREVENTING CROSS-LINKING IN A
WAY YOU CAN INHIBIT HIV BY
PUTTING IN CYSTEINE RESIDUES IN
THE RIGHT PLACES, CROSS-LINK THE
HEXAMER.
I'M GOING TO CHANGE GEARS AND
BRIEFLY MENTION SOME OTHER
FACTORS IN WHICH THE GAG GENE IS
INVOLVED IN RESTRICTION.
THE FIRST IS A FORMER
RESTRICTION SEEN JAAGSIEKTE.
IT APPEARS TO BE A DEFECTIVE
GAGPOLY PROTEIN THAT INTERFERES
WITH LATE REPLICATION OF THE
RELATED VIRUSES.
MAYBE ALAN CAN TELL US HOW THIS
MIGHT WORK.
AND SEEMS TO BE ASSOCIATED WITH
TRAFFIC AT THE VIRUS GENE.
THAT'S ONE CASE WHERE GAG IS
IMPORTANT FOR RESTRICTION.
ANOTHER IS IN THE YEAST, TY1,
YEAST LIKE US DOESN'T WANT WILD
VIRUS INTERFERING AND MUTATING.
THEY HAVE A WAY OF REGULATING,
SEEMS TO BE MEDIATED BY A
PROTEIN THAT'S ENCODED WITHIN
THE GAG GENE.
AND IT INHIBITS VIRUS PARTICLE
FORMATION.
AND IN COLLABORATION WITH I
THINK SOME OF YOUR FORMER
COLLABORATORS, DAVID GARFINKEL
WHO USED TO BE HERE, WE'VE DONE
STRUCTURAL ANALYSES THAT WOULD
SAY THAT THE INHIBITION ARISES
BECAUSE YOU'RE APPROACHING AND
COMPETING WITH ASSEMBLY PROCESS,
SEEMS TO BE INVOLVING THE SAME
RESIDUES AS WHEN AN MLV
ASSEMBLES, THE SAME REGION OF
C-TERMINAL REGION OF THE PROTEIN
IMPORTANT FOR ASSEMBLY.
I'M SURE THAT THERE'S BEEN MORE
TALK ABOUT THESE GAG GENES
ACTING AS SAFETY FACTORS.
IT IS INTERESTING THAT GAG SEEMS
TO BE INVOLVED IN MULTIPLE
DIFFERENT WAYS, BECAUSE IT DOES
DIFFERENT THINGS FOR THE VIRUS.
THE CELL DOES USE IT IN ORDER O
INHIBIT VIRUS REPLICATION, ONE
WONDERS HOW MANY MORE OF THESE
THINGS ARE OUT THERE AND WHETHER
OTHER GAGS PLAY PHYSIOLOGICAL
ROLES OF ONE KIND OR ANOTHER,
AND THIS REMAINS TO BE SEEN.
I WANT TO CLOSE BY SAYING I TOO
HAVE BECOME A RETIRED GENTLEMAN
OF LEISURE.
[LAUGHTER]
MY LAB CLOSED, AND I BECAME AN
EMERITUS SCIENTIST AT THE CRICK
A FEW WEEKS AGO.
THIS PICTURE WAS TAKEN ON TOP OF
THE CRICK, JUST BEFORE COVID,
I'VE LISTED HERE PEOPLE WHO DID
TIME IN MY LAB.
[LAUGHTER]
I'D LIKE TO THANK THEM.
I HOPE I HAVEN'T MISSED ANYBODY
OUT.
IT'S BEEN A WONDERFUL TIME.
AND I'D LIKE TO CLOSE WITH A
FINAL SLIDE OF A GATHERING OF
RETROVIROLOGYISTS.
ONE OF THE -- THIS HAS BEEN ONE
OF THE NICE THINGS ABOUT BEING A
MEMBER OF THIS GROUP, IT'S BEEN
A PRIVILEGE TO BE PART OF THIS
GROUP TRYING TO WORK OUT
INTERESTING BIOLOGY.
AND GETTING TOGETHER
PERIODICALLY.
SO WE'VE GOT STEVE, WE'VE GOT
JOHN AND ALAN, MYSELF, IF YOU
LOOK CAREFULLY YOU CAN FIND
STEVE AND KARIN, I THINK MONICA.
IT WOULD HAVE BEEN NICE TO HAVE
ALL THE SPEAKERS IN ONE PLACE AT
ONE TIME.
I DON'T KNOW IF IT'S REASONABLE.
ANYWAY, THANK YOU VERY MUCH.
ALAN, IT'S BEEN A REAL PLEASURE
>> [OFF MICROPHONE].
>> IT HAS, BUT IT'S NOT VERY
GOOD.
IT'S NOT A VERY GOOD VIRUS.
AND THE ONE THAT WE HAVE IS
Fv1 IS AWAY FROM.
WE TRIED TO DO THAT.
THIS IS -- THEY HAVE
RECONSTRUCTED THE VIRUS, THE
TIME IS UP, PRETTY AWFUL,
EXPERIMENTS ARE DIFFICULT.
WE'VE TRIED.
>> YOU SPECULATE Fv1 MIGHT
PREVENT ENCODING, IS IT POSSIBLE
IT MIGHT, YOU KNOW, INDUCE
PREMIUM ENCODING?
>> SEQUESTERING IS A REASONABLE
POSSIBILITY.
THERE ARE VERY OLD DATA FROM THE
VIRUS LAB, SLIGHTLY DIFFICULT TO
INTERPRET.
WHICH SUGGESTS THAT FULL LENGTH
DNA IS MADE NORMALLY AND GETS
INTO THE NUCLEUS, BUT IT'S --
THE DATA -- IT'S NOT A VERY
CLEAR PAPER.
BUT YES, I MEAN, SO, WE KNOW
TRIM5 IN SIMPLEST MANIFESTATION
ACTS BY CAUSING PREMATURE
DISASSEMBLY BUT IT'S CLEAR IF
YOU BLOCK TRIM5 FROM ITS
ACTIVITIES YOU KILL CAN BLOCK
BOTH REPLICATION AND IT BECOMES
MUCH CLOSER TO Fv1.
>> VERY NICE.
FV EXPRESSED IN -- (INAUDIBLE).
>> IT'S NOT INDUCIBLE BY
INTERFERON.
IT'S EXPRESSED ONLY IN VERY LOW
LEVELS.
AND I SUSPECT, I NEVER HAVE
FORMALLY SHOWN THIS, IT MIGHT BE
TOXIC IF OVEREXPRESSED.
IT WAS INTERESTING WHEN WE FIRST
CLONED Fv1, INTO PLASMID
EXPRESSION VECTOR, THIS IS WORK
DONE BY KATE BISHOP, SHE ASSAYED
EIGHT CLONES WITH RESISTANCE.
FIVE DELETED Fv1.
SO WHICH IS WHY ALL THE ASSAYS
WE DO NOW ARE BASED ON TRANSIENT
EXPRESSION.
AND IT'S CLEAR THAT
OVEREXPRESSING CAN INTERFERE
WITH A READOUT, COMPARED TO THE
NATURAL LEVELS.
IF YOU LOOK AT THE NATURAL
LEVELS, YOU CAN BARELY SEE IT.
>> (INAUDIBLE) THIS STUDY
SUGGESTS MOST OF THE RETROVIRUS
IS ENDOGENOUS -- (INAUDIBLE).
>> I DON'T KNOW.
I MEAN, IF YOU WANT -- I DON'T
THINK THERE WOULD BE ANY MARKET
TRYING TO ENCOURAGE PEOPLE TO
REINVESTIGATE PRECISELY WHERE
THE TRANSCRIPTION OCCURS IN MLV
BUT MAYBE IT WOULD BE
INTERESTING.
AGAIN, AN EXPERIMENT TO SEE.
>> SO DOES IT ONLY WORK ON -- -
(INAUDIBLE).
>> THE FOAMY VIRUS IS THE
INTERESTING ONE TO CONSIDER,
BECAUSE THE DETERMINED
SPECIFICITY FOR Fv1
SPECIFICITY WITH FOAMY IN THE
N-TERMINAL REGION, NOT IN THE
CAPSID.
WE KNOW THAT THIS FOAMY VIRUS IS
NOT CLEAVED.
AND WE KNOW THAT THIS THING --
OUTSIDE THE CAPSID, WHETHER IT'S
OBSCURED, INTERNAL REGIONS, OR
WHETHER THE N-TERMINAL REGION IS
ITSELF HELICAL, HEXAGONAL, I
DON'T KNOW.
IT WOULD BE VERY INTERESTING TO
LOOK AGAIN.
BUT PART OF THE PROBLEM IS THAT
WE CANNOT EXPRESS Fv1.
SO BINDING STUDIES ARE VERY
DIFFICULT.
NOW, WE HAVE -- EVERY FIVE YEARS
OR THREE YEARS, EVERY TIME WE
HAVE AN IDEA HOW WE MIGHT DO
THAT, WE'VE GOT ANOTHER ONE AT
THE MOMENT, BUT --
>> EXPRESSION MAY BE DIFFICULT
BUT ALPHAFOLD DOES SEEM TO BE TO
DO INTERESTING THINGS.
MULTIMERS, HAVE YOU CONSIDERED
--
>> WE'VE CONSIDERED IT.
PART OF THE PROBLEM, WELL, NOT
WITH ALPHAFOLD ITSELF BUT WITH
THIS MOLECULE IS IT HAS THIS
LONG AND SPIRAL CODE AT THE
N-TERMINAL, THERE'S ABOUT 150
AMINO ACIDS OF UNSTRUCTURED
MATERIAL, AND THEN YOU HAVE THIS
DOMAIN THAT I SHOWED AT THE END
HERE.
NOW, IF -- AND I THINK Fv1
MUST BE WORKING AT LEAST AS A
DIMER IF YOU DIMERIZE, THE WAY
YOU POSITION THE OTHER TWO, IT'S
DIFFICULT.
I SUSPECT -- NO, WE HAVE TO TRY,
TRY MORE.
MAYBE WE DO IN THEORY HAVE OUR
NEIGHBORS, PERHAPS I COULD
ENCOURAGE THEM TO TAKE ON THIS
AS A CHALLENGE, THAT THIS IS AN
INTERESTING MOLECULE.
BUT, NO, I AGREE.
WE HAVE ONLY JUST STARTED
THINKING ABOUT THIS.
WE HAVE THE ALPHAFOLD MODELS ARE
NOT -- ARE NOT NEW BUT TO TRY
AND FIT THIS AND PIN IT BY THE
AMINO ACIDS THAT WE HAVE FOR THE
VARIABLE REGIONS IS I THINK NEW,
TRYING TO DO THAT, WE JUST
STARTED.
>> I'M MORE CONFUSED.
I WAS TOLD MLV DOESN'T GET INTO
THE NUCLEUS AND WAIT FOR THE
NUCLEAR ENVELOPE TO BREAK DOWN.
WHAT DOES IT MEAN, NUCLEAR
ENTRY?
>> WELL, IT BOOKS A LATE STAGE
IN THAT PURPOSE WITH THE
REPLICATION CYCLE.
I'M SORRY.
TEXT BOOKS FOR THE 1990s.
I DON'T KNOW IF -- IF YOU
PREVENT THE CAPSID COMING APART.
>> RIGHT, YEAH.
>> WHICH IS COINCIDENT OR JUST
PRECEDES NUCLEAR ENTRY, PERHAPS
YOU HAVE A SIMILAR PHENOTYPE TO
BLOCKING NUCLEAR ENTRY.
>> RIGHT.
YOU'RE LOOKING AT TWO CIRCLES.
I THINK THE ANSWER IS EVEN IN
RAPIDLY DIVIDING CELLS WHERE THE
NUCLEAR MEMBRANE IS BREAKING
DOWN, Fv1 BLOCKS WHAT WOULD
NORMALLY HAPPEN SO YOU DON'T GET
CIRCLES, YOU GET FULL LENGTH
LINEAR.
>> FULL LENGTH LINEAR, IN THE
NUCLEUS, YOU KNOW, IN TERMS
OF -- IN THE NUCLEAR FRACTION.
HELLO BACK, EVERYONE.
IT'S A PLEASURE NOW TO INTRODUCE
ONE OF THE WONDERFUL POSTDOCS
WHO HAS COME THROUGH MY LAB,
THAT'S DELPHINE MURIAUX.
SHE CAME FROM FRANCE, AND SHE
WENT BACK TO FRANCE TWICE.
SHE HAD A PERMANENT JOB.
AND SHE WENT BACK TO TAKE THAT
PERMANENT JOB, BUT SHE CAME BACK
TO MY LAB, SUPPOSEDLY
TEMPORARILY, AND NOW SHE'S IN A
MUCH BETTER POSITION.
SHE NOW IS RUNNING A SHOP OF HER
OWN IN MONTPELLIER AND HAS DONE
A LOT OF WONDERFUL WORK ON HIV
ASSEMBLY, I THINK DOING A LOT OF
VIRAL PHYSICS AND I THINK WE'LL
HEAR A LITTLE ABOUT THAT TODAY.
DELPHINE, THANK YOU FOR COMING.
IT'S BEEN WONDERFUL KNOWING YOU
ALL THESE YEARS, AND LOOKING
FORWARD TO YOUR TALK.
>> THANK YOU VERY MUCH, ALAN.
HAVEN'T BEEN HERE FOR 20 YEARS,
AND THERE ARE MANY FRIENDS OF
MINE IN THIS ROOM, AND SO I
THANK A LOT THE ORGANIZER TO
INVITING ME TODAY.
I HOPE YOU ENJOY THE TALK.
ALAN, THANK YOU SO MUCH FOR ALL
THESE YEARS.
SO I JUST PUT TO SHOW YOU MY
STAY IN ALAN'S LAB WAS ALWAYS
SECURE, I CAME BACK TO FRANCE
BUT DIDN'T LIKE IT.
TOO MUCH TEST, COULDN'T DO WHAT
I WANTED.
I ASK IF I COULD COME BACK,
SURE.
I ROLLED BACK MY H1 VISA ON THE
SIDE.
I WEPT BACK TO FRANCE, TOOK THE
POSITION, I SAY KNOW, WENT BACK,
I TOOK BACK MY H1 VISA.
STAY FOR FOUR YEARS.
>> BE CAREFUL WHAT YOU SAY.
>> AND THEN, YEAH, WHEN I CAME
BACK TO FRANCE THE FIRST TIME I
HIDE IT.
THERE WAS THIS LITTLE GUY, I
DON'T KNOW WHO HE WAS, THAT SAY,
BUT I DON'T UNDERSTAND WHAT
HAPPENED IN 99.
I SAY, OKAY.
TWO CHOICE.
EITHER I LIE OR I PAY WITH
TRUTH.
IT WENT ONE, TWO, THREE.
WELL, IT'S TRUE, I WENT BACK TO
NIH BECAUSE I COULDN'T DO WHAT I
WANTED IN FRANCE.
BUT NOW I'M BACK.
IT'S UP TO YOU.
I GOT THE POSITION.
FOR ME WAS RESTRAINING IN GO
BACK IN 1999, WE DID A LOT.
I TRIED TO SUMMARIZE.
IN 1995 IN COLD SPRING HARBOR,
IN 1996 MY FORMER BOSS INVITED
TO MY Ph.D. DEFENSE, YOU WERE
MY REVIEWER, RIGHT?
DURING MY Ph.D. DEFENSE.
AND AT THIS TIME WE SHARED THE
TEST FOR DIMERIZATION AND
NUCLEOCAPSID.
>> THAT'S WHAT YOU SAY.
[LAUGHTER]
I DIDN'T HEAR A WORD OF THE
DEFENSE BECAUSE IT WAS --
>> IT WAS IN FRENCH.
>> THE WINDOWS WERE OPEN,
BECAUSE IT WAS HOT.
>> IT WAS HOT.
>> EVERYBODY WAS TALKING FRENCH
BESIDES.
>> YES.
BUT I DID ANSWER THE QUESTION IN
ENGLISH, NO?
NOT PERFECTLY.
[LAUGHTER]
THE SLIDES WERE IN ENGLISH.
>> OKAY.
[LAUGHTER]
>> WE SHARE THE SAME INTERESTS,
RIGHT?
THERE WAS A BIOCHEMIST, KNEW
NOTHING ABOUT VIROLOGY, I WAS
VERY HAPPY TO GET TO ALAN'S LAB.
HE REALLY TAUGHT ME VIROLOGY.
YOU'RE THE ONE WHO TAUGHT ME
VIROLOGY.
SO, I THINK WE TRIED ANOTHER
ROLE OF JUST GENOME PACKAGING.
AND THEN WE DID ALSO CONTINUED
TO WORK ON NUCLEOCAPSID MUTANT
AND FOUND THAT THERE WERE SOME
MUTANTS THAT WERE SEDATING RAG,
HIGHLIGHTING THE FACT
TRANSLATION AND PACKAGING WERE
LINKED TOGETHER.
AND WE TRIED TO UNDERSTAND THAT
BETTER.
AND, YEAH, WE CONTINUED TO WORK
ON NUCLEOCAPSID ASSEMBLY AND
FOUND THAT IF YOU REMOVED THE
DOMAIN ACTIVITY PROTEIN WAS
STILL BINDING TO MEMBRANE BUT
WOULD NOT FORM STRUCTURES THAT
WERE LOOKING LIKE VIRAL
PARTICLE.
THAT WAS PART OF THE WORK WE
DID, AND OTHERS ALSO.
THEN I WANT TO SAY ALSO
OVERTHINKING, HAVE TIME TO GO AT
NIGHT WHEN EVERYBODY WAS GONE,
TALK TO THE BOSS, I WOULD GO
INTO HIS OFFICE.
THE NICE OFFICE.
[LAUGHTER]
WE WERE ORGANIZED.
[LAUGHTER]
AND THEN MY COLLEAGUE.
IT WAS REALLY AMAZING.
THAT'S ME PAINTING.
REALLY NICE.
SO I TRIED TO LIST MAYBE I
FORGET A FEW THINGS BUT ALAN
TAUGHT ME EVERYTHING ON MULV
INFECTION, TRIED TO SHOW ME TO
MAKE -- CONVINCE ME ABOUT
RESTRICTION FACTOR BUT I'M VERY
SORRY, I STILL DON'T UNDERSTAND.
[LAUGHTER]
AND YEAH, I'M SORRY, I'M SORRY.
YOU KNOW, I TRIED TO GET AWAY.
IT WAS A CO-FACTOR.
AND THEN I LEARNED TO DO VIRUS,
BUT YOU'RE THE ONE DOING THAT.
AND WE DID A LOT OF -- WHEN I GO
BACK TO FRANCE, THAT'S WHY I DID
ACTIVITY.
I WENT NOT TO DO THAT AGAIN.
AND ALSO I WANT TO THANK NCI,
TRAINING MANY METHODOLOGY.
WONDERFUL.
AND I THANK YOU FOR SCIENTIFIC
EDUCATION, LATE IN THE EVENING,
BECAUSE IN THE MORNING, WE'RE
NOT MORNING PERSONS.
AND TO HAVE BETTER ENGLISH, I
REMEMBER COMING WITH MY PAPER,
VERY HAPPY, AND THEN CAME BACK
ALL RED.
SO BUT I TRIED.
BUT I'M NOT AS GOOD AS YOU.
YOU SHOULD WRITE A BOOK.
AND ALSO MANY, MANY WE HAVE OUR
MEETING, THIS IS REALLY PART OF
MY FAMILY, I ENJOYED SEEING
TODAY.
YOU TAUGHT ME THINKING, I THINK,
TRUST, SELF-EMPOWERMENT, DEAF
CONFIDENCE, TRUST.
THANK YOU FOR THAT.
AND ALSO WE HAD FRIENDSHIP,
RIGHT?
AND WE WERE CELEBRATING ALL
TOGETHER, VERY FUN, ALAN IS NOT
ALWAYS SERIOUS, AS YOU CAN SEE
HERE.
[LAUGHTER]
AND STILL ON TODAY, WHO IS THIS
GUY BEHIND HERE?
THIS WAS THIS YEAR, COLD SPRING
HARBOR, VERY HAPPY AND WE HAD
THIS BIG PICTURE WITH ERIC,
FORMER TRAINEES, FROM THE
PROGRAMS, CONTINUING.
THANK YOU FOR ALL OF THAT.
AND THEN I TRAVELED BACK TO
FRANCE.
STILL CONTINUING THE WORK ON THE
ASSEMBLY, NUCLEOCAPSID, AND THEN
I TEN YEARS AGO, 12 YEARS AGO IN
MONTPELLIER TO GET CLOSER TO THE
VIRUS THAT I LIKE, THEY HAD A
VERY GOOD MICROSCOPE CENTER.
I END UP, BUT WHEN I WENT BACK
TO THE LAB I CONTINUED ON MULV
ASSEMBLY, SO WE DID THAT WORK,
WHERE WE SHOWED ALSO GAG WAS
TARGETING, THE DIFFERENCE WAS
THE FACT THAT THE FIRST SETTING
WAS SELECTING THE GAG AND
BINDING TO THE MEMBRANE BY P2,
WHICH DOESN'T NEED THAT, SO WE
CONTINUED THAT WORK.
AND GET INTERESTED IN THE LIPID
MEMBRANE, BINDING OF GAG, AND
RELATIONSHIP.
WITH ALAN ON THE INTERPLAY
BETWEEN GAG AND SERINE SITE,
ALSO A PROTEIN, THAT -- SO THAK
YOU.
UNFORTUNATELY IN FRANCE COULDN'T
GET ANY FUNDING.
I HAD TO SWITCH TO HIV.
AND WHEN I MOVED TO MONTPELLIER,
WE END UP TOOLS AND DIFFERENT
SCALE OF INVESTIGATING INFECTED
CELLS, UP TO SINGLE VIRUSES BY
MICROSCOPY DEVICE, FLUORESCENT
DEVICE, AND WE END UP WORKING ON
THIS DIFFERENT VIRUSES OVER
TIME.
ESPECIALLY NUCLEAR RESCUE.
TODAY I WILL FOCUS ON HIV.
I END UP ALSO IN 2017 BEING HEAD
OF THE TEAM IN MONTPELLIER WITH
TRAINING AND IMAGING, I SPENT MY
TIME PUTTING ALL THIS
MICROSCOPY, NOW THERE'S NO MORE
ROOM ANYMORE.
TO STUDY MAINLY HIV REPLICATION
IN T CELLS.
SO IN THE LAB WE'RE MAINLY
INTERESTED IN T CELLS.
FLUORESCENT HIV.
TO STUDY THAT.
SO, WE ARE INTERESTED IN LATE
PHASE OF THE HIV REPLICATION, SO
AS I SAY, WORK VERY INSPIRING TO
ME.
YOU CAN SEE DIRECTLY I'M ON THE
LINE, AND WE'RE INTERESTED IN
WHAT P2 CLUSTERING BY GAG WOULD
TRIGGER IN A T CELL.
WHAT THE ACTING UNDERNEATH
REGULATION CHANGE, AND HOW THIS
WOULD INTERFERE WITH THE NORMAL
BRAIN, THE FORMATION OR DOING
HIV PREDICTION.
SO MUCH INSPIRED BY THE WORK IN
ERIC FREED'S LAB, TOGETHER
POSTDOC AT THE SAME TIME, THIS
WAS THE PAPER THAT INSPIRE US.
BUT AT THAT PERIOD OF TIME,
CORRECT ME, ERIC, WE DIDN'T KNOW
REALLY GAG WAS BINDING TO
PREEXISTING P2 HUB DOMAINS IN
THE MEMBRANE, OR IF GAG WAS
GENERATING THAT.
SO, THAT'S WHAT -- YEAH,
INSPIRING ME A LOT, LIPIDOMIC
EXTRACTION, COMPARING
COMPOSITION OF RETROVIRAL
PATHOGENS, AS GOING BACK TO
PLASMA MEMBRANE, INITIAL VERY
TYPICALLY THAT PSP 2 ENRICHED IN
THE MEMBRANE.
SO HOW COME?
SO, WHAT WE DID, WE SET UP
TOOLS, BASICALLY SOME TOOLS, AND
WITH THE GAG PROTEIN, TRIED TO
MEASURE LIPID DOMAIN BY DYNAMICS
AND SEE IF GAG WAS A TRIGGER OF
THIS PHENOMENON.
SO FOR EXAMPLE HERE FLAT
MEMBRANE, SCIENTIFIC COMPOSITION
UP TO PLASMA MEMBRANE, MANY
COMPOSITION, WE HAVE THE GAG
PROTEIN, THAT WE LABEL IN GREEN.
REPEAT P2 LABELED IN RED.
YOU CAN SEE OVER TIME, YOU CAN
SEE THE P2 IS CLUSTERING OVER
TIME.
AND YOU HAVE PERFECT
CO-LOCALIZATION BETWEEN THIS
CLUSTER, SAYING THAT GAG WAS THE
TRIGGER OF THE CLUSTERING AND
SETTING MAYBE NOT AS FAR AS WE
CAN LOOK PREEXISTING DOMAIN.
SO GAG WAS GENERATING THIS
DOMAIN BUT WE HAD TO PROVE THAT
IN T CELLS, SO THAT'S HOW WE
ASSOCIATED WITH JACOB AND
CHRISTIAN, AND WE SHOWED THAT IF
WE TRANSFECT A T CELL WITH HIV
GAG PROTEIN ONLY OR IF WE INFECT
THE CELL WITH HIV CLONE WE CAN
SEE EACH DO THE AT RESOLUTION,
AND THEN WE LABELED THE T CELL
WITH DIFFERENT LIPID, LIKE P2,
FLUORESCENT, APPLIED COUPLING TO
MICROSCOPY AND MEASURE DYNAMIC
OF THE REPEAT OUTSIDE THE
BURDEN, INSIDE THE BURDEN.
FROM THAT WE CAN EXTRACT
CONFINEMENT INDEX OF LIPID.
WHAT YOU CAN SEE EVEN IN THE
NICE T CELL WE COULD SEE P2 WAS
HIGHLY TRAPPED BY THE GAG
PROTEIN, EITHER INFECTED CLONE
OR IN GAG EXPRESSION T CELL.
CHOLESTEROL ALSO.
CHOLESTEROL WERE MORE TRAPPED
WHEN YOU HAVE ENVELOPE.
DIDN'T FIND PE TRAPPING FOR P2
AND CHOLESTEROL, WHICH WAS
MATCHING.
WE SAY THAT P2 AND CHOLESTEROL
ACTUALLY ARE THE LIPID TRAPPED
BY INFECTED T CELL.
BUT THAT DOESN'T MEAN -- THE
PROTEIN BY ITSELF IS CONSTANTLY
CHANGING GRADIENT OF THE
MEMBRANE, THIS HAS STRONG
REPERCUSSION, P2 IS A MAIN
SIGNALING LIPID IN T CELL.
IT CAN INDUCE DIVERSITY.
NOW I SPEND MY TIME TO TRY TO
IDENTIFY OR UNDERSTANDING WHICH
FACTOR ARE INVOLVED IN THIS
FORMATION.
AND HERE, BEFORE WE WERE PLUG
DOWN DIFFERENT PATHWAY OF
SIGNALING LIKE THE ONE, AND WE
SHOW THIS PATHWAY WAS REQUIRED
IN T CELL.
YOU HAVE ONE, IRSP53, WE SHOW
THAT PAPER ACTIVATING WHEN GAG
WAS EXPRESSED.
SO THIS IS PARTICULAR, PROTEIN
IS ABLE TO GENERATE OUTWARD, AND
THE SIZE OF THIS PROTRUSION,
DIAMETER EQUIVALENT TO DIAMETER
OF A VIRUS.
SO, WE DECIDED TO STUDY IRSP 53
AND IRTKS, IN THE HIV CONTEXT.
THERE'S A POINTER.
OKAY.
YOU WILL BE ABLE TO SEE VIRUS
WAS REDUCED, AND HAD NOTHING, A
CLOSE PROTEIN AND COULD FIND BY
ELECTRON MICROSCOPY THANKS TO
PHILIP THAT THE BED WAS NOT
COMPLETE.
IT WAS LIKE THIS, THE CELL WAS
NOT DYING, SO WE THOUGHT THAT
IRSP 53 HAD A ROLE IN ASSEMBLY,
TRIED TO SEE IF IT WAS WITH GAG,
AS YOU CAN SEE HERE IT WAS.
THEN WE DID DIFFERENT MUTANT OF
GAG AND TRIED TO IDENTIFY THE
DOMAIN REQUIRED FOR THIS
INTERACTION.
ACTUALLY I HAVE TO SAY SIGNAL
CLEAR, BUT WHAT WE CAN SEE IS
THAT IF WE HAVE THE HVR
RELATION, NOT BINDING TO THE
MEMBRANE, AND THE MUTATION, THE
INTERACTION.
IN OUR OPINION THIS INTERACTION
BETWEEN GAG AND IRSP 53 OCCUR AT
THE CELL MEMBRANE, AND WE REDUCE
A LITTLE BIT THIS INTERACTION SO
EITHER WAY, EITHER WE PULL THE
HYPOTHESIS IT OCCURS BEFORE BUT
HAVE NOT PROVEN THAT SO WE
CONTINUE TO WORK ON THAT.
THEN IF IRSP 53 WAS REQUIRED TO
THE SITE, WE USED MICROSCOPY, SO
THEN WE EXPRESSED THE GAG
PROTEIN THAT IS ABLE TO BRING,
AND YOU HAVE ONLY THE PLASMA
MEMBRANE UNDERNEATH THAT YOU CAN
SEE HERE.
FROM BLINKING, RESOLUTION
20-NANOMETER, PLATFORM IN GREEN.
WE CAN COME WITH THE ANTIBODY
LABELING IRSP 53 AND COMPARE
SIGNALING DIFFERENCE TO THE
BUDDING SITE.
THANKS TO CYRIL, AND HIS
WORKFLOW.
SO WE COULD DATA MINE THE CENTER
AND DISTANCE OF 300-NANOMETER,
FOR IRSP 53, INDEED IT IS
CLOSELY RELATED TO GAG ASSEMBLY
SIDE, SO YOU CAN SEE HERE, AND
ACTUALLY IT SEEMS TO SURROUNDING
THE ASSEMBLY BUDDING SITE, HAVE
THIS PATTERN.
SOME DON'T HAVE IT.
IT'S COMPLICATED SO WE BELIEVE
THAT THE TOP, THERE'S A TOP
RECRUITMENT OF IRSP 53 TO THE
BUDDING SITE AND DON'T KNOW THE
CORRELATION WITH THE PROTEIN.
WANTED IT FLOW IF IRSP 53 HAS
ANY ROLE IN GAG TO MEMBRANE
BINDING.
WE SET UP IN VITRO MODEL, WE HAD
LABEL WITH FLUEROSOL, GOOD
CODING IN P2, IRSP 53-I-BAR
DOMAIN, TO SEE MEMBRANE BINDING.
SO HERE OUR RESULT.
WE SHOW WHEN WE HAVE GAG, EVEN
IF CONTAIN P2, THERE'S NO MATCH
GAG BINDING.
IF YOU ADD THE I-BAR DOMAIN, WE
BELIEVE THE TWO PROTEINS WITH
INTERCONNECTED.
AND WE DID A MOVIE ON THAT.
SO HERE WE CAN SEE THE MEMBRANE.
IRSP53 I-BAR, AND IF YOU PUT BAG
ON IT, YOU CAN SEE THAT GAG
PREFERS TO BIND WHERE YOU HAVE
THE IRSP53 IN PINK.
GAG PROTEIN LOCATE EXACTLY AT
THE TIP.
SO TWO PROTEIN ARE CERTAINLY
LINKED TOGETHER, AND WE TRIED TO
CONTINUE TO UNDERSTAND THIS
MOLECULAR PATHWAY.
THEN WE WANTED TO KNOW IF IT
COULD BE INCORPORATED IN GAG,
AND WE DID THAT WITH IRSP 53,
GFP, OR DOMAIN, OR PH-GFP.
YOU CAN SEE 53 IS THE MOST
INCORPORATED PROTEIN, AND COULD
FIND SOME IN THE HIV VIRUS BUT
VERY FEW IN HIV VIRUS SO MAYBE
THERE'S ANOTHER REGULATION HERE
THAT ALLOWS ITS JOB AT ONE POINT
BUT THERE IS A REGULATION WE
DON'T FULLY UNDERSTAND TODAY.
WE FINISH WITH THE WORK OF A
TALENTED PERSON, RAYANE DIBSY,
NOW IN POSTDOC TRAINING AT
PASTEUR INSTITUTE.
IF YOU IMAGINE THIS PATHWAY IN
THE CELL, VERY USEFUL, IF GAG
INTERACT WITH P53 YOU WILL
INTERFERE WITH THIS PATHWAY,
THERE'S TWO POSSIBILITIES.
WE DON'T KNOW EXACTLY THE
CHOLINERGIC PATHWAY ACTIVATION,
BUT WE TRY MIMIC IF WE BLOCK THE
PATHWAY AT THE END OF THIS
SIGNALING, WHAT HAPPEN?
SO FOR THAT WE USE CK-666, THAT
BLOCK UP TO THREE COMPLEX.
WHEN DO YOU THAT, IT IS LESS
THAN.
WHEN YOU DO THAT IN T CELL WE
SEE THAT AT CERTAIN POINT, WE
HAVE A STRONG INCREASE IN
RELEASE, SAYING IF YOU HAVE LESS
DENSE ACTING, AND ANOTHER DRUG
WOULD NOT HAVE THIS EFFECT,
TRIED TO CHARACTERIZE THAT.
SO WE SHOW THAT JURKAT T CELL,
PRIMARY T CELL, EACH TIME HAVE
INCREASE.
WE HAVE INCREASING RELEASE.
AND THEN TRIED TO SEE IF IT WAS
INCREASE IN THE PLATFORM, SO WE
APPLIED THE SAME STORM/TIRF
MICROSCOPY DEVICE.
FIRST YOU CAN SEE T CELL HAVE
LESS DENSE ACTIN.
YOU CAN SEE THE CLUSTER
INDIVIDUAL PLATFORM WE CAN SEE
THAT WE HAVE INCREASE IN NUMBER
OF PLATFORM.
AND THEN IF WE RELATE TO THE
MESH WORK OF ACTIN WE HAVE A
NUMBER OF CLUSTER FROM ASSEMBLY
INCREASE FOR NUMBER OF ERROR.
GAG CAN ACCESS BETTER P2, BETTER
MEMBRANE, YOU CAN HAVE BETTER
ASSEMBLY.
WE TRY TO MIMIC THAT IN VITRO.
SO THAT WE TOOK OUR BIOLIMITED
MEMBRANE WITH M1 AND PUT
DIFFERENT ACTING MESHWORK
DENSITY OF SURFACE OF MEMBRANE
AND LOOK AT P2 DEFICIENT.
WHAT WE OBSERVE P2 DEFICIENT WAS
DECREASING, AND WHAT HAPPENS
AGAIN.
IF WE DECREASE THE DYNAMIC IN
THE MEMBRANE GAG DIFFUSION DROPS
SO IF YOU HAVE LESS ACTIN YOU
HAVE MORE ACCESS TO P2
DIFFUSION, NUCLEATION OF GAG TO
MAKE A PARTICULAR ASSEMBLY WOULD
REQUIRE THAT.
SO THEN WE WANTED TO KNOW IF ANY
CELLULAR INHIBITOR OF ARP2/3
WOULD BE INVOLVED, WE TRIED SOME
AND COULD SEE HERE THAT WE HAD
GAG WITH SOME, ONE WAS ARPIN,
AND WE CAN SEE THIS COMPUTATION
WAS NOT EXISTING IF WE TAKE A
MINUS GAG NOT BINDING MEMBRANE
SO WE BELIEVE ARPIN WOULD BE
RECRUITED BY THE GAG PROTEIN OR
TRAPPED BY THE GAG PROTEIN, IN
ORDER TO HAVE LESS BRANCH
ACTIVITY.
SO WE CONFUSE THIS
IMMUNOPRECIPITATION BETWEEN GAG
AND ARPIN.
WHEN WE LOWER EXPRESSION, WE
LOWER HIV PARTICULAR, AND
FINALLY WE COULD SEE THAT WHEN
GAG IS EXPRESSED IN T CELL WE
INCREASE ARPIN IN THE MEMBRANE
FACTION, AND WE INCREASE ARPIN
AT THE T CELL SURFACE, AS YOU
CAN SEE HERE BY MICROSCOPY.
WE THINK THAT GAG IS ABOUT TO
ACTIVATE SOME OF THE ACTIN
PATHWAYS TO INHIBIT THE MESHWORK
TO HAVE MORE PARTICULAR
ASSEMBLY.
SO NOW WE WANT TO KNOW WHAT ARE
THE RECRUITMENT INVOLVING
POLYMERIZATION, THE
SPATIOCOMPONENTS, AND HOPEFULLY
NEXT YEAR.
HERE IS CONCLUSION, CONVINCE YOU
MANY OF US IN THE ROOM THAT WE
SHARE THE INTERESTS IN
UNDERSTANDING THE LIPID AND ML
RELATIONSHIP TO MAKE PARTICLES
OF THE CELL MEMBRANE IN
DIFFERENT CELL TYPE, DIFFERENT
SYSTEM, DIFFERENT GAG.
I WANT TO THANK MY TEAM BECAUSE
WITHOUT THEM NOTHING IS
POSSIBLE, BECAUSE I WISH I COULD
STILL DO EXPERIMENTS.
BUT ACTUALLY THE LAST EXPERIMENT
WAS THE MICROSCOPY, WE HAVE MORE
STUDENTS AT THAT TIME, SO WE DID
ALL THE EXPERIMENTS.
BUT TEN YEARS AGO NOW.
EIGHT YEARS AGO.
I WANT TO THANK THE SPONSORS AND
PLEASE COME AND VISIT
MONTPELLIER, A CITY BY THE SEA.
I WANT TO FINISH TO THANK ALAN
FOR ALL HIS YEARS OF EXCELLENT
SCIENCE, FOR FRIENDSHIP, AND
NON-SCIENTIFIC DISCUSSION ON
MUSIC, ART, AND THIS WAS A
SLIDE, ALSO I REMIND YOU OF
THIS.
YES, MARK.
>> ALL THIS WAY FROM
MONTPELLIER.
YOU MAY GO BACK TO (INAUDIBLE).
>> I KNOW YOU TRY TO CONVINCE
ME.
>> ALL THE GAMMA RETROVIRUSES IN
THE HUMAN GENOME INCLUDING GAG
PROTEIN HAVE NOW HUGE TREND ON
HOW GAG IS INVOLVED IN THE
FORMATION OF VESICLES.
>> YEAH.
>> THAT'S WONDERFUL.
>> THE OTHER ONE, SO AS
CHRISTINE -- (INAUDIBLE) ALL
THOSE RECEPTORS USED BY
ENVELOPES, LET'S SAY
TRANSPORTERS, REGULATE PATHWAYS,
I THINK THERE'S A WAY TO
ASSEMBLE IN THE HUMAN CELL ON
THE ONE HAND THE EQUIVALENT OF
ENVELOPES THAT WOULD BIND
TRANSPORTERS AND GAG, THE GAG
FORMATION.
>> YES, THAT'S ONE SIDE.
THE OTHER SIDE I WOULD SAY,
WE'RE SHOWING CERTAIN
POPULATIONS OF EXTRACELLULAR
VESICLES, AND THERE'S A
BEAUTIFUL WORK, THEY ARE
INTERACTING.
TO SEND DISTANT SIGNAL FOR
MEMORY, SO, THIS IS, YOU KNOW, A
VERY OPEN PERIOD.
I LIKE THE IDEA OF RETROVIRUSES
USING THAT PATHWAY TO GET OUT
AND TO SEND A SIGNAL,
TRANSPORTATION, TO LONG DISTANCE
IN FORMATION.
>> NEXT SPEAKER IS RIGHT NOW IN
URUGUAY, I BELIEVE.
HE WAS SUPPOSED TO BE HERE BUT
UNITED AIRLINES DIDN'T
COOPERATE.
NO, SORRY, SORRY.
NEXT SPEAKER IS HERE.
IT'S KARIN MUSIER-FORSYTH, A
NAME FAMILIAR TO MOST OF US I'M
SURE.
I WILL JUST SAY THAT KARIN WAS
TRAINED IN tRNA BIOCHEMISTRY
AND INTERACTION OF tRNAs
WITH CELLULAR PROTEINS.
AND I FIRST BECAME AWARE WITH
HER FIRST RETROVIRUS PAPER, I
THINK IT WAS THE FIRST
RETROVIRUS PAPER, AND IT STRUCK
ME FROM THE FIRST PAPER HERE IS
SOMEONE WHO IS REALLY INTERESTED
IN HOW THE VIRAL PROTEINS ARE
MANIPULATING THE RNAs,
INTERACTING WITH RNAs, IT WAS
A FASCINATING PAPER, AND SHE'S
CONTINUED TO DO FASCINATING
WORK, EVER SINCE.
WE'VE REALLY KNOWN EACH OTHER
QUITE A WHILE NOW, HAD A NUMBER
OF PAPERS TOGETHER.
AND TODAY SHE'S GOING TO TELL US
ABOUT WORK, REALLY AMAZING WORK.
WITHOUT FURTHER ADO, THANK YOU
FOR COMING, KARIN, AND THANK YOU
FOR BEING PART OF THIS EVENT.
>> IT'S AN HONOR AND PLEASURE TO
BE HERE TO CELEBRATE ALAN'S LONG
CAREER.
WE DID MEET IN 1997, MY FIRST
COLD SPRING HARBOR MEETING.
AND BASICALLY WE'VE BEEN TALKING
EVER SINCE.
AT THAT MEETING I ALSO MET
WEI-SHAU, STEVE AND EVERYBODY
HERE, A LOT OF PEOPLE HERE.
TWO YEARS LATER HAD A
SABBATICAL.
I DECIDED TO DO IT HERE AND BE
IMMERSED IN RETROVIROLOGY.
THAT'S WHAT I DID.
AND SO I THINK IT WAS THE FALL
OF 1999, I SPENT FIVE MONTHS
HERE, INTERACTED, MANY MEALS
WITH ALAN, LOTS OF CONVERSATIONS
WITH ALAN AND CONTINUED TO THIS
DAY.
I'M HAPPY TO TELL YOU ABOUT
COLLABORATION WITH WEI-SHU AND
HER TEAM, MY TEAM IS HERE ON THE
LEFT.
MIKE IS ASSISTANT PROFESSOR, AND
JONATHAN WAS A TRAINEE, Ph.D.
WITH ME, NOW POSTDOC WITH
WEI-SHAU.
HOW DO I ADVANCE SLIDES?
OKAY.
WE ALL KNOW ALL THE CURRENT HIV
THERAPEUTICS ACT MAINLY ON
ENZYMES, WE'RE INTERESTED IN
EXPANDING THE RANGE OF DRUGGABLE
TARGETS TO RNA.
IT'S A TEMPLATE FOR TRANSLATION,
GAG AND GAGPOL, AND GENOME, SO
PRESUMABLY IDENTICAL MOLECULE IS
PACKAGED AS A DIMER.
WHAT'S CLEAR IN RECENT YEARS IS
THAT THIS DIFFERENCE IN THE
STATE OF THE FULL LENGTH
INSPLICED RNA IS DETERMINED AT
THE LEVEL OF TRANSCRIPTION.
AND SO IF YOU LOOK AT WHAT HOST
RNA POLYMERASE TRANSCRIBED
YOU'LL SEE THERE'S A MIXTURE OF
SPECIES, THE VIRUS ENCODES 3G,
YOU GET 3Gs IN A CAP, TWO,
ONE, A BUNCH OF MISCELLANEOUS
OTHER THINGS.
A NOBODY OF LABS HAVE SHOWN IF
YOU LOOK IN THE CELL THAT MOST
OF THE RNA IS 3G AS YOU WOULD
PREDICT BUT THERE'S SIGNIFICANT
AMOUNT OF 2G, 1G, OTHER SPECIES
AS WELL.
HOWEVER, IF YOU LOOK IN THE
VIRUS PARTICLE, WHAT YOU SEE IS
IT'S NOW ALMOST -- PREDOMINANTLY
1G WITH A LITTLE BIT OF 3G,
SELECTED MECHANISM OF PACKAGING
GOING ON HERE.
THAT'S THE BASIS OF THE PROJECT.
HOW ARE THE 1 Gs PACKAGES AND
ARE 3Gs PREFERENTIALLY
TRANSLATED.
I NEED TO INTRODUCE OR REMIND
YOU OF WHAT THE IMPORTANT RNA
STRUCTURAL ELEMENTS IN THE FIVE
PRIME INTRANSLATED REGION.
WE SAW THESE STRUCTURES, HERE IS
THE TAR/POLY-A, TAR LOOP, POLY-A
LOOP, PRIMER BINDING SITE
DOMAIN.
AND THEN THIS IS THE SITE
PACKAGING SIGNAL WITH THE
PALINDROMIC LOOP, IMPORTANT FOR
INITIATING DIMERIZATION, AND A
NUMBER OF SINGLE-STRANDED GENES
ARE IMPORTANT FOR BINDING.
WHAT I'LL FOCUS ON TODAY IS
WHERE YOU HAVE THE VARIABLE
NUMBER, HOW THEY DETERMINE THE
STATE OF THIS GENOMIC RNA.
AND AS MANY OF YOU KNOW, MANY
FIVE PRIME UNTRANSLATED REGIONS
OF HUMAN GENES, ALL KINDS OF
GENES, ARE REGULATED BY
SO-CALLED RIBAL STRICTURES, THIS
EXPRESSION WOULD OCCUR.
HOWEVER, IF A LIGAND BINDS THAT
CAN CHANGE THE CONFIRMATIONAL
STATE, TURNING THE GENE OFF.
SO WE KNEW ABOUT THIS PARADIGM
AND WHAT WE WERE WONDERING IS,
WELL, MAYBE IT'S NOT SMALL
MOLECULE BINDING BUT MAYBE IT'S
THE NUMBER OF GENES REGULATING
THE CONFIRMATION OF THIS RNA.
AND SO THAT IF YOU HAVE 3 Gs,
THIS IS A CONFIRMATION OF
PREFERENTIAL TRANSLATION, IF YOU
HAVE 1G, FORMS A CONFIRMATION
THAT'S PREFERENTIAL TO THE
PACKAGING.
SO TO LOOK AT THIS, THIS WAS
PUBLISHED A NUMBER OF YEARS AGO,
I'M GOING TO SUMMARIZE.
WE PERFORMED RNA STRUCTURE
PRONING TECHNIQUE CALLED SHAPE,
AND WE RAN -- FIRST OF ALL I CAN
SHOW WHEN YOU TAKE THE 1G RNA
AND RUN IT ON A NATIVE GEL, WHAT
YOU SEE RIGHT AWAY IS THAT IT'S
MOSTLY DIMERIC SPECIES WITH A
LITTLE BIT OF MONOMER, BUT IF
YOU TAKE THE EXACT SAME 3G RNA,
FOLD IT UNDER THE SAME
CONDITIONS, WE SEE MAINLY
MONOMERIC SPECIES.
AND SO WE CUT OUT THESE BANDS
THROUGH GEL AND ASSOCIATED IN
CHEMICAL REAGENTS THAT PROBE RNA
STRUCTURE AND WHAT WE FOUND WAS
THAT THE 1G RNA, PRIMARILY
DIMERIC, HAS THIS SECONDARY
STRUCTURE FAIRLY SIMILAR TO WHAT
I SHOWED EARLIER.
YOU CAN SEE THE TAR CONFORMS,
POLY-A CONFORMS, YOU GET INTO
THE PRIMER BINDING SITE.
I'M CIRCLING THESE, CRITICAL FOR
GAG BINDING, PACKAGING RNA,
EXPOSED AND AVAILABLE IN STINGEL
STRANDED REGIONS.
IF YOU TAKE THE 3G RNA, WE'VE
ADDED TWO MORE Gs.
WHAT YOU'LL SEE HAPPEN IN THE
NEXT SLIDE THAT NEXT G IS PAIRED
WITH C, AND WHAT YOU GET, WHAT
WAS SURPRISING, COMPLETE
REARRANGEMENT OF THE RNA,
STEALING NUCLEOTIDE FROM POLY-A,
RESULTING IN UNFOLDINGS, DOWN
EXTREME INTERACTION, FURTHER
DOWNSTREAM EFFECTS, NOW Gs
WHICH WERE CLOSE TOGETHER AND
SINGLE-STRANDED HERE ARE NOW
FURTHER APART AND PARTIALLY
SEQUESTERED.
AND DIMERIZATION INITIATION SITE
IS NO LONGER IN A PALINDROMIC
LOOP.
THIS HELPED TO EXPLAIN WE THINK
WHY THIS IS FOR PACKAGING, HAS
EXPOSED DIS, HAS THE G BINDING
SITES FOR PACKAGING EXPOSED, AND
WHAT WE DIDN'T HAVE PREDICTED
INTACT POLY-A HAIRPIN.
THOSE ELEMENTS ARE MISSING IN
THE 3G RNA NOT PREFERENTIALLY
PACKAGED.
AGAIN, WE IDENTIFIED TWO SETS OF
MUTANTS THAT ELIMINATED SELECTED
1G RNA PACKAGING.
WE DESTABILIZED BOND OF POLY-A.
USE ARTIFICIALLY DESTABILIZED,
YOU SEE IN CONTRAST TO WILD TYPE
WITH THIS DRAMATIC DIFFERENCE IN
STRUCTURE OF THE 3G AND 1G AND
PREFERENTIAL PACKAGING.
1G RNA NOW YOU ELIMINATE THAT
PREFERENTIAL PACKAGING AND WHEN
WE PROBE THE STRUCTURE FOR
EXAMPLE ON THE NATIVE GEL NO
MATTER IF YOU HAVE 3G OR 1G,
THEY ALL LOOK 3G LIKE.
WE'VE ELIMINATED THAT STRUCTURAL
DIFFERENCE AND ELIMINATED
SELECTIVE PACKAGING.
ANOTHER SET HAD TWO NUCLEOSIDES
INSERTED, THIS GENE TO BASE PAIR
WITH THIS, ANY TWO NUCLEOSIDES
ELIMINATED SELECTED 1G PACKAGING
IN CELLS AND ALSO ELIMINATED
STRUCTURAL DIFFERENCES BETWEEN
RNAs.
NOW THEY ALL LOOK LIKE THEY ARE
1G AND THERE'S NO PREFERENTIAL
PACKAGING.
WE FOUND THAT SELECTED 1G
PACKAGING IS THE CONFIRMATIONAL
ENSEMBLES THAT HAVE DIFFERENCES,
IN PARTICULAR DIFFERENCES IN
POLY-A HAIRPIN STABILITY.
YOU HAVE DIS EXPOSURE, EXPOSED
GAG BINDING SITES, AND
PACKAGING, BUT IF THE POLY-I
HAIRPIN IS DISRUPTED
ARTIFICIALLY OR THROUGH
INTERACTION THE DIS IS
SEQUESTERED, DNA BINDING SITES
ARE SEQUESTERED.
GAG SHOULD BIND AND ASSOCIATE
WITH THE 1G RNA WHICH HAS
EXPOSED GAG BINDING SITE.
WE'VE TRIED HARD TO LOOK AT
DIFFERENCES IN AFFINITY, WE
DON'T SEE ANY DIFFERENCES..
GANG IS PROMISCUOUS BINDING
PROTEIN, HAS PREFERENCES TO
SINGLE-STRANDED GENE BUT IF WE
LOOK AT BINDING AFFINITY WE
DON'T SEE DIFFERENCES.
WHAT WE DECIDED TO DO, WE
PREDICT THAT GAG WOULD ASSEMBLE
MORE READILY ONTO THIS RNA AND
SO WE USED MASS PHOTOMETRY, A
SOLUTION-BASED MASS MEASUREMENT
TECHNIQUE, YOU HAVE YOUR
MOLECULES IN SOLUTION, SHINE
INCIDENT LIGHT ON THE MOLECULE.
THEY WILL SCATTER THE LIGHT AS
THEY HIT THE GLASS SURFACE SO
YOU CAN MEASURE THAT SCATTERING.
THIS IS ALL CALIBRATED SUCH THAT
THE SCATTERED LIGHT IS BASICALLY
IN RELATION TO THE MOLECULE AND
CALIBRATED WITH PROTEIN RNA
STANDARDS.
YOU TAKE MOVIES, BUILD UP
HISTOGRAMS, CAN SEE DIFFERENT
SIZES OF COMPLEXES FORMING.
WE USED OUR 1G AND 3G RNAs,
WHICH WE MIMICKED BY PUTTING 2G
AND 4G, I FORGOT TO SAY THAT.
AND THAT'S WHY THIS IS LABELED
2G, 4G, OVERLATE MASS
SPECTROMETRY DATA HERE, THE
ORANGE -- HERE WE HAVE THE COUNT
VERSUS MASS, EACH PEAK IS A
POPULATION OF MOLECULES WITH
THIS MASS.
WHAT YOU CAN SEE IS THAT IN A
CASE OF THE 4G, WE SEE THE RNA
MONOMER, RNA DIMER, AND WE ADD
GAG, WE ONLY SEE A LITTLE BIT OF
COMPLEX FORMATION.
BUT UNDER THE EXACT SAME
CONDITIONS WHEN WE LOOK AT GAG
INTERACTING WITH THE 2G RNA, WE
AGAIN SEE THE MONOMERIC AND
DIMERIC RNA BUT SEE BUILDUP OF
GAG ON RNA WHERE WE CAN MEASURE
1G, 2G, 3G, 4G, 5G, 6G AND
HIGHER ORDERS OF COMPLEXES.
THIS SHOWS GAG ASSEMBLES MORE
FAVORABLY ON 1G.
THE OTHER PREDICTION FROM THIS
EXPERIMENT IS THAT YOU -- WE SEE
DIFFERENCES IN POLY-A HAIRPIN
STABILITY APPEAR TO IMPACT
PACKAGING.
WE WANTED TO MEASURE POLY-A
HAIRPIN STABILITY.
WE USED THERMAL MELTING
EXPERIMENTS, WHERE WE JUST
LOOKED, IF YOU'RE FAMILIAR WITH
THESE, YOU CAN LOOK AT THE
ABSORBANCE AS A FUNCTION OF
TEMPERATURE AND GET THESE
CURVES, MID-POINT IS MELTING
TEMPERATURE, YOU CAN TAKE THE
FIRST DERIVATIVE OF THIS AND IN
THIS CASE THE PEAK IS THE
MELTING TEMPERATURE.
I'LL BE SHOWING YOU SHE'S TYPES
OF CURVES.
AND THIS IS DONE BY MICHAEL IN
MY LAB, UNDERGRADUATE.
WHAT HE LOOKED AT WAS THEY ARE
-- THERMAL STABILITY OF TAR.
IF YOU USE THE PROGRAM LIKE RNA
STRUCTURE TO PREDICT THE
DIFFERENCES IN STABILITY
PRE-DISTRICT THE -- PREDICT THE
ENERGY OF THE 4G IS MORE STABLE.
WE TAKE THE FIRST DERIVATIVE,
SLIGHT INCREASE IN STABILITY FOR
THE 4G WHICH HAS THAT GC PAIR.
WANTED TO LOOK AT TAR POLY-A,
HERE IS TAR.
POLY-A HAIRPIN, 2G AND 4G
VERSIONS.
IF YOU USE RNA STRUCTURES IT
DEPREDICTS IF THESE ARE EXACTLY
THE SAME IN STABILITY.
IF YOU DO THE MEASUREMENT,
THAT'S NOT ALL YOU SEE.
WE SEE A MUCH MORE STABLE
OVERALL PROFILE FOR 2G, AND IF
YOU TAKE THE DERIVATIVE YOU CAN
TEASE OUT DIFFERENT MELTING
TEMPERATURES.
WE SEE TWO MELTING TEMPERATURES,
ONE FOR TAR, ONE FOR POLY-A.
AGAIN THE TAR IS MORE STABLE FOR
THE 4G RNA.
BUT IN CONTRAST TO THE POLY-A
HAIRPIN IS MUCH LESS STABLE FOR
THE 4G RNA.
THIS IS ABOUT A 90-DEGREE
DIFFERENCE IN MELTING
TEMPERATURE.
IF YOU LOOK AT THE CONTEXT OF
THE CELL, THE GENOME, THIS IS A
SITUATION WHERE WE SEE SELECTIVE
PACKAGING.
AGAIN IN COLLABORATION WITH
WEI-SHU AND JONATHAN WE MADE
DIFFERENT TRANSCRIPTION START
SITES, MEASURED HAIR SPIN
STABILITY.
WHEN WE CHANGED TO A, COMPARED
TO NOT NOW 3G BUT GDA, WE SEE
SOME DIFFERENCES IN MELTING BUT
LOOK AT THE FIRST DERIVATIVE,
NOW THERE'S STILL A LITTLE
DIFFERENCE IN TAR BUT NOW THE
POLY-A HAIRPIN STABILITY IS
IDENTICAL, NO LONGER HAS THAT
DIFFERENCE.
AND WHEN JONATHAN LOOKED AT
PACKAGING CELLS, WHAT HE SAW IS
NO PREFERENTIAL PACKAGING OF ANY
OF THESE RNAs, ESPECIALLY NOT
OF THE 1A OR 2A.
AND SO BUT WE HAVE IDENTIFIED
ALSO A COMPENSATORY MUTATION,
BASICALLY IF YOU HAVE THE SAME
VARIANTS THAT NOW YOU INCLUDE AN
ADDITIONAL C57 MUTATION, NOW
WHEN YOU MEASURE MELTING
TEMPERATURE OF THE TAR POLY A
YOU SEE WE'VE RESTORED
DIFFERENCES IN POLY-A HAIRPIN
STABILITY AND JONATHAN MEASURES
STABILITY.
AGAIN, THIS IS CONSISTENT WITH
THE DIFFERENCES IN POLY-A
HAIRPIN STABILITY.
WHAT ABOUT TRANSLATION?
WE UNDERSTAND PACKAGING WELL,
WHAT ABOUT TRANSLATION?
WE PERFORMED IN VITRO AND
CELL-BASED STUDIES TO LOOK AT
THIS.
FOR THE IN VITRO STUDIES WE MADE
A REPORTER CONSTRUCT, WHICH HAD
THE FIVE PRIMES UNTRANSLATED
REGION, A LITTLE BIT OF GAG, AND
DOWNSTREAM LUCIFERASE GENE,
TRANSCRIBED IN THE PRESENCE OF
AUTHENTIC CAP ANALOG, GET BE CAP
RNA.
WE TAKE THESE RNAs AND PUT
THEM INTO IN VITRO TRANSLATION,
AND LOOK AT THE READOUT.
BASED ON THE READOUT WE CAN SEE
IN A CAP-DEPENDENT MANNER, WE
SEE GREATER TRANSLATION WITH THE
CAP 3G CONSTRUCT OVER THE CAP 1G
CONSTRUCT.
AND SO THIS IS -- THIS WAS
EXCITING, AND WE WANTED TO MAKE
SURE -- NOW WE WANT TO TEST OUR
MUTANTS IN THE SAME SYSTEM.
HERE AGAIN IS WHAT I JUST SHOWED
YOU, DIFFERENCE IN READOUT
BETWEEN THE 1G AND 3G WILD TYPE.
AND AGAIN YOU CAN SEE THE
DIFFERENCE IN STRUCTURES ON THE
GEL.
IF WE LOOK AT MELTED DIS
VARIANT, THIS DOES NOT DIMERIZE
AS EFFICIENTLY BUT HOPEFULLY
YOU'LL NOTICE CONFIRMATIONAL
DIFFERENCES IN THE MONOMERS AND
WITH STILL SEE WE RETRAIN
PREFERENTIAL TRANSLATION OF 3G
RNA.
WE LOOK AT THE MUTANTS I TOLD
YOU ABOUT EARLIER, SO WHAT WE
CALL THE GU OR ADD-GG, NOW
ELIMINATE THE STRUCTURAL
DIFFERENCES AS YOU CAN SEE ON
THE NATIVE GEL BETWEEN 1G AND 3G
REELIMINATE TRANSLATIONAL
DIFFERENCES, NOW TRANSLATED
EQUALLY EFFICIENTLY IN THIS IN
VITRO ASSAY.
WHAT ABOUT IN CELLS?
I WANT TO REMIND EVERYBODY
TRANSLATION HAPPENS, YOU'LL BE
SEEING SOME LABELS ON THE GEL,
SMALL RIBOSOME SCANS, AUG START
CODON, LARGE SUBUNIT JOINS, YOU
GET MONOSOMES, ELONGATION
CONFIDENT.
YOU WILL START BUILDING UP AS
TRANSLATION OCCURS LIGHT
POLYSOMES, TRANSLATING mRNAs
WILL HAVE MEDIUM AND HEAVY
POLYSOMES BUILDING ON THE
mRNA.
AND SO WEI SUE'S LAB TOOK CELLS,
TREATED WITH CYCLOHEXIMIDE,
COLLECTED THE CELL LYSATE,
LOADED ON SUCROSE DENSITY GRADE
WENT.
FRACTIONATED THEM SO WHAT YOU
SEE HERE, ANALYZE THE RNA BY
MASS GENERATION FIVE PRIME RACE.
RESULTS ARE THAT IF YOU LOOK AT
WHAT'S IN THE TOTAL RNA AS I
SHOWED BEFORE IT'S MOSTLY 3G
WITH A LITTLE BIT OF 1G.
IF YOU LOOK AT THE FREE RNA
THAT'S NOT BEEN LOADED ON
POLYSOMES, YOU CAN SEE THAT THAT
PROFILE DOESN'T CHANGE SO THERE
DOESN'T SEEM TO BE PREFERENTIAL
LOADING OF THE 1G OR 3G.
AND THEN IF YOU LOOK AT THE VERY
FRACTION, WHAT YOU CAN SEE IS
THAT THE 1G RNA IS PRESENT IN
THE 4DF, 6 DF, EVEN THE LIGHT
POLYSOMES.
ONCE YOU GOAT MEDIUM AND HEAVY
IT'S SIGNIFICANTLY LESS
DOMINANT.
SO THIS SUGGESTS THE VERY
ACTIVELY TRANSLATING GENES ARE
NOT THE 1G RNA.
WE WANTED -- HERE IS WHAT I
SHOWED YOU AND QUANTIFICATION,
YOU CAN SEE PRESENT TO THE LIGHT
FRACTIONS BUT THEN NOT
REPRESENTED IN THE MEDIUM AND
HEAVY FRACTIONS.
WE WANT TO LOOK AT MUTANTS.
WHAT WE SAW WITH G MUTANT AND
ADD-GG, ALL LOADED EQUALLY WELL.
CHANGING THE STRUCTURE REALLY
HAS IMPACTS TRANSLATION IN
CELLS.
AND SO TO FINISH WITH THINGS
ONGOING STUDIES WE'RE NOW
WORKING ON, IF THE 1G -- SORRY,
THE CAP IS BOUND BY EIF BINDING
COMPLEX, THAT COMPLEX CONTAINS
EIF 4E, IN ORDER TO LOCATE THE
CAP IT'S BEEN SHOWN IN RECENT
YEARS IT REALLY NEEDS 4G
SCAFFOLD PROTEIN, AND THE
HELICASE TO BIND FOR EFFICIENT
LOADING OF RIBOSOMES, THE FIRST
STEP.
LOOK AT CAP BINDING OF 1G AND 3G
WITH THE COMPLEX, WHICH THIS IS
WORK DONE IN COLLABORATION,
PURIFYING 4G FOR US, IN INSECT
CELLS, YOU SEE SIMILAR BINDING
BETWEEN THE CAP 1G AND CAP 3G
RNA CONSISTENT WITH RIBOSOME
PROFILES, NO PREFERENTIAL
LOADING OF RIBOSOMES AND NO
PREFERENTIAL BINDING OF THE
COMPLEX EITHER.
AND SO IN COLLABORATION WE'RE
STARTING SINGLE MOLECULE
EXPERIMENTSES TO LOOK AT
SCANNING OF RIBOSOMES OF THE ONE
RESULT HERE AGAIN HE'S LOOKING
AT THE FRET SYSTEM SET UP SO YOU
HAVE A CAP AND THE ACCEPTOR IS
DOWNSTREAM OF THE 5 PRIME ATR
WITH OLIGO, IN THE FOLDED
CONFIRMATION YOU'D EXPECT A HIGH
FRET BUT ONCE THIS COMPLEX BINDS
AND FACILITATES UNWINDING OF THE
RNA YOU WOULD EXPECT LOW FRETS.
WE CAN COMPARE THIS FRET
EFFICIENCY UPON THIS CAP BINDING
COMPLEX BY NAME, TO ABATE A
GLOBIN UNSTRUCTURED RNA.
WE START WITH A HIGH FRET, AS
YOU ADD THE EIF 4 COMPLEX YOU
SAY TRANSITION TO COMPLEX IN THE
LOW FRET STATE.
HOWEVER WITH 1G AND 3G RNA
HIGHLY STRUCTURED YOU SEE MORE
HETEROGENEITY BUT ALSO MUCH
SLOWER UNWINDING OF THESE RNAs
AS EXPECTED.
AGAIN, WE SEE VARIABLES,
DIFFERENCE BETWEEN ABILITY OF
THIS COMPLEX TO UNWIND THE 1G
AND 3G.
THEY BOTH TAKE 20 OR 30 SECONDS
TO BE UNWOUND.
WE'RE INTERESTED IN LOOKING AT
SCANNING.
I DON'T HAVE DATA BUT HYPOTHESIS
BASED ON THE DATA SO FAR IS THAT
BOTH RNAs CAN LOAD THE CAP
BINDING COMPLEX AND 4 DIS
RIBOSOMES.
RESULTING IN EFFICIENT, YOU
KNOW, AN EFFICIENT TRANSLATION,
THOUGH TRANSLATION DOES OCCUR.
YOU HAVE MORE ON THE LEFT
STRUCTURED 3G RNA.
AND SO THAT'S ALL THE SCIENCE I
WANTED TO TELL YOU ABOUT.
AGAIN, I MENTIONED A LOT OF
THESE PEOPLE, SHOWED PICTURES ON
THE FIRST SLIDE.
I WON'T BELABOR THIS TO GET TO
THE IMPORTANT PART.
THAT'S ALAN.
A FEW THINGS I'VE LEARNED FROM
ALAN OVER THE YEARS, NOT A TOP
TEN BUT A TOP FIVE LIST.
TOP FIVE, PUT YOUR BOOK DOWN IN
GRACE AUDITORIUM BEFORE
BREAKFAST.
THIS WAS LITERALLY THE FIRST
THING I LEARNED FROM ALAN.
AND THE BEST PLACE IS RIGHT
THERE.
AND ALAN DOESN'T SIT IN THE
FRONT ROW.
HE SITS ABOUT FOUR BACK.
IF YOU LOOK AT THAT PICTURE THAT
SOMEONE SHOWED ALREADY THAT'S
EXACTLY WHERE I WAS SITTING.
I ALWAYS LIKED SITTING CLOSE TO
ALAN ESPECIALLY WHEN I FIRST
STARTED, I'M NOT TRAINED AS A
VIROLOGISTS.
I WOULD SAY DO YOU BELIEVE THAT?
AND WE WOULD HAVE QUIET
CONVERSATIONS.
NEXT IS ASK SIMPLE QUESTIONS.
IMPORTANT THOUGH.
THESE PAPERS WERE HIGHLIGHTED
HERE TODAY.
WE ALL ADMIRE THE PAPERS.
THE QUESTION IS WHAT DO
RETROVIRAL PACKAGES?
DELPHINE SHOWED RIBOSOMES,
SIMPLE QUESTION, AMAZING ANSWER.
WHAT IS THE KEY COMPONENT OF
RETROVIRAL ASSEMBLY IN VITRO?
ERIC OR SOMEONE MENTIONED
iNOSITOL PHOSPHATES, NOW WE
KNOW HOW IMPORTANT THEY ARE FOR
ASSEMBLY IN CELLS.
BE SKEPTICAL.
THERE'S A DIFFERENCE BETWEEN
WHAT YOU KNOW AND WHAT YOU THINK
YOU KNOW.
AND RELATED TO THAT, DON'T
FORGET HISTORY.
IF YOU WEREN'T THERE FOR ALAN'S
COLD SPRING HARBOR KEYNOTE TALK
IN 2016, FIND IT ON YouTube
AND LISTEN TO THAT, I LEARNED A
LOT FROM IT.
AND THEN THIS ONE.
IT'S OKAY TO JUST LOOK OUT THE
WINDOW.
THIS IS RELATED TO ONE TIME ALAN
AND I HAD BEEN AT MANY
CONFERENCES, MANY TIMES IN
EUROPE OR WHATEVER.
YEAH, I DON'T MIND LONG FLIGHTS,
GET WORK DONE, WRITE PAPERS.
HE SAID, YOU KNOW, I JUST LIKE
TO LOOK OUT THE WINDOW.
THIS IS A PICTURE I TOOK LAST
YEAR ON THE WAY TO COLD SPRING
HARBOR, A BEAUTIFUL DAY, I
SHOULD CAPTURE THAT.
OVER THE YEARS WE HAD PUBLISHED
EIGHT MANUSCRIPTS AND ARTICLES
OF VARIOUS KINDS TOGETHER.
WE SHARED COMMON INTERESTS, WHEN
WE HAD AN OPPORTUNITY WE TRIED
TO COLLABORATE TOGETHER, AND
IT'S BEEN A LOT OF FUN.
WE HAVE A LOT OF FOND MEMORIES
LIKE I SAID ALL OVER EUROPE AND
EVERYWHERE.
BUT THIS WAS THE BOSTON DUCK
BOAT TOUR IN 2019 AT THE
RETROVIRAL ASSEMBLY MEETING.
AND WE WERE SITTING RIGHT BEHIND
I THINK KATHY AND BEN, THAT WAS
FUN.
LOTS OF FUN DINNERS TOGETHER.
THIS WAS AFTER THE MEETING WITH
OUR MUTUAL AND DEAR FRIEND
JUDITH.
WE INTERACTED A LOT, COLD SPRING
HARBOR, HERE AT MY SABBATICAL
AND MEETINGS.
THIS IS ONE MEETING I DIDN'T GO
TO BECAUSE I CAN'T FIND MYSELF
BUT I FOUND ALAN, YOU'LL NOTICE
IN THE CENTER.
SO YOU KNOW HOW THEY SAY LET'S
GET UP, GET A PICTURE, WE
ASSEMBLE.
EVERY PHOTO I FOUND, YOU SEE HIM
ON THE BOTTOM HERE?
IN THE CENTER AGAIN.
THIS IS MINNEAPOLIS MEETING.
HERE'S A COUPLE MORE.
THIS ONE HE'S MAYBE OFF CENTER
BUT PRETTY CLOSE, A STRANGE
L-SHAPED ROOM.
LAST ONE, BOSTON, THERE HE IS.
TO THE PERSON, I FOUND IT.
THIS REALLY IS BECAUSE HE'S A
CENTRAL IF NOT THE CENTRAL
PLAYER IN THIS FIELD OF
RETROVIRAL ASSEMBLY SO IT'S
APPROPRIATE.
I COULDN'T FIND THE LAST PHOTO
FROM THE MEETING, MANY OF YOU
WERE THERE IN SALT LAKE CITY.
I E-MAILED, DO YOU HAVE THE
GROUP PHOTO?
WE FORGOT TO TAKE ONE.
THEN I REMEMBERED FOR THOSE THAT
WERE THERE, THIS PHYSICIST HAS
CONNECTIONS WITH ASTRONOMERS.
AFTER THE POSTER SESSION,
ASTRONOMY SET UP TELESCOPES IN
PITCH DARK UTAH AND LOOKED UP AT
CELESTIAL BODIES.
THIS IS APPROXIMATELY WHAT WE
SAW.
IN THE CENTER WE ALSO HAVE ALAN.
[LAUGHTER]
SO, ALAN, THANK YOU FOR BEING
THE CENTER OF OUR UNIVERSE AND
THE RETROVIRUS FIELD, AND
CONGRATULATIONS.
>> SO YOUR COMMENTS ABOUT HIV,
HAVE YOU EVER BEEN ABLE TO
MEASURE EFFECTS OF, SAY, THESE
MUTATIONS ON VIRUS REPLICATION?
>> YES.
SO, WEI-SUH CAN ANSWER BETTER
THAN I CAN BUT BASICALLY IF YOU
HAVE -- IF YOU ALTER DISTANCE,
HAVE BEEN ABLE TO MAKE A 1G ONLY
VIRUS AND 3G ONLY VIRUS, ONLY
EXPRESS AND ARE REPLICATION
COMPETENT.
IF YOU COMPETE THEM WITH THE
WILD TYPE, THE WILD TYPE ALWAYS
WINS OUT.
THERE'S A FITNESS DEFECT.
AND SO, YEAH, ANYTHING ELSE,
WEI-SUH?
OKAY.
THE VIRUS HAS OPTIMIZED -- TAKEN
ADVANTAGE OF THE SLOPPINESS OF
THE HOST RNA POLYMERASE.
I GUESS THAT'S STILL A
POSSIBILITY BUT WE DON'T KNOW
THAT.
ADAM?
>> REAL BEAUTIFUL WORK,
REMARKABLE PHOTOGRAPH.
IS THERE A WAY YOU CAN
MANIPULATE IT?
>> I BET YOU COULD.
WE HAVEN'T TRIED THAT.
DIMERIZATION, THE VARIANT THAT
DOESN'T -- IT STILL FORMS A
DIMER, STILL PACKAGES
SELECTIVELY AND EVERYTHING BUT
WE DO THINK DIMERIZATION IS
IMPORTANT.
WE'VE NEVER TRIED THAT
SPECIFICALLY.
>> (INAUDIBLE).
>> UH-HUH, YEAH.
>> WHAT WAS THE RATIO OF
INVESTIGATORS TO AMINO ACIDS AT
THE MEETING?
[LAUGHTER]
>> ABOUT TWO INVESTIGATORS FOR
EVERY AMINO ACID.
I SHOULD SAY MAYBE ERIC WOULD
HAVE A SLIDE, THE NEXT ONE IS
GOING TO BE IN PRAGUE, END OF I
BELIEVE -- IS IT MIDDLE OR END
OF SEPTEMBER?
>> THIRD WEEK.
>> THIRD WEEK IN SEPTEMBER,
2025.
WE'LL GET AN E-MAIL OUT,
ORGANIZING IT, HOPEFULLY NEXT
WEEK FOR THOSE ON THE LIST.
>> BEAUTIFUL.
I'M TRYING TO REMEMBER ABOUT
PACKAGING, MLBs, DO OTHER
VIRUSES DO SELECTIVE PACKAGE?
>> THERE'S ALSO GOING TO BE TWO
PULLS, AND WITH HIV IT WAS NEVER
THOUGHT TO BE THE CASE.
BUT IN RECENT YEARS IT'S
SHOWN -- THE MECHANISM IS
DIFFERENT IN MLV.
WE DON'T THINK IT'S RELATED TO
THE FIVE PRIME END.
YES, IT'S BEEN SHOWN IN OTHER
VIRUSES.
DELPHINE?
>> I WAS WONDERING IF YOU HAVE
DONE OR INTEND TO DO STRUCTURE
WITH SENTAG DYNAMIC OF
TRANSLATION IN THE CELL.
>> YES.
>> AND YOU CAN SEE MAYBE IT'S --
>> IT'S IN MY PROPOSAL.
YEAH, NO, THOSE ARE EXACTLY THE
THINGS WE WANT TO DO.
WE UNDERSTAND THE BASICS.
WHAT ARE THE DIFFERENCES IN THE
DYNAMICS, KINETICS, PROTEIN
BINDING DIFFERENCES,
METHYLATION, MODIFICATION, WE
HAVE THE TOOLS BUT THE 1G,
3G-ONLY VIRUS, TO LOOK AT THAT.
>> YOU COULD SEE THE RNA -- I
MEAN TRANSLATION, BUT THEN DO
YOU THINK THIS GAG WOULD BE THE
CHAPERONE OR ONE OR THE OTHER OF
THE SPECIES OVER TIME?
>> A TRANSLATION CHAPERONE?
WHAT DO YOU MEAN?
>> WE KNOW GAG, LIKE I SAID IN
VITRO, WE ALSO WANT TO LOOK AT
ANY IN CELLS BUT DEFINITELY
ASSEMBLE MORE READILY IN THE
FIVE PRIME OF THE 1G RNA, I
IMAGINE THE SAME THING IS TRUE
IN CELLS.
WE HAVEN'T LOOKED-IT.
I DON'T THINK IT'S A SWITCH.
I THINK IT'S TWO SPECIES, RIGHT?
ONE OF THEM, EXACTLY, YOU KNOW,
ONE OF THEM IS BOUND BY GAG
READILY AND GUIDED TO THE
MEMBRANE FOR ASSEMBLY.
THE OTHER ONE -- BOTH LOADED BY
RIBOSOMES.
THE 1G IS NOT AS EFFECTIVE, IT'S
A DISTRIBUTION OF ENSEMBLES OF
RNA STRUCTURES, DISTRIBUTION
SHIFTED AND FOR THESE DIFFERENT
FUNCTIONS.
>> I KNOW THERE'S DIFFERENCES IN
CAP BINDING COMPLEXES.
>> YES.
>> SO IS ANYTHING KNOWN IN T
CELLS SPECIFICALLY ABOUT --
>> EXACTLY WHAT THOSE ARE IN T
CELLS?
I DON'T KNOW.
I THOUGHT YOU WERE GOING ASK
ABOUT THE TRIMETHYL G CAP.
IT'S BEEN PROPOSED AND SHOWN
PRETTY CLEARLY TO BE SOMETIMES
GIVE US TRIMETHYL G CAP AND BIND
ALTERNATIVE COMPLEX AND WE'RE
LOOKING AT THAT BUT IN TERMS OF
IN T CELLS WHAT THE ISOFORMS ARE
WE HAVEN'T LOOKED.
I I SHOULD STOP THERE.
>> THANK YOU FOR A BEAUTIFUL
TALK, KARIN.
WE'VE HEARD THE FIRST PART OF MY
INTRODUCTION OF THE NEXT
SPEAKER, BY MISTAKE, BUT THE
NEXT SPEAKER IS MAURICIO
COMAS-GARCIA, WHO WAS ALSO A
POSTDOC IN MY LAB, ACTUALLY
PRETTY RECENTLY.
HE CAME FROM A LAB REALLY FROM A
PHYSICS BACKGROUND, FROM A
COMMUNITY OF PHYSICISTS WHO HAD
DECIDED TO STUDY VIRUSES.
CENTERED AROUND UCLA, WHO WAS
HIS THESIS ADVISER, MAURICIO
CAME TO MY LAB AND REALLY WORKED
OUT THE TECHNIQUES TO MEASURE
BINDING OF GAG TO DIFFERENT
RNAs, UNDER DIFFERENT
CONDITIONS.
I THINK HE DID BEAUTIFUL WORK
FOLLOWING THE FOOT STEPS OF WHAT
KARIN HAD ALREADY PUBLISHED,
EXPANDED THE VIEW WITH
FASCINATING RESULTS.
I WON'T TALK ABOUT THAT, I'LL
JUST SAY HE DID BEAUTIFUL WORK
IN MY LAB AND WAS DELIGHTED TO
GET A NICE JOB IN HIS HOME
COUNTRY, WHICH IS MEXICO, AND SO
HE'S NOW PROFESSOR IN SON SAN
LUIS POTOSI CONTINUING TO DO
GREAT WORK IN A NUMBER OF
DIRECTIONS.
SORRY HE WOULDN'T BE HERE IN
PERSON BUT THERE HE IS.
GOOD MORNING, BUENOS DIAS.
LOOK FORWARD TO YOUR TOOK.
>> IT'S A SHAME UNITED AIRLINES
DIDN'T GET IT TOGETHER.
AND NOW I GUESS YOU CAN SEE MY
SCREEN RIGHT NOW.
OKAY.
SO, FIRST THIS IS ALAN WHEN HE
CAME TO VISIT AFTER I MOVED.
SIX MONTHS.
I'M DEEPLY SADDENED NOT ABLE TO
ATTEND TO THE MEETING IN PERSON.
BUT WE'RE HERE TO CELEBRATE YOU,
YOUR REMARKABLE SCIENTIFIC
CAREER, MOST IMPORTANTLY I'M
HERE TO THANK YOU FOR EVERYTHING
YOU'VE SAID TO ME.
I'M SORRY I WON'T BE TALKING
ABOUT HIV.
BUT BE SURE EVERYTHING I WORKED
ON IS A SEQUENCE OF YOUR
PREVIOUS RESEARCH.
YOU TAUGHT ME TO BE KIND,
POLITE, PASSIONATE.
YOU TAUGHT ME THAT WORDS MATTER,
ONE SHOULD BE CAREFUL WHEN
WRITING, HAVING SCIENTIFIC
DISCUSSIONS.
BUT HE ALSO TAUGHT ME TO BE
VERY, VERY SKEPTICAL.
YOU TAUGHT ME IT'S OKAY TO SAY I
DON'T KNOW.
YOU TAUGHT ME THAT ONE OF THE
GREATEST PLEASURES IN LIFE IS TO
DEEPLY UNDERSTAND WHAT YOU
STARTED, WHAT YOU THINK IS
INTERESTING.
IF I HAD TO CHOOSE MY CAREER
PATH AGAIN, FOR SURE I WOULD
CHOOSE WORKING WITH YOU.
HOWEVER, I MIGHT HAVE FOUND A
WAY TO STAY LONGER IN YOUR LAB.
IN FACT A COUPLE WEEKS AGO, I
SAW BILL, HE TOLD ME AFTER
SPENDING A FEW DAYS WORKING
TOGETHER, I'M GLAD YOU WENT TO
WORK WITH ALAN.
NOW YOU ARE REAL VIROLOGIST,
WHATEVER THAT MEANS, I DON'T
KNOW WHAT THAT MEANS.
ALAN, THERE ARE NO WORDS TO
EXPRESS HOW MUCH I ADMIRE YOU, I
OWE YOU MORE THAN I COULD EVER
GIVE YOU BACK.
ONE OF MY FAVORITE MOVIES, OH
CAPTAIN, MY CAPTAIN, YOU HAVE A
FRIEND FOR LIFE.
SO I WANT TO FIRST -- I WROTE A
FEW WEEKS AGO, I'M SORRY, I
WON'T TALK ABOUT HIV, BUT I'LL
TALK ABOUT ALPHAVIRUSES.
AND ALPHAVIRUSES ARE VERY, VERY
INTERESTING BECAUSE THEY ALLOW
ME TO KEEP STUDYING ASSEMBLY IN
A VERY DIFFERENT WAY FROM WHAT
DID I WITH ALAN.
THESE VIRUSES ARE AMAZING, TWO
LAYERS, SEPARATED BY A LIPID
BILAYER.
YOU CAN SEE HERE THE CORE
CONTAINS NUCLEIC ACID, 240
COPIES OF THE CAPSID PROTEIN.
OUTER LAYER CON TAINTS 80
TRIMERS OF HETERODIMERS OF THE
GLYCOPROTEINS.
TODAY I'M GOING TO TELL YOU
ABOUT A SMALL PROJECT TO
UNDERSTAND THE ASSEMBLY OF THE
CORE BECAUSE WHEN YOU TAKE THE
CURRENT DATA AND REMOVE DENSITY
FOR THE PLASMA MEMBRANE, WELL
VIRAL MEMBRANE AND GLYCOPROTEINS
YOU SEE THESE WIFFLE BALLS WITH
LARGE HOLES AND DISTANCES WHICH
BAFFLES ME.
TWENTY YEARS AGO OR MORE,
ANOTHER PERSON I ADMIRE A LOT
PUBLISHED THIS PAPER, SHE WAS
TRYING TO RECONSTRUCT AGAIN THE
CORE OF TWO ALPHAVIRUSES.
THIS IS AMAZINGLY SHE WAS ABLE
TO ABLE BECAUSE SHE COULDN'T DO
IT TO RESOLUTION BETTER THAN 30
ANGSTROMS, AT THAT TIME IT WAS 8
ANGSTROMS.
OFTEN SHE WAS COMPLETELY RIGHT,
INABILITY TO IMPROVE RESOLUTION
SUGGESTS CORE-LIKER PARTICLES
VARY SLIGHTLY IN STRUCTURE AMONG
THEMES SELVES AND MAY DEVIATE
FROM ACT ICOSAHEDRAL SYMMETRY.
IF YOU REMOVE THE GLYCOPROTEINS,
ISOLATE BEFORE THEY TARGET
GLYCOPROTEINS HOW THAT STRUCTURE
IS NOT SYMMETRIC?
SHE SHOWED A FEW YEARS LATER
PARTICLES ARE NOT EVENLY CLOSE
TO RESEMBLE SPEARICAL PARTICLES.
HIV MAKES A LOT OF MISTAKES, I
WONDER ARE THESE VIRUSES MAKING
MISTAKES LIKE HIV?
SO HERE IS A CORE WE'RE
STUDYING.
WE'RE ASSUMING FIRST IN HEXAMERS
AND PENTAMERS, THERE ARE A
COUPLE INTERACTIONS, INTERNAL
INTERACTIONS, AND IN THE HEXAMER
BECAUSE IT'S ASYMMETRIC, THE
INTERACTION IS NOT AT THE SAME
DISTANCE AS THIS.
WHEN YOU LOOK AT THE PENTAMER,
SAME PRINCIPLE, YOU SEE
INTERACTIONS ARE SIMILAR TO EACH
OTHER AND AGAIN THE SAME BASIC
AND AMINO ACIDS SEEM TO BE
PARTICIPATING IN THE ASSEMBLY OF
THE CAPSOMER, WE WANTED TO KNOW
IF THESE -- WHICH IT MAKES
SENSE, WE WANT SOMETHING STABLE,
NEUTRAL PH, BUT IT CAN FALL
APART WHEN IT BECOMES ACIDIC.
OUR IDEA, BECAUSE ALAN SAID IT
RIGHT, I WAS A PHYSICIST, FISCAL
'DEMIST, WE WANTED TO STUDY
INTERACTIONS RESPONSIBLE FOR
ASSEMBLY OF CAPSOMER.
BEFORE DOING MUTATIONS, WE
LOOKED AT OVER 1800 UNIQUE
SEQUENCES AND THIS IS AN RNA
VIRUS, QUITE SURPRISING,
CONSERVED IN 99.4% OF ALL
SEQUENCES FOR PATIENTS.
SO I THOUGHT, OKAY, THAT'S FINE.
HIGHLY CONSERVED.
THAT'S A GOOD STARTING POINT TO
DOING MUTATIONS.
BUT I'M ALWAYS SECOND GUESSING
MYSELF.
AND THEN, OKAY, WAIT, BEFORE
DOING ANY MUTATION LOOK AT
DIFFERENT ALPHAVIRUSES, THIS IS
JUST A LIST OF PHYLOGENETIC
TREE.
CryoEM OR X-RAY STRUCTURES.
LEFT-HAND SIDE IS ONE OF THE
CryoEM STRUCTURES, CAPSID
PROTEIN, I JUST HIGHLIGHTED FOUR
AMINO ACIDS I'M TALKING ABOUT
TODAY.
THERE ARE ANOTHER FOUR THAT HAVE
SOMETHING ELSE TO DO BUT I WON'T
TALK ABOUT TODAY.
AND THEN ON THE RIGHT IT'S AN
OVERLAP OF THE STRUCTURAL FOR
THE CAPSID PROTEIN OF CHIKV, AND
THEY ARE CLOSE TO EACH OTHER
GENETICALLY, THERE'S A
PERFECT -- ALMOST PERFECT MATCH
IN STRUCTURE.
WHEN YOU SEE BLUE AMINO ACID YOU
SEE BLUE AMINO ACID OR REGULAR
RED.
NOW WE HAVE STRUCTURE FOR EIGHT
OR NINE -- I FORGET HOW MANY
ALPHAVIRUSES.
WE USE ALPHAFOLD 2 AND 3 TO
PREDICT THE REST OF THE
STRUCTURE, ONE OF THE MOST
DISTANT ALPHAVIRUSES, AND
THERE'S SOME CHANGES FOR EXAMPLE
LYSINE, HERE HIGHLY CONSERVED IN
OLD WORLD ALPHAVIRUSES, NOW
INSTEAD OF BEING POSITIVE, IT'S
NEGATIVE.
WE START USING THIS APPROACH OF
MATCHING STRUCTURES, BECAUSE IF
YOU LOOK AT THE PRIMARY SEQUENCE
OF CAPSID PROTEINS THEY ARE
DIFFERENT FROM EACH OTHER.
WE STARTED OVERLAPPING ALL THE
STRUCTURES SO WE COULD USE THEM
AS PINPOINTS TO BE ABLE TO DO A
PHYLOGENETIC ANALYSIS THAT I
WON'T TALK ABOUT TODAY.
ALPHAVIRUSS, YOU CAN DIVIDE THEM
AS OLD WORLD OR NEW WORLD.
FOR THOSE WHO WE KNOW THE
MAMMALIAN HOST, BY MOSQUITOES,
INVERTEBRATES, VERTEBRATES.
OLD WORLD MEANS ANY ALPHAVIRUS
THAT FITS THE WINDOW OF
MAMMALIAN HOST.
THAT IS FOUND MANY CONTINENT
EXCEPT AMERICAS, NEW WORLD JUST
RESTRICTED TO AMERICAS.
WE HAVE A FEW ALPHAVIRUSES FOUND
IN MOSQUITOES, NO IDEA THE
VERTEBRATE HOST.
ALSO ALPHAVIRUSES THAT AFFECT
FISH AND SEA LIONS.
WHAT'S SURPRISING FOR OLD WORLD,
100 CONSERVED, DOESN'T MATTER IF
THE VIRUS IS FROM AFRICA OR
NETHERLANDS OR NEW ZEALAND,
THESE FOUR AMINO ACIDS ARE
CONSERVED.
IF YOU TAKE NEW WORLD
ALPHAVIRUS, IF TAMPA, AND FROM
VENEZUELA, THEN THESE ARE
INTERACTIONS OF THE CAPSOMER NOT
CONSERVED.
THOSE WHO DON'T KNOW THE
VERTEBRATE HOST, THREE OF FOUR
CON SEARCHED.
SUGGESTING STRONG EVOLUTIONARY
PRESSURE ON INTERACTIONS, WHY
ARE NOT INNER INTERACTIONS
HIGHLY CONSERVED?
WE FOUND HIGHLY AMOUNT OF
CONSERVATION, THEN I WAS BRAVE
ENOUGH TO START DOING
MUTAGENESIS THANKS TO MITCH IN
ALAN'S LAB, HEAD OF MOLECULAR
CLONING.
WE CONSTRUCTED A PLASMATE, WE
WAIT 24 HOURS, WATCH THEM WELL,
WAIT ANOTHER 24 HOURS, TAKE THE
SUPERNATANT AND FREEZE IT, TAKE
IT AND INFECT NAIVE CELLS, DO
THESE IN 48 WELL PLATES.
AND WE'RE LUCKY TO HAVE THESE
AUTOMATIC MICROSCOPES BECAUSE
IT'S FLUORESCENTS MICROSCOPE,
BECAUSE MUCH FLUORESCENT GENE WE
CAN DO SEVERAL RAL REPLICATES FR
EACH EXPERIMENT.
WHATEVER WE FIND INTERESTING WE
TAKE TO ELECTRON MICROSCOPE.
SO THIS IS INTERACTION BETWEEN
AMINO ACID HERE CONSERVED IN
93%, THIS ONE 98%, WE CHANGE
BASIC AMINO ACID OR REVERSE THE
CHARGES SO THERE'S NO
ELECTROSTATIC REPULSION BUT NOW
DON'T HAVE POSITIVE AND NEGATIVE
INTERFACE.
ALL THIS WORK HAS BEEN DONE SO
FAR, MOST BY UNDERGRAD STUDENTS.
IT WAS VERY INTERESTING BECAUSE
HERE FOR THE MUTANTS YOU SEE
REPULSION, SO THAT MAKES SENSE.
HERE YOU SEE REPULSION.
MAKES SENSE.
BUT HERE THERE'S NO REPULSION
BUT NOW WE DESTROY ABILITY OF
THE CAPSID PROTEIN TO BEHAVE
LIKE A DIPOLS, DEPENDS ON THE
ANGLE, VERY IMPORTANT FOR PART
OF THE STORY.
THEN WE SAY, OKAY, WHAT'S WRONG
WITH THESE VIRUSES?
WITHIN THE LAB, THANKS TO
TRAINING I RECEIVED AT NCI.
YOU CAN SEE BETWEEN TWO CELLS,
BUDDING PARTICLES.
AND THE COOL THING ABOUT ALPHA
VIRUSES, HOW CAN I FIND A CELL
THAT IS INFECTED?
THINGS IN THE CIRCLES ARE TYPE 2
WHERE THE GENOME IS REPLICATED,
ISOLATED FROM THE REST OF THE
CELL, SO CGAS CANNOT DETECT OR
STING, I DON'T REMEMBER, DOESN'T
MATTER, BUT FOUND THE VIRAL RNA.
IN THIS USEFUL BECAUSE WE SEE OR
LOOK FOR CELLS THAT HAVE THESE
REPLICATION SITES AND WE LOOK TO
THE -- MOVE AND LOOK TO THE
PLASMA MEMBRANE TO SEE IF WE CAN
FIND VIRUSES.
FOR THE MUTANTS WHERE WE CHANGE
POSITIVE FOR NEGATIVE AMINO
ACID, OUTER INTERACTION, WE HAD
TO LOOK FOR A LOT OF CELLS UNTIL
WE FOUND CYTOPATHIC VESICLES,
THIS IS REALLY BEAUTIFUL.
LARSON IN SWEDEN HAS DONE
BEAUTIFUL WORK.
OUR SURPRISE WE FOUND TUBES,
LONG TUBES.
THESE TUBES ARE NOT PRESENT IN
UNINFECTED CELLS, OR EVEN
INFECTED CELL WITH WILD TYPE,
TUBES ARE REALLY COOL BECAUSE --
THIS GOES BACK TO CASPER, WELL,
GOES BACK TO IF YOU WANT TO MAKE
THIS, YOU ONLY NEED 12 PENTERS
PENTAMERS.
IF YOU WANT TO MAKE LARGER YOU
NEED HEXAMERS, A DISCRETE
NUMBER, I WON'T DESCRIBE HOW
THAT HAPPENS.
IF YOU ONLY HAVE HEXAMERS YOU
GET TWO POSSIBLE STRUCTURES,
PLANAR SHEETS OR TUBES, LIKE
CARBON NANOTUBES.
SO OUR HYPOTHESIS NOW WE'RE
DOING SIMULATIONS WITH ROY AND
VICTOR, IN MADRID, TRYING TO SEE
IF WE CAN SIMULATE ASSEMBLY
SUGGESTING MUTATION HERE BY
CHANGING POSITIVE FOR NEGATIVE
AMINO ACID IS DETABLIZING
ABILITY OF THE PROTEIN TO FORM
PENTAMERS BUT NOT HEXAMERS.
THIS IS A SIMULATION FROM ORAZIO
VICTOR, ALL SIMULATIONS,
PRELIMINARY RESULTS.
AND THE PENTAMER IS NOT -- WILD
TYPE IS NOT VERY STABLE.
THAT HAS TO DO WITH ARTIFACTS
THAT I DON'T HAVE THE TIME TO GO
OVER THAT.
WHEN YOU LOOK AT THE MUTANT, IT
IMMEDIATELY FALLS APART.
YOU CAN SEE IT FALLING APART,
CONFIRMING THAT THE PENTAMER
UNDER THIS SIMULATION IS
UNSTABLE.
STILL WAITING FOR HORACI, TO
HAVE RESULT FOR HEXAMERS AND
THEN DO SIMULATION TO UNDERSTAND
HOW THIS FORMS HEXAMERS.
IT MAKES SENSE.
REMEMBER, THIS AMINO ACID IS
HIGHLY CONSERVED.
SO THAT'S WHY YOU DON'T SEE
ANYTIME NATURE, THESE MUTATIONS.
NOW WHEN WE LOOK AT THE PARTNER
OF 172 AND CHANGE THAT'S ACID R
A BASE, ANOTHER PROJECT WE'RE
STUDYING BUDDING WITH U.C.
RIVERSIDE, SO WE HAVE
CHARACTERIZED BUDDING OF THE
VIRUS QUITE EXTENSIVELY AND HAVE
PAPERS SOON TO BE REVIEWED SOON.
WHAT WE FIND THE CORES STOP AT
PLASMA MEMBRANE, TURNS OUT THE
LAB IN SANFORD SHOWED THESE HERE
AND HERE, IN THE CYTOPLASM, NOT
FORMING HEXAGONAL SHEETS.
WHEN THEY INTERACT THE CORE --
IT'S WELL KNOWN THAT
GLYCOPROTEINS IN ORDER TO --
THEY ARE TOUCHING THE CAPSID
PROTEIN AND CORE VERY STRONGLY,
THEY NEED TO BIND.
SO WHILE WE'RE ASSUMING, THESE
CORES THAT WE'RE FORMING CANNOT
REANEAL THEMSELVES.
WE LOOK AT SOME MUTATIONS IN THE
PENTAMERS AND HEXAMERS, STILL
MISSING A FEW MUTATIONS, LYSINE
TO USINE IS NOT BAD, WE FIND
THIS IN NEW WORLD ALPHAVIRUSES,
SURPRISED CHANGING BASIC FOR
ACIDIC WAS NOT THAT BAD.
BUT IF YOU LOOK, IF WE DO THE
OPPOSITE CHANGE IN 184, CHANGE
ACID FOR HYDROPHOBIC, COULD BE
LOSING A PROTEIN, THE VIRUS IS
PRETTY MUCH DEAD.
SEEMS TO ME THAT ONE AMINO ACID
IS MORE CRITICAL THAN THE OTHER,
AND WE HAVE NO IDEA WHAT THAT
MEANS.
SO, WE'RE THINKING OF WHAT'S
GOING ON, AND WE SAY, WELL, THIS
IS A PHYSICAL CHEMISTRY PROJECT,
BUT THESE ARE VIRUSES SO LET'S
THINK ABOUT THE HOST.
IT TURNS OUT THE OLD WORLD
ALPHAVIRUSES ARE NON-LETHAL.
THE DISEASE IS NASTY BUT YOU
WON'T DIE FROM CHIKUNGUNYA.
IF YOU'RE INFECTED, A HORSE
INFECTED WITH A NEW WORLD
ALPHAVIRUS, YOU MIGHT DIE MOST
LIKELY FROM ENCEPHALITIS, HIGHLY
LETHAL.
THEY ARE DIFFERENT IN THE WAY
THEY BEHAVE IN MAMMALS.
SO A COUPLE YEARS AGO KAEBLER AT
GUTTERS ARE DID CryoEM OF
CHIKUNGUNYA, AND HE WAS AN
HONEST MICROSCOPIST.
BEFORE DOING RECONSTRUCTION
REALIZED THERE WERE FOUR
POPULATIONS OF PARTICLES, IN
BLACK NORMAL SIZE, THEN SMALLER
ONE, ONE IN YELLOW, LARGER ONES
IN GREEN, AND MULTI-CORE IN RED.
TURNS OUT HE MADE THESE
INTERESTING OBSERVATIONS THAT HE
HASN'T TAKEN FURTHER FROM THIS
TABLE THAT CHIKUNGUNYA MAKES A
MISTAKE IN 4% OF THE PARTICLES
BUT VEEV HIGHLY LETHAL IN TEN
AND EEEV IN 12%.
WHEN YOU LOOK AT INTERACTIONS,
YOU SEE EEEV INSTEAD OF HAVING
STRONG ELECTROSTATIC
INTERACTIONS NOW YOU CHARGE
POLAR AMINO ACID FOR
NON-POLAR -- SORRY, NON-POLAR,
NOW HAVE YOU TWO HYDROPHOBIC
INTERACTIONS.
WE HAVE PROPOSED, WELL, RECENTLY
POSTED THIS QUESTION IN JOURNAL
OF VIROLOGY THAT ARE MISTAKES IN
ASSEMBLY A WAY TO ATTENUATE
HIGHLY PATHOGENIC ALPHAVIRUSES
AND IS INTERACTION BETWEEN TWO
AMINO ACIDS UPON FOR FIDELITY OF
ASSEMBLY PROCESS OR DO WE NEED
TO FIND MORE AMINO ACIDS?
AND WE'RE LOOKING AT ANOTHER
FOUR SET OF AMINO ACIDS BUT I
DON'T HAVE ENOUGH TIME TO TELL
YOU ABOUT THOSE.
SO JUST TO FINISH HOPEFULLY ON
TIME, WE HAVE FOUND HIGHLY
CONSERVED -- THAT THESE ARE
HIGHLY CONSERVED AND PRETTY MUCH
ANY MUTATIONS HERE KILL THE
VIRUSES, I SHOWED THREE BUT WE
HAVE MORE.
THAT THE INNER INTERACTIONS ARE
ONLY CONSERVED OLD WORLD
ALPHAVIRUSES, NOT CONSERVED IN
OTHER ALPHAVIRUSES SO THE VIRUS
IS TOLERANT TO MUTATIONS HERE,
NOT TO ALL OF THEM.
IN FACT, THEY ARE NOT CONSERVED
THE SAME WAY.
184 IS MORE CONSERVED THAN 177,
AND 184 IS LESS TOLERANT TO
MUTATION THAN 177.
RELATION BETWEEN DEGREE OF
CONSERVATION OF THESE AMINO
ACIDS IN NATURE AND WHAT ARE WE
DOING IN THE LAB.
AND THE FINAL QUESTION THAT IS
REALLY PUZZLING, ARE MAY TAKES
IN ASSEMBLY A WAY TO ATTENUATE
HIGHLY PATHOGENIC VIRUS, NOW
CHIKUNGUNYA, ALSO MUTATING VEEV
TO MAKE IT LOOK LIKE CHIKUNGUNA
BUT ONLY THE CAPSID PROTEIN TO
SEE IF WE CAN PROVE THE
HYPOTHESIS.
THESE ARE THE PEOPLE HERE.
CAROLINA, MIGUEL, WORK DONE IN
PART WITH JUAN AND ROYA AND
VICTOR.
THIS IS FUNDING AGENCIES THAT
SUPPORT OUR PROJECTS.
I HAVE TO THANK YOU FOR
EVERYTHING, ALAN.
GOOD LUCK.
AND I COULDN'T -- I COULDN'T
HAVE MORE LUCK BECAUSE WHEN I
MET YOU MY LIFE REALLY, REALLY
CHANGED.
AND I HOPE I HAD -- I WAS NOT
OVER TIME.
AND THANK YOU VERY MUCH.
I DON'T KNOW IF YOU HAVE ANY
QUESTIONS.
>> CALL OUT IF YOU HAVE
QUESTIONS.
YEAH.
>> NICE TALK.
WE FOUND THAT DEFECTS CAN
FACILITATE DISASSOCIATION.
SO, ARE YOU SEEING YOUR
PARTICLES WITH LESSER ABILITY TO
ASSEMBLY ALSO ABLE TO
DISASSEMBLE MORE READILY.
>> GOOD QUESTION.
WHEN THEY HAVE PROTEINS, NO.
WE HAVE DONE LOTS OF
EXPERIMENTS.
BUT WHEN WE REMOVE THE
GLYCOPROTEINS, THEN THEY WANT TO
MAKE LOTS OF MISTAKES, THEY FALL
APART IMMEDIATELY, THE ONES THAT
MAKE MISTAKES.
IT'S MORE ASSEMBLY.
ONE CAVEAT, WE'RE NOW DOING
EXPERIMENTS, HOW THEY
DISASSEMBLE IN THE LYSOSOME
BECAUSE THEY MIGHT FALL APART
EITHER IN THE LYSOSOME,
RELATIVELY HIGHER PACE, COULD BE
CAUSING LESS INFECTIOUS.
I DON'T KNOW IF THAT MAKES
SENSE.
>> OTHER QUESTIONS, YES?
>> YEAH, SO I DIDN'T QUITE
UNDERSTAND WHY THERE'S SO MUCH
MORE VARIABILITY IN THE CAPSID
PROTEIN OF THE ALPHAVIRUSES IN
THE NEW WORLD VIRUSES VERSUS OLD
WORLD.
>> OH, SO WE DON'T KNOW.
WE HAVE FINISHED NOW A
PHYLOGENETIC ANALYSIS, AND IT IS
AMAZING.
ALPHAVIRUSS STARTED AND WE HAVE
NOW RECONSTRUCTED SEVERAL
ANCESTORS AND STARTED VARIABLE.
OUR GUESS WAS AT THE BEGINNING
HIGHLY PATHOGENIC, AT SOME
POINT, SOMETHING HAPPENED THAT
ALPHAVIRUSES BECAME LESS
PATHOGENIC.
AND A WAY TO -- OUR HYPOTHESIS
IS THAT THAT LED THEM TO
STABILIZE A CAPSID AND THAT
ALLOWED THEM TO SPLIT ALL
OVER -- SPREAD ALL OVER THE
WORLD.
INTERACTIONS IN PAKISTAN AND
UGANDA, IT'S UNCANNY, SUSTAINED.
BUT NOT IN THE AMERICAS.
AND WE THINK THAT HAS TO DO WITH
DEGREE OF PATHOGENICITY, THEY
MADE MISTAKES, IN AMERICA, TO
HAVE ENOUGH PROBABILITY TO
INFECT ANOTHER HOST.
>> SO IS IT HUMAN HOST OR ARE
THESE IN MONKEYS, NEW WORLD
MONKEYS?
>> OH, IN THE NEW WORLD IT'S
MAINLY HORSE.
HORSES, THIS IS A BIG PROBLEM
FOR PEOPLE THAT LIKE RACES.
BUT THEY CAN INFECT MONKEYS.
THEY CAN INFECT EVEN SEA LIONS.
TROPISM IS REALLY WIDE.
>> THANK YOU.
>> THANK YOU VERY MUCH.
THANK YOU, ALAN.
>> THANK YOU TO ALL THE SPEAKERS
FOR THE MORNING SESSION, REALLY
FANTASTIC.
WE'RE ON BREAK UNTIL 2:15.
THERE'S A CAFETERIA TO YOUR LEFT
AS YOU LEAVE THE MAIN DOORS
HERE.
WE'LL SEE YOU BACK HERE AT 2:15
THE NEXT SPEAKER IS A GOOD
FRIEND, AN OLD FRIEND, AND ONE
OF THE FEW COLLEAGUES WHO
PERSISTS IN STUDYING MURINE
LEUKEMIA VIRUS DESPITE THE
DIFFICULTIES IN GETTING FUNDING
FOR A VIRUS THAT DOESN'T INFECT
HUMANS.
MONICA HAS MADE MANY SEMINAL
CONTRIBUTIONS TO MURINE LEUKEMIA
VIRUS BIOLOGY. I ALWAYS
PARTICULARLY GO BACK TO HER WORK
ON THE FACT THAT, UNLIKE THE
PARADIGM HIV, MURINE LEUKEMIA
VIRUS DO NOT ENTER THE NUCLEUS.
THEY HOOK UP BY A COMPLETELY
DIFFERENT KIND OF MECHANISM. I
FEEL THAT A LOT OF MY WORK
RELATES TO THAT BASIC FACT ABOUT
MLV, WHICH SHE ELUCIDATED.
ANYWAY, I DON'T KNOW WHAT SHE'S
GOING TO TALK ABOUT TODAY. HER
TITLE IS MYSTERIOUS AND ELUSIVE,
FROM MLV TO EX MACHINA. WE'LL
FIND OUT WHAT THAT MACHINES.
WELCOME, MONICA. >> SO I
DECIDED NOT TO TALK ABOUT MY
RESEARCH DIRECTLY AND TALK MORE
ABOUT MY COMMUNICATIONS WITH
ALAN THROUGH E-MAILS AND THROUGH
STUDYING MLV.
I THINK THIS IS PROBABLY THE
LAST COLLECTION OF PEOPLE WHO
HAVE ACTUALLY WORKED ON THE
VIRUS AND STILL MIGHT REMEMBER
THE RESTRICTION MAP.
SO ADD AN AS RESEARCHER, MENTOR,
FRIEND, AND MOVIE CRITIC
BECAUSE, AS YOU'LL SEE, A LOT OF
THE E-MAILS WERE ABOUT WHAT
MOVIES WE'RE WATCHING.
EVERYONE HAS SEEN THIS TYPE OF
PICTURE BEFORE.
THIS IS A HYBRID BECAUSE I KNOW,
JONATHAN, THAT WAS A GREAT
PICTURE, BUT I KNOW YOU NEVER
SAT IN THE FRONT ROW, SO I
PLACED YOU IN YOUR CORRECT SPOT.
IN THE BACK RIGHT SECTION
CORNER.
WE MISS YOU THERE.
AND STEVE ALSO WAS PLACED IN THE
FRONT, BUT USUALLY IF YOU WERE
LOOKING FOR STEVE, HE'S SORT OF
IN THE BACK RIGHT HAND CORNER
WHEN HE ACTUALLY SHOWS UP.
AND OF COURSE, WE HAVE THE FRONT
ROW PEOPLE, WHO STILL ARE IN THE
FRONT ROW, BUT IF YOU REALLY
WERE LOOKING FOR ALAN, HE REALLY
WAS IN THAT RIGHT HAND SECTION,
A FEW ROWS IN, A FEW ROWS BACK,
AND THAT'S WHERE I'VE BEEN
SITTING RECENTLY.
I APOLOGIZE TO EVERYONE ELSE WHO
ACTUALLY SITS IN OTHER PLACES,
BUT IF YOU'RE ON THE LEFT-HAND
SIDE OF THE AUDITORIUM, I
USUALLY DON'T KNOW WHERE YOU
SIT.
(LAUGHTER)
IF YOU'RE LOOKING FOR ALAN IN
THE AUDITORIUM, THAT'S WHERE YOU
WOULD FIND HIM.
EVERY YEAR I REALLY WOULD --
THIS IS TRUE.
EVERY YEAR I WOULD GO TO COLD
SPRING HARBOR AND SAY TO MYSELF,
I WONDER WHAT ALAN REIN IS
WORKING ON.
IT WAS ALWAYS SOMETHING NEW AND
DIFFERENT.
IT WASN'T LIKE YOU'RE WORKING ON
RT ALL THE TIME.
HE ALWAYS CAME UP WITH SOMETHING
DIFFERENT.
AND THE KEY POINT FOR ME IS LIKE
SOMETIMES, ESPECIALLY IF YOU
WORK ON MLV AND YOU SEE A PAPER
LIKE THIS AND IS SAYS, OH,
GLYCOGAG EFFECTS OF VIRUS ENTRY,
DEPENDING WHAT SET OF
GLYCOPROTEINS YOU WORK ON, IT
SENT ME INTO A PANIC BECAUSE I
WORK ON GLYCOPROTEIN PARTICLES,
AND I WORK ON TARGETING
RETROVIRAL PAR I CAN LS BY USING
DIFFERENT ENVELOPES.
AND WE USE DIFFERENT PLASMA
SYSTEMS, AND I'M SITTING THERE
SAYING, OH, MY GOD, WHICH ONE OF
OUR PLASMA SYSTEMS EXPRESS
GLYCOGAG?
SO HERE ARE TWO FREQUENTLY USED
EXPRESSION SYSTEMS.
ONE WAS FROM THE COSSET LAB, AND
IT'S CALLED PCEB.
THE OTHER ONE IS A PCGP FROM THE
KINGSMEN LAB, AND WHICH ONE OF
THESE OR NEITHER OF THESE OR ALL
OF THEM EXPRESS GLYCOGAG?
ANYBODY?
YOU HAVE A 50 PERCENT CHANCE OF
GETTING THE RIGHT ANSWER.
THE BOTTOM ONE, OKAY, YES.
GIVE HER THE AWARD.
BECAUSE IT'S HOW THEY WERE MAD.
SO PCEB WAS MADE FROM MAKING A
DELETION.
IT'S THE MSC SITE TO PST SITE,
AND IF YOU'RE A REAL EXPERT,
YOU'LL SAY THERE ARE TWO PSII
SITES, WHICH ONE?
WHEREAS CGP WAS MADE BY MOVING A
FRAGMENT TO EXPRESS IT AT THE
CMV PROMOTER.
AND JUST FORTUITOUSLY, THE SITE
THAT THEY PICKED WAS JUST A
STREAM OF WHERE GLYCOGAG STARTS.
JUST TO BRING YOU BACK TO WHAT
RESEARCH WAS LIKE IN THE EARLY
'80s, WHEN I JOINED STEVE'S
LAB, THIS WAS THE SEQUENCE THAT
HE GAVE US.
IF YOU NOTE THAT GAG, THERE'S A
FAINT ERROR, SO IF YOU REALLY
WANTED TO KNOW THE SEQUENCE OF
GAG, YOU HAD TO COUNT THAT IN
THREES, AND YOU CAN SEE VERY
HIDDEN THERE PENCILED IN, IT
SAYS WHERE GLYCOGAG STARTS, AND
YOU COULD SEE HERE THAT BAL 1,
WHICH IS NOW MSC 1 TO PST, IF
YOU DID THAT INCISION, WRITTEN
IN MY HANDWRITING ON THE LEFT,
YOU CUT OUT GLYCOGAG.
BUT IF YOU STARTED THAT XMA 1
SITE, YOU WOULD JUST LIKELY HAVE
GLYCOGAG INCLUDED IN IT.
SO IF YOU WERE INTERESTED IN
WORKING WITH A VECTOR, YOU
SHOULD BE WORKING IN ONE THAT
STUDIES PARTICLE ENTRY, IT'S THE
BOTTOM ONE TO USE.
OF COURSE I DIDN'T REMEMBER
THAT, AND WE'RE STILL WORKING
WITH PCEB, AND I WENT INTO A
PANIC AGAIN.
ALAN FREQUENTLY GETS ASKED TO
WRITE STORIES ABOUT SCIENTISTS
WHO PASSED AWAY, AND HE DID
WRITE THIS ONE ABOUT STEPHEN
ROROSZLAN, AND HE SENT THIS TO
PEOPLE WHO HAD STORIES ABOUT
STEPHEN OROSZLAN, AND I WROTE
THIS STORY ABOUT, WHEN WE FIRST
STARTED IN STEPHEN GOFF'S LAB,
WE WEREN'T VERY ADVANCED ABOUT
WHERE PROTEINS STARTED AND
ENDED.
SO WE HAD NO IDEA WHERE
PROTEINED STARTED AND ENDED, SO
WE PICKED A SEQUENCE AND PUT IT
ON THE END AND TRIED TO PICK THE
MOST STABLE PROTEIN, FIGURING
THE MOST STABLE SOLUBLE PROTEIN
IS PROBABLY THE RIGHT THING, AND
THAT'S WHAT WE WORKED WITH.
THEN I WENT TO COLD SPRING
HARBOR FOR THE FIRST TIME, AND
STEPHEN OROSZLAN WAS THERE,
DESCRIBING, I THINK, THE
TERMINII OF RT, AND I
IMMEDIATELY HAD TO FIND HIM.
I FOUND HIM IN THE HALLWAY IN
FRONT OF THE BAR, WE ALL KNOW
WHERE THAT IS, AND I CORNERED
HIM TO FIND OUT WHERE THE REAL
SEQUENCE WAS, AND HE TOLD ME
THERE WOULD BE A MANUSCRIPT
COMING OUT VERY SOON.
IT WAS ACTUALLY RELEASED RIGHT
AFTER THE MEETING, SO HE
ACTUALLY DID KNOW THE ANSWER,
BUT HI TO WAIT FOR THE PAPER TO
COME OUT.
IT TURNS OUT WE WERE PRETTY
CLOSE TO THE REAL AUTHENTIC
TERMINI.
I WROTE THAT STORY TO ALAN, AND
I COULD HEAR HIS VOICE SAYING,
MONICA, THANKS.
THAT'S A VERY NICE LITTLE
ANECDOTE.
THE REST OF IT BASICALLY SAYING
IT'S PROBABLY NOT GOING TO BE
INCORPORATED INTO IT.
(LAUGHTER)
BUT THE BOTTOM LINE IS ALSO A
CLASSIC ALAN.
THANKS AGAIN AND BEST REGARDS.
SO THIS WAS THE SUMMER OF THE
THING THAT ALAN WROTE ABOUT
STEVE OROSZLAN, AND THREE
QUARTERS OF IT IS ABOUT HIV, BUT
THE VERY LAST PARAGRAPH IS ABOUT
DESCRIBING WHEN STEVE OROSZLAN
FOUND A SEQUENCE OF P12, AND IT
HAD A PPPY SFENS IN IT, AND
STEVE SAID HE ELICITED A LOUD
SIGH OF AMAZEMENT AND INCLUDING
AN EXPLETIVE, TURNS OUT THAT'S
THE LAST DOMAIN AND P12.
SO I WAS AT LEAST HAPPY THAT P12
WAS INCORPORATED INTO THAT
STORY.
THEN I PUBLISHED OUR WORK ON
P12, IN WHICH WE TOOK OUT THE
REGION WE THOUGHT WAS THE
TETHERING DOMAIN AND INSERTED IN
TETHERING DOMAINS FROM LOTS OF
DIFFERENT VIRUSES AND FOUND OUT
THAT THEY SUBSTITUTED.
BUT MORE TOUCHING WAS THAT ALAN
WROTE THE COMMENTARY ABOUT THIS
STUDY, AND IT REALLY WAS
SIGNIFICANT TO ME THAT ALAN
WROTE THIS COMMENTARY ABOUT OUR
WORK, SO THANK YOU.
I HAVE SOME MORE POLITE E-MAILS
FROM ALAN.
USUALLY WHEN HE'S LOOKING FOR A
REAGENT, AND YOU CAN TRY TO
GUESS WHAT HE'S WORKING ON
BECAUSE I HAD NO IDEA WHY I WAS
ASKING FOR THESE REAGENTS.
THIS IS ONE HE WROTE TO LORRAINE
AND MYSELF, LOOKING FOR
REPLICATION COMPETENT MLV WITH A
XENOTROPIC EMV.
AND I DON'T HAVE IT.
HERE'S A CLASSIC ALAN ANSWER,
OKAY, BEST REGARDS ANYWAY.
HERE'S ANOTHER ONE FROM OCTOBER
9th, 2020.
LOOKING, ASKING ABOUT P50.
FOR THOSE OF YOU WHO WORKED IN
MLV, THERE'S THIS P50.
IT HAS MA, P12, CA, AND SPLICES
INTO THE C TERMINUS OF IN.
SO HE ASKED ME IF I HAD ANY
ANTISERA TO TRY, WHICH IS
ANTI-MA OR ANTI-IN.
IF YOU LOOK AT THE DATE, IT'S
OCTOBER 9th, 2020, WHEN BOTH OF
US WERE AT HOME BEING LOCKED OUT
OF OUR LABS DURING COVID.
I SAID, ALAN, IT'S GOING TO TAKE
A LITTLE DIGGING IF I STORE IT
ANYWHERE.
I'VE BEEN WORKING AT HOME SINCE
MARCH.
I LITERALLY WAS GIVEN TWO HOURS
TO LEAVE THE LAB AND NEVER COME
BACK FOR A YEAR.
I ACTUALLY DIDN'T TAKE MY OWN
IPs WITH MLV SERA.
ANYWAY, THAT'S HOW WE TRIED TO
DO SCIENCE DURING COVID.
HE SAID, I KNOW WHAT YOU MEAN.
I'M HOME SEPARATED FROM MY
FILES.
I NEVER DID -- I APOLOGIZE.
I NEVER DID GO BACK TO LOOK FOR
THOSE, BUT I DO WANT TO HAVE A
COMMERCIAL HERE SAYING, IF
YOU'RE LOOKING FOR MLV
ANTIBODIES, CHRISTINE KOZACK HAS
FULL FROM THE ONES FROM THE
REPOSITORY.
IF YOU ARE LOOKING FOR THOSE,
THEY'RE GREAT ANTIBODIES.
THEY'RE PROBABLY NICE VIALS, AND
WE'D HATE TO HAVE THEM THROW
OUT.
IF PEOPLE KNOW WHAT TO DO WITH
THIS SERUM, WE'D CERTAINLY LIKE
THEM TO HAVE A HOME BEFORE
CHRISTINE RETIRES OR ANYONE
ELSE.
I DO HAVE AN APOLOGY.
I'M SURPRISED THAT ALAN ASKED ME
FOR REAGENTS BECAUSE ACTUALLY IN
2015 I HAD SENT HIM SOME SAMPLES
OF SOME P12 MUTANTS, TRYING TO
LOOK AT THE SHAPE OF THE CAPCID.
UNFORTUNATELY, THIS IS WHAT I
GOT BACK WHERE THEY SAID VERY
LAST SIX IMAGES DISPLAYED,
POSSIBLE MICROPLASM OR YEAST
CONTAMINATION.
SO I AM HORRIFIED STILL TO THIS
DAY THAT I SENT SAMPLES THAT
MIGHT NOT HAVE BEEN AS CLEAN AS
THEY EVER WERE.
LITERALLY, MY LAB HAS NEVER
TAKEN ANY CULTURE AT A PLASMA
SCENE SINCE THEN BECAUSE WE
DON'T EVER WANT THAT TO HAPPEN.
MY APOLOGIES IF WE CONTAMINATED
ANYTHING.
I'M STILL SHAKING FROM THAT.
HERE'S ANOTHER E-MAIL THAT WAS
JUST LAST YEAR.
WE WERE AT THE 40th ANNIVERSARY
OF HIV, AND I WAS SPEAKING WITH
AT ALLEGE ARE ALICE AND WES.
THE SOLUTION WAS LET'S ASK ALAN
REiNS.
THIS WAS AT 4:54 COFFEE BREAK.
I SENT HIM AN E-MAIL.
LITERALLY FOUR MINUTES LATER WE
GOT THE ANSWER.
THE ANSWER TO THE QUESTION IS
YES, AND I'M HOPING TO PUBLISH
THE PAPER BEFORE I DIE OF OLD
AGE, GLAD SOMEONE IS INTERESTED.
SO I'M HAPPY TO SAY THAT THIS
PAPER DID COME OUT IN MBIO IN
JULY.
SO, YES, THERE'S THE ANSWER.
>> HE'S WITH US.
>> RAISE YOUR HAND.
THANK YOU.
THIS IS A SLIGHT DEVIATION FROM
THE TOPIC.
I WENT TO MY SCHOOL'S TRYING TO
DISCUSS OUR NEW ENTERING CLASS
OF MEDICAL STUDENTS, AND THIS
WAS ONE OF THEIR SLIDES ABOUT A
SNAPSHOT OF THE PEOPLE GOING OR
ENTERING ROBERT W. JOHNSON
MEDICAL SCHOOL CLASS, AND THE
QUESTION WAS HOW MANY PEOPLE
SPEAK DIFFERENT LANGUAGES AT
HOME.
THEY SAID 40 PERCENT OF THE
CLASS SPEAKS ONE OR TWO
DIFFERENT LANGUAGES AT A NATIVE
OR ADVANCED LEVEL AT HOME.
IT'S REALLY AN IMPRESSIVE STORY,
AND IT BASICALLY SHOWS YOU WHO'S
GOING IN TO BE CLINICIANS THESE
DAYS.
SO WHEN I SAW THIS, I'M SURE
EVERYONE ELSE HERE IS THINKING,
OH, WHAT LANGUAGES WERE SPOKEN
IN MY HOUSE?
BOTH OF MY PARENTS WERE BORN IN
BROOKLYN, WHICH SOME PEOPLE
MIGHT THINK IS A FOREIGN
LANGU
LANGUAGE, AND IF YOU REALLY WANT
TO TALK ABOUT IT, WE CAN HAVE IT
OVER COFFEE.
DESPITE BEING BORN IN BROOKLYN,
MY MOM WAS A YIDDISH-IST, AND
SHE TRIED HER HARDEST TO KEEP
THIS LANGUAGE FROM DYING, SO SHE
SENT ME TO YIDDISH SCHOOL FOR 12
YEARS, MAYBE 15 YEARS.
WHEN ALAN HEARD THIS, HE STARTED
SENDING ME NEWSLETTERS FROM KLEZ
CALIFORNIA, AND I THOUGHT I KNEW
EVERY YIDDISH ORGANIZATION EVER
BECAUSE OF MY MOM, BUT I
ACTUALLY HAD NEVER HEARD OF KLEZ
CALIFORNIA.
HE SENT ME ONE, AND I WAS
LOOKING THROUGH IT.
SOME OF YOU MIGHT HAVE HEARD OF
PETE SEGER.
THE OTHER PERSON IN THE PICTURE
IS RUTH RUBIN, WHO IS A VERY
FAMOUS FOLKLORIST IN YIDDISH
MUSIC, AND MY MOM KNEW HER AND
WORKED WITH HER DIRECTLY.
SHE REALLY IS ONE OF THESE
FAMOUS PEOPLE.
WE HAD HER BOOKS AT HOME.
WHEN I SAW THIS, I WENT AND I
WATCHED THIS LINK, AND THEY WERE
SINGING THIS SONG.
JUST LIKE LOOKING BACK, WHY WAS
I DOING THIS?
WHAT WAS MY FRAMEWORK THAT I
STARTED TO GO BACK AND LOOK AT
YIDDISH SONGS FROM THE EARLY
1900s?
I LOOKED AT THE DATE OF THE
E-MAIL I SENT TO ALAN, NOVEMBER
7, 2022.
IT SAYS, WITH THE ELECTIONS
TOMORROW, IT'S HARD TO WATCH TV,
AND I'VE BEEN RESORTING TO EASY
WATCHING LIKE BRITISH BAKING
SHOW TWO.
>> BAKEOFF, ISN'T IT?
>> IN ENGLAND, IT'S CALLED THE
BAKEOFF, BUT IT'S THE BRITISH
BAKE SHOW TWO.
AND IT JUST REALLY HIT ME
BECAUSE HERE I AM TWO YEARS
LATER, I CAN'T WATCH THE NEWS.
I'VE BEEN WATCHING THE BRITISH
BAKE SHOW TWO, AND THERE'S A NEW
EPISODE TONIGHT.
SO IF YOU ALSO CAN'T WATCH THE
NEWS, I'D HIGHLY RECOMMEND
WATCHING THE SHOW.
UNLIKE AMERICAN TV, WHERE PEOPLE
COMPETE WITH EACH OTHER AND BACK
STAB EACH OTHER.
IN THIS SHOW, THEY ACTUALLY DO
COMPETE EACH OTHER, BUT THEY
ACTUALLY HELP EACH OTHER AND
THEY LIKE EACH OTHER.
I REALLY THINK THAT'S WHERE WE
SHOULD BE GOING.
I ALSO HEAR PUT A MOVIE
RECOMMENDATION THAT I WATCHED,
WHICH IS THE PATIENT, WHICH ALSO
WE COMMUNICATE WHERE YOU CAN
STREAM IT, SO IT WAS ON HULU.
IF YOU'VE EVER WATCHED THAT, IT
WAS PRETTY INTENSE IN OTHER
WAYS.
SO I WAS LOOKING FORWARD TO SOME
MORE SUGGESTIONS, AND HERE I AM.
SO WHAT ABOUT THIS SONG, WHY DID
I KNOW THIS SONG?
THIS IS THE TRANSLATION OF IT.
IT TALKS ABOUT THESE TWO TOWNS
THAT HAVE VERY FUNNY NAMES,
SVASTOPOL AND SIFARUPPUL AND
THIS TOWN NEARBY CALLED
ZHANKOYE, AND IT'S A FARM WHERE
GRANDPA DRIVES THE TRACTOR AND
GRANDMA RUNS THE CREAM
EXTRACTOR.
I SAID, WHERE IS THIS IDYLLIC
PLACE, AND YOU LOOK AT THE MAP,
AND WE'RE RIGHT IN THE MIDDLE OF
CRIMEA AND UKRAINE, NOT QUITE
THESE DAYS AN IDYLLIC PLACE.
SO I WANT TO THANK ALAN FOR
GOING DOWN THIS TRIP TO MEMORY
LANE.
I HEARD A STATEMENT, WHEN IT
COMES TO TIME TRAVEL, SCIENTISTS
ALWAYS CHOOSE THE FUTURE BECAUSE
WE CANNOT GO BACK.
SO NOW WE'LL GET INTO SOME OTHER
MOV
MOVIES.
ACTUALLY COMING HERE, I WAS
TRYING TO LOOK UP MY TSA NUMBER
BECAUSE SOMEHOW IT WASN'T ON MY
TICKET AND I WASN'T GOING TO
TAKE MY SHOES OFF AND TAKE MY
COMPUTER OUT.
I WAS SEARCHING THROUGH OLD
NOTES THAT I HAD ON MY PHONE,
AND I ACTUALLY FOUND THE NOTE
FROM 2016 THAT SAYS REIN EX
MACHINA.
THAT WAS ONE OF THE FIRST MOVIES
THAT HE HAD SUGGESTED THAT I
WATCH.
I DON'T KNOW HOW MANY OF YOU
WATCHED IT.
NO?
ANYWAY, IT'S A GREAT MOVIE.
IT'S AI BEFORE THERE WAS AI, SO
IF YOU THINK OF 2001 AND HAL AS
DEPICTING THE FUTURE, WELL, EX
MACHINA IS REALLY PREDICTING
WHERE WE ARE THESE DAYS.
THE RUNNING LINE IS TO ERASE THE
LINE BETWEEN MAN AND MACHINE IS
TO OBSCURE THE LINE BETWEEN MEN
AND GODS.
DON'T YOU WANT TO WATCH THAT
MOVIE?
MIRACULOUSLY, IT'S NOW STREAMING
ON AMAZON.
IT JUST CAME BACK TO BE ON
AMAZON.
SO IF YOU DON'T WANT TO WATCH
THE BRITISH BAKE SHOW, YOU CAN
WATCH EX MACHINA AND AFTER THAT
RECOMMENDATION FROM ALAN, I
TRUST ALL OF HIS
RECOMMENDATIONS.
HERE'S MORE MOVIE SUGGESTIONS
FROM ALAN.
AND SOME OF HIS COMMENTS.
OUTLANDER IS COMPELLING, NOT TO
SAY ADDICTIVE, BUT I THINK HE
DOESN'T WATCH IT ANYMORE.
AND THEN THE TRIAL OF THE
CHICAGO SEVEN ON NETFLIX, TOOK
ME RIGHT BACK TO 1968.
AMAZING, MUST WATCH.
THAT WAS A VERY GOOD MOVIE, I
DID WATCH IT.
THE DANISH GIRL, NOT TO BE
MISSED.
I AGREE.
THE SIXTH SENSE IS DYNAMITE.
THEN SOME OTHER ONES THAT I HAD
FROM DIFFERENT E-MAILS,
OPPENHEIMER, THE 3 BODY PROBLEM,
AMERICAN FICTION, DISOBEDIENCE,
THE QUEENS GAMBIT, AND ALAN
WROTE, THE SOPHIE LOREN MOVIE,
AND IF YOU ACTUALLY GOOGLE THE
SOPHIE LOREN MOVIE, YOU'LL
ACTUALLY FIND THE TITLE OF IT.
IT ACTUALLY DOES WORK.
SO I WATCHED THEM, AND I AGREE.
SORT OF IN CONCLUSION, I THINK
ALAN IN HIS RESEARCH AND IN HIS
MOVIES ARE ALWAYS ON THE CUTTING
EDGE, AND AS YOU KNOW, THE NOBEL
PRIZE IN CHEMISTRY THIS YEAR WAS
ABOUT AI AND THE DEVELOPMENT OF
AI MODELS TO STUDY PROTEIN
PREDICTIONS.
I'M NOT TALKING ABOUT MY OWN
WORK, BUT WE ACTUALLY DURING
COVID, BECAUSE WE COULDN'T DO
WET BENCH WORK, WE ACTUALLY
STARTED TO DO PROTEIN PREDICTION
STUDIES, AND WE HAVE A PAPER NOW
COMING OUT -- WELL, IT WAS
SUBMIT
SUBMITTED.
STUDYING HOW RECEPTOR
TRANSPORTERS WHO HAVE TWO
DIFFERENT CONFIRMATIONS.
IF YOU USE ALPHA FOLD, YOU'LL
PROBABLY ONLY GET ONE OF THE TWO
CONFIRMATIONS, AND WE WORKED OUT
A WAY, A METHOD TO GET BOTH
INWARD AND OUTWARD CONFIRMATION
FOR THIS -- FOR ANY PROTEIN, BUT
WE STARTED THIS BECAUSE WE HAD
IDENTIFIED -- RERANDOMIZED THE
RECEPTIVE BINDING DOMAINS OF
LEUKEMIA VIRUSES, WE IDENTIFIED
FUNCTIONAL ENVELOPES, AND WE
IDENTIFIED THE RECEPTORS.
ONE OF THEM IS THIS SLC-14
PROTEIN.
IT'S A RECEPTOR, AND WE STILL
DON'T KNOW WHAT IT TRANSPORTS,
BUT WE STARTED TO DO STRUCTURAL
WORK, PREDICTIONS OF IT DURING
COVID, AND NOW WE HAVE THIS
METHOD THAT LOOKS PRETTY GOOD
AND WE'RE ABLE TO PREDICT THE
DIFFERENT CONFORMATIONS.
SO I WANT TO THANK ALAN FOR
OPENING MY EYES TO AI BACK IN
2015 WITH EX MACHINA, AND I JUST
APPRECIATE EVERYTHING HE'S DONE
AS A RESEARCHER, MENTOR, FRIEND,
AND MOVIE CRITIC.
THANK YOU.
>> WHAT ARE YOU WATCHING NOW?
>> THE GREAT BRITISH BAKING
SHOW EVERY FRIDAY.
>> WHO ARE YOU HOPING TO WIN
THE BAKEOFF?
>> I DO LIKE DYLAN.
>> HE'S CUTE.
>> THAT WAS ABOUT THE NICEST
TALK I'VE EVER HEARD.
THANK YOU.
SO OUR NEXT SPEAKER IS ONE OF A
SERIES OF SPEAKERS WHO NEEDS NO
INTRODUCTION.
IT'S STEVE GOFF, WHO HAS BEEN AT
COLUMBIA FOR PROBABLY A FEW
DECADES.
HE AND I WERE BOTH INTERESTED IN
HOW RETROVIRUSES WORK BEFORE HIV
CAME ALONG, AND THEREFORE, WE
BOTH BEGAN WORKING WITH MURINE
LEUKEMIA VIRUSES.
I'LL JUST SAY IN 25 WORDS OR
LESS THAT STEVE HAS REALLY FOUND
ONE AMAZING SURPRISE AFTER
ANOTHER.
HE ALWAYS, LIKE MONICA STRANGELY
WAS SAYING ABOUT ME, HE ALWAYS
COMES UP WITH SOMETHING QUITE
NEW AND UNEXPECTED, CONNECTIONS
BETWEEN THE VIRUSES THAT WE KNOW
A LOT ABOUT AND THE CELLS WHICH
ARE STILL LARGELY BLACK BOXES,
AT LEAST COMPARED TO THE
VIRUSES.
I THINK I'LL JUST LEAVE IT AT
THAT.
STEVE, THANK YOU FOR COMING.
I CAN'T IMAGINE WHAT YOU'RE
GOING TO SAY, BUT I'M LOOKING
FORWARD TO IT.
>> ALAN THANK YOU.
IT IS REALLY A PLEASURE.
LET'S GET STARTED.
I'M GOING TO DO TWO THINGS IN
THE NEXT FEW MINUTES.
I'LL SPEND SOME TIME KIND OF
DOING A LITTLE HISTORY, AND IF
THERE'S AN IMPORTANT TAKE AWAY
FROM THIS, IT'S HOW MUCH ALAN
HAS BEEN A ROLE MODEL FOR ME,
FOR EVERYTHING I'VE DONE HAS
REALLY FOLLOWED IN ALAN'S FOOT
STEPS.
THEN I'LL TRY TO GIVE YOU A
BRIEF SCIENCE TALK ABOUT THINGS
THAT ARE GOING ON IN THE LAB,
WHICH I THINK ALAN WOULD LIKE TO
HAVE ALWAYS ALWAYS REAL SCIENCE
BUILT IN.
THIS IS THE TRUTH.
THIS IS ALWAYS THE TRUTH BECAUSE
WE OFTEN HAVE THINGS THAT WE'RE
INSPIRED BY BECAUSE WE WOULDN'T
KNOW THIS IS A COOL TOPIC TO
WORK ON IF IT WEREN'T FOR ALAN.
A FEW EXAMPLES ARE NC MUTANTS,
THE GAG-POL READTHROUGH,
MYRISTATE AND L DOMAINS.
ALL OF THESE YOU'VE HEARD ABOUT,
AND ALL OF THESE ALAN INSPIRED
US TO WORK ON BECAUSE HE MADE
SOME DISCOVERY ABOUT THESE.
I THINK LOOKING BACK AT THE
HISTORY PART, THE FIRST TIME I
MET ALAN MAYBE, YOU MAY CORRECT
ME, WAS WHEN I WAS JUST STARTING
OUT AS A POST DOC IN DAVID
BALTIMORE'S LAB.
THIS WOULD HAVE BEEN 1979, AND I
THINK HE WAS ON A SITE VISIT
PANEL REVIEWING A GRANT, AND I
THINK FOR SOME REASON HE HAD ME
PRESENT A REVIEW STUDY TO THE
PANEL.
SO I WAS TERRIFIED, OF COURSE.
THIS WAS ME JUST STARTING INTO
RETROVIRUSES FROM SB-40.
SO OF COURSE I HAD READ ALL OF
ALAN'S PAPERS AND WAS
INTERESTED, ACTUALLY EVEN THEN,
IN THINGS LIKE FV-1 WHICH
INTRIGUED ME WITH MLV, AND THERE
WAS THIS GUY ALAN REIN, WHO I
HAD TO PRESENT AND BE REVIEWED
BY.
SO I DID, AND DAVID OF COURSE
GOT THE GRANT, THANK YOU, BUT
I'M SURE THROUGH NO VALUE OF
ANYTHING I SAID BECAUSE I HAVE
NO CLUE WHAT I SAID IN THAT
PRESENTATION, BUT THERE WAS
ALAN.
OVER THE YEARS SINCE THEN, I
HAVE GOTTEN REAGENT AFTER
REAGENT AFTER REAGENT FROM HIM,
AND EVERY TIME HE SENDS ME
EXACTLY WHAT I ASK FOR, AND IT'S
ALWAYS PERFECT AND IT'S JUST
WHAT I NEEDED.
WHEN I STARTED OUT, THE FIRST
THING I FELT I HAD TO DO WAS SET
UP A PLAQUE ASSAY BECAUSE THAT'S
WHAT I THOUGHT YOU NEEDED FOR
VIROLOGY.
THAT IN THOSE DAYS WAS THE XE
PLATH ASSAY, WHICH WAS A TRICKY
ASSAY, AND ALAN SENT THE XE
CELLS AROUND AND THE VERY TRICKY
PROTOCOL HOW TO DO IT, AND WE
GOT GOING.
THAT WAS HOW WE STARTED OUT
ASSAYING VIRUS.
IT WAS SO HARD THAT I WANTED
BETTER ASSAYS, SO WE DID SET UP
BETTER ASSAYS LATER THAT WERE
EASIER.
BUT ALL OF THE CONCEPTS THAT
FLOWED INTO MY WORK WERE THE
VERY FIRST RECOMBINANT CLONES
THAT ALLOWED YOU TO DO THINGS,
AND ALAN AND I RAN WITH THAT AS
FAR AS WE COULD, AND IT'S BEEN A
GOOD RIDE.
LET ME TALK SOME OF THE
EXAMPLES.
YOU'VE SEEN SOME OF THIS BEFORE.
IN THE EARLY ABILITY TO MAKE
MUTANTS AND CLONE DNA WAS WHAT
ALLOWED FOR THE WORK TO SHOW
THAT NC WAS REALLY IMPORTANT AND
WHAT IT DID AND HOW IT WAS
INVOLVED IN PACKAGING, AND THIS
WAS ONE OF THE BIG PAPERS FROM
BOB AND ALAN, AND WE FOLLOWED
CLOSELY UP, ALWAYS BEHIND OF
COURSE, TO DO SIMILAR THINGS
WITH CLAUDE TO LOOK AT NC
MUTANTS.
WE KNEW IT WAS GOING TO BE A
GOOD THING TO DO BECAUSE OF
ALAN.
HERE'S ANOTHER ONE WHERE IN VERY
EARLY DAYS WITH STEVE OROSZLAN,
BASED FIRST ON THE PROTEIN
SEQUENCES REALLY, THAT THIS
GAG-POL READTHROUGH THING HAD TO
BE IN FRAME AND INVOLVE
READTHROUGH OF THE STOP CODON,
AMAZING.
ONE OF THE THINGS THAT ALWAYS
BLEW ME AWAY, THIS WAS NOT
UNIVERSAL FOR ALL RETROVIRUSES,
SOMETIMES THEY DID FRAME
SHIFTING INSTEAD OF READTHROUGH,
THAT'S CRAZY, BUT THAT'S THE WAY
IT IS.
IT JUST SHOWS YOU THAT DIFFERENT
VIRUSES DO DIFFERENT THINGS, THE
SAME THING IN DIFFERENT WAYS.
WITH THAT, OF COURSE THEN YOU
HAVE A CLONE, SO YOU CAN
MANIPULATE THE NATURE OF THE
READTHROUGH FEATURES, AND ALAN
DID THAT WITH QD-11.
AND THERE WAS A READTHROUGH FOR
THAT, AND THOSE WERE SEMINAL
PAPERS, AND IN MY LAB THEY WERE
ABLE TO FOLLOW UP AND DO MORE.
FROM FEELING YOUR WAY AROUND,
LIKE FEELING THE ELEPHANT, YOU
LEARN WHAT'S REQUIRED.
IN THE END, YEARS LATER, THAT IS
ANOTHER COMMON THING THAT YOU
GET CONNECTED TO SERIOUS
STRUCTURE PEOPLE WHO HELP YOU
TRANS
TRANSLATE THE MUTATIONAL STUDY
YOU DID TO TURN THE LIGHT ON AND
ACTUALLY SEE THE STRUCTURE, AND
WE CONNECTED WITH PEOPLE LIKE
VICKY AND MIKE, AND THAT BECOME
THE PSEUDOKNOT, AND WE LEARNED
WHICH OF THESE FEATURES ARE
REALLY IMPORTANT.
IT HAS SOME AMAZING FEATURES.
IT TURNS OUT TO BE, THE
STRUCTURE ITSELF, TURNS OUT TO
BE INCREDIBLY PH DEPENDENT
BECAUSE THERE'S A SINGLE
PROTONAT
PROTONATED BASE THERE ON THE
RIGHT THAT CHANGES THE STRUCTURE
COMPLETELY BECAUSE OF FUNCTION.
THAT'S A CONCEPT OF ENSEMBLES IN
OUR STRUCTURES THAT MY NEW
COLLEAGUE AL HASHIMA MAKES FOR A
LIVING, AND THIS PSEUDOKNOT IS
THE PERFECT EXAMPLE OF THAT.
THERE'S STILL TONS TO LEARN
ABOUT HOW THIS WORKS, HOW IT
INTERACTS WITH THE RIBOSOME, HOW
IT TELLS THE RIBOSOME TO DO ALL
THE THINGS IT MUST BE TELLING IT
TO DO, BUT AGAIN THIS ALL
STARTED WAY BACK WITH OROSZLAN
AND ALAN'S PROTEIN SEQUENCE.
ANOTHER ONE IS THE WHOLE
INSTALLATION STORY, THE VERY
TERMINUS OF GAG.
THE GAG PROTEIN IS NOT
MYRISTYLATABLE, SO THE GAG DOES
NOT GET TO THE MEMBRANE AND DOES
NOT BUILD PARTICLES.
IT'S PROBABLY ONE OF THE
STRONGEST PHENOTYPES FOR
ANYTHING WITH A SINGLE AMINO
ACID CHANGE.
THAT JUST CRYSTALLIZED US TO
KEEP WORKING ON IT OVER THE
YEARS.
ONE OF THE HOLY GRAILS WAS,
WELL, IS THIS A PURELY
SPONTANEOUS THING THAT A
MYRISTYLATED PROTEIN IS PUT INTO
A CARRIER.
WE WENT LOOKING AND NEVER FIND A
CARRIER PROTEIN THAT DID IT, BUT
WE DID FIND BY PROTEOMICS, A
BINDING PROTEIN.
IT HAS THE PERFECT POCKET, IT
BINDS MYRISTASTE.
IT DOESN'T BIND NON-MYRISTATED
PROTEINS.
IT'S PROBABLY GOT A BROAD ROLE,
AND THAT CONNECTED US TO
STRUCTURE PEOPLE SAYING, WELL,
WE CAN CRYSTALLIZE THESE.
AND THERE'S THE MYRISTATE
SITTING IN THIS POCKET.
ALL AMINO ACIDS MAKE CONTACT,
AND THEY TOTALLY IMPACT THE
CO-IT OF THESE PROTEINS THAT SIT
IN THIS BLOCK.
THESE ARE THE KINDS OF THREADS
THAT ALL EMANATE FROM A STARTING
POINT.
I THINK THIS IS STILL RIPE FOR
LOTS MORE WORK.
IT LOOKS LIKE, IF ANYTHING, THIS
IS PROBABLY NOT A CARRIER
PROTEIN THAT DELIVERS IT TO THE
MEMBRANE.
IT'S PROBABLY NOT A NEGATIVE
REGULATOR OF MYRISTOYL
FUNCTIONS, INCLUDING A KNOCKOUT
WHERE THE CELLS MAKE THEM WAY
BETTER AT ASSEMBLING THE
PARTICLES, WHICH IS COOL.
THERE'S MORE AND MORE.
I HAVE TO SAY, IN SPITE OF ALL
THESE OVERLAPS, ALAN AND I HAVE
HARDLY EVER PUBLISHED TOGETHER.
THIS IS ONE OF THE FEW.
PEOPLE LIKE JOHN WILS AND OTHERS
DISCOVERED THE INTERCHANGE
L-DOMAINS, THE FAMOUS SEQUENCES
IN GAG THAT ARE NECESSARY TO
EXCHANGE PARTICLES.
YOU COULD INTERCHANGE THEM,
EXCHANGE THEM BETWEEN VIRUSES,
AND WE FOUND THAT WAS TRUE WITH
MLV, AND ALAN AND I GENERATED A
WHOLE SERIES OF CONSTRUCTS THAT
JUST GENERALIZED THAT PHENOMENON
TO MANY SITES FOR BINDING TO THE
MANY COMPONENTS OF THE S-CORE
MACHINERY.
I'M VERY PROUD TO HAVE THAT ONE
PAPER IN COMMON.
JUST TO WRAP UP THIS PART, I
THINK IT'S IMPORTANT TO REFLECT
ON WHY AND IN WHAT WAY ALAN HAS
BEEN SO IMPORTANT TO US ALL,
FIRST OF ALL, PROFESSIONALLY.
THE FIRST THING IS HE'S REVIEWED
MOST OF OUR PAPERS PROBABLY OF
ALMOST EVERYBODY IN THE ROOM.
CRITICALLY AND VERY OFTEN
POSITIVELY, THANKFULLY, AND HE'S
BEEN AN EDITOR OF ALL THE
IMPORTANT VIROLOGY JOURNALS LIKE
JV AND VIROLOGY.
AS AN EDITOR, I USE HIM ALL THE
TIME BECAUSE HE ALWAYS GIVES ME
BACK FAIR AND ACCURATE REVIEWS
OF ALL THE PAPERS THAT I GIVE
HIM, WHICH IS GREAT.
ALAN RUNS THE BIG MEETINGS LIKE
COLD SPRING HARBOR AND THE MANY
NUCLEAR CAPSID SYMPOSIA THAT
WILL BE RUN IN PRAGUE NEXT YEAR.
HE DOES GIVE AMAZING
PRESENTATIONS, INCLUDING AT THE
WEST COAST RETROVIRUS MEETING,
AND AS ALREADY MENTIONED, AT THE
2016 TALK, WHICH IS A CAREER
LIFETIME TALK, IT'S MENTIONED IN
LINKABLE, AND I WOULD ENCOURAGE
EVERYBODY TO SEE HIS TALK
BECAUSE IT'S REALLY A WALK DOWN
THE LANE OF MLV AND THE HISTORY.
TERRIFIC.
NOW THAT'S ALL I WANTED TO SAY
ABOUT MY MEMORIES OF THE TIME
WITH ALAN.
I JUST WOULD END THAT PART AND
JUST SAY THAT THE MOST CRITICAL
THING TO ME IS HE'S ALWAYS THERE
WHENEVER I NEED SOMETHING, WHICH
IS OFTEN.
I CANNOT TELL YOU HOW MANY TIMES
I'VE ASKED HIM FOR PROBABLY THE
SAME AMYTROPHIC GENOME, WHICH HE
SENDS TO ME.
HE CAN FIND THINGS IN HIS
FREEZER AND SEND THEM TO ME,
WHICH IS FABULOUS.
WE'VE SENT HIM A FEW THINGS,
WHICH IS NOT AS MANY AS HE'S
SENT TO US.
BEYOND THAT PROFESSIONAL THING,
IT IS ALSO IMPORTANT TO SAY HE'S
BEEN IMPORTANT TO ME PERSONALLY
AS A GOOD FRIEND.
I HAVE ALWAYS BEEN DELIGHTED TO
BE ABLE TO MEET UP WITH HIM,
USUALLY AT COLD SPRING HARBOR,
AND DISCUSS EVERYTHING ABOUT OUR
CAREERS AND OUR THOUGHTS.
SO THANK YOU, ALAN.
LET ME TURN TO A QUICK LITTLE
BIT OF SCIENCE BECAUSE I THINK
IT'S A GOOD THING TO DO.
ONE OF THE PROJECTS GOING ON IN
THE LAB IS ABOUT SUMOYLATION, SO
I'LL TELL YOU ABOUT THAT.
HOW DID I GET INTO THAT?
ACTUALLY, ONE OF THE WAYS I GOT
INTO THIS HAD TO DO WITH
JONATHAN A LITTLE BIT, WHICH WAS
IN CAPSID.
WE WERE LOOKING AT CAPSID
MUTANTS, AND ONE OF THE THINGS
WE WANTED TO KNOW IS WHAT ARE
SOME OF THE PROTEINS THAT
INTERACT WITH MLV CAPSID?
WE DID PROTEOMIC SCREENS AND GOT
A LOT OF HITS.
SOME OF THEM HAD TO DO WITH
UBIQUITINYLATION AND
SUMOYLATION.
WHICH IS ALL TRUE.
IT'S A BIG DEAL.
I THINK INCOMING CAPSIDES ARE
RECOGNIZED BY THE
UBIQUITINYLATION MACHINERY AND
SOMETIMES SUMOYLATED, BUT I
ALWAYS HAD THAT IN THE BACK OF
MY HEAD TO FOLLOW UP ON.
ACTUALLY WITH JONATHAN IN THAT
VEIN, WE DID A LOT OF TWO HYBRID
SCREENS.
WE DID CAPSID/CAPSID THAT
INTERACTS NICELY.
WE DID A LOT OF CAPSID SCREENS
THAT HAD TO DO WITH THESE GUYS
WITH THE UBIQUITINYLATION, AND
WE HAD FB-1, WHICH IS HORRIBLE
BIOCHEMICALLY, BUT IT TURNS OUT
TO BE WELL BEHAVED IN THE TWO
HYBRID SCREEN SYSTEM.
IT'S GIVEN US LOTS OF HITS.
IT DIMERIZED BEAUTIFULLY IN THAT
SYSTEM, WHICH WE USED THROUGH
MUTATIONS, AND IT INTERACTS WITH
THIS WHOLE UBUIQUITINE
MACHINERY.
IT TURNS OUT SOMEDAY, IF WE'RE
NOT CUT OFF FROM FUNDING, WE
SHOULD LOOK AT THIS MORE.
SO WE DECIDED TO LOOK AT SUMO
RECENTLY.
A NEW POST DOC LOOKED IT UP AND
SAID WE'RE GOING TO DO A NEW
PROTEOMIC SUMOYLATION WITH HIV.
IF YOU DON'T KNOW ALL ABOUT
SUMO, IT'S A UBIQUITIN LIKE
MODIFICATION.
IT CAN BE MONOSIMULATED OR
MULTIPLE.
THERE ARE FIVE SUMOs IN MAMMALS
AND PEOPLE.
THERE'S SUMO-1, WHICH IS
DISTINCT.
SUMO-2 AND 3, WHICH ARE ALMOST
IDENTICAL AND QUITE DIFFERENT
FROM SUMO-1, AND DIFFERENT GUYS.
THE WAY THEY'RE PUT ON PROTEINS
IS SIMILAR TO THE WAY UBIQUITIN
IS PUT ON PROTEINS.
THERE'S LIGASES, E1, 2, AND 3,
WHICH IS ALL IN PARALLEL TO WHAT
WE KNOW ABOUT UBIQUITIN.
THERE ARE MANY, MANY PROTEINS
THAT ARE SUMOYLATED.
WHEN ERR, THEY'RE NOT TAGGED AT
ALL THE WAY UBIQUITIN TAGS ARE.
THE MOST COMMON FEATURE THEY
INTERMEDIATE IS THE DELIVERY
INTO THE NUCLEUS, AND THEY HAVE
THESE SUMO-INTERACTION MOTIFS,
THESE SIMS, AND THEY DO A LOT OF
DIFFERENT THINGS IN THE CELL.
SO IT'S A BIG MODIFICATION.
THERE'S CERTAINLY A LOT OF
OPPORTUNITIES IN THE HIV
LIFECYCLE WHERE YOU COULD
IMAGINE SUMO BEING IMPORTANT.
IT'S BEEN LOOKED AT IN VERY FEW
CASES.
SO P6 IS SUMOYLATED, BUT IT
DOESN'T SEEM TO DO ANYTHING.
SUMO IS A BIG REGULATOR OF NFAT,
AND THERE'S A LOT OF ROOM FOR
SUMO TO BE DOING OTHER STUFF.
SO IN THE LAB THEY DECIDED TO
LOOK AT IT IN A PROTEOMIC WAY.
THE PROBLEM WITH SUMO IN TERMS
OF A PROTEOMIC SCREEN IS THAT
THE USUAL WAY YOU LOOK AT
UBIQUITINYLATION IS YOU DO
REGULAR TRADITIONAL MASS SPEC,
AND THE BEAUTY OF UBIQUITIN IS
IT'S CLOSE TO THE C TERMINUS.
SO WHEN YOU MOVE YOUR LYSATE FOR
THE MASS SPEC, YOU LEAVE A
LITTLE GLUSIN AT THE SITE OF THE
ADDITION, AND YOU CAN SEE IT AS
A LITTLE BLIP IN THE PEPTIDE OF
THE PRODUCT.
THAT'S GREAT.
UNFORTUNATELY, SUMO 1 AND SUMO 2
HAVE THE FIRST CLEAVABLE SITE
NOWHERE NEAR THE C TERMINUS, AND
THE STUB THAT IS LEFT IS TOO BIG
TO ALLOW YOU TO IDENTIFY THE
TARGET PROTEIN THAT WAY.
SO THE ANSWER, WHICH WE DIDN'T
HAVE TO INVENT, IS THAT YOU
CHANGE, YOU MAKE A SUMO
MODIFIED, SUMO 1 OR SUMO 2 WITH
A TRIPSIN CLEAVABLE AMINO ACID
NEAR THE C TERMINUS, AND NOW THE
STUB IS SHORT ENOUGH TO SEE THIS
ON THE MASS SPEC PRODUCTS.
DMITRA MADE CELL LINES WITH
THESE VARIANTS.
THEY'RE EXPRESSED AT THE
ENDOGENOUS LEVELS, WHICH IS A
BIG DEAL.
YOU'RE NOT OVEREXPRESSING
ANYTHING.
THE MODIFIED SUMO BEHAVES WELL,
GOES EVERYWHERE IT SHOULD GO,
AND NOW YOU TAKE THOSE CELLS AND
ASK WHAT HAPPENS WHEN YOU INFECT
WITH HIV.
SO SHE DID THAT, SHE TOOK -- AND
SHE MADE VERSIONS, I SHOULD SAY,
THAT ARE FLAG TAGGED OR HIS
TAGGED WHICH IS HELPFUL.
YOU HAVE THE CELL LINES READY TO
GO, INFECT THEM WITH HIV, MAKE
LYSATES.
YOU CAN PULL THE TAGS OUT, WHICH
HELPS, YOU DON'T HAVE TO, AND
SEND THE WHOLE DAMN THING OFF TO
THE MASS SPEC PEOPLE AND THEY
COME BACK WITH A LIST OF
EVERYTHING THAT'S SUMOYLATED.
AND WE ASK THOSE, OKAY, WHAT'S
GONE UP WITH SUMOYLATION AND
MAYBE WHAT'S GONE DOWN.
THERE YOU GO.
YOU GET A HUGE HIT LIST, A BUNCH
OF THINGS ARE UP, AND SOME
THINGS ARE WAY UP.
IT'S REALLY SOME OF THEM ARE
VERY STRIKINGLY INCREASED IN THE
SUMOYLATION UPON INFECTION.
A SMALLER NUMBER ARE DOWN, AND I
THINK THERE'S A TON OF STUFF
HERE FOR US TO WORK ON FOR A
LONG TIME, BUT THEY'RE
INTERESTING.
THERE'S A LOT OF OVERLAP BETWEEN
SUMO 1 AND SUMO 2, BY THE WAY.
A LOT OF PROTEINS ARE SUMOYLATED
BY EITHER ONE.
THERE ARE SOME THAT ARE
SIGNIFICANTLY ONLY SUMOYLATED BY
ONLY SUMO 1 OR ONLY SUMO 2.
LOTS OF THESE ARE INTERESTING.
NOW WHAT DO YOU DO?
YOU'VE GOT THIS LIST.
WELL, YOU LOOK AT THE LIST AND
SAY, WHAT DO WE GOT HERE?
WHAT HAS COME, AND WHAT MIGHT BE
INTERESTING TO LOOK AT?
YOU ARE ENCOURAGED BY THE FACT
THAT YOU SEND THESE SAMPLES TO
MULTIPLE MASS SPEC FOR YOU, AND
THEY COME UP WITH THE SAME THING
MOST OF THE TIME OR SOME OF THE
TIME, AND THAT'S GOOD.
WE DID THAT.
SO WHAT DO WE GOT?
THERE WERE SOME INTERESTING
THEMES THAT CAME UP IN THIS
SCR
SCREEN, AND THE MOST STRIKING IS
THERE'S A WHOLE LOT OF HNRNPs.
HNRNP 1 AND 3, I'M GOING TO TALK
ABOUT.
THEY COME UP WITH SUMO 1 AND
SUMO 2.
THESE ARE A LOT.
THESE ARE TWO LOGS AND MORE UP
IN INFECTED CELLS, SO HUH, DON'T
KNOW.
THERE ARE OTHER INTERESTING
THINGS.
THERE'S A LOT OF NUPs, NUCLEAR
CORE PROTEINS, WHICH MAY BE
INTERESTING.
BUT WE'RE LOOKING AT THOSE GUYS.
EVEN WHEN YOU LOOK KIND OF IN
THE GO CATEGORIZATION OF WHAT
THINGS ARE INCREASED, YOU REALLY
GET A LOT OF RNA HANDLING GUYS,
RNA SPLICING GUYS LIKE THE RNPs,
A LOT OF RNA PROCESSING GUYS, A
LOT OF GENES FLAGGED HAVING TO
DO WITH THE METABOLISM OF RNA.
I SERIOUSLY BELIEVE IN INFECTION
THE VIRUS IS SCREWING AROUND IN
VARIOUS WAYS WITH THIS RNA
MACHINERY.
SO THESE ARE THE DOTS FOR THOSE
TWO GENES, THOSE PARTICULAR GUYS
THIS IS THE AVERAGE OF
TRIPLICATES BY THE MASS SPEC
GUYS.
NOW WE CAN GO BACK AND LOOK AT
ACTUAL PROTEIN WORKUPS, WHICH IS
TRUE.
IF YOU LOOK AT UNINFECTED AND
INFECTED FOR SUMO 1 FOR CONTROL,
YOU HAVE YOUR CONTROLS, AND WHAT
YOU SEE IS THE PROTEIN IS HUGELY
SUMOYLATED AFTER INFECTION.
THAT UPPER BAND WITH THE ARROW
IS MONOSUMOYLATED.
THE UNSUMOYLATED DOESN'T CHANGE.
THE PROTEIN LEVEL IS NOT
AFFECTED.
WE HAVE THESE ADDED BANDS,
PROBABLY THOSE ARE DI AND
TRI-SUMOYLATED VERSIONS, PRETTY
DRAMATIC.
JUST TO REMIND YOU WHAT'S
POSSIBLE, WHICH DON'T KNOW WHICH
IS HAPPENING, AND WE DON'T KNOW
ALL THE PLACES WHERE THOSE
MODIFICATIONS ARE PROBABLY
HAPPENING.
THEN IF YOU EXPOSE IT MORE, YOU
CAN MAKE IT LOOK EVEN MORE
BETTER.
YOU CAN MAKE THE FACTORS LOOK
THE WAY YOU WANT THEM.
FOR SUMO 1, IT'S TRUE.
FOR HNRNPA3.
THERE'S A BIG BASAL LEVEL
SUMOYLATION, BUT THEN THEN
THERE'S A BIG INCREASE AFTER
INFECTION.
YOU CAN SEE THAT OVER THERE.
AND WE KNOW WE CAN SHOW THAT AT
LEAST THESE BANDS ARE SUMO
ADDITION BECAUSE THERE'S A
DYNAMITE SUMO INHIBITOR.
TACK THE DRUG AND THROW IT ON,
AND IT TOTALLY BLOCKS THAT
CHANGE AFTER INFECTION.
IT'S CLEAR THAT THIS IS
SUMOYLATION.
SO THAT WAS INTERESTING.
YOU KNOW, WE DON'T KNOW REALLY A
WHOLE LOT MORE YET.
WHAT WE KNOW IS THE TIMING.
IT'S JUST KIND OF INTERESTING.
IT'S NOT EARLY IN INFECTION.
IT'S QUITE LATE.
SO HERE WE'VE LOOKED AT
DIFFERENT TIMES AFTER
INFECTION -- 6, 12, 24, 48 --
AND IT REALLY KICKS IN ONLY
AROUND 24 HOURS AFTER INFECTION.
SO IT'S PROBABLY REQUIRING A
VIRAL INTEGRATION AND
EXPRESSION.
SO SOME LATE PROTEIN, SOME
ASPECT RELATIVELY LATE IN
INFECTION IS INDUCING THIS BIG
MODIFICATION OF THE HNRNPs, AND
THAT'S TRUE OF BOTH OF THOSE TWO
PARTICULAR PROTEINS.
IT'S NOT ONLY HELA CELLS WHERE
WE'VE DONE THE MOST, BUT IT'S
TRUE IN CELLS, AND IT SEEMS TO
BE QUITE GENERAL.
WE'LL BE ASKED IN PRIMARY
T-CELLS ANY DAY NOW, BUT WE
HAVEN'T DONE THAT, BUT I BET IT
WILL BE TRUE BECAUSE IT SEEMS TO
BE TRUE FOR BOTH.
WHAT ARE THESE PROTEINS DOING?
THEY'RE KNOWN TO BE HELPFUL IN
SPLICING, AND THAT'S WHERE WE'LL
GO.
WE'VE DONE A LITTLE WORK ON
THAT, SO YOU ALL KNOW IT'S
COMPLICATED.
THERE'S TWO DONORS, ALL THESE
SPLICE ACCEPTORS, AND THE RESULT
IS A MESS OF SPLICING.
THERE'S ALWAYS OFTEN THIS FIRST
DONOR TO A BUNCH OF ACCEPTORS,
AND THEN THERE'S THIS SET OTHER
DONOR THAT CAN BE USED OR NOT TO
ITS ACCEPTOR, AND THERE'S
ALTERNATE ACCEPTORS OVER THERE
THAT MAKE MORE ALTERNATE
PROTE
PROTEINS.
SO WHEN YOU ADD UP ALL THESE
COMBOS, THERE'S AT LEAST 50 OR
MORE SPLICE VARIANT MRNAs THAT
ARE FORMED ON THE VIRUS.
THE RATIO OF ALL OF THEM ARE
PRETTY STEREOTYPICAL ACTUALLY,
BUT I'M SURE UNDER COMPLICATED
CONTROL, AND I DON'T THINK WE
REALLY KNOW MUCH ABOUT IT.
A LITTLE IS BEING KNOWN ABOUT
THE SITES OF THE ACCEPTORS AND
THERE ARE NOW A FAIR NUMBER OF
MUTANTS THAT FOOL AROUND WITH
THE ACCEPTOR USE.
I THINK OF THE -- A LOT OF THAT
WORK BEING DONE BY ANN EMERY
DOWN THERE AND RON SWANSTROM'S
LAB.
WE HOOKED UP WITH HER AND
KNOCKED DOWN OUR TWO FAVORITE
SUMOYLATED PROTEINS, AND THAT'S
WHAT HAPPENS.
SHE DOES THE ANALYSIS BY DEEP
SEQUENCING WITH RANDOM PRIMERS
UP ON TOP, AND SHE DOES IT WITH
SPECIFIC PRIMERS THAT READ OUT
INDIVIDUAL SPLICE SITES.
I WON'T WALK THROUGH IT EXCEPT
TO SAY THERE ARE PRETTY BIG
EFFECTS, SO WHEN YOU KNOCK DOWN
SOME OF THE HNRNPs, JUST LIKE
OUR FAVORITE GUYS, WE SEE THESE
OFTEN BIG INCREASES IN SPLICE
USAGE OF SOME OF THE PARTICULAR
SPLICE, PARTIALLY SPLICED SITES
UP ON THAT TOP RIGHT.
AND BIG CHANGES IN PARTICULAR
SPLICES, USES OF ACCEPTOR 2,
ACCEPTOR 3 WHEN YOU KNOCK THOSE
DOWN.
THE FUTURE IS CLEAR OF WHAT WE
WILL BE WANTING TO DO, WHICH IS
TRY TO UNDERSTAND IF SUMOYLATION
IS A DEAL IN THIS, AND IF THAT'S
PART OF WHAT THE VIRUS IS DOING
TO CONTROL ITS OWN SPLICING.
WE'RE GOING TO ASK DOES THE
HIV-INDUCED SUMOYLATION WHICH WE
KNOW IS HAPPENING REALLY
REGULATING THE FUNCTIONALITY OF
THESE GUYS OR IS IT JUST
HAPPENING?
AND WE'LL FIND OUT BECAUSE WE'RE
GOING TO MAKE NON-SUMOYLATEDABLE
MUTANTS OF THE PROTEINS AND SEE
WHAT'S ADDED.
BUT SPLICING AND VIRUS
REPLICATION.
I'LL STOP THERE AND THANK ALAN
AGAIN.
HERE'S THE CURRENT GANG.
I THINK THERE ARE SO MANY
AVENUES OF RESEARCH WE HAVE YET
TO RUN WITH, SO MANY OF WHICH
ARE STARTED BY ALAN.
WE'LL KEEP RUNNING AS LONG AS WE
CAN.
THANKS.
I'LL STOP THERE.
>> I'M SURE YOU'RE IN THE
PRESENCE OF FINDING OUT WHICH OF
THESE RETROVIRAL PROTEINS
CONTRIBUTE TO THIS?
>> I DON'T.
THE CHEAP WAY, WE'RE GOING TO
OVEREXPRESS EACH ONE FIRST
ALONE, NO VIRUS OTHERWISE, AND
WE'LL SEE IF THAT'S IT.
AND THEN OF COURSE WE HAVE VIRUS
RESTRAINTS THAT LACK, YOU NAME
IT, BPU, BPR, WHATEVER, WE'LL
FIND OUT.
>> IT'S A BLUNT TOOL, IF YOU
LOOKED AT WHAT THE SUMOYLATION
INHIBITORS DO TO HIV-1?
>> YES, AND THE ANSWER IS
COMPLICATED.
THOSE DRUGS ARE FABULOUSLY
POTENT, AND THEY ARE TOTALLY
BLOCKING THE VIRUS, BUT THEY'RE
PROBABLY DOING MORE THAN ONE
THING.
THEY DO, AS I SHOWED YOU, BLOCK
THE SUMOYLATION OF THESE
FACTORS, YES, LATE IN INFECTION,
BUT THEIR BIG BLOCK, IS IT
SOMETHING ELSE?
IT'S SUPER EARLY, AND IN FACT,
THEIR MOST POTENT FUNCTION
REQUIRES THE PRETREATMENT OF THE
CELLS FOR LIKE 12 HOURS BEFORE
YOU INFECT THEM.
AND IF YOU DO THAT BEFORE AN
INFECTION IS THREE LOGS DOWN,
WHOPPING, BUT I GUESS IT'S NOT
THOSE PROTEINS.
>> AND THE CELLS ARE FINE?
>> THEY DON'T LOVE IT, BUT
THEY'RE OKAY FOR -- THEY'RE NOT
DEAD.
THEY'RE OKAY FOR A FEW DAYS, AND
THEY DON'T KNOW THEY'RE DEAD
YET.
(LAUGHTER)
AND THE INFECTION, THE VIRUS IS
STILL ABLE TO DO THINGS IN THOSE
CELLS, YEAH.
>> SO IN TERMS OF THE
SUPPRESSING BY INTERACTING WITH
THE SERUM, I DON'T KNOW WHETHER
YOU DEMONSTRATE THIS FACT?
>> YES, THAT'S THE $64,000
QUESTION.
EVERYBODY WOULD AGREE THEY'RE
IMPORTANT IN THAT PROCESS
SOMEHOW, AS REGULATORS OF
SPLICING, AND WHETHER THE
SUMOYLATION OF THEM IS GOOD OR
BAD FOR THAT, WE DON'T KNOW.
THAT'S WHAT WE NEED TO FIND OUT.
I HOPE I'LL TELL YOU SOON.
>> ONE OF THE THINGS THAT SEEM
TO BE UPREGULATED FOR
SUMOYLATION IS WHAT'S SITTING ON
THE EXIT TUNNEL.
HAVE YOU LOOKED TO SEE ABOUT
TRANSLATIONAL?
>> WE HAVEN'T.
AND ONE OF THE GREAT THINGS
ABOUT PUTTING UP A SLIDE WITH
ALL THE HIT LIST, PEOPLE ALWAYS
GO, OH, MY PROTEIN IS THERE.
AND THEN YOU GO, GREAT, CAN WE
WORK ON THAT TOGETHER?
SO HERE WE ARE.
>> (NO MICROPHONE).
>> IT'S UBC-9, I THINK.
I CAN'T REMEMBER.
THEY'RE KNOWN, THE SUMO E1, E2,
E3 ARE KNOWN FOR SUMOS.
I DON'T REMEMBER FOR SURE,
THOUGH.
I'D HAVE TO THINK.
AND THE DRUGS ARE KNOWN WHAT
THEY DO IN THAT CASCADE.
I THINK THEY DO IT EARLY IN THE
CASCADE.
>> YOU'RE IN THE CENTER OF THAT
PICTURE TOO.
>> THESE ARE USUALLY TAKEN AT
BIRTHDAYS.
I DON'T KNOW.
>> THANK YOU, RECEIVE, FOR A
WONDERFUL TALK.
OUR NEXT SPEAKER NEEDS NO
INTRODUCTION.
STEVE HUGHES HAS BEEN HERE IN
FREDERICK ALMOST AS LONG AS I
HAVE, AND LIKE ME, HE'S BEEN
STUDYING RETROVIRUSES ALL THESE
YEARS, AND I THINK AS TIME HAS
GONE ON, WE'VE BECOME BETTER AND
BETTER FRIENDS.
I THINK I'LL LEAVE IT AT THAT.
STEVE?
>> I WAS TRYING TO THINK BACK
ABOUT WHEN I FIRST MET ALAN, AND
IT'S GOT TO HAVE BEEN MORE THAN
FOUR DECADES AGO, AND IT MAY
WELL HAVE BEEN IN COLD SPRING
HARBOR, BUT I CERTAINLY MET HIM
WHEN I CAME TO FREDERICK AND
BEGAN TO REALLY BENEFIT FROM
HAVING HIM AS A FRIEND AND A
COLLEAGUE.
I'M GOING TO TELL A FEW STORIES
OF ALAN'S, AND I HOPE TO DO
REASONABLE JUSTICE TO THEM, THAT
I PARTICULARLY LIKE.
EACH ONE OF THOSE HAS TAUGHT ME
SOMETHING.
I'VE TAKEN AWAY A GREAT DEAL OF
POSITIVE FROM MY LONG
ASSOCIATION WITH ALAN.
ONE OF THE INTERESTING THINGS OF
COURSE IS THAT THE IDEA OF
ASSEMBLY, PUTTING THE VIRUS
TOGETHER, ISN'T THE SAME
NECESSARILY AS HAVING THE VIRUS
READY TO BE INFECTIOUS.
YOU SOMEHOW HAVE TO CONVERT THE
PROBLEM FROM PUTTING THINGS
TOGETHER INTO BEING ABLE TO
SOMETIMES TAKE THEM APART AGAIN.
FOR RETROVIRUSES AND OTHER
VIRUSES AS WELL, THE PROPERLY
ASSEMBLED POLYPROTEIN PRECU
PRECURSORS, THE IMMATURE VIRION
NEED TO BE CONVERTED FROM
PROTEASES INTO MATURE VIRION,
AND THAT INVOLVES PROTEASES.
ONE OF THE BIZARRE THINGS ABOUT
MLV IS THE MATURATION OF
ENVELOPE -- AND THIS IS
DIFFERENT FROM MLV THAN IT IS
FOR THE OTHER VIRUSES -- IT'S A
PRO-STAGE VIRUS AND IT INVOLVES
TWO DIFFERENT PROTEASES.
THERE'S A HOST PROTEASE THAT
SEPARATES THE SURFACE AND THE
TRANSMEMORY PROTEIN, AND THAT
CLEAVAGE IS NECESSARY, BUT IT'S
NOT SUFFICIENT TO GIVE YOU
FUSIGENIC ABILITY.
AND THE SECOND, AND THIS IS THE
STORY OF ALAN'S THAT I LIKE SO
MUCH, THERE'S A SECOND CLEAVAGE
WHICH REMOVES ABOUT A 2 KB FROM
THE END OF TRANSMEMORY THAT'S
CARRIED OUT BY THE MLV PROTEASE.
AND IT TURNS OUT THIS IS A VERY
IMPORTANT SAFETY FEATURE THAT
PREVENTS THEM FROM BEING
FUSIGENIC BEFORE THAT LITTLE
PIECE IS REMOVED.
IN FACT, BECAUSE PROTEASE IS
ACTIVATED WHEN THE VIRION IS
RELEASES, THIS MEANS THE MLV ON
THE SURFACE OF CELLS IS NOT
FUSIGENIC, SO WHEN THAT PROTEIN
IS EXPRESSED WITHOUT PROTEASE,
THERE'S NO SYNCYTIA.
ALAN SHOWED THAT VERY ELEGANTLY,
AND I'LL SHOW YOU A SLIDE ON
THIS IN JUST A MOMENT.
IF YOU EXPRESS MLV THAT LACKS
THE P2E SAFETY PEPTIDE, THEN THE
PROTEIN IS FUSIGENIC ON THE
SURFACE OF THE CELLS AND MAKES
THE SYNCYTIA.
THESE ARE DESCRIBED IN TWO OF MY
FAVORITE ALAN PAPERS.
THE FIRST IS ACTUALLY THE
ORIGINAL DEMONSTRATION THAT THE
MLV PROTEASE IS INVOLVED, AND
THE SECOND WHICH IS ACTUALLY THE
ONE THAT'S, I THINK, GENERALLY
BETTER KNOWN, AND THIS WAS, I
BELIEVE, MENTIONED BY ERIC, IS
THE PAPER THAT SHOWS THAT THAT
REMOVAL IS, IN FACT, REMOVING
THE LITTLE SAFETY BIT.
SO THIS SLIDE THAT'S NEAREST ME,
THE SLIDE THAT'S ON THE RIGHT,
MY RIGHT, SHOWED THAT THE
ENVELOPE IF EXPRESSED WITHOUT
PROTEASE, THE CELLS ARE
PERFECTLY HAPPY, BUT IF YOU
EXPRESS A DELETED PART OF THE
PROTEIN THAT'S GOT THAT LITTLE
BIT LOPPED OFF, THEN YOU MAKE
LOTS OF SYNCYTIA.
THE MESSAGES HERE, THE MOST
FUNDAMENTAL MESSAGES, THE ONE I
LIKE THE BEST, IS THIS IS ONE OF
THE CLEAR DEMONSTRATION OF HOW,
IN FACT, ELEGANTLY AND CAREFULLY
THE VIRUSES, EVEN REALLY WHAT WE
THINK OF AS VERY SIMPLE VIRUSES,
HOW CAREFULLY THEY MANAGE THEIR
MATURATION AND HOW EXQUISITELY
APPROPRIATE THEIR INTERACTIONS
WITH THE HOST CELLS ARE.
IT TURNS OUT THAT OTHER
INTERACTIONS, LIKE THE AVIAN
VIRUSES I PRIMARILY WORKED WITH
BEFORE HIV, AND HIV ITSELF, ALSO
REGULATE THE FUSIGENICITY OF
THEIR ENVELOPE PROTEINS ON THE
SURFACE OF THEIR PRODUCER CELLS.
BUT, IN FACT, THE UNDERLYING
MECHANISMS BUT WHICH
RETROVIRUSES CONTROL THIS USE
PARTICULAR TRICKS.
THIS IS BEAUTIFUL TO HAVE PEOPLE
THAT LOVE NON-HIV RETROVIRUSES
SPEAK BECAUSE ONE OF THE
MESSAGES I THINK THIS SHOULD
BRING HOME TO ALL OF THIS, AND
ONE OF THE GREAT THEMES, I
THINK, OF ALAN'S CAREER, IS IN
SPITE OF THE FACT THAT PEOPLE
THAT GIVE OUT THE MONEY DON'T
SEEM TO THINK SO, STUDYING
RETROVIRUSES OTHER THAN HIV IS
STILL VERY VALUABLE.
AND ONE OF THE THINGS THAT IS
SORT OF AN ASTOUNDING ASIDE IS
THAT, IN FACT, ALTHOUGH WHILE
HIV DOESN'T USE THE PROTOIATION
AS A WAY TO REGULATE HOW FUSION
IS CONTROLLED, THERE'S ACTUALLY
A BEAUT OF HIV THAT WAS STUDIED
BY ABDUL WAHIM WHEN HE WAS IN
ERIC'S LAB, THAT DOES USE THIS
MECHANISM.
I'M NOT GOING TO TRY TO SORT
THROUGH THAT, BUT IF YOU'RE
INTERESTED, YOU CAN ASK EITHER
ABDUL OR ERIC, AND I WOULD
RECOMMEND THAT YOU DO SO BECAUSE
IT'S A VERY CUTE STORY.
ONE OF THE STORIES -- AGAIN, ONE
OF MY -- THESE ARE ALL SOME OF
MY FAVORITE ALAN STORIES, AND
I'M NOT TRYING TO SUGGEST TO YOU
THAT I PICKED THE THINGS THAT IN
SOME COSMIC SENSE ARE THE MOST
SCIENTIFICALLY IMPORTANT,
ALTHOUGH THIS ONE, I'M GOING TO
ARGUE, THAT'S ENORMOUS IMPACT,
BUT THESE HAVE BEEN, FOR ME,
VERY PERSONAL LY ENLIGHTENING.
AND EACH ONE OF THESE RESONATES
WITH ME, AND I'LL TRY TO EXPLAIN
WHY.
THE ONE I'M GOING TO TALK ABOUT
NOW IS THE DEMONSTRATION THAT
THE MINOXIDIL PHOSPHATES, IP5 OR
IP6 ARE IMPORTANT IN THE
ASSEMBLY, THE PROPER AND NORMAL
ASSEMBLY OF HIV-GAG.
WE NOW KNOW THAT THE ROLE OF IP5
AND IP6 IS IMPORTANT NOT ONLY IN
THE APPROPRIATE ASSEMBLY OF GAG,
BUT IT'S ALSO SUBSEQUENTLY
IMPORTANT IN THE PROPER ASSEMBLY
OF THE CAPSID PROTEIN TO FORM
THE CAPSID WITHIN THE VIRION.
IT TURNS OUT -- AND I SUSPECT
MOST OF THE PEOPLE IN THE
AUDIENCE KNOW THIS -- THAT THE
NEGATIVELY CHARGED PHOSPHATES OF
IP5 AND IP6 INTERACT WITH
POSITIVELY CHARGED AMINO ACIDS,
AND THEY STABILIZE HEXAMERIC
FORMS OF EITHER GAG OR CAPSID.
THESE SUBSEQUENT RECOGNITIONS OF
THE IMPACT OF IP5 AND IP6 ON
BOTH GAG ASSEMBLY AND CAPSID,
AND EXACTLY HOW THEY DO IT --
AND I'LL SHOW YOU THE STRUCTURAL
INFORMATION IN A LIMITED WAY IN
JUST A MOMENT -- ALL OF THESE
SUBSEQUENT DISCOVERIES REST ON
THE INITIAL DEMONSTRATION OF
ALAN'S LABORATORY THAT THE IP5
AND IP6 HAS AN IMPORTANT ROLE IN
THE ASSEMBLY OF GAG IN-VITRO.
YOU'VE ACTUALLY SEEN THIS
REFERENCE BEFORE, AND THIS IS --
I WILL REMIND YOU AGAIN THIS IS
NOT QUITE 25 YEARS AGO.
SO THIS IS A LONG WAY BACK, AND
IT'S TAKEN ALL THIS TIME, I
THINK, FOR PEOPLE TO PROPERLY
APPRECIATE HOW IMPORTANT IP5 AND
IP6 ARE IN WHAT WE NOW KNOW
ABOUT HIV, AND IT TURNS OUT
SUBSEQUENTLY IN OTHER
RETROVIRUSES AS WELL.
THIS IS A SLIGHTLY COBBLED
TOGETHER SLIDE, AND I APOLOGIZE
THAT IT'S NOT A BETTER ONE.
IT'S MY FAULT, NOT ALAN'S.
WHEN ALAN AND HIS COLLEAGUE, IN
THIS PARTICULAR CASE, STEVE
CAMPBELL, BEGAN TRYING TO
ASSEMBLE GAG PARTICLES IN A TEST
TUBE, USING A SLIGHTLY MODIFIED
FORM OF GAG, WHEN THEY FIRST
STARTED DOING THAT, THE FIRST
THING THEY SAW WAS THAT THEY
COULD GET LITTLE VIRUS-LIKE
PARTICLES, BUT THEY'RE VERY MUCH
THEIR OWN SIZE FOR THE
EXPERIMENT.
THEY'RE TOO SMALL, CONSIDERABLY
TOO SMALL.
THIS IS A BIT OF A CHEAT BECAUSE
THEY'RE SLIGHTLY COBBLED
TOGETHER, SO I WON'T SWEAR TO
YOU THE SIZE RATIOS ARE EXACTLY
RIGHT.
BUT THE IDEA THAT THEY'RE WAY
TOO SMALL IS CORRECT.
BUT THE THING THAT WAS
ASTOUNDING WAS IF IF
RETICULOCYTE IS ADDED, YOU GET
THE SIZE CORRECT, AND YOU KNOW
THE SIZE IS CORRECT BECAUSE
THERE'S HIV CORE FOR COMPARISON.
SOMETHING MAGICAL IN THE RABBIT
RETICULOCYTE WAS ABLE TO CONVERT
THE WRONG ASSEMBLY INTO THE
RIGHT ASSEMBLY, AND THEY SHOWED
THAT MAGIC SOMETHING WAS IP5 AND
IP6, AND INSTEAD OF ADDING THE
RETICULOCYTE, YOU ADD SIMPLY THE
APPROPRIATE CO-FACTOR.
THIS IS A DEMONSTRATION OF
WHAT'S GOING ON TAKEN FROM A
REVIEW THAT WAS WRITTEN BY ALAN.
SO YOU HAVE THE IMMATURE VIRION
HERE AND YOU HAVE THE LITTLE
PIECE THAT'S CAPSID.
OVER HERE YOU SEE THE C-TERMINAL
DOMAIN OF CAPSID EMBEDDED IN
GAG.
IT'S GOT A LITTLE BIT OF A SPACE
FOR PEPTIDE 1 ATTACHED TO IT,
AND IT'S A LITTLE HARD TO SEE.
BUT IP6 IS RIGHT THERE, AND THE
PHOSPHATES OF IP6, IN THIS CASE,
ARE INTERACTING WITH A RING OF
AMINO ACIDS HOLDING THE HEX
TOGETHER, IN THIS CASE,
HEXAMERIC GAG.
IN A SLIGHTLY DIFFERENT FASHION,
THE SAME SMALL MOLECULE IS ABLE
TO BRING TOGETHER THE HEXAMERIC
ARRAY OF CAPSID ITSELF.
THERE IT'S A LITTLE EASIER TO
SEE, THE IP6 IN THE MIDDLE WITH
THE PHOSPHATES AROUND THE EDGE
AND THE 6 CAPSIDS.
IN THIS CASE, THIS IS THE MATURE
CAPSID IN THE MATURE VIRION.
AND ONE OF THE THINGS THAT'S
INTERESTING ABOUT THIS TO ME IS,
IN FACT, ALTHOUGH THE SIMILAR
TYPE OF INTERACTION IS TAKING
PLACE IN BOTH CASES, IN WHICH
YOU HAVE THE PHOSPHATES BEING A
CHARGE, A RING OF CHARGE THAT
BRINGS TOGETHER THE SIX
COMPARTMENTS, WHETHER IT'S GAG
OR CAPSID, EXACTLY WHERE THOSE
ARE IN CAPSID IS DIFFERENT.
SO IF WE'RE TALKING ABOUT IN THE
CONTEXT OF GAG, THESE TWO
LYSATES, ONE HERE AND ONE HERE
RIGHT NEXT TO SB 1, AND YOU LOOK
AT THE MATURE GAG, IT'S IN A
SLIGHTLY DIFFERENT PLACE AND
DIFFERENT AMINO ACIDS.
IT'S THE SAME THEME, BUT IT'S
MAKING USE OF THE INTERACTIONS,
WHICH IS A SIMILAR INTERACTION,
INVOLVING DIFFERENT AMINO ACIDS.
AND ONE OF THE THINGS THAT'S
SORT OF CUTE ABOUT THAT, WHICH
HAS ONLY RECENTLY BEEN
RECOGNIZED, I THINK, IS THE
INTERACTION WITH GAG THAT BRINGS
THE IP5 AND IP 6 INTO THE VIRION
WHERE IT CAN ACT TO NUCLEATE THE
ASSEMBLY OF THE CAPSID AT THE
TIME AND PLACE THAT IT IS
NEEDED.
OF COURSE, IN A SITUATION IN
WHICH THE IP5 AND IP6 IS NOT
BROUGHT IN WHICH THE INTERACTION
WITH GAG, IT WON'T BE THERE FOR
CAPSID TO PROPERLY ASSEMBLE.
SO I THINK THE KEY IDEA HERE, AT
LEAST FOR ME, IS THAT THE
DISCOVERY OF IP5 AND IP6, THE
DISCOVERY OF THOSE TWO PROTEINS,
THOSE TWO SMALL MOLECULES, PLAY
A CRUCIAL ROLE IN GAG ASSEMBLY,
PAVED THE WAY FOR OUR CURRENT
UNDERSTANDING MEMORY OF HOW GAG
ASSEMBLES, BUT IN FACT HOW
CAPSID ASSEMBLES, AND THOSE
DISCOVERIES UNDERLIE OUR CURRENT
UNDERSTANDING OF HOW REVERSE
TRANSCRIPTION TAKES PLACE IN
CAPSIDS.
THAT ALSO HAS FACILITATED SOME
REALLY IMPORTANT STRUCTURAL
WORK, SOME OF WHICH YOU'VE SEEN
FROM ME, OR MORE LIKELY IN TALKS
GIVEN BY OTHERS.
THE INTERESTING THING ABOUT THIS
FROM MY OWN PERSONAL PERSPECTIVE
IS THAT, WHEN THE GAG ASSEMBLY
WORK WAS BEING DONE, ALAN'S LAB
AND MY LAB HAD HELD JOINT LAB
MEETI
MEETINGS, AND IT WAS STEVE
CAMPBELL WHO DID THE ACTUAL WORK
ON THE ASSEMBLY OF GAG.
WHEN HE DISCOVERED THAT THERE
WAS SOMETHING MAGIC IN RETIC
RETICULOSITE GLYSATES, HE WAS
GOING TO IMPORT HIM.
I DECIDED TO KEEP MY BIG MOUTH
SHUT, AND I DIDN'T TELL THEM HOW
HORRIBLE I THOUGHT HE WAS GOING
TO BE TO PULL OUT THIS FACTOR
FROM EITHER ONE.
I WAS QUITE CONFIDENT IT WAS
GOING TO BE SOME BIG, LARGE,
COMPLICATED PROTEIN THAT WAS
GOING TO SOMEHOW MODIFY THE
STRUCTURE OF THE GAG BITS, AND I
WAS JUST ALL WRONG.
THE OTHER THING THAT WAS
IMPRESSIVE WAS IN VERY SHORT
ORDER, THEY PULLED OUT THE
MOLECULE AND IDENTIFIED IT.
THIS WAS A SEMINAL DISCOVERY
THAT'S REALLY SET THE STAGE FOR
A LOT OF WHAT'S GOING ON NOW
BOTH IN TERMS OF CAPSID AND
REVERSE TRANSCRIPTION AND A
WHOLE BUNCH OF OTHER THINGS.
SO THIS IS ONE OF THE ONES I
REALLY LIKE.
AND BOTH OF THE THINGS I'VE TOLD
YOU SO FAR IN THE LITTLE
VIGNETTES ARE LITTLE VIGNETTES
THAT OTHERS HAVE MENTIONED, BUT
ONE OF THE ONES THAT ACTUALLY NO
ONE HAS TALKED ABOUT -- AND THIS
IS ACTUALLY A MEASURE OF THE
FACT THAT THERE'S LOTS OF THINGS
TO TALK ABOUT WHEN WE'RE TALKING
ABOUT ALAN'S CAREER -- IS THAT,
IN FACT, XMRV -- AND SOME OF YOU
MAY HAVE HAD EITHER GOOD OR BAD
MEMORIES OF THIS.
XMRV IS A LABORATORY
CONTAMINANT.
IT'S NOT A HUMAN PATHOGEN.
AS I SAID, SOME OF YOU MAY
REMEMBER THE CLAIMS THAT ARE NOW
CLEARLY FALSE THAT A MURINE
RETROVIRUS XMRV WAS FOUND IN
PROSTATE TUMORS AND ASSOCIATED
WITH CANCER AND ALL SORTS OF
OTHER MAGIC THINGS DEPENDING ON
WHAT DAY OF THE WEEK IT WAS.
I DON'T MEAN TO IMPLY THAT ALAN
WAS THE ONLY LAB IN DRP TO WORK
ON THIS, THERE WERE A NUMBER OF
DRPs AND PIs THAT SORTED OUT ALL
THESE CLAIMS, BUT WHAT WAS
REALLY IMPORTANT IS SOMEONE
NEEDED TO LOOK CAREFULLY AT THE
PROSTATE CANCER CLAIMS.
ALAN MADE USE OF HIS CONTACTS
WITHIN THE NCI TO GET PROSTATE
TUMOR SAMPLES, AND HE WAS VERY
BOTHERED BY THE FACT THAT, WHEN
THE PROSTATE SAMPLES WERE
CAREFULLY ANALYZED BOTH BY PCR
AND ANTIBODY STAINING, ALL THE
CONTROLS WORKED AND ALL THE
PROSTATE SAMPLES WERE NEGATIVE.
THIS IS THE MOST RELEVANT PAPER
THAT DESCRIBES THAT.
THIS IS ONE OF THE PIECES OF
INFORMATION, THIS IS AN ANTIBODY
STAIN, AND YOU CAN SEE THAT THE
CONTROLS AT THE TOP HAVE THINGS
THAT OBVIOUSLY STAIN STRONGLY
WITH ANTIBODY THAT RECOGNIZES
XMRV, AND SURE ENOUGH, BOTH THE
NORMAL PROSTATE AND THE TERMINAL
SAMPLES ARE ALL NEGATIVE.
IT'S IMPORTANT TO STATE, THOUGH
I'M NOT GOING TO SHOW ANY OF THE
DATA, THAT ALL OF THE DATA ALAN
AND HIS COLLEAGUES OBTAINED
USING PCR WAS EQUIVALENTLY
NEGATIVE.
THERE WERE TWO THINGS THAT VERY
MUCH IMPRESSED ME ABOUT THE WORK
THAT WAS DONE WITH THE PROSTATE
SAMPLES IN ALAN'S LAB.
THE FIRST WAS, IN CONTRAST TO
WHAT A NUMBER OF OTHER LABS, NOT
HERE IN FREDERICK BUT ELSEWHERE,
WERE DOING, ALL THE RESULTS WERE
NEGATIVE.
WHICH OF COURSE TURNS OUT TO BE
THE RIGHT ANSWER.
THE SECOND THING THAT WAS
IMPRESSIVE WAS HOW WORRIED ALAN
WAS BECAUSE HE DIDN'T WANT TO
TRY TO REACH A CONCLUSION THAT
WAS BASED ON NEGATIVE DATA.
AND THAT, I THINK, HAS A REALLY
IMPORTANT MESSAGE THAT RESONATES
VERY STRONGLY WITH ME, AND THAT
IS YOU'VE GOT TO BE YOUR OWN
HARSHEST CRITIC, AND YOU'VE GOT
TO BE REALLY CAREFUL WITH
NEGATIVE DATA.
ALAN, AFTER HAVING DONE HEROIC
WORK ON THIS AND HAVING SPOKEN
WITH HIS DRP COLLEAGUES, WAS
CONVINCED, IN FACT, THAT THE
ANSWER WAS NO, WHICH IT IS.
NOW, YOU JUST HEARD ABOUT ALAN'S
AFFECTION FOR GREAT MOVIES.
MY SESSIONS WITH HIM WAS MORE
OVER BOOKS THAN MOVIES.
HE HAS A REAL PASSION FOR GREAT
BOOKS, AND AGAIN AS OTHERS HAVE
MENTIONED IN OUR DISCUSSIONS, I
THINK I'VE GAINED MUCH MORE FROM
HIM IN TERMS OF THE BOOKS THAT
WE'VE READ AND THE THINGS THAT
WE'VE SUGGESTED THAN HE EVER
GAINED FROM ME.
BUT I THINK HE HAD SOME
PARTICULAR AFFECTION FOR I GUESS
WHAT I WOULD CALL RECENT SOCIAL
HISTORY.
THESE ARE A SERIES OF THREE
BOOKS BY TAYLOR BRANCH THAT ALAN
RECOMMENDED TO ME.
THEY RECOUNT THE HISTORY OF THE
CIVIL RIGHTS MOVEMENT IN THE
UNITED STATES THROUGH THE LIFE
OF MARTIN LUTHER KING.
THEY'RE BY TAYLOR BRANCH.
THESE ARE SOME OF THE MOST
MOVING BOOKS I'VE EVER READ, AND
THEY ARE SEMINAL, THEY ARE
BEAUTIFULLY WRITTEN, AND THEY
ARE COMPLETELY RELEVANT TODAY.
I WOULD RECOMMEND THEM TO EACH
OF YOU.
IN FACT, I THINK THE COUNTRY
WOULD BE A BETTER PLACE IF EVERY
MAN, WOMAN, AND CHILD OVER THE
AGE OF ABOUT 12 WOULD READ THEM.
THEN I THINK YOU MAY HAVE SEEN
ALAN IN AN ORIOLES CAP.
I WOULD TEND TO SAY THAT HIS
ORIOLES CAPS TEND TO BE OF
SLIGHTLY HIGHER MILEAGE THAN
THIS ONE.
THE INTERESTING THING IS FROM
TIME TO TIME I'VE BEEN STANDING
NEAR ALAN OFTEN IN PLACES LIKE
COLD SPRING HARBOR WHERE HE'S
KNOWN, BUT PEOPLE DON'T KNOW HIM
PERSONALLY QUITE AS WELL AS I
DO.
SOMEBODY WILL COME UP TO HIM,
AND HE'S GOT AN ORIOLES BASEBALL
CAP, AND THEY'LL SAY, DO YOU
LIKE THE ORIOLES?
AND ALAN WILL GET THAT BEAUTIFUL
SMILE, WHICH HE ACTUALLY HAS ON
HIS FACE RIGHT NOW, AND HE'LL
SAY, I LIKE THE BIRD.
I THINK THAT SUMS IT UP EXACTLY.
ALAN DOES LOVE BIRDS, AND THAT'S
BEEN MENTIONED, AND HE'S
INCREDIBLY KNOWLEDGEABLE OF
BIRDS IN GENERAL.
WALKING WITH HIM AND HAVING HIM
TELL YOU WHICH BIRD IT IS
LISTENING TO THE BIRD SONG IS AN
EDUCATION IN ITSELF.
IN SPITE OF SOME OF HIS BETTER
EFFORTS, I LIKE BIRDS VERY MUCH,
BUT MY KNOWLEDGE OF BIRDS IS
THEY'RE PRETTY AND I LIKE TO SEE
THEM FLY AND SOME OF THEM ARE
TASTY.
SO I'LL FINISH BY TRYING TO PICK
A BIRD THAT HAS SOMETHING THAT
BOTH HE AND I CAN LIKE.
THIS IS A BIRD THAT'S A
FISHERMAN.
ALAN, IT'S BEEN A TREMENDOUS
PLEASURE.
I'VE LEARNED A GREAT DEAL FROM
OUR LONG ASSOCIATION, AND I HOPE
THAT LEARNING PROCESS IS NOT
DONE, BY WHICH I MEAN, OF
COURSE, THAT WE SHOULD STAY IN
CONTACT SO WE CAN RECOMMEND
OTHER GOOD THINGS FOR ME TO READ
AND HELP ME CONTINUE MY
UNDERSTANDING OF VIRUSES AND HOW
THEY WORK.
THANK YOU SO MUCH.
I WILL ANSWER QUESTIONS IF
ANYONE THINKS THEY HAVE THEM,
BUT NOT ABOUT MLV.
>> I WANT TO SAY ABOUT XMRV
THAT, YES, I PRODUCED CLEARCUT
NEGATIVE DATA, BUT THE ANSWER TO
THE PUZZLE CAME FROM JOHN AND
RENEE.
THEY REALLY PUT IT TO REST IN A
WAY THAT SIMPLY FINDING NEGATIVE
DATA COULD NOT.
>> AND I WOULD POINT OUT THAT
ALAN'S RESPONSE SAYS SOMETHING
ELSE ABOUT HIM THAT I ALSO LIKE.
HE'S VERY GENEROUS WITH CREDIT
FOR OTHERS. THANK YOU.
>> OUR NEXT SPEAKER PROBABLY
NEEDS NO INTRODUCTION.
IT'S JOHN COFFIN, WHO HAS BEEN
AT THE FOREFRONT OF SO MANY
DIFFERENT AVENUES OF RETROVIRUS
RESEARCH THAT I WOULD NOT
ATTEMPT TO TELL YOU MORE ABOUT
IT.
JUST ALWAYS BRINGS A WONDERFUL,
ENLIGHTENED PERSPECTIVE.
HE ALWAYS INCLUDES A WONDERFUL
EVOLUTION IN WHATEVER HE'S
THINKING ABOUT, AND I HAVE
CERTAINLY LEARNED MORE THAN I
COULD SAY FROM MY LONG
ASSOCIATION WITH JOHN.
SO I THINK I'LL JUST LEAVE IT
WITH THAT.
JOHN?
>> THANK YOU, ALAN.
THIS HAS BEEN A WONDERFUL EVENT,
I HAVE TO SAY.
THE STORY I'M ABOUT TO TELL YOU,
I HOPE WILL SOMEWHAT CLOSE THE
CIRCLE BETWEEN WHAT JONATHAN
SAID EARLIER TODAY AND GOING ON
TO A DIFFERENT MODEL.
BEFORE I DO THAT -- AND SOME OF
YOU MAY RECOGNIZE THESE TWO GUYS
HERE.
SOME OF THE OLDER ONES AMONG
YOU.
BEFORE I DO THAT, I'VE GOT TO
FIGURE OUT HOW TO ADVANCE HERE.
WHEN I FIRST MET ALAN,
PRESUMABLY AT A COLD SPRING
HARBOR MEETING, AS THEY WERE
CALLED AT THE TIME, TUMOR VIRUS
MEETING.
BEFORE THAT, WE WERE ACTUALLY
CUSTOMER INS IN AN INTERESTING
WAY, IS THAT IS THAT OUR THESIS
MENTORS WERE RESPECTIVELY HARRY
RUBIN AND HOWARD TENEMAN.
THOSE OF YOU WHO KNOW ABOUT THE
HISTORY OF THIS, HARRY AND
HOWARD WERE TOGETHER IN A LAB,
AND THEY DID THE SEMINAL
EXPERIMENT THAT ACTUALLY
REFERRED HOWARD TO DETERMINE
RETROVIRUS MUST REPLICATE BY A
STABLED CELLULAR INTERMEDIATE,
WHICH HE REFERRED TO AS THE PRO
VIRUS.
THEN HE INFERRED FROM THAT THE
PRO VIRUS HAD TO BE DNA, EVEN
THOUGH IT WAS WELL-KNOWN ALREADY
EVEN AT THE TIME, THIS WAS LATE
'50s AND EARLY '60s, THAT THE
VIRUS GENOME WAS RNA.
THERE WAS NO DNA IN THE VIRUS.
IT HAD ALREADY BEEN WELL
ESTABLISHED.
BUT THAT KIND OF INFERENCE DID
NOT GO SIT WELL WITH HARRY, WHO
WAS NOT AN INFERENTIAL
SCIENTIST.
HE HAD TO SEE SOLID DATA
INCONTROVERTIBLE BEFORE HE WOULD
BELIEVE ANYTHING.
THEY HAD A FAMOUS FEUD ABOUT
THIS FOR QUITE A NUMBER OF YEARS
UNTIL -- THAT WAS UNTIL THERE
WAS IN 1970, THERE WAS A CANCER
CONGRESS IN HOUSTON, IF I
REMEMBER CORRECTLY, AND HOWARD
PRESEN
PRESENTED THE DISCOVERY REVERSE
TRANSCRIPTICS AND ALL THE
EVIDENCE FOR THAT IN A SESSION
AT THE MEETING WHERE HARRY WAS
THE CHAIR.
I REMEMBER HIM COMING BACK ALL
GLOWING BECAUSE HARRY KEPT
SAYING, I DON'T BELIEVE IT, I
DON'T BELIEVE IT, AND HOWARD
WOULD SAY THIS IS THE DATA, AND
SURE ENOUGH, IT WAS THE DATA.
SURE ENOUGH, THE REST IS
HISTORY.
IN ANY CASE, THEY NEVER DID
SPEAK TO EACH OTHER AGAIN AS FAR
AS I KNOW.
>> HARRY LEFT THE FIELD.
>> EXACTLY.
HE GOT MUCH MORE INTO CELL
BIOLOGY KINDS OF THINGS.
BUT ALAN AT COLD SPRING HARBOR
WAS HARDLY ALAN'S FIRST COLD
SPRING HARBOR EXPERIENCE, AND I
HAD THE GOOD FORTUNE OF BEING
ABLE TO CONTACT THE COLD SPRING
HARBOR PHOTOGRAPHER, WHO WE ALL
KNOW ACTUALLY FROM THE MEETING,
AND THE ARCHIVIST WHO MAINTAINS
A VERY GOOD ARCHIVE OF PAST
MEETINGS, AND ASKED THEM TO FIND
ME PICTURES OF ALAN REIN, AND
THEY SAID, WELL, WE'VE GOT ONE
FROM 1961.
I DON'T KNOW IF ALAN HIMSELF HAS
A COPY OF THIS.
SO THAT WAS ALAN, AS I THINK
ABOUT A SENIOR IN COLLEGE DOING
A PROJECT AT COLD SPRING HARBOR.
AS YOU CAN SEE FROM THE
PARAPHERNALIA AROUND THERE, IT
WAS A FLY PROJECT HE WAS WORKING
ON.
HE COULD TELL YOU ALL ABOUT IT
PROBABLY IF YOU ASK.
ACTUALLY, I GOT ABOUT 60 OR SO
PICTURES FROM THEM, WHICH I
CALLED OUT A FEW TO SHARE WITH
YOU.
THIS ACTUALLY WAS A MEETING THAT
I ORGANIZED, ALONG WITH IRVIN
CHEN IN THE MIDDLE, AND THIS WAS
A PICTURE OF THE SESSION CHAIRS
AT THE MEETING, AND ALAN IS IN
THE BACTERIO.
TWO OF THE PEOPLE YOU MIGHT
RECOGNIZE, NANCY HOPKINS OVER
THERE ON THE RIGHT THERE.
BY THE WAY, WHO WILL BE GIVING A
TALK IN A COUPLE OF WEEKS -- I
THINK IN BETHESDA ACTUALLY --
BUT IT'S VERY WORTH TUNING INTO
TO HEAR WHAT SHE HAS TO SAY.
I'VE HEARD IT BEFORE.
DAVID HO IS THERE, A NUMBER OF
PEOPLE YOU KNOW, DON BLAIR,
KAREN IS THERE.
MUCH YOUNGER, THIS WAS 1991,
THIS MEETING.
AND HERE'S ANOTHER MEETING.
HERE'S ALAN WITH TAYE ZHANG.
OH, YOU'RE ABSOLUTELY RIGHT.
THIS WAS SAID TO ME AS BEING
ALAN, BUT YOU'RE ABSOLUTELY
RIGHT.
THAT'S ALAN.
AND WITH DAVID BALTIMORE AND ANN
SKULKA AT THE 40th ANNIVERSARY
OF REVERSE TRANSCRIPTICS, SO IT
WOULD HAVE BEEN 2010.
AND HERE HE IS AT POSTER
SESSIONS AT COLD SPRING HARBOR
LOOKING EITHER SKEPTICAL, AS HE
SOMETIMES DOES, OR ENJOYING A
GOOD JOKE.
THEN PROBABLY IN THE MIDDLE OF
THE ROOM OUTSIDE AWAY FROM THE
POSTERS.
AND FINALLY, ALAN ALWAYS DOES
TEND TO STAND OUT IN A CROWD A
LITTLE BIT.
YOU'RE VERY EASY TO SPOT IN THIS
PICTURE, HARDER TO SPOT ARE THE
THREE GUYS IN THE FRONT THERE
THAT YOU MIGHT RECOGNIZE.
>> THIS IS THE ONE WITH THE
SILVER HAIR?
>> THAT'S YOUNG BURTOWSKY, A
STAPLE AT COLD SPRING HARBOR.
AT THE COCKTAIL PARTY, WE ALWAYS
TALK TO HIM ACTUALLY.
AND THIS IS THANKS TO THE COLD
SPRING HARBOR LABORATORY PEOPLE
WHO PROVIDED THESE PICTURES.
THEY HAVE A MARVELOUS COLLECTION
OF PICTURES FROM THESE MEETINGS.
BACK TO THE STORY.
WE ALL KNOW NOW ABOUT ENDOGENOUS
RETROVIRUSES, AND JONATHAN GAVE
A LOVELY INTRODUCTION AT THE
BEGINNING.
THIS IS STILL NOW A VERY LARGE
AREA OF STUDY NOW.
WHAT THESE VIRUSES HAVE DONE IN
TERMS OF THEIR EFFECTS ON THE
EVOLUTION AND GENE EXPRESSION
AND ALL KINDS OF OTHER ASPECTS
OF US BEING US, THERE IS, AS I
SAY, ONE OF THE REMARKABLE
FINDINGS OF THE HUMAN GENOME
PROJECT IS THERE'S MORE VIRUSES
IN US THAN US IN US.
WE HAVE SOMETHING LIKE 80,000
ENDOGENOUS PRO VIRUSES COMPARED
TO LIKE 20,000 GENES.
THESE ARE KNOWN TO DO LOTS OF
THINGS, LIKE BRINGING IN NEW
GENES, MODIFYING GENE
EXPRESSION.
THEY CAN BECOME PARTS OF A LONG
LINE OF CODING RNAs AND THINGS
LIKE THAT.
IN CASE OF MICE THEY CAN
REPLICATE AND CAUSE DISEASE,
WHICH IS NOT TRUE IN HUMANS,
DESPITE THE EFFORTS OF MANY
PEOPLE TO TRY TO SHOW THAT IT
IS.
AND TO OUR INTEREST, THEY
PROVIDE A FOSSIL RECORD OF THE
HOST-VIRUS INTERACTION GOING
BACK MANY MILLIONS OF YEARS.
JONATHAN WAS TALKING ABOUT 47
MILLION, I THINK, IN HIS.
WE DON'T BACK QUITE THAT FAR
WITH THE ONES WE'RE LOOKING AT,
BUT WE GO BACK A LONG WAYS.
AND I THINK THAT ONE OF THE MOST
OVERLOOKED THINGS IS BECOMING,
THANKS TO JONATHAN AND THE WORK
OF MANY OTHERS, UNDERSTAND IS
PROTECTION FROM INFECTION BY
RELATED EXOGENOUS VIRUSES.
I WOULD ARGUE THAT, PROBABLY THE
REASON THESE VIRUSES HAVE GONE
EXTINCT IS LARGELY DUE TO THIS
HAPPENING.
SOONER OR LATER A PRO VIRUS GETS
INTEGRATED INTO THE GERMLINE OF
THE HOST, AND IT BLOCKS THE
INTERACTION OF THE INCOMING
VIRUS IN SOME IMPORTANT HOST
CELL MACHINERY OR DOES SOMETHING
ELSE OF INTEREST THAT PREVENTS
INJECTION OF THAT HOST BY THE
SAME VIRUS FROM OUTSIDE.
IN FACT, IN LOOKING AT MLV SOME
YEARS AGO, WE PUBLISHED THAT YOU
COULD ACTUALLY SEE THIS BACK AND
FORTH AT THE LEVEL RECEPTOR
REACTION, LOOKING AT ENDOGENOUS
MLVs.
I'M NOT GOING TO GO INTO THAT
STORY.
I WANT TO GO INTO A RATHER
DIFFERENT STORY.
THIS JUST SHOWS A VERY, VERY
PARTIAL TREE, IF YOU LIKE,
PHILOGENY OF ENDOGENOUS
RETROVIRUSES COMPARED TO THE
RELATED EXOGENOUS.
YOU CAN SEE THE X IS MARKED
EXOGENOUS VIRUSES.
THE BLUE SQUARES MARK ONES THAT
ARE FOUND IN HUMANS, ONLY A VERY
SMALL FRACTION OF THE 80,000 OR
SO THAT WE CARRY.
AS YOU CAN SEE, THERE JUST HAVE
BEEN LOTS AND LOTS OF THESE.
EACH OF THESE, WE CONSIDER,
REPRESENTS A WAVE OF INJECTION
THAT A PANZOOTIC INFECTION THAT
IT CAME AND THEN WENT EXTINCT
AND IS LEFT TO US ONLY BY THIS
REMARKABLE FOSSIL RECORD.
A FAIR AMOUNT OF THE WORK OF MY
LABORATORY UP IN BOSTON IS
DEVOTED TO TRY TO INTERPRET
THIS.
I'M GOING TO TELL YOU
PARTICULARLY ONE PRO VIRUS AT
ONE LOCATION, THAT'S IN A GROUP
OF VIRUSES CALLED HERV-K
(HML-2).
IT'S THE MOST RECENTLY ACQUIRED
AND THE MOST RECENTLY EXTINCT
VIRUS.
THESE VIRUSES WERE STILL BEING
INTRODUCED INTO THE HUMAN GENOME
UP UNTIL MAYBE AS LITTLE AS HALF
A MILLION YEARS AGO.
AS FAR AS WE COULD TELL, THERE
WERE NO MORE INTRODUCTIONS SINCE
THEN.
AS I SAID AGAIN, THEY'VE ALL
GONE EXTINCT.
SO THERE ARE ABOUT 100 PRO
VIRUSES.
EACH OF US CARRIES ABOUT 1 HOW
MANY OF THESE PROVIRUSES, MOST
OF THEM IDENTICAL FROM ONE OF US
TO ANOTHER, AT LEAST IN THEIR
LOCATION.
THERE ARE A FEW THAT ARE
POLYMORPHIC IN LOCATION.
THIS IS THE ONLY GROUP OF
ENDOGENOUS VIRUSES IN HUMANS, AS
FAR AS WE KNOW, THAT HAS
POLYMORPHIC INSERTION OF THAT
SORT.
I HAVE A FEW THAT YOU MIGHT NOT
HAVE, AND EACH OF YOU MAY HAVE A
FEW THAT I DON'T HAVE.
AS YOU CAN SEE FROM LOOKING AT
THESE, THESE ARE JUST GENOME
MAPS REFERRED, MANY OF THEM BY
PULLING THEM OFF A HUMAN GENOME
SEQUENCE EARLY ON.
MANY OF THEM ARE DEFECTIVE.
ALL OF THEM ARE DEFECTIVE IN ONE
WAY OR ANOTHER, MANY OF THESE BY
DELETIONS AND STOP CODON
INSERTIONS AND SO ON AND SO
FORTH.
I WANT TO FOE UBIQUITOUS YOUR
ATTENTION TO TWO OF THESE.
THE ONE I'M REALLY INTERESTED IN
IS THE ONE AT CHROMOSOME 3Q12.3.
WE IDENTIFY THESE BY THEIR
CHROMOSOMAL BAND POSITION.
THERE'S PRETTY MUCH ONLY ONE PER
BAND, MAYBE TWO OR THREE.
IN FACT, WE FIND THAT IN MUCH
BETTER WAYS THAN ANY OF THE
PREVIOUS NOMENCLATURES.
WE'RE NOT QUITE ALONE IN THIS, A
LOT OF PEOPLE PICK IT UP.
BUT WE STILL USE OLD
NOMENCLATURES.
IF YOU READ A PAPER ON THIS
SUBJECT, YOU NEED A TRANSLATOR
WITH YOU.
FORTUNATELY, THIS PAPER ACTUALLY
SERVES AS THIS BECAUSE WE HAVE
SYNONYMS NEXT TO ALL OF THESE.
AND THE OTHER PROVIRUS I'M GOING
TO MENTION IS THIS ONE AT
22Q11.23.
NOW, YOU'LL NOTE I HAVE THESE IN
DIFFERENT COLORS, AND THAT'S
BECAUSE, IF YOU LOOK AT MY LAST
POINT THERE, THIS GROUP OF
VIRUSES, HERV-K (HML-2) GROUP
CAN BE SUBDIVIDED INTO THREE
TAXA, LTRSA, 5B, AND 5HS.
THE OTHER TWO TAXA, THEY'RE
KNOWN INTEGRATIONS FOR MORE THAN
THE LAST 5 MILLION YEARS HAVE
FOUND THEIR WAY INTO THE
GERMLINE.
IF YOU LOOK AT THE THREE ON THE
LEFT, THE ONES IN BLUE NOW JUST
FOR THE GAG GENE, BUT ANY OTHER
TREE WOULD LOOK THE SAME.
THE ONES IN BLUE ARE THE MOST
RECENT, AS YOU CAN TELL IN THE
TERMINAL, THE BRANCH ON THESE,
BRANCH LENGTHS ON THESE IS MORE
OR LESS PROPORTIONAL TO THE AGE
OF THE PROVIRUS, AND THESE GO
BACK MANY MILLIONS, 25 OR MORE
MILLION YEARS.
THE 5B PROVIRUSES OR THE OTHER
I'LL CALL YOUR ATTENTION TO IN
PINK, AND THEY ARE -- THEY
APPEAR TO BE ANCESTRAL TO THE
OTHER TWO GROUPS. THE 5A I'M
NOT GOING TO TALK ABOUT DOWN AT
THE BOTTOM, THE GREEN ONES.
NOW, ALL OF THESE, I SAID, ARE
DEFECTIVE.
THEY ALL HAVE VARIOUS KINDS OF
DELETIONS.
PAUL'S LAB AND ALSO TERRY
HINMAN'S LAB IN FRANCE, HAD THE
ORIGINAL INSIGHT THE VIRUS THAT
GAVE RISE TO THE RECENT ONES IS
PRETTY MUCH ESSENTIALLY THE
SAME, AND MOST OF THESE
MUTATIONS HAVE ARISEN IN THE
MANY MILLIONS OF YEARS FOR THE
MOST PART SINCE THEY WERE
INTEGRATED.
THEREFORE, THE CONSENSUS
SEQUENCE BETWEEN ALL OF THOSE
SHOULD REPRESENT SOMETHING CLOSE
TO THE ORIGINAL VIRUS.
SO THEY CREATED THIS VIRUS
CALLED KCON, WHICH I'LL USE A
LOT, WHICH WAS DONE BY TAKING
THE CONSENSUS OF ALL OF THE
SEQUENCES SHOWN UP AT THE TOP
AND PUT THAT ON A NON-ROOTED
TREE, YOU CAN SEE THAT SITS MORE
OR LESS AT THE CENTER, WHICH IS
WHERE YOU WOULD EXPECT IT TO BE.
THAT ACTUALLY CREATES AN
INFECTIOUS VIRUS.
IF YOU JUST REFLECT THE GAG OR
GAG GENES, YOU GET THIS NICE
VIRUS LIKE PARTICLES.
SO THAT ACTUALLY HAS BEEN
EXTREMELY USEFUL TO US FOR THE
EXPERIMENTATION.
I DON'T KNOW WHAT TO TELL YOU.
YOU CAN AGE THE INSERTION OF
ENDOGENOUS PROVIRUS BY LOOKING
AT PHILOGENETIC DISTRIBUTION FOR
THE ONES IT'S IN, OR ONES THAT
ARE OLD ENOUGH TO BE IN THE
SPECIES.
THESE TWO PROVIRUSES, AS YOU CAN
SEE, ARE ALSO FOUND IN
CHIMPANZEES, AND IN PARTICULAR
IN GORILLAS, BUT NOT IN
ORANGUTANS.
THAT DATES THE COMMON ANCESTOR
AND PROBABLY ALMOST CERTAINLY
THE INTEGRATION GAVE RISE TO
THIS, BACK INTO SOME COMMON
ANCESTOR OF HUMANS AND GORILLAS.
THAT IS ON THE ORDER OF 8 TO 12
MILLION YEARS AGO.
WE'LL SEE -- IN ROUND NUMBERS,
WE'LL SAY 10 MILLION YEARS AGO.
THESE ARE ABOUT 10 MILLION YEARS
OLD.
THE 3Q 12.3 HAS AN INTERESTING
PROPERTIES.
THE STRUCTURE OF THE VIRUS, THE
GAG GENE IS COMPLETELY INTACT.
IT'S BEEN PRESERVED INTACT FOR
10 MILLION YEARS SITTING IN THE
GENOME.
HOWEVER, IT WAS DERIVED -- IF
YOU LOOK AT THAT, THERE WAS A
RECOMBINATION EVENT THAT
GENERATED THIS PROVIRUS
ORIGINALLY THAT BROUGHT IN THIS
OLDER TYPE B PROVIRUS, AND
CROSSOVER WAS IN THE MIDDLE OF
THE GAG TO THE MIDDLE OF POL.
THAT WAS ALMOST IDENTICAL
BETWEEN THE TWO PROVIRUSES.
THE OTHER PROVIRUS, THE 22Q11.23
IS STILL IN OUR GENOME AND HAS A
COMPLETELY INTACT GAG GENE.
SO THERE'S RATHER STRONG
EVIDENCE THERE'S SOMETHING
SELECTIVELY ADVANTAGEOUS ABOUT
HAVING THESE GAG GENES IN OUR
GENOME.
ANOTHER INTERESTING PROPERTY IS
OF 3Q12.3 IS THAT THE 5 PRIME
LTR BUT NOT THE 3 PRIME LTR
CONTAINS THE FOUR PAIR BASE
MUTATION THAT, AS IT HAPPENS
CREATES THE BINDING SITES FOR
HOX-PBX TRANSCRIPTION FAMILY
PROTEIN.
THIS IS A COMMON TRANSCRIPTION
FACTOR, AND IT APPEARS A LOT OF
TIMES.
IT APPEARS IN CANCER AS
IMPORTANT.
IT APPEARS IN LOTS OF
DIFFERENTIATION AS IMPORTANT.
AS I WILL SHOW YOU IN THIS
SLIDE, THIS IS DONE SOME YEARS
AGO BY MEGAN IN MY LAB, IF YOU
LOOK AT -- WHAT SHE DID IS JUST
COMPARED WHAT THAT TRANSCRIPTION
FACTOR DOES IN A STANDARD
PROMOTER BASHING ASSAY AND PUTS
IT NEXT TO A VOCIFEROUS GENE.
IF YOU LOOK AT THE BOTTOM, THE
3Q PRIME WILD TYPE LTR, IF YOU
MAKE NO OTHER CHANGE, IT BRINGS
DOWN THE EXPRESSION A LOT.
ANOTHER RELATED PROPERTY OF THIS
PROVIRUS WAS WORKED OUT BY AIDAN
BURN A FEW YEARS AGO WHEN HE
LOOKED AT THE EXPRESSION OF ALL
ENDOGENOUS PROVIRUSES OF THESE
GROUPS ACROSS THE LARGE GTEX
DATABASE OF LARGE RNASEQ DATA
FROM TISSUES FROM 100 OR MORE
INDIVIDUALS, AND AS YOU CAN SEE,
3Q12.3 IS ONE THAT'S BY FAR THE
MOST HIGHLY EXPRESSED.
THE ONE EXPRESSED MORE IS HIGHLY
DEFECTIVE.
IT DOES SOMETHING, BUT IT HAS
NOTHING TO DO WITH BEING A
VIRUS.
THIS HAS A TECH ADD GENE, AND
THAT GENE IS HIGHLY SPECIFIC,
NOT IN CHIMPANZEES.
SO IT OCCURRED IN THE LAST 5
MILLION YEARS.
AND THAT MUTATION FURTHER, THAT
MUTATION FURTHER IS FIXED IN THE
HUMAN GENOME.
THE OTHER THING THAT IS
REMARKABLE IS THAT GAG -- IF YOU
LOOK AT THAT GAG GENE ACROSS
5,000 ALLELES IN THE HUMAN 1,000
GENOME DATABASE, THE CAPSID
DOMAIN HAS NOT A SINGLE
MUTATION.
THERE'S NO SINGLE CHANGE IN THE
CAPSID DOMAIN.
THERE ARE VERY FEW OVERALL, AND
IT'S OPEN TO EVERYBODY.
THAT FOR US IS RATHER STRONG
EVIDENCE THAT THIS IS DOING
SOMETHING INTERESTING AND MAYBE
EVEN IMPORTANT.
SO OUR HYPOTHESIS GIVEN WHAT
I'VE TOLD YOU BEFORE ABOUT
ENDOGENOUS PROVIRUSES IS THAT
THIS MAY HAVE EVOLVED INTO SOME
KIND OF RESTRICTION FACTOR FOR
REPLICATION OF THAT RELATED
VIRUS, AND OUR SPECULATIVE
HYPOTHESIS, BECAUSE WE'LL NEVER
BE ABLE TO ACTUALLY PROVE IS, IS
THAT WE HAVE EVIDENCE OF THIS
GROUP OF PROVIRUS, THIS GROUP OF
VIRUSES WENT EXTINCT AS A VIRUS
ON THE ORDER OF HALF A MILLION
YEARS AGO.
OUR SPECULATIVE HYPOTHESIS IS IT
WAS THE ORIGIN OF THIS MUTATION
AND ITS SELECTION THAT SPREAD
THROUGH THE POPULATION THAT
ACTUALLY LED TO THE EXTINCTION
OF THE VIRUS.
I'LL JUST LEAVE YOU WITH THAT AS
A SPECULATION RIGHT NOW.
SO THE REST OF WHAT I'LL TELL
YOU IS ABOUT TRYING TO PROVE
THIS OR TEST THIS HYPOTHESIS AND
THEN CHECK IF IT TESTS OUT
BECAUSE YOU MIGHT GUESS IT DID,
CHECK THE MECHANISM OF IT.
THE GENERAL EXPERIMENT IS TO
EXPRESS THE KCON, EITHER
PSEUDOVIRUS OR EXPRESSIVE VIRUS
BY ADDING AN EXTRA GENE AND TEST
WITH THE 3Q12.3 GAG GENE BECAUSE
THE GAG GENE IS MOST CERTAINLY
DOING THE ACTION HERE, IF IT'S
GOING TO DO ANYTHING, AND
CO-TRANSEFFECT AND CHECK FOR
NON-INFECTIOUS VIRUSES, OR IF
YOU ADD AN ENVELOPE GENE, TO
ACTUALLY LOOK FOR AN INFECTIOUS
VIRUS.
AT THE SAME TIME, WE CAN DO LOTS
OF OTHER STUDIES TO SEE WHAT'S
GOING ON.
THIS CURVE IS PLOTTED PERHAPS A
LITTLE BIT BACKWARDS, BUT YOU'LL
NOTICE NOW AT THE -- SO THIS
SHOWS THAT THE HERV-K, THE
3Q12.3 RATHER STRONGLY BLOCKS
PRODUCTION, IN THIS CASE FOR AN
INFECTIOUS VIRUS, AS YOU'LL SEE
THE SAME IF YOU LOOK FOR
PARTICLE FORMATION, AND UP TO
ABOUT -- I'M GOING BACKWARDS
THERE.
UP TO ABOUT 1 IN 16 IS ENOUGH TO
GIVE ABOUT A 50 PERCENT
INHIBITION.
ONE GAG MOLECULE PER 16 OR
ONE -- OR ABOUT PER 3 HEXAMERS,
IF YOU LIKE, IS ABOUT THE RATIO,
AND SOME VIRUSES YOU GET EVEN
MORE.
JUST TO REMIND EVERYBODY OF
ERIC'S VERY POPULAR SLIDE, ON
THE TINY CORNER OF THIS SLIDE,
JUST TO MAKE SURE WE ALL
UNDERSTAND -- I'M SURE YOU ALL
KNOW THIS ACTUALLY.
BUT THE GAG CONSISTS OF TOLEDO
MAINS, MATRIX, CAPSID, AND
NUCLEOCAPSID PRINCIPALLY, PLUS
SOME OTHERS INVOLVED WITH MATRIX
DOMAINS, BUT I'M NOT GOING TO
TALK ABOUT THAT.
THAT GOES WITH MATRIX ASSEMBLING
THE MEMBRANE, AND CAPSIDS
ASSEMBLING EACH OTHER INTO
HEXAMERS GENERALLY, AND THEN
WRAPPING THE MEMBRANE AROUND,
BUDDING OCCURS, AND IT BUDS OFF
AFTER INCLUDING MACHINERY TO
CLOSE OFF THE MEMBRANE AT THE
END.
THE BUDS IN THE MATURE PARTICLE
AND ONLY AFTERWARDS GETS
PROCESSED INTO ITS FINAL MATURE
STRUCTURE.
WHAT IS QUITE REMARKABLE IS THAT
THE DIFFERENCE IN STRUCTURE
BETWEEN THE MATURE AND THE
IMMATURE, THAT WAS ALREADY
MENTIONED A LITTLE BIT THAT T
THE -- THAT THERE'S QUITE A
SUBSTANTIAL REARRANGEMENT
BETWEEN HEXAMERS, BUT THEY'RE
QUITE A MATURE ORGANIZATION.
IT'S STRIKING THAT THE IMMATURE
PARTICLES, ALTHOUGH THEY CAN'T
BE COMPLETE PARTICLES, FOR
REASONS I'M NOT GOING TO GO
INTO, BUT THEY'RE ALWAYS ALMOST
PERFECTLY SPHERICAL IN THE
APPEARANCE, IN THE MICROGRAPH,
AND YOU'VE ALREADY SEEN SOME OF
THAT.
WHEREAS THE MATURE PARTICLES
HAVE THIS -- OFTEN HAVE SOME --
ARE NEVER ACTUALLY PERFECTLY HEX
GON
HEXAGONAL SORT OF STRUCTURES
THAT YOU SEE WITH MANY OTHER
VIRUSES.
THEY'RE ALWAYS EITHER THIS
BULLET-SHAPED CAPSID OR
CONE-SHAPED CAPSID THAT YOU SEE
IN HIV, OR SOME OTHER STRUCTURE
WHICH IS NOT -- WE DON'T -- IT'S
NOT VERY WELL DEFINED FOR OTHER
RETROVIRUSES.
IT'S KIND OF LUMPY AND UGLY BY
AND LARGE.
IT'S NEVER PERFECT LY ICOSOHEDRL
IN APPEARANCE.
THAT'S MADE POSSIBLE BY THE
DIFFERENCE BETWEEN THE NTD THAT
YOU CAN SEE DOWN IN THE LOWER
LEFT THERE BEING -- I'M SORRY,
BY THE CAPSID PROTEIN BEING
DIVIDED INTO THE N-TERMINAL
DOMAIN AND THE C-TERMINAL
DOMAIN.
THE C-TERMINAL DOMAIN OF THE
CAPSID INTERACTS CAPSID
MOLECULES WITH ONE ANOTHER TO
FORM HEXAMERS, AND THE INTERNAL
DOMAIN INTERACTS HEXAMER TO
HEXAMER TO FORM THE STRUCTURE,
AND THAT'S AN OVERSIMPLIFIED
VERSION OF WHAT'S GOING ON.
AND THAT'S MADE POSSIBILITY BY
THE STRUCTURE OF THIS LINKER
DOMAIN SO THERE CAN BE DIFFERENT
LEVELS OF INTERACTION BETWEEN
DIFFERENT HEXAMERS, AND THAT
ALLOWS THE PARTICLES TO ASSUME
OTHER THAN THIS ICOSALHEDRAL
STRUCTURE.
AND YOU SEE IT STARTS WITH THE
EXPERIMENT IN BLUE THAT STARTS
WITH KCON, THE WILD SIDE GAG
PROTEIN, AND WITH SUBSTITUTIONS
OF THE VARIOUS DOMAINS, CAPSID,
NUCLEOCAPSID, AND SO ON.
IF YOU SUBSTITUTE JUST THE
INTERIM PLUS THE LINKER, THEN
YOU GET A STRONG INHIBITION, AND
THE OTHER PARTS YOU SEE LESS
INHIBITION OR NO INHIBITION.
THAT LOCALIZES THE EFFECT OF
THIS PARTICULAR REGION.
WHEN YOU LOOK HARD, PARTICULARLY
AT THE LINKER SEQUENCE, ONE
MUTATION STANDS OUT, AND THAT'S
THE DELETION OF THE LUCYRINE.
AND THAT DELETION IN THE
STRUCTURE OF THE IMMATURE KCON
PROTEIN FORMS A VERY SPECIFIC
HYDROGEN BOND WITHIN A HELICAL
REGION IN THE INTERNAL DOMAIN.
THAT HYDROGEN BOND IS NOT SEEN
IN THE MORE MATURE PROTEIN.
YOU CAN SEE A COUPLE OF OTHERS,
BUT NOT THAT ONE.
IF YOU REPAIR THE SERINE
MUTATION -- SORRY.
IF YOU TAKE THE WILD TYPE VIRUS
AND INTRODUCE THESE VARIOUS
MUTATIONS INTO IT, YOU FIND ON
THE FAR RIGHT INTRODUCING THIS
SERINE DELETION RECAPITULATES
MOST OF THE ACTIVITY IN THIS
PROTEIN.
NOT QUITE ALL, SO WE THINK THERE
ARE OTHER MUTATIONS THAT ARE
ALSO IMPORTANT, BUT WE HAVEN'T
TRACKED THOSE DOWN YET.
THOSE ARE THE OTHER DIFFERENCES
THAT WE SEE REALLY MATTER VERY
MUCH AT ALL AS FAR AS THE
INHIBITION OF ACTIVITY IS
CONCERNED.
SO WE WANTED TO SEE WHAT THIS
LOOKED LIKE IN TERMS OF WHAT THE
VIRUS IS ACTUALLY DOING AS THE
BUDDING IS TRYING TO OCCUR, AS
THE REPLICATION IS TRYING TO
OCCUR, AND OF COURSE AS THE TAG
LINE OF AN OLD JINGLE THAT I
THINK YOU HAD TO LIVE IN NEW
ENGLAND TO HEAR, IT'S WHO YOU
GONNA CALL?
OF COURSE THE ANSWER IS OBVIOUS,
RIGHT?
IT'S ALAN.
WE CALL OUR VIRUS ASSEMBLY GURU
HE
HERE, AND ALAN CALLS FERRI
SOHELLAN, OUR MICROSCOPE EXPERT,
AND WE SENT ALL THE SAMPLES OF
THIS.
THE FIRST IS ALL THE WILD TYPE
SAMPLES, THE KCON WILD TYPE
PROTEIN, AND YOU SEE THE
PERFECTLY GOOD PARTICLE
ASSEMBLY.
THEY ALWAYS APPEAR PERFECTLY
CIRCULAR, IF YOU LIKE PERFECTLY
SPHERICAL, AND QUITE UNIFORM IN
SIZE.
UNLIKE THE FINAL PRODUCTS.
HOWEVER, WHEN YOU LOOK AT 3Q12.3
GAG ALONE, REMEMBER IS THE
MUTANT PROVIRUS, YOU DON'T SEE
ANYTHING LIKE THAT.
YOU JUST SEE THESE SORT OF --
LOOKS LIKE THE THICKENINGS OF
THE MEMBRANES WHERE IT'S TRYING
TO BUD.
THEY HAVEN'T ACTUALLY PROVEN
YET, WE HAVEN'T DONE ANY
EXPERIMENTS TO SHOW DIRECTLY
THOSE ARE GAG PROTEINS, BUT IT'S
HARD TO IMAGINE THEY'RE ANYTHING
ELSE BECAUSE YOU DON'T SEE THIS
EVER UNLESS YOU HAVE THIS IN
THERE.
BUT NOW IF YOU TRANSFECT WITH
THE WILD TYPE PROTEINS, PROTEINS
FROM KCON, WHAT YOU GET ARE WHAT
WE LIKE TO CALL VUPs, OR VIRUS
UNLIKE PARTICLES, AND YOU GET
THIS BIZARRE KIND OF ATTEMPT TO
BOND BUT NOTHING -- THEY NEVER
CLOSE.
THEY JUST SORT OF GROW AROUND
INTO THESE UGLY STRUCTURES.
IT'S AS THOUGH YOU HAD A MIXTURE
OF THINGS WITH THOSE THAT CAN'T.
THAT'S WHAT YOU GET.
WE HAVEN'T PROVEN THAT'S WHAT IT
LOOKS LIKE.
WE HAVE COLLABORATION SET UP TO
DO REAL STRUCTURAL ANALYSIS ON
THESE THINGS, BUT THAT IS OUR
HYPOTHESIS AT LEAST, AND I THINK
IT'S A PRETTY GOOD ONE, TO
UNDERSTAND WHAT'S GOING ON.
THAT ALLOWS THE FORMATION OF
THESE IMMATURE PARTICLES.
IF YOU DON'T HAVE IT, THEN YOU
JUST CAN'T -- THE VIRUS PROBABLY
CAN'T PULL TOGETHER STRONG
ENOUGH TO FOLD A MEMBRANE AROUND
IT WITHOUT THAT PARTICULAR
LINKAGE.
SO IT STARTS OUT, BUT IT CAN'T
GET VERY FAR.
IF YOU MIX IT WITH GOOD, THEN
YOU JUST GET THESE MISSES.
ERIC ACTUALLY YEARS AGO
PUBLISHED WITH HIV WHAT
HAPPENS -- THERE'S A HOMOLOGOUS
SERINE IS HIV, AND IF YOU LOOK
AT THIS, YOU GET THE SIMILAR
BUDDING TYPE PHENOTYPE FROM IT.
IT WILL BE INTERESTING TO SEE
WHAT THE DELETION OF THAT LOOKED
LIKE TOO, BUT THAT HASN'T BEEN
DONE YET.
INTERESTING NOW, 22Q11.3, WHICH
HAS IN THE UPPER RIGHT, THE
IDENTICAL AMINO ACID SEQUENCE.
THIS HAS BEEN PRESERVED FOR 10
MILLION YEARS IN OUR ANCESTRAL
GENOMES, DIFFERENT FROM ANOTHER
AMINO ACID THAT WE DON'T THINK
IS MEANINGFUL, ALTHOUGH WE
HAVEN'T PROVEN IT YET, BUT
THERE'S NO REASON TO BELIEVE
THAT POSITION HAS ANY EFFECT ON
IT.
THAT DOES NOT HAVE THIS EFFECT.
IF YOU LOOK AT THE BOTTOM, YOU
CAN SEE IT DOES NOT BLOCK THE
BUDDING AND RELEASE.
SO WHY DOES IT DO THAT?
THIS IS THE GAG GENE OF THE
ANCESTRAL VIRUS.
YOU LOOK, AND YOU DON'T SEE
ANYTHING HAPPENING.
YOU LOOK AT THE TOP PICTURE, AND
YOU DON'T SEE ANYTHING HAPPENING
WITH THE MEMBRANE.
YOU DO SEE BLOBS AND STUFF
INSIDE.
WHEN WE LOOK AT THE BLOBS OF
HIGHER MACK -- MAGNIFY , WHAT
YOU SEE, IF YOU REMEMBER YOUR
HISTORICAL SEROLOGY, SOMETHING
YOU CALL A PARTICLES.
MANY RETROVIRUSES, INCLUDING
MPNV AND OTHERS, DO NOT MATURE
THE VIRUS MEMBRANE THE WAY HIV
AND MOST OTHER RETROVIRUSES WE
WORK WITH DO, THEY MATURE AS
INTERCELLULAR PARTICLES,
INTERCELLULARPLASMIC PARTICLES
THAT THEN FIND THEIR WAY TO THE
MEMBRANE AND WRAP THE MEMBRANE
AROUND THEM.
THAT SORT OF SEEMS TO BE WHAT'S
HAPPENING HERE, BUT THESE NEVER
GET OUT TO THE MEMBRANE, AND
THEY'RE INEFFECTIVE IN DOING
THAT.
YOU SEE THAT, THEY SEEM NOT TO
CLOSE COMPLETELY EITHER.
YOU APPROACH THE IDENTICAL AMINO
ACID SEQUENCE, LIKE THE ONE I
SHOWED YOU BEFORE.
AND THEN NOT AN IMPORTANT ONE,
AS FAR AS WE CAN TELL.
THE DIFFERENCE IS IN THE MATRIX
PROTEIN OF THE ANCESTRAL VIRUS.
MOST MATRIX PROTEINS WE'VE
TALKED ABOUT AT THE AMINO
TERMINUS HAVE DONE ONLY GLY
GLYCOLSYLATION SITE, AND THAT
ALLOWS THE ASSOCIATION AND THE
BUDDING PROCESS TO GO.
HOWEVER, IF YOU LOOK AT THE
LTR5B, IF YOU LOOK, THERE ARE
MORE ACIDIC AMINO ACIDS IN THE
REGION THAN BASIC AMINO ACIDS.
AND THE PREDICTED PK OF THIS IS
ACTUALLY QUITE ACIDIC.
THERE'S NO WAY THIS PROTEIN IS
GOING TO ASSOCIATE WITH THE
MEMBRANE THE SAME WAY.
THAT'S WHAT HAPPENS.
THESE DON'T GO TO THE MEMBRANE
THEY ASSOCIATE.
THEY DON'T GO TO THE MEMBRANE AT
THAT POINT.
HOW THEY DO GET THERE EVENTUALLY
IN THE REAL VIRUS, I HAVE NO
IDEA, BUT THEY DON'T ASSOCIATE
WITH IT THERE.
I'LL TELL YOU HOW WE INTERPRET
ALL THIS IN EVOLUTIONARY TERMS
AT THE END, BUT FIRST I WANT TO
POINT OUT SOMETHING ELSE.
IF YOU LOOK AT THE TWO LITTLE
SETS OF BARS, IF YOU DO THE SAME
KINDS OF EXPERIMENTS WITHIN MLV,
THE GAMMA RETROVIRUS, OR THE
ALPHA RETROVIRUS, THIS HAS NO
EFFECT ON THESE RETROVIRUSES,
WHICH MAKES SENSE BECAUSE
COEXISTENCE WOULD BE NECESSARY
FOR THIS EFFECT TO OCCUR.
IF YOU LOOK AT MPMV, THIS IS A
VIRUS THAT ALSO ACCESSIBLES
INTRACELLULAR, YOU CAN SEE THIS
IS INHIBITED BY THE 22Q11.23,
WHICH HAS THE INTERPLASMIC
ASSEMBLY AS WELL.
THIS IS THE SAME KIND OF
COASSEMBLY DEFECT, JUST IN
INTERCYTOPLASMIC.
REMARKABLY, AND WHY I WAS ABLE
TO GET A GRANT ON THIS SUBJECT,
THE -- THIS PROTEIN DOES BLOCK
THE 3Q12.23 PROTEIN DOES BLOCK
THE HIV ASSEMBLY.
AGAIN, YOU SEE IT'S ALMOST
EXACTLY THE SAME KIND OF CURVE I
SHOWED YOU BEFORE WHEN YOU
TITRATE IT AGAINST HIV, AND THE
LTR RELEASE.
SO IT BLOCKS HIV PARTICLE
MATURATION WHEN DOSED IN THE
INTENDED MANNER.
IF YOU LOOK AT PICTURES --
HERE'S HIV ALONE ON THE LEFT.
HERE'S THE GAG PROFILE, SO IT
GETS MATURE PARTICLES.
IF YOU ADD THAT WITH KCON GAG,
WHICH ACTUALLY HAS BEEN SHOWN BY
OTHERS, INCLUDING A PAPER WITH
ALAN REIN, TO BE SOMEWHAT
INHIBITORY, BUT NOWHERE NEARLY
AS MUCH AS 3Q12.3.
YOU GET PERFECTLY GOOD LOOKING
STRUCTURES, BUT YOU GET THE SAME
KIND OF ASSEMBLY DEFECTS ON THE
RIGHT THAT WE SAW WITH THE WILD
TY
TYPE.
HERV-K.
SO IN ALMOST ALL THE EXPERIMENTS
I SHOWED YOU BEFORE, EXCEPT FOR
THE END, SET UP A NANO-BRET
ASSAY, WHICH USES BIOLEUKEMIA --
BIOLUMINESCENCE ASSAY.
SO YOU HAVE TWO POTENTIAL
INTERACTORS, ONE HAS THE
NANO-LUKE TAG AND THE OTHER HAS
A HALO TAG.
THE EMISSIONS FROM THE NANO-LUKE
CAN THEN STIMULATE THE HALO AND
GIVE THE HALO SPECTRAL
FLUORESCENCE, AND YOU CAN USE
THAT AS AN INTRACELLULAR MEASURE
OF INTERACTION, AND YOU CAN SEE
FROM THE BARS THAT THE HALO
ALONE DOES NOTHING OF
SIGNIFIC
SIGNIFICANCE.
THEN ON THE RIGHT, THE OTHER
CONTROLS, MLV AND MPNV DON'T
HAVE ANY EFFECT, SHOW NO
INTERACTIONS, WHEREAS BOTH THE
3Q12.3 GAG HALO, THE KCON GAG
HALO INTERACTS WITH ITSELF, AS
YOU'D EXPECT.
THE 3Q12.3 HALO INTERACTS, AND
HIV INTERACTS WITH MORE WHAT
SEEM TO BE THE SAME LEVEL.
THE FACT THAT THEY INTERACT WITH
QUITE SURPRISING, THESE TWO
CAPSID PROTEINS HAVE VERY LITTLE
SIMILARITY IN SEQUENCE, BUT THEY
ARE QUITE SIMILAR IN STRUCTURE
AT THE CRITICAL JUNCTION WHERE
THE HEXAMER IS FORMED IN THE
IMMATURE FORM.
YOU CAN SEE THE GRAY TRACE ON
THE RIGHT IS HIV.
THE ORANGE IS HML-2.
THEY'RE REMARKABLY SIMILAR IN
THEIR STRUCTURE EVEN THOUGH THE
AMINO ACID SEQUENCE IS
DIFFERENT.
WE IMAGINE THIS DOES ALLOW AT
LEAST SOME COASSEMBLY OF THE
TWO, AND THAT IS WHAT ALLOWS IT
TO BE PROTECTIVE, IF YOU LIKE,
EVEN THOUGH, IF YOU LOOK AT THE
SEQUENCE OF THESE, AS SHOWN BY
THE TREE THERE, THERE'S REALLY
NO PARTICULAR SPECIAL SEQUENCE
RELATIONSHIP BETWEEN THESE
PROTEINS OVERALL.
NOW TO THE SORT OF BOTTOM LINE
HERE.
WE INFERRED FROM ALL OF THIS
WITH A LITTLE BIT OF SPECULATION
MIXED IN AN EVOLUTIONARY PATHWAY
BY WHICH THIS HAS COME TO BE.
YOU START OUT WITH THE LTR-5B,
THE ANCESTRAL VIRUS, TO THE HS
GROUP, AND REPLICATED IN OUR
ANCESTORS GOING BACK 25 MILLION
YEARS.
I ONLY SHOW WHAT'S HAPPENED IN
THE LAST 10 MILLION YEARS.
AT SOME POINT, AND ALSO WE THINK
IN THE HUMAN, COMMON ANCESTORS
HUMANS AND GORILLAS, BUT PRIOR
TO THE OTHER ONE, THIS MUTATION
INSERTED, WE SPECULATE THAT THIS
WAS THEN PROTECTED AGAINST
REPLICATION OF THE 5B TYPE
VIRUSES WHICH HAD THIS
INTRACELLULAR PLASMIC ASSEMBLY.
PERHAPS IN RESPONSE TO THIS, THE
5HS TYPE, WHICH HAS THE
CYTOPLASMIC ASSEMBLY MAY HAVE
EVOLVED BY MUTATIONS OF
DUPLICATION, AS YOU CAN SEE IN
THE PREVIOUS SLIDE, IN THE
MATRIX N-TERMINAL REGION TO GIVE
IT MEMBRANE ASSOCIATED ASSEMBLY,
AND THAT HAS ALLOWED IT TO
ESCAPE FROM THE EFFECTS OF THIS
INHIBITORY EFFECT MIGHT HAVE
BEEN.
THAT WAS THEN IN THE COURSE OF
THE REPLICATION THAT VIRUS, THE
RECOMBINATION OCCURRED, WHICH
BROUGHT IN THE CAPSID,
PARTICULARLY THE CAPSID DOMAIN
FROM THE RESIDENT 5B VIRUS.
THAT THEN REPLICATED ALL THE WAY
UP AND PROBABLY ALSO GAVE AT
LEAST SOME PROTECTION INTO
HUMANS, AND THEN AT SOME POINT
IN THE HUMAN SPECIFIC ANCESTRY
IN THE LAST 5 MILLION YEARS OR
SO, THAT DOES DUPLICATION IN THE
LTR OCCURRED, AND THAT GAVE IT A
MUCH BETTER EXPRESSION.
AND WE SPECULATE IT MAY HAVE
CONTRIBUTED AT LEAST TO THE
EXTINCTION OF THAT AS A VIRUS.
THAT IS OUR SORT OF CURRENT
HYPOTHETICAL FRAMEWORK FOR
THINKING ABOUT HOW THE EVOLUTION
OF THIS VIRUS, THIS PARTICULAR
VIRUS AND RESTRICTION FACTOR
MIGHT HAVE OCCURRED.
MANY PEOPLE IN THE LAB HAVE MADE
THIS POSSIBLE.
THIS IS A LONG SERIES OF
STUDENTS AND POST DOCS WHO
WORKED ON THIS OVER THE YEARS,
RAVI, MEAGHAN, FARRAH, AIDAN,
AND MOST OF THEM, ALL THE WORK I
SHOWED YOU, IS NOW THE WORK OF
MIKE FREEMAN, NOW A POST DOC IN
THE LAB, WITH THE ENORMOUS
SUPPORT TO GET THE BEAUTIFUL
ELECTROMICROGRAPHS.
WITH PARTICULAR THANKS OVERALL
TO ALAN REIN, WHO EVER SINCE HE
JOINED THE DRP BACK IN '98 OR SO
HAS BEEN A REALLY VALUED
COLLEAGUE AND SOURCE OF
INSPIRATION ON CRITICAL SCIENCE.
I'LL THANK YOU.
>> YOU'VE SHOWN THIS DATA.
I DON'T REMEMBER THE RESULTS.
HOW BROAD IS THE CELLULAR TISSUE
DISTRIBUTION OF THE INTERFERING
RETROVIRUS?
>> IT'S EVERYWHERE.
THE ONLY PLACE IN THE DATA SET
IT DIDN'T SHOW UP WAS IN WHOLE
BLOOD, WHICH YOU WOULD ARGUE
COULD WELL BE ALLOWING -- IF IT
HAD BEEN EXPRESSED THERE WELL
ENOUGH, WE MIGHT NEVER HAVE HAD
THE HIV PANDEMIC.
NOW WE'RE GETTING REALLY
SPECULATIVE OF COURSE.
IN FACT, MY -- ONE OF THE
STRETCH AIMS OF MY GRANT IS TO
TRY TO GET IT EXPRESSED AGAIN TO
SEE IF WE CAN ACTUALLY SEE WHAT
THE EFFECT WOULD BE.
>> HOW CAN THE ASSAY WITH THE
CYTOPLASMIC PROTECTION DESCRIBED
BY ERIC HUNTER SEVERAL YEARS
AGO, AND IT'S FURTHER DOWN --
>> I WOULD HAVE TO DO OTHER
THINGS TO MPNV TO MAKE IT
NECESSARY.
>> ARE THERE CHANGES IN THAT
REGION?
>> TO BE HONEST WITH YOU, WE
HAVEN'T LOOKED.
THAT'S A VERY GOOD POINT.
WE SHOULD TAKE A LOOK AT THAT.
>> WITH THE MYRISILATION --
>> WE HAVEN'T LOOKED TO SEE,
BUT THE SIGNAL IS THERE.
THANK YOU VERY MUCH.
>> OUR NEXT SPEAKER HAS A TRULY
UNIQUE DISTINCTION.
HE HAS BEEN IN THE FIELD LONGER
THAN I HAVE.
THE REASON I KNOW THAT IS WHEN I
JOINED RUBIN'S LAB IN THE SUMMER
OF 1962, PETER WAS JUST LEAVING.
WE OVERLAPPED FOR PROBABLY A FEW
WEEKS IN HARRY RUBIN'S LAB WHEN
HE WAS JUST LEAVING TO FORM HIS
OWN LAB.
SO IT'S BEEN MANY, MANY DECADES,
AND HE HAS PERFORMED, MADE MANY
CRUCIAL DISCOVERIES PURSUING THE
VIROLOGY OF THE ALPHA
RETROVIRUSES AND THEN ACTUALLY
HE WAS A PIONEER IN GOING AROUND
AND DIGGING VIRUSES OUT OF
PEOPLE'S FREEZERS AND
DISCOVERING NEW ONCOGENES THAT
WAY.
IN MORE RECENT YEARS, I THINK
HE'S SPECIALIZED IN LIPID
SIGNALING, WHICH REALLY ARISES
FROM HIS DISCOVERY OF YET
UNEXPECTED NEW ONCOGENES AND
CONSIDERING THE POSSIBLE
APPLICATIONS OF HIS DISCOVERIES
ON LIPID SIGNALING IN CANCER.
WITHOUT FURTHER ADO HERE,
WELCOME TO THE MEETING.
>> THANK YOU FOR YOUR KIND AND
GENEROUS INTRODUCTION.
I AM HONORED AND DELIGHTED TO
SPEAK AT THE SYMPOSIUM
CELEBRATING ALAN REIN'S WORK.
ALAN AND I SHARE EARLY
SCIENTIFIC ROOTS, AS YOU JUST
LEARNED.
WE WERE BOTH WORKING ON THE
NEUROSARCOMA VIRUS IN THE SAME
LAB IN BERKELEY.
IN THIS SYMPOSIUM I WILL BE THE
ONLY SPEAKER TALKING MAINLY
ABOUT AVIAN VIRUSES, AND AS THE
LAST SPEAKER, I HAVE DECIDED TO
TAKE A LIGHT HEARTED APPROACH
AND DISTILL IT INTO SEVEN
SIMPLIFIED LESSONS LEARNED
DURING A LIFELONG SCIENTIFIC
EDUCATION.
I NEED TO EMPHASIZE THESE ARE
LESSONS LEARNED.
THEY ARE NOT MEANT AS CAREER
ADVICE.
SO LET ME BEGIN SCREEN SHARING.
LESSON NUMBER ONE, YOU SHOULD
JOIN AN EMERGING, NOVEL FIELD,
AND APPLY NEW CONCEPTS AND
GENERATE NEW TECHNOLOGIES.
DON'T JOIN A MATURE FIELD.
I THINK ALL OF YOU HAVE APPLIED
THAT LESSON IN YOUR CAREERS WHEN
YOU JOINED HIV.
THE EMERGING FIELD OF MY TIME
WAS A QUANTITATIVE ANALYSIS FOR
TUMOR VIRUS REPLICATION AND A
VIRUS-INDUCED CELLULAR
TRANSFORMATION.
THAT IS WORK THAT START ED AT CL
TECH WITH HARRY RUBIN AND HOWARD
TENEMAN.
AND THE BASIC TECHNOLOGY WAS THE
FOCUS ASSAY OF ROUS SARCOMA
VIRUS.
THE FIRST ASSAY THAT ALLOWED YOU
TO QUANTIFY ONCOGENIC
TRANSFORMATION.
HERE YOU SEE AN ILLUSTRATION OF
THIS NORMAL AVIAN FIBROBLAST TO
THE LEFT AND ROUS SARCOMA VIRUS
TRANSFORMED FIBROBLASTS TO THE
RIGHT.
YOU COULD REALLY HAVE THIS
TRANSFORMATION IN VERY DISCRETE
FOCI, AND THE FOCI, A SINGLE
FOCUS WAS THE RESULT OF
INFECTION WITH A SINGLE VIRAL
PARTICLE.
SO IT WAS QUANTITATIVE FROM THE
START.
IT WAS BROUGHT ON BY THE WORK
BEING DONE AT CAL TECH.
LESSON NUMBER 2, TRUST YOUR
INSTINCTS.
CHICKEN TUMOR VIRUSES ARE
SIGNIFICANT FOR HUMAN MEDICINE.
THERE WAS ALWAYS THIS TENDENCY
OF DOWNPLAYING THE AVIAN VIRUSES
BECAUSE THEY COULDN'T POSSIBLY
BE RELEVANT FOR HUMANS IN
MEDICINE.
SO THE FIRST IMPORTANT FEATURE
THAT SETS AVIAN TUMOR VIRUSES
APART IS THAT MOST ROUS SARCOMA
VIRUSES ARE NON-DEFECTIVE.
THEY CONTAIN ALL THE VIRAL GENES
PLUS THE ONCOGENE.
THAT'S THE ONLY WAY YOU CAN DO
GENETICS IN A CONVENTIONAL WAY.
AND ONLY AFTER THE ROUS SARCOMA
VIRUS WAS FOUND TO BE
NON-DEFECTIVE WAS A GENETIC
APPROACH TO THIS GENOME
POSSIBLE.
AND THE SECOND ADVANTAGE IS
AVIAN RETROVIRUSES USE TEN
DIFFERENT SURFACE GLYCOPROTEINS,
AND IT GIVES YOU ENORMOUS
RICHNESS AND WEALTH OF ALL KINDS
OF TOOLS AND APPROACHES.
EACH GLYCOPROTEIN TARGETS A
SPECIFIC CELL SURFACE RECEPTOR,
AND WHEN THE GLYCOPROTEIN
OCCUPIES THAT RECEPTOR, THAT
INDUCES VIRAL INTERFERENCE.
LESSON 3 IS TAKE THESE
OBSERVATIONS AND BUILD NEW TO
TOOLS, AND FOR THE AVIAN VIRUSES
IT WAS CLEAR THAT GLYCOPROTEIN
ALLOWED YOU TO CLASSIFY THE
VIRUSES INTO SUBGROUPS.
THAT WAS COMPLEMENTED BY FINDING
CHICKEN LINES THAT ARE RESISTANT
TO INFECTION BY A SINGLE VIRAL
SUBGROUP BECAUSE THEY LACK THAT
PARTICULAR RECEPTOR, AND THIS
INTERPLAY OF VIRAL GLYCOPROTEIN
AND CELLULAR RECEPTOR COULD BE
MADE INTO AN ASSAY OF VIRAL
INTERFERENCE.
SO YOU HAVE TO REMEMBER THIS IS
65 YEARS AGO, AND IT WAS THE
BEGINNING OF THE MONOLAYER.
PLAQUE AND FOCUS ASSAYS HAD ONLY
BEEN DISCOVERED.
ANTIBODIES COULD BE GENERATED,
BUT ONLY FROM ANIMALS, THERE WAS
NO BIOCHEMISTRY AND NO MOLECULAR
SEQUENCING.
LET'S LOOK AT THE VIRAL SURFACE
GLYCOPROTEINS.
A LOT OF THEM FROM A TO J, SOME
ARE EXCLUSIVELY AVIAN HOST
RANGE, SOME CAN INFECT MAMMALIAN
CELLS, AND SOME ARE AVIAN
ENDOGENOUS RETROVIRUSES.
WHAT'S INTERESTING IS THERE IS
AN EVOLVING SUBGROUP THAT IS THE
PREDOMINANT PATHOGEN IN THE
POULTRY INDUSTRY IN CHINA, AND
IT IS SPREADING VERY RAPIDLY
OVER THE GLOBE.
YOU CAN USE THESE SUBGROUPS TO
DESIGN INTERFERENCE ASSAYS, AND
INTERFERENCE WITH SUBGROUP
SPECIFIC.
IF A CELL IS INFECTED WITH A
SUBGROUP A VIRUS, IT WOULD BE
SPECIFICALLY RESISTANT TO A
CHALLENGE WITH A VIRUS, AND B
MAKES RESISTANT TO B AND SO ON.
HERE IS AN EXAMPLE.
IT'S AN AVIAN LEUKOSIS VIRUS
THAT INFECTS CELLS WITHOUT ANY
MORPHOLOGICAL EVENT.
YOU WOULD NOT BE ABLE TO TELL
THE INFECTED FROM THE
NON-INFECTED CULTURES WITHOUT
ANY SORT OF BIOCHEMICAL TOOLS,
WHICH DIDN'T EXIST AT THAT TIME.
BUT IF YOU HAVE AN AVIAN
LEUKOSIS VIRUS A IN INFECTED
CELL LINE AND THEN CHALLENGE
WITH A, YOU WILL EXPERIENCE
INTERFERENCE.
THERE'S NO INTERFERENCE WITH
SUBGROUP B, C, OR D.
THAT WAS USED IN, I THINK,
IMPORTANT EXPERIMENT THAT SHOWED
THAT EARLY DNA SYNTHESIS
REQUIRED IN RETROVIRAL INFECTION
IS VIRUS SPECIFIC.
SO YOU TAKE YOUR CELLS AND
INFECT THEM WITH RSV A AS ZERO
HOURS.
AT 24 HOURS YOU INHIBIT THE DNA
SYNTHESIS, SO YOU GIVE VIRUS A
TIME TO MAKE DNA.
THEN YOU CHALLENGE AT 96 HOURS
WITH RSV C, AND THEN YOU ANALYZE
THE PROGENY.
RSV A CAN BE MADE, BUT RSV C
CANNOT BE MADE.
SO THESE TWO VIRUSES CANNOT
SHARE THE SAME DNA.
DNA MADE EARLY IN INFECTION IS
VIRUS SPECIFIC.
SO THAT BRINGS ME TO LESSON 4.
YOU SHOULD EARLY ON GET TO KNOW
PEOPLE IN THE FIELD AND ASK FOR
THEIR REAGENTS.
I DID THAT EARLY ON, AND THEY
TURNED OUT TO BE VERY
INTERESTING.
THE PRAGUE STRAIN OF RSV WAS THE
FIRST, REPRESENTATIVE OF A NEW
SUBGROUP C.
THEN I GOT THE CARR-Z IRBGS LBER
STRAIN OF RSV, AND
NEUROFIBROMATOSIS A NEW SUBGROUP
D.
THEN SCHMIDT-RUPPIN SENT ME HIS
VIRUS, AND IT WAS ALSO IN
SUBGROUP D.
AND THEN SOMEONE SENT ME THE
SARCOMA VIRUS, WHICH WAS VERY,
VERY SIMILAR TO OUR SARCOMA
VIRUS.
I SUBMITTED IT WAS IN
CZECHOSLOVAKIA AND CALLED IT
VIRUS B77 AND ALSO SUBGROUP C.
SO YOU COULD ENRICH YOUR
INVENTORY AND YOUR TOOLS BY
COLLECTING SOME VIRUSES FROM
OTHER LABS.
THAT BRINGS ME TO LESSON 5,
ATTRACT GREAT POST DOCS.
I WANT TO BRIEFLY MENTION THREE
EXAMP
EXAMPLES.
I'LL START AT THE BOTTOM BECAUSE
IT'S AN HIV MEETING AND YOU ALL
WILL KNOW ERIC HUNTER.
YOU ALSO WILL KNOW ROBIN WEISS
BECAUSE HE ALSO WORKED WITH HIV.
AND THEN THERE'S KUMAO
TOYOSHIMA.
KUMAO WAS ESSENTIALLY THE
EMPEROR OF TUMOR VIROLOGY IN
JAPAN.
HE RECEIVED THE HIGHEST
DISTINCTION AWARD FROM THE
JAPANESE GOVERNMENT.
HE PASSED AWAY TWO YEARS AGO.
ROBIN WEISS IS A MEMBER OF THE
NATIONAL ACADEMY OF SCIENCES
USA.
HE IS A MEMBER OF THE AMERICAN
PHILOSOPHICAL SOCIETY, AND OF
COURSE HE IS A MEMBER OF THE
ROYAL SOCIETY IN LONDON.
ERIC HUNTER STILL VERY ACTIVE,
AND HE COMES TO A MEETING EVERY
YEAR IN JANUARY OUT HERE IN SAN
DIEGO, AND WE ALWAYS MEET AND
HAVE DINNER TOGETHER.
LESSON 6 IS TO INTERROGATE
NATURE, GO OUT AND COLLECT
VIRUSES FROM THE ANIMAL SOURCE.
SO I WENT INTO THESE CHICKEN
SLAUGHTER HOUSES WITH PERMISSION
OF THE DEPARTMENT OF AGRICULTURE
AND COLLECTED TUMORS AND
ISOLATES VIRUSES.
I ALSO GOT VIRUSES FROM OTHER
COLLECTORS SOME EXAMPLES OF
IMPORTANT ONCOGENES THAT EMERGED
FROM THESE SPONTANEOUS TUMORS,
MC29, WHICH IS AN AVIAN LEUKEMIA
VIRUS ISOLATED OR ACTUALLY
COLLECTED BY JOE BEARD AT DUKE
UNIVERSITY THAT CONTAINED MYC.
MYC IS PERHAPS THE EMPEROR OF
ONCOGENES, VERY, VERY IMPORTANT
IN HUMAN MEDICINE.
THEN AVIAN SARCOMA VIRUS 17
YIELDED JUN, AN IMPORTANT
REGULATOR OF TRANSCRIPTION.
AN NOT COGENE THAT IS DERIVED
FROM OTHER AVIAN SARCOMA VIRUS
IS QIN.
IT IS NOW CALLED FOX G.
IT WAS THE FIRST EXAMPLE OF A
FOX PROTEIN ISOLATED FROM A
VERTEBRATE.
AND FINALLY, AVIAN SARCOMA VIRUS
16 YIELDED PI 3-KINASE.
SO THAT BRINGS ME TO THE FINAL
LESSON, WHICH IS LESSON 7,
COLLABORATE, COLLABORATE,
COLLABORATE.
SCIENCE IS DRIVEN BY ADVANCES IN
TECHNOLOGY.
YOU WILL NOT BE ABLE TO MASTER
ALL NEW TECHNIQUES.
YOU HAVE TO COLLABORATE WITH
EXPERTS.
I'D LIKE TO CITE BRIEFLY THREE
EXAMPLES.
ONE IS THE DISCOVERY OF THE
FIRST ONCOGENE COLLABORATION.
THE SECOND IS -- OOPS.
LET ME GO BACK.
GENERATING THE SRC PROBE BY
SUBTRACTIVE HYBRIDIZATION.
IT'S A CO-STUDY WITH THE BISHOP
LAB, AND ONE OF THE MAJOR FORCES
IN THIS LAB WAS STEPHEN HUGHES.
SO I WANT TO SAY HI TO STEVE.
HE IS PROBABLY STILL IN THE
AUDIENCE.
AND THE FINAL IS ON THE CRYO-EM
STRUCTURE OF WILD TYPE AND
MUTANT PI 3-KINASES.
SO THE SRC PARADIGM IS REALLY
BASED ON THE NON-DEFECTIVENESS
OF ROUS SARCOMA VIRUS.
THIS VIRUS HIGHLY SIMPLIFIED
GENOME STRUCTURE CONTAINS ALL
THE BIOGENES PLUS SRC.
AND SRC IS LOST IN REPLICATION,
AND THIS DELETION MUTANT
ACTUALLY REPLICATES FASTER THAN
THE ORIGINAL COMPLETE ROUS
SARCOMA VIRUS.
EVENTUALLY YOU CAN SEPARATE
DELETION FROM TRANSFORMING
VIRUS.
IF YOU THEN COMPARE SIZE OF RNA,
YOU'LL FIND THAT THE RNA OF THE
TRANSFORMING VIRUS IS LARGER
THAN THE RNA OF THE
NON-TRANSFORMING VIRUS.
THE CONCLUSION, SUGGESTED
CONCLUSION IS THAT THE
DIFFERENCE IS IN THE ONCOGENIC
TRANSFORMATION IN THE ONCOGENE.
THAT BRINGS ME TO THE
SUBTRAC
SUBTRACTIVE HYBRIDIZATION THAT
BISHOP PERFORMED WITH THIS SAME
SET OF VIRUSES.
YOU HAVE THE RNA OF A
NON-DEFECTIVE ROUS SARCOMA
VIRUS.
YOU TRANSCRIBE IT WITH REVERSE
TRANSCRIPTORS INTO SINGLE STRAND
DNA, USUALLY IN SMALL PIECES,
AND HYBRIDIZE THAT RNA IN
TRANSFORMATION-DEFECTIVE ROUS
SARCOMA VIRUS GENOME.
THAT CAPTURES ALL THE VIRAL
SEQUENCES, AND YOU ARE LEFT WITH
SINGLE STRAND OF DNA THAT IS
SPECIFIC FOR THE ONCOGENE.
THAT'S THE SRC ROPE.
AND THAT LED TO THIS REALLY
IMPORTANT CONCLUSION THAT ALL
RETROVIRAL ONCOGENES ARE
CELLULAR GENES ARE HITCHHIKERS
ON THE VIRAL GENOME.
THAT BRINGS ME TO THE
COLLABORATION OF PI3-KINASE.
PI3-KINASE IS A DIMERIC LIPID
KINASE.
THE ISOFORM IMPORTANT FOR HUMAN
CANCER IS THE A ISOFORM.
IT CONSISTS OF CATALYTIC
SUBUNITS AND REGULATORY
SUBUNITS.
THERE HAVE BEEN MANY, MANY
ATTEMPTS TO GET THE CRYO-M
STRUCTURE OF THAT PROTEIN, AND
ALL OF THEM FAILED.
WHEN WE STARTED, WE WERE TOLD TO
FORGET ABOUT IT, THAT WE WERE
NEW BYES IN THIS FIELD, WE WOULD
NEVER SUCCEED BECAUSE MANY OF
THE REAL EXPERTS HAD NOT BEEN
ABLE TO DO IT.
BUT THAT ADVICE IGNORED THE FACT
THAT IN THE U.S. ACCESS TO
CRYO-EM IS VERY LIMITED AND VERY
EXPENSIVE, AND WE COULDN'T GET
ENOUGH TIME IN CRYO-EM SCRIPTS
TO DO THAT WORK.
SO EVENTUALLY I GOT TOGETHER
WITH A FRIEND IN SHANGHAI, MING
HOO WON, AND WE DECIDED TO DO IT
HERE, THREE SCOPES, UNLIMITED
TIME, WE COULD TRIAL AND ERROR
APPROACH IT, USE TRIAL AND ERROR
APPROACHES TO SEE WHAT WORKS.
THERE WERE LOTS OF DIFFICULTIES.
FIRST OF ALL, NCI WOULDN'T
APPROVE, AND WE HAD TO BEG AND
BEG AND BEG, AND EVENTUALLY OUR
REQUEST WENT UP ALL THE WAY TO
THE EMBASSY IN BEIJING, THE U.S.
EMBASSY IN BEIJING, AND THEY
SAID, OKAY, YOU CAN GO AHEAD.
THEN WE GOT APPROVAL, AND WE
STARTED WORK WITH THE SHANGHAI
TEAM.
MONTHS OF MONTHS OF MONTHS OF NO
SUCCESS, AND FINALLY WE HIT THE
RIGHT FREEZING CONDITIONS, THE
RIGHT BUFFER CONDITIONS, AND WE
WERE SUCCESSFUL.
I JUST WANT TO SHOW YOU THE
ELECTRON DENSITY MAP OF P
PI3-KINASE.
WE WERE ABLE TO IDENTIFY EACH OF
THE DOMAINS OF THESE PROTEINS,
AND NOT ONLY THE WILD TYPE, BUT
ALSO THE CANCER ASSOCIATED
MUTANTS, AND WE USED THAT
INFORMATION TO GENERATE
MUTANT-SPECIFIC LIGANDS THAT
COULD BE DEVELOPED INTO
INHIBITORS.
THAT'S REALLY ALL I WANT TO SAY,
AND I WANT TO END BY SAYING
HAPPY RETIREMENT, ALAN.
THANK YOU.
>> THANK YOU, PETER.
THANK YOU FOR A WONDERFUL TALK.
EXCELLENT ADVICE TO ALL YOU
YOUNG KIDS DOWN THERE.
I'M WISHING YOU REALLY HAPPY,
PRODUCTIVE YEARS AHEAD.
AND WHAT'S NEXT ON THE PROGRAM
IS CLOSING REMARKS.
I HAVE A FEW THINGS I WANT TO
SAY.
FIRST OF ALL, I COULD NEVER
BELIEVE THAT I WOULD LIVE TO SEE
AN EVENT LIKE THIS.
I REALLY APPRECIATE ALL THE KIND
THINGS THAT EVERYONE HAS SAID
AND THE FACT THAT AND THE EFFORT
THAT WAS MADE TO PRODUCE THIS
EVENT.
THANK YOU TO YOU ALL FOR COMING.
I SPECIFICALLY WANT TO THANK THE
PEOPLE WHO MADE THIS EVENT
POSSIBLE.
THAT'S ERIC WHO SUGGESTED IT,
WHICH AGAIN WAS A COMPLETE
SURPR
SURPRISE, AND SPECIFICALLY ANNA
NORRIS AND ALSO TERRY BURDETT.
NO DOUBT OTHER PEOPLE BEHIND THE
SCENES WHOSE NAMES I MAY NOT
EVEN KNOW.
SECONDLY, I WANT TO GIVE CREDIT
TO A NUMBER OF OTHER PEOPLE FOR
DIFFERENT CONTRIBUTIONS TO MY
TRAJECTORY AND TO THE FACT THAT
I'M HERE TODAY.
I HAVE BEEN VERY FORTUNATE IN MY
CHOICE OF BOSSES, AND I WANTED
TO MENTION, FIRST OF ALL, BOB
BASSEN, WHO INTRODUCED ME TO THE
MURINE LEUKEMIA VIRUS AND WAS
ALWAYS GENEROUS AND SUPPORTIVE.
I'M SURE FRANCOIS WOULD AGREE.
SHE MAY HAVE GONE TO BED BY NOW,
BUT ANYWAY, I'M SURE SHE WOULD
AGREE.
WE WERE ALL VERY CLOSE TO BOB.
HE AND, AS I THINK FRANCOIS
MENTIONED, HE DIED A YEAR AGO
QUITE UNEXPECTEDLY.
SO I'M SORRY HE'S NOT HERE.
AFTER BOB, I WAS HIRED IN
FREDERICK BY REGULAN, WHO AGAIN
WAS HELPFUL AND SUPPORTIVE AND
HELPED ESTABLISH GOOD RESEARCH
HERE IN FREDERICK.
THEN GEORGE VANDRUD, WHO PUT
FREDERICK ON THE MAP AS A FIRST
CLASS SITE FOR RESEARCH.
GEORGE HAD A VERY BROAD VISION
ABOUT CANCER RESEARCH AND HOW TO
FOSTER RESEARCH.
ONE OF THE THINGS GEORGE DID WAS
FOSTER THE CREATION OF THE DRP,
AND THAT WAS REALLY THE CREATION
OF JOHN COFFIN, SO JOHN SET UP
THE DRP.
AND STEVE AND I WERE ALREADY
HERE WHEN THE DRP WAS FORMED.
EVERYONE ELSE IN THE DRP WAS
REALLY RECRUITED BY JOHN OR
SUBSEQUENTLY, BUT MAINLY
RECRUITED BY JOHN.
THEN EVENTUALLY JOHN HANDED THE
BATON TO STEVE, AND EVENTUALLY
AFTER THAT, STEVE HANDED THE
BATON TO ERIC, AND ALL THREE OF
THEM, ONE AFTER THE OTHER, HAVE
BEEN AGAIN IMMENSELY SUPPORTIVE
AND HELPFUL AND REALLY FOSTERED
GOOD RESEARCH AND GOOD FREE
THINKING.
SO I WANT TO THANK ALL OF THEM.
I ALSO JUST WANT TO MENTION A
COUPLE OF PEOPLE WHO HAVE MADE
ENORMOUS CONTRIBUTIONS IN THE
RESEARCH IN THE LAB, SO I HAD
TWO TECHNICIANS, ONE WHO JOINED
IN 1989 AND ONE WHO JOINED IN
1990, AND THE ONE WHO JOINED IN
1989 RETIRED A FEW YEARS AGO,
JANE MIRRO, AND THE ONE WHO
JOINED IN 1990 JUST RETIRED
ABOUT A YEAR AGO, AND SHE IS
HERE TODAY, THAT'S DIMITRIA
HARVIN, KNOWN TO US AS MEECH.
THESE TWO KEPT THE LAB RUNNING
AND KEPT THE EXPERIMENTS GOING,
AND REALLY BEAR A LOT OF
RESPONSIBILITY FOR THE
ACCOMPLISHMENTS OF THE LAB.
THEN I MUST MAKE SPECIAL MENTION
OF SID DABEH, SO HE JOINED THE
LAB IN 2001, AND HE CAME WITH
TREMENDOUS EXPERTISE ABOUT
PROTEIN STRUCTURE, PROTEIN
FUNCTION, PROTEIN PURIFICATION,
AND HE HAS REALLY, I THINK,
PLAYED A CRITICAL ROLE IN ALL
THE WORK IN THE LAB SINCE HE
JOINED IN 2001.
SO MUCH OF THE WORK IN THE LAB
WAS WORKING DIRECTLY WITH
PROTEINS PURIFIED FROM BACTERIA.
THAT WAS VERY IMPORTANT.
SHIFT IN DIRECTION OF OUR
RESEARCH, BUT NOT ALL OF IT.
SOME OF IT WAS GOOD OLD
FASHIONED VIROLOGY PERFORMED IN
THE MILLION CELLS, BUT SID ALSO
PLAYED A CRITICAL ROLE, JUST AS
ANOTHER CRITICAL THINKER AND
TRYING TO UNDERSTAND WHAT'S THE
RIGHT EXPERIMENT?
WHAT DOES THIS RESULT MEAN?
WHAT DO WE DO NEXT?
SO I WANT EVERYONE TO UNDERSTAND
HOW IMPORTANT SID'S WORK HAS
BEEN, SID HAS BEEN IN THE
ACCOMPLISHMENTS OF THE LAB FOR
THE LAST 20-PLUS YEARS.
BEYOND THAT, I WANTED TO -- I
HAVE BEEN, NEEDLESS TO SAY,
RETROSPECTING, INTROSPECTING,
HOW DID I GET HERE?
WHAT LED ME TO THIS JUNCTURE OF
HAVING SPENT ALL THESE YEARS
STUDYING WHAT WE NOW CALL
RETROVIRUSES?
SO I WILL TAKE A COUPLE OF
MINUTES TO RETRACE MY STEPS FOR
YOU.
I'VE THOUGHT ABOUT THEM, AND
POSSIBLY WHAT I'M GOING TO SAY,
ALTHOUGH VERY SIMPLISTIC, SIMPLE
MINDED, NAIVE, MAY NEVERTHELESS
RESONATE WITH MANY OF YOU.
SO I WENT TO HIGH SCHOOL IN THE
1950s, AND FOR CONTEXT, THE
WATSON-CRITZ STRUCTURE, WHICH IS
A FOUNDATION OF WHAT WE DO EVER
SINCE, WAS PUBLISHED IN 1953, AS
YOU KNOW.
AND I THINK I PROBABLY READ
ABOUT THE DNA STRUCTURE IN
SCIENTIFIC AMERICAN, AND I TOLD
MY PARENTS THAT I WANTED TO BE A
BIOCHEMIST.
THEY SAID, YOU DON'T WANT TO BE
A BIOCHEMIST.
THEY KNEW SOMEONE WHO WAS A
BIOCHEMIST.
AND HE SAID, HE TOLD THEM --
>> DON'T LET ALAN BE A
BIOCHEMIST.
>> DON'T LET ALAN BE A
BIOCHEMIST.
BIOCHEMISTRY, HE SAID -- THIS IS
A QUOTE -- IS A LIFE OF
PROGRESSIVE POVERTY.
ALAN SHOULD BE A DOCTOR INSTEAD.
(LAUGHTER)
BUT NO, I WANTED TO BE A
BIOCHEMIST COME HELL OR HIGH
WATER.
I WANTED TO BE A BIOCHEMIST.
SO THAT'S WHAT I WANTED TO DO.
WHY IS THAT?
WHY DID I WANT TO BE A
BIOCHEMIST?
IT'S BECAUSE, I THINK -- WELL, I
TOOK, LIKE EVERYBODY ELSE, I
TOOK CHEMISTRY IN HIGH SCHOOL.
I LIKED CHEMISTRY IN HIGH
SCHOOL.
YOU FIND OUT THE ATOMS, THE
MOLECULES, THEY FOLLOW VERY
SIMPLE RULES.
THAT'S WHAT CHEMISTRY IS.
WHAT ARE THE ATOMS AND MOLECULES
GOING TO DO BECAUSE OF COURSE
THEY FOLLOW THE RULES.
THAT'S WHAT CHEMISTRY IS.
AT THE SAME TIME, IF YOU STUDY
THE MOLECULES IN LIVING THINGS,
THEY SEEM TO ACT WITH A PURPOSE.
THEY SEEM TO HAVE FUNCTION.
YOU KNOW MOLECULES, IT'S LIKE
THEY KNOW WHAT THEY'RE DOING.
BUT SINCE YOU KNOW A LITTLE BIT
ABOUT CHEMISTRY, YOU KNOW THEY
DON'T KNOW WHAT THEY'RE DOING.
THEY'RE JUST FOLLOWING RULES.
I THINK THIS BASIC PARADOX IS
WHAT ATTRACTED ME.
HOW IS THIS POSSIBLE THAT THESE
MOLECULES, WHICH ARE ONLY
FOLLOWING RULES, SEEM TO KNOW
WHAT THEY'RE DOING AND ACT WITH
PURPOSE?
SO NOT TAKING MY PARENTS' GOOD
ADVICE, I WENT TO COLLEGE.
I STUDIED BIOLOGY IN COLLEGE,
AND THEN WHEN I WENT TO GRADUATE
SCHOOL, I CHOSE BERKELEY, AND
THE REASON I CHOSE BERKELEY WAS
I DID NOT KNOW EXACTLY WHAT I
WANTED TO STUDY, AND I FIGURED
BERKELEY WOULD HAVE GOOD
POSSIBILITIES IN JUST ABOUT
ANYTHING I WANTED TO STUDY.
I WAS KEEPING MY OPTIONS OPEN BY
GOING TO BERKELEY.
THINK THAT WAS EXACTLY RIGHT.
I STARTED OFF IN BERKELEY IN A
VERY FAMOUS, RESPECTED LAB,
STUDYING SEA URCHIN EMBRYOLOGY,
WHICH WAS VERY INTERESTING.
BUT THERE WAS A GUY UP THE HILL,
HARRY RUBIN, STUDYING THESE
AMAZING VIRUSES.
YOU JUST PUT THE VIRUSES ON THE
CELLS, A COUPLE DAYS LATER,
THEY'RE CANCER CELLS.
ISN'T THAT FANTASTIC?
SO I WAS JUST -- IT WAS JUST UP
THE HILL, AND I SWITCHED TO
RUBIN'S LAB.
HERE WE ARE, ALL THESE DECADES
LATER.
STILL TALKING ABOUT WHAT WE NOW
CALL RETROVIRUSES.
I THINK I BECAME A VIROLOGIST
BECAUSE VIROLOGISTS JUST
EPITOMIZE THIS PARADOX, THIS
CONTRADICTION.
ON THE ONE HAPPENED, THEY'RE
JUST A FEW MOLECULES.
THEY'RE JUST SITTING THERE.
ON THE OTHER HAND, AS WE ALL
KNOW, AS WE'VE SPENT OUR LIVES
STUDYING, THOSE MOLECULES SURE
SEEM TO ACT WITH A PURPOSE.
SO IT WAS THE FASCINATION WITH
THIS PARADOX THAT HAS HAS DRIVEN
ME ALL THESE YEARS.
I STILL FIND IT FASCINATING, AND
I THINK THAT'S WHAT I WANTED TO
SAY.
THANK YOU ALL FOR COMING AND ALL
THE KIND WORDS.
I WANT TO ESPECIALLY THANK ERIC.
I DID NOT EXPECT AN EVENT LIKE
THIS, AND IT'S REALLY BEEN MORE
THAN I CAN -- I APPRECIATE IT
MORE THAN I CAN EXPRESS.
THANK YOU AGAIN.
NOW ERIC HAS SOME CLOSING,
CLOSING REMARKS.
>> YOU'RE ALL PROBABLY SAYING,
WAIT A SECOND, ERIC'S NOT ON THE
PROGRAM.
HE ALREADY TALKED TODAY, AND I
REALLY WANT TO GO TO STEINHART'S
AND GET A BEER.
I WILL BE VERY BRIEF.
I JUST WANTED TO JOIN ALAN IN
THANKING ALL OF YOU FOR COMING
FROM NEAR AND FROM FAR.
THOSE OF YOU WHO JOINED REMOTELY
AND MIGHT STILL BE ON THE CALL,
A LOT OF GOOD DISCUSSION IN THE
AUDIENCE.
MOST OF ALL, I'D LIKE TO THANK
ALAN.
I THINK WE GOT A FLAVOR TODAY OF
THE RESPECT, ADMIRATION, AND
AFFECTION THAT THE ENTIRE
COMMUNITY HAS FOR ALAN.
NOW I'D REALLY LIKE TO THANK YOU
FOR BEING MY COLLEAGUE ALL THESE
MANY YEARS.
I'VE REALLY CHERISHED IT.
I WISH YOU THE BEST OF LUCK IN
YOUR FUTURE, AND I HOPE FOR MANY
MORE INTERACTIONS IN THE YEARS
AHEAD.
ONE LAST THING OTHER THAN
MENTIONING AGAIN THERE IS A
GATHERING AT STEINHART'S.
CORINNE MENTIONED THE RETROVIRUS
ASSEMBLY MEETING IN PRAGUE, THE
DATES ARE SEPTEMBER 16-19 OF
NEXT YEAR.
YOU CAN GOOGLE PRAGUE RETROVIRUS
ASSEMBLY MEETING, AND IT WILL
COME RIGHT UP, AND YOU CAN
REGISTER THERE.
I HOPE TO SEE MANY OF YOU THERE.
AND THANK YOU AGAIN VERY MUCH.