WELCOME.
GOOD AFTERNOON, EVERYONE.
THANK YOU FOR JOINING IN THIS
SESSION OF THE WEDNESDAY
AFTERNOON LECTURE SERIES.
AND IT'S A REAL PLEASURE AND
HONOR FOR ME, I'M IN THE
INTRAMURAL DIVISION OF NICHD, TO
INTRODUCE TODAY'S SPEAKER,
HARMIT MALIK, WHO COMES TO US
FROM THE FRED HUTCH CANCER
RESEARCH CENTER IN SEATTLE,
WHERE HE'S PROFESSOR AND
COASSOCIATE PROFESSOR AND
INVESTIGATOR OF THE HOWARD
HUGHES MEDICAL INSTITUTE.
HARMIT STUDIES THE CAUSE AND
CONES QENSES OF GENETIC
CONFLICTS THAT TAKE PLACE
BETWEEN DIFFERENT GENOMES, AND
HE'S INTERESTED IN UNDERSTANDING
THESE WHAT HE CALLS MOLECULAR
ARMS RACES AND HOW THEY DRIVE
INNOVATION FROM THE PERSPECTIVE
OF BOTH EVOLUTIONARY BIOLOGY AND
HUMAN DISEASE.
HARMIT AND HIS COLLEAGUES HAVE
USED AN EVOLUTIONARY LENS TO
DISSECT AND DISCOVER ADAPTATION
STRATEGIES AND THIS WORK HAS
HELPED FOUND THE FIELD.
THE OTHER AREA OF RESEARCH IN
HARMIT'S LAB IS THE STUDY OF
RAPID EVOLUTION IN GENES
INVOLVED IN ESSENTIAL CELLULAR
PROCESSES SUCH AS CHROMOSOME
SEGREGATION AND HIGH TOE CON ANL
BIOLOGY.
HE PROPOSED A MODEL -- THE
UNEXPECTEDLY RAPID EVOLUTION OF
16 DRCENTOMERIC PROTEINS.
HE RECEIVED HIS
UNDERGRADUATE DEGREE FROM THE
IIT BOMBAY, AND IN HIS FINAL
YEAR WAS INTERESTED IN A NEW
COURSE TITLED AN INTRODUCTION TO
MOLECULAR BIOLOGY.
HIS CORE REQUIREMENTS PREVENTED
HIM FROM ACTUALLY TAKING THAT
COURSE FOR CREDIT BUT HIS
TEACHER OFFERED TO TEACH HIM THE
COURSE ONE ON ONE IN THE
AFTERNOON PROVIDED HARMIT KEPTD
KEPTUP WITH THE READINGS.
HARMIT DID THAT AND OF COURSE
MORE BECAUSE THAT TOGETHER WITH
THE SIMULTANEOUS READING OF THE
SELFISH GENE BY RICHARD DAWKINS
SPARRED A DEEP INTEREST IN
TRANSPOSABLE ELEMENTS THAT LED
HIM TO A PH.D. AT THE UNIVERSITY
OF ROCHESTER FOLLOWED BY A
POSTDOC AT THE FRED HUTCH, WHERE
HE STARTED HIS WORK ON EVOLUTION
OF CENTROMERES.
HE STARTED HIS LAB IN 2003 AT
THE HUTCH AND HAS STAYED THERE
EVER SINCE.
HE HAS RECEIVED A WIDE NUMBER OF
AWARDS, I'M ONLY GOING TO NAME A
FEW.
THE PTHE WAS RECIPIENT OF THEAWD
COMPANY RESEARCH AWARD FROM THE
AMERICAN SOCIETY OF
MICROBIOLOGY.
HE'S AN HHMI INVESTIGATOR AND I
WANT TO SPECIFICALLY MENTION THE
AWARD FOR EXCELLENCE IN LIFE
SCIENCES NAMED AFTER THE
FOUNDING FATHER OF MOLECULAR
BIOLOGY RESEARCH IN INDIA.
HE WAS INDUCTED INTO THE
NATIONAL ACADEMY OF SCIENCES AND
THE AMERICAN ACADEMY OF ARTS AND
SCIENCES.
HARMIT IS ALSO VERY KEEN TO
PROMOTE INCLUSION IN SCIENCE AND
IS ACTIVE ON SOCIAL MEDIA
ENCOURAGING PEOPLE TO GET
INVOLVED IN SCIENCE AND BREAKING
DOWN THE BARRIERS OF ENTRY INTO
SCIENCE.
THEY BUILD ROLE MODELS AND BUILD
COMMUNITY THAT IS PARTICULARLY
INSPIRING FOR YOUNGER
RESEARCHERS AND FOR THAT, WE
HAVE TO BE COLLECTIVELY THANKFUL
FOR FOLKS LIKE HARMIT.
THANK YOU FOR COMING, LOOK
FORWARD TO YOUR SEMINAR.
[APPLAUSE]
>> THANK YOU VERY MUCH.
I FEEL A LITTLE BUI BIT CHEATED
BECAUSE I WAS ASSURED THERE
WOULD BE NO MORE THAN 20 PEOPLE
IN THE AUDITORIUM AND I'M PRETTY
SURE THAT ISN'T TRUE.
I WAS TOLD THERE ARE GOING TO BE
COFFEE AND COOKIES AFTER THE
THING, SO YOU JUST HAVE TO WAIT
FOR ABOUT AN HOUR.
IT IS ALSO THE LONGEST DAY OF
THE YEAR AND I HOPE TO NOT MAKE
IT SEEM EVEN LONGER.
[LAUGHTER]
SO TODAY I'M ACTUALLY GOING TO
TELL BUT SOME OF THE WORK THAT
WE'RE DOING IN MY LAB ON HOST
APT VIANTIVIRAL PROTEINS.
I JUST WANT TO ADVERTISE IF YOU
ARE KEEN TO GET MORE OF THIS,
I'M ALSO GIVING A LECTURE IN THE
NHGRI TOMORROW AND YOU ARE
WELCOME TO ATTEND THAT AS WELL.
SO BEFORE I GET INTO THE MEAT OF
TODAY'S TALK, I JUST WANTED TO
GIVE YOU AN INTRODUCTION INTO
WHAT INSPIRES ALL OF THE WORK
THAT GOES ON IN MY LAB.
WHICH IS REALLY ABOUT GENETIC
CONFLICTS.
AND THE IDEA OF GENETIC
CONFLICTS REALLY EMERGES FROM A
VERY UNUSUAL SOURCE, SO I
THOUGHT I'D SPEND A FEW MINUTES
DISCUSSING THAT.
WHICH IS THE FICTIONAL CHARACTER
THE RED QUEEN IN THE BOOKS ABOUT
ALICE'S ADVENTURES IN WONDERLAND
WHERE AT ONE POINT IN RESPONSE
TO ALICE'S COMPLAINT, THE RED
QUEEN RETORTS THAT IT TAKES ALL
THE RUNNING YOU CAN DO IN
WONDERLAND TO KEEP IN THE SAME
PLACE.
THOSE OF YOU WHO RUN LABS CAN
TOTALLY APPRECIATE THE RED QUEEN
INTERACTION AS LIKE A REALLY
IMPORTANT LESSON FOR LIFE.
BUT UNSURPRISINGLY THIS ALSO WAS
A VERY IMPORTANT LESSON FOR
EVOLUTIONARY BIOLOGY AND LED TO
THE FORMULATION OF THE RED QUEEN
HYPOTHESIS, WHICH I LIKE TO
THINK ABOUT AS THE SECOND SORT
OF INCARNATION OF DAR VINNIAN
THINKING IN EVOLUTIONARY -- IN
THE ROLE OF NATURAL SELECTION
AND HOW ORGANISMS ADAPT TO THEIR
ENVIRONMENTS, THEY HAD
PREDOMINANTLY FOCUSED ON THEIR
ADAPTATION TO THEIR ABIOTIC
ENVIRONMENT, AND IT WAS
EXPLICITLY POINTED OUT THAT A
LARGE PART OF THAT ENVIRONMENT
IS ALSO MADE UP OF COMPETING
SPECIES WHICH ARE OFTEN
COMPETING FOR THE SAME FITNESS
SPACE.
SO IT WAS IMPOSSIBLE, FOR
EXAMPLE, TO CONSIDER WHAT WAS
GOING TO HAPPEN IN THE FITNESS
OF THE SNOW LEOPARD POPULATION
IF YOU FAIL TO CONSIDER WHAT WAS
HAPPENING IN THE SNOW HARE
POPULATION BECAUSE THE ADAPTIVE
STRATEGIES DIRECTLY TIED IN WITH
THE ADAPTIVE STRATEGIES OF THE
OTHER.
THE RED QUEEN HYPOTHESIS IS A
VERY IMPORTANT -- EVERYTHING
FROM ECOLOGY TO PUBLIC HEALTH
AND WHAT LABS LIKE MINE ARE VERY
INTERESTED IN IS ADOPTING THIS
INTO MOLECULAR TERMS.
SO I'M GOING TO MAKE A VERY
SMALL LEAP BETWEEN ARMS RACES
THAT OCCUR BETWEEN PREY AND
PREDATORS VERSUS ARMS RACES THAT
OCCUR BETWEEN ANTIVIRAL PROTEINS
AND TODAY'S LECTURE IS GOING TO
BE EXCLUSIVELY ON INNATE
ANTIVIRAL PROTEINS, AND VIRAL
PROTEINS.
I'LL JUST POINT OUT THAT IN THIS
HIGH RESOLUTION STRUCTURE OF AN
ANTIVIRAL PROTEIN RECOGNIZING A
VIRAL PROTEIN BY SOME SORT OF
EPITOPE RECOGNITION, IT'S GOING
TO ENCOUNTER THE VIRUSES AS SOON
AS THEY ENTER THE CELL,
EXTINGUISH THE VIRUS FROM THE
CELL EVENTUALLY FROM THE
ORGANISM, AND EVENTUALLY FROM
THE SPECIES.
THIS, OF COURSE, PUT A LOT OF
DARWINIAN PRESSURE TO RAPIDLY
EVOLVE TO A NEW STATE THAT IS NO
LONGER CAPABLE OF BEING
RECOGNIZED OR ANTAGONIZED, AND
THIS CAN HAPPEN AT THE LEVEL OF
A SINGLE AMINO ACID MUTATION,
WHICH, GIVEN THE VERY RAPID
MUTATION RATES AFFORDED TO
VIRUSES, CAN HAPPEN VERY
QUICKLY.
OVER TIME THIS WILL ACTUALLY
FORCE THE ANTIVIRAL PROTEIN TO
RE-ESTABLISH ITS ANTAGONISM --
MUTATION BUT THIS IS A MUCH
SLOWER STEP BECAUSE WE NEED TO
RELY ON GENERATIONAL MUTATION
TIME WHICH IS, OF COURSE, MUCH
SLOWER IN SPECIES SUCH AS OURS.
SO A QUESTION THAT OFTEN ARISES
IS HOW IS IT POSSIBLE TO THINK
ABOUT THESE AS CONTEMPORANEOUS
ARMS RACES WHERE ONE PARTY IS SO
MUCH MORE ADAPTED EVOLUTION AND
MUTATION THAN THE OTHER.
AND THE RESPONSE IS THAT IF THIS
IS A 1 TO 1 INTERACTION IT WOULD
BE GAME OVER, JUST BECAUSE THE
INHERENT ASYMMETRIC NATURE OF
THESE ARMS RACES BUT LUCKILY FOR
US, IT'S NOT A ONE TO ONE
INTERACTION.
IN FACT, WE HAVE NEARLY 200
ANTIVIRAL PROTEINS ENCODED AND
OFTEN TRIGGERED BY THE
INTERFERON SYSTEM, THAT
BASICALLY ARE DEDICATED TO THE
JOB OF INTERROGATING INCOMING
PATHOGENIC VIRUSES AND BACTERIA,
AND BASICALLY EXPUNGING THEM.
AND REALLY FOR THE HOST TO WIN,
IT REALLY ONLY NEEDS TO WIN ONE
OF THESE INTERACTIONS IN ORDER
TO DO ITS JOB.
WHEREAS FOR A VIRUS, EVEN THE
VERY RAPIDLY INVOLVING RNA
VIRUS, IT NEEDS TO RUN THE
ENTIRE GAUNTLET OF ANTIVIRAL
PROTEINS PROVIDED AND THAT'S
WHERE THE MATH BEGINS TO ADD UP.
ANTIVIRAL PROTEINS ALSO DON'T
ENCOUNTER JUST ONE PATHOGENIC
WILD VARIANT AS WE'VE ALREADY
SEEN MULTIPLE PATHOGENIC
VARIANTS OF THE SAME VIRUS
MULTIPLE TIMES.
AND IF YOU USE PUBLIC TRANSPORT,
YOU PROBABLY ENCOUNTER THEM ON A
WEEKLY BASIS OR IF YOU HAVE KIDS
IN DAYCARE.
THOSE ARE THE REAL FRONT LINES
OF INFECTIOUS DISEASE IN OUR
SPECIES.
AND SO REALLY YOU BEGIN TO
APPRECIATE THAT ANTIVIRAL
PROTEINS AND VIRAL PROTEINS ARE
LOCKED IN THIS MANY TO MANY
INTERACTION BUT EVEN IF YOU WERE
TO FOCUS ON THE FOCAL 1 TO
1 INTERACTION YOU'LL APPRECIATE
THAT THIS IS A RELENTLESS ENGINE
FOR FORWARD EVOLUTION.
AND WHAT DRIVES THIS ENGINE IS
THE FACT THAT ONE PARTY IS
ALWAYS ON THE LOSING SIDE OF
THIS ARMS RACE, RESULTING IN
GENETIC INNOVATION BEING
CONSTANTLY SPURRED BY THE NATURE
OF THIS INTERACTION.
SO THERE'S ALWAYS GOING TO BE AN
EVOLUTIONARY ADVANTAGE TO BE
GAINED BY THIS INNOVATION, AND
WHAT LABS LIKE MINE ARE TRYING
TO DO IN A NUTSHELL IS LOOK
BACKWARDS IN THE SEQUENCES OF
VIRAL PROTEINS AND THE SEQUENCES
OF ANTIVIRAL PROTEINS TO TRY TO
RECONSTRUCT THE STEPS THAT TOOK
PLACE IN THE COURSE OF THESE
ARMS RACES AND UNDERSTAND THAT
BIOCHEMICAL AND BIOLOGICAL
OUTCOMES.
ONE SPECIAL TYPE OF INNOVATION
THAT IS QUITE EASY TO IMAGINE IS
FOCUSING ON PROTEIN CODING IMEEN
INNOVATION AND THIS IS JUST A
BRIEF PRIMER FOR THOSE OF YOU
WHO DON'T THINK ABOUT THIS ON AA
DAILY BASIS WHERE IF YOU WERE TO
CONSIDER A HYPOTHETICAL PIECE OF
A PROTEIN CODING GENE, WE CAN
CLASSIFY ALL OF THE NUCLEOTIDE
SUBSTITUTIONS THAT OCCUR HERE
OVER EVOLUTIONARY TIME INTO
THOSE THAT WILL NOT ALTER THE
AMINO ACID ENCODED, SILENT OR
SYNONYMOUS CHANGES VERSUS THOSE
THAT INSTANTLY ALTER THE GENE
BECAUSE OF THE NATURE OF THE
AMINO ACID SUBSTITUTION.
WE CALL THESE NON-SYNONYMOUS OR
REPLACEMENT CHANGES.
NOW NORMALIZED AS DS AND DN, WE
HAVE DIFFERENT WAYS OF VIEWING
WHAT ARE THE EVOLUTIONARY EVOLUS
THAT HAVE DRIVEN EACH OF THESE
CHANGES.
WE MAKE THE ASSUMPTION THAT
SILENT OR SYNONYMOUS CHANGES ARE
REALLY NOT VISIBLE TO NATURAL
SELECTION.
WE DO SO IN SPITE OF THE FACT WE
KNOW THIS ASSUMPTION IS WRONG,
THERE ARE MANY, MANY GOOD
EXAMPLES WHERE SYNONYMOUS
CHANGES WITH CAN AFFECT PROTEIN
TRANSLATION RATES AND EVEN
PROTEIN STABILITY BUT WE
NONETHELESS MAKE THIS ASSUMPTION
BECAUSE AS A FIRST ORDER OF
APPROXIMATION, AN AVERAGE SILENT
CHANGE IS MUCH MORE BENIGN THAN
A REPLACEMENT CHANGE.
AS A PROXY OF ALMOST LIKE A
CLOCK-LIKE FASHION, THE LONGER
TIME OF DIVERGENCE, THE MORE
YOUR DS IS GOING TO DIVERGE.
YET AS NATURAL SELECTION WILL
DICTATE WHAT WILL HAPPEN WITH
THE DN TERM, IF YOU DO NOT
TOLERATE AMINO ACID CHANGES
YOU'LL BASICALLY HAVE A DN WHICH
IS MUCH LESS THAN YOU'D EXPECT
AR AS IF YOU FAVOR REPLACEMENT
CHANGES YOU'D HAVE A DN THAT'S
MUCH HIGHER THAN WHAT YOU
EXPECT.
THOSE OF YOU USED TO LOOKING AT
PROTEIN ALIGNMENTS INHERENTLY
USE THIS TOOL, EVEN THOUGH YOU
MIGHT NOT BE THINKING OF THIS AS
THE DN/DS TOOL, BECAUSE WHEN
YOUR EYE IS DRAWN TO THOSE
POSITIONS THAT ARE COMPLETELY
FROZEN IN EVOLUTION, DESPITE
MILLIONS OF YEARS OF DIVERGENCE,
LET'S SAY CATALYTIC RESIDUES OF
AN ENZYME, WHAT YOU'RE DOING IS
MAKING A MATHEMATICAL
CALCULATION OF THE NUMBER OF
MUTATIONS THAT MUST HAVE
OCCURRED THAT ALTERED THE AMINO
ACID, AT THOSE CRITICAL
POSITIONS, BUT ALL OF THOSE WERE
PURGED BECAUSE THEY WERE
DELETERIOUS FOR FUNCTION.
IN OTHER WORDS, WE USE THIS
PROXY OF HOW SELECTION HAS ACTED
TO PURIFY THE POPULATION OF
PRESUMED DELETERIOUS AMINO ACID
CHANGES OR A PROXY OF HIGH
EVOLUTIONARY CONSERVATION FOR
FUNCTION.
SO IF IT'S HIGHLY CONSERVED IN
EVOLUTION, DESPITE MILLIONS OF
YEARS OF DIVERGENCE, IT MUST BE
IMPORTANT TO HAVE ESSENTIALLY
SURVIVED MUTATION.
BUT WE SOMETIMES MAKE THE
MISTAKEN ASSUMPTION THAT THINGS
THAT ARE NOT CONSERVATIVE
EVOLUTION MUST, THEREFORE, NOT
BE IMPORTANT.
IN FACT, MY ENTIRE LAB STUDY
CASES WHERE THE RATE OF AMINO
ACID CHANGE MUCH EXCEEDS WHAT
YOU WOULD EXPECT BY CHANCE
ALONE, AND THAT'S ACTING NOT
BECAUSE YOU SUDDENLY BECOME MUCH
MORE SENSITIVE TO DS-TYPE
CHANGES, BUT THAT'S OCCURRING
BECAUSE SELECTION IS NOW ACTING
POSITIVELY TO ACCELERATE THE
RATE OF FIXATION OF AMINO ACID
ALTERING MUTATIONS BECAUSE THEY
WERE BENEFICIAL IN THE CONTEXT
THAT THEY OCCURRED.
WE WOULD MAKE THE CASE TODAY
THAT JUST LIKE HIGH EVOLUTIONARY
CONSERVATION IS A PROXY FOR
FUNCTIONAL IMPORTANCE, THIS
CONSTANT RELENTLESS SIGNATURE OF
INNOVATION AT THE AMINO ACID
LEVEL IS ALSO A PROXY FOR
FUNCTIONAL IMPORTANCE, BUT THIS
ONE IS A I LO LOT MORE SPECIFIT
YES NE TGENETIC CONFLICTS.
WE CAN USE THIS TO APPLY TO ALL
PROTEIN CODING GENES IN THE
GENOME, WE CAN DO THIS IN OUR
SPECIES, WE CAN DO THIS IN
DROSOPHILA OR WHATEVER YOUR
FAVORITE SPECIES IS.
WE SEE THE OVERWHELMING
SIGNATURE IS THAT OF PURIFYING
SELECTION.
ONCE AGAIN, IF THE ONLY EFFECT
WAS ABOUT MUTATION, THEN ALL OF
THE GENES WOULD BE PILED UP
AROUND THIS DOTTED LINE OF DN/DS
EQUALS 1, BUT BECAUSE MOST
PROTEIN CODING GENES ARE
BASICALLY IN THIS LEFTWARD
HISTOGRAM, THIS GAP BETWEEN THE
BOTTOM LINE ON THE HISTOGRAM
REALLY INDICATES HOW NATURAL
SELECTION I HAS ACTUALLY PURGED
ALL OF THE AMINO ACIDS THAT HAVE
PROBABLY TAKEN -- AMINO ACID
MUTATIONS TAKING PLACE PRESUMED
DELETERIOUS AND PURGED OUT WHICH
IS WHY WE DON'T SEE THEM
ANYMORE.
BUT EVEN IN THIS WHOLE SORT OF
CRUDE DN/DS AVERAGE, YOU CAN SEE
THERE'S A BLIP OF GENES WHICH IS
VERY MUCH IN THE -- BREAKING THE
SPEED LIMIT, IF YOU WILL, THAT
IS IMPOSED BY MUTATION.
UNSURPRISINGLY IMMUNITY GENES
ARE UNSURPRISINGLY REPRESENTED
IN THIS CATEGORY.
INNATE IMMUNE GENES THAT DEFEND
AGAINST VIRUSES AND BACTERIA ARE
HEAVILY OVERREPRESENTED HERE.
THAT'S WHAT I'M GOING TO FOCUS
ON FOR TODAY'S LECTURE.
WEE ALSO INTERESTED IN CATEGORY
OF GENES THAT DON'T MAKE ANY
SENSE -- THAT'S ACTUALLY THE
TOPIC FOR TOMORROW'S LECTURE.
RETURNING TO THIS SORT OF HOW TO
THINK ABOUT PURIFYING SELECTION
AND POSITIVE SELECTION ON A SORT
OF BIOCHEMICAL BASIS, LET'S
IMAGINE AN INTERACTION BETWEEN
TWO HOST PROTEINS WHOSE
INTERACTION IS REALLY IMPORTANT.
LET'S SAY FOR A SIGNAL
TRANSDUCTION PATHWAY.
NOW WE CAN IMAGINE MUTATIONS
WILL OCCUR EITHER IN ONE PROTEIN
OR THE OTHER PROTEIN THAT MIGHT
PERTURB THIS INTERACTION BUT
THAT WOULD BE DL DELETERIOUS
BECAUSE YOU'RE ESSENTIALLY
BREAKING THE SIGNAL TRANSDUCTION
PATHWAY AND GIVEN BOTH ARE RARE
IN THE POPULATION, IS
EXCEEDINGLY RARE, WHICH MEANS
THAT YOU WILL COME BACK MILLIONS
OF YEARS LATER AND FIND THAT
PURIFYING SELECTION IS LARGELY
MAINTAINED THE INTERACTION
INTERFACE BECAUSE THAT IS SO
IMPORTANT IN THE COURSE OF
EVOLUTION.
SO THIS PARTICULAR PROTEIN HAS
BASICALLY BEEN LEFT UNCHANGED BY
NATURAL SELECTION.
IN CONTRAST, IF YOU IMAGINE WHAT
HAPPENS IN A HOST-VIRUS ARMS
RACE, HERE WE BASICALLY BOUNCE
BETWEEN TWO STATES BETWEEN THE
HOST RINNING VERSUS THE VIRUS
WINNING, AND BECAUSE WHAT'S IN
THE BENEFIT OF THE HOST IS
ACTUALLY TO THE DETRIMENT OF THE
VIRUS AND VICE VERSA, SO ONCE
AGAIN WE'VE GOT A
PROTEIN-PROTEIN INTERACTION
INTERFACE, BUT THIS ONE CANNOT
STAY STABLE AND IT'S CONSTANTLY
CHISELED AWAY BY ADAPTIVE
EVOLUTION, EITHER ON THE HOST
SIDE OR THE VIRUS SIDE, AND
MOREOVER, IF YOU'RE EVEN PAYING
ATTENTION TO THIS CARTOON
EXAMPLE, YOU CAN APPRECIATE THAT
WE DON'T EXPECT RAPID EVOLUTION
TO BE RANDOMLY DISTRIBUTED OVER
THESE PROTEINS, BUT I IN FACT,
CONCENTRATED ALL THOSE RESIDUES
OR DOMAINS -- THE BINDING
AFFINITY BETWEEN THE HOST AND
PROTEIN INTERACTIONS.
SO MANY YEARS AGO, ARMED WITH
NOTHING MORE THAN THIS, WE
WONDERED IF WE COULD ACTUALLY
SIMPLY ASK WHERE POSITIVE
SELECTION OCCURS ON THE SURFACE
OF THE PROTEINS AS A FIRST ORDER
PREDICTOR OF WHAT THE
INTERACTION SURFACE IS BETWEEN
HOST AND VIRAL PROTEINS MIGHT BE
LIKE.
SO IN OTHER WORDS, RATHER THAN
FOCUSING ON THE MOST -- PARTS OF
PROTEINS LIKE MOST MOLECULAR
BIOLOGISTS, HERE WE'RE ACTUALLY
FOCUSED ON THE LEAST -- PARTS OF
THE PROTEIN IN ORDER TO
DETERMINE WHAT THE SPECIFICITY
DOMAINS MIGHT BE.
ALONG THE WAY WE REALIZED WE
LEARNED A LOT ABOUT
SUSCEPTIBILITY DETERMINANTS WHEN
WE DO THIS.
SO THERE ARE MANY INSTANCES
WHERE ONE PARTICULAR VIRUS IS
HIGHLY PATHOGENIC TO ONE SPECIES
BUT VERY CLOSELY RELATED SPECIES
IS ALMOST COMPLETELY IMMUNE.
AND WE REALLY SORT OF
HYPOTHESIZED IN THE EARLY DAYS
OF GENOMICS, THIS MUST BE
BECAUSE OUR GENE REPERTOIRES ARE
ACTUALLY DIFFERENT.
IT TURNS OUT THEY'RE NOT TOTALLY
DIFFERENT, THEY'RE BASICALLY
ALMOST THE SAME, BUT THE --
WE'VE EACH ENCOUNTERED A
DIFFERENT PRIVATE HISTORY OF
PATHOGENIC VIRUSES.
AND WHAT'S REALLY SPECTACULAR
IS, WE CAN COMPLETELY GRAFT THIS
TUNING FROM ONE SHE SHE'S INTO
THE OTHER JUST BY MAKING SIGNAL
AMINO ACID CHANGES AT THE
CRITICAL INTERFACES AND THAT'S
WHAT I'M GOING TO SHOW YOU
TODAY.
WE'VE DONE THIS FOR NEARLY A
DOZEN ANTIVIRAL PROTEINS.
THEY ALL HAVE COMPLETELY
DIFFERENT MECHANISMS.
SOME OF THEM ARE UBIQUITIN
LIGASES, SOME OF THEM ARE DEAM
NAISES, SOME OF THES ARE PROTEIN
KINASE.
THEY ALL WORK AGAINST COMPLETELY
DIFFERENT CLASSES OF VIRUSES
ENTIRELY.
AND YET IN EACH CASE, WE CAN USE
THIS POSITIVE SELECTION DRIVEN
APPROACH TO CORRECTLY PREDICT
THE INTERACTION SURFACE AND
GRAFT CHANGES THAT WILL MAKE THE
HOSE MORE FROM A LOSING SIDE TO
A WINNING SIDE, WHICH MEANS THAT
REALLY THE RATE LIMITING STEP TO
DICTATE THE COURSE OF THE VIRUS
INFECTION IN ALL OF THESE CASES
IS WHAT IS THE BINDING AFFINITY
OF THE INITIAL INTERACTIONS
BETWEEN THE VIRAL AND THE
IMMUNITY PROTEINS, WHICH IS WHY
THIS APPROACH IS ACTUALLY SO
CONSEQUENTIAL.
IF YOU'D LIKE TO READ A LITTLE
BIT MORE ABOUT THIS, HERE'S A
SLIGHTLY OUT OF DATE REVIEW
WRITTEN BY A FORMER POSTDOC,
MATADOR TEE, IMATT DOHERTY, IN .
TODAY I'M GOING TO PRESENT TWO
VIGNETTES.
THE FIRST PROTEIN I'VE CHOSEN TO
TALK TO YOU ABOUT IS A PROTEIN
CALLED TRIM5ALPHA, BECAUSE THIS
IS ACTUALLY WHAT I USE TO GET
INTO THE BUSINESS MYSELF WHEN I
WAS STARTING MY LAB MORE THAN
TWO DECADES AGO NOW ALMOST.
SO TRIM5 WAS ACTUALLY DISCOVERED
IN A BEAUTIFUL SCREEN DONE, ALL
MONKEYS LIKE RHESUS MACAQUES
CANNOT GET INFECTED WITH HIV-1
AND THIS PROTECTION IS ACTUALLY
AT THE CELLULAR LEVEL.
IT TURNS OUT WHEN HIV-1 ENTERS
THE SHELL AND SHEDS ITS ENVELOPE
AT THE CELL SURFACE, IT ENTERS
THIS PROTEINACEOUS -- TRIM 5 IS
ACTUALLY A CYTOPLASMIC SENT NIL
WHICH IS BASICALLY ALWAYS
EXPRESSED, ALWAYS AROUND, AND IT
INTERCEDES AND FORMS A
LATTICE-LIKE STRUCTURE, LEADING
TO THE RAPID DEGRADATION OF THIS
RETROVIRAL CAPSID.
PREVENTING THE VIRUS FROM
UNDERGOING ITS OTHERWISE NORMAL
CYCLE.
THE ENTIRE SUCCESS OF TRIM 5 IS
DICTATED BY THE KINETICS OF THE
INTERACTION OF THE TRIM
5 PROTEIN TO THE CAPSID CORE.
SO THE REASON RHESUS IS SO
SUCCESSFUL IS IT HAS EXTREMELY
HIGH AIF I WANT FOR THE CAPSID.
-- WHICH IS PROTECTIVE AGAINST
MANY RETROVIRUSES BUT OUR
VERSION IS NOT EFFECTIVE AT
CORRECTLY BINDING THE HIV-1
CAPSID, WHICH IS WHY WE DO NOT
HAVE BIOLOGICALLY MEANINGFUL
PROTECTION AFFORDED BY THE HUMAN
VERSION OF TRIM 5.
SO MANY YEARS AGO, ARMED WITH
THIS SORT OF PARADIGM OF OUR
GENETIC CONFLICTS AND ARMS
RACES, THIS TIME BETWEEN TRIM
5 AND THE CAPSID GENE OF HIV-1,
WE THOUGHT WE COULD ACTUALLY
PREDICT WHAT THE INTERACTION
INTERPHASE BETWEEN TRIM 5 AND
CAPSID WOULD BE, AGAIN BY TAKING
A LOOK AT WHERE THE RAPID
EVOLUTION OF TRIM 5 MIGHT HAVE
TAKEN PLACE.
AND TO OUR SURPRISE, EVEN THOUGH
TRIM 5 IS NEARLY 600 AMINO
ACIDS, ALMOST ALL OF THE RAPID
EVOLUTION IN TRIM 5 OCCURS IN
THIS VERY TIGHT WIN DOLE, 11
AMINO ACID STRETCH IN HUMANS,
WHICH IS A 13 AMINO STRETCH IN
RHESUS.
SO IT'S ALMOST AS IF YOU
BASICALLY FOCUSED ALL OF THE
ENERGY OR ALL OF THE SUBSEQUENT
INTERACTIONS DOWN TO JUST ONE
DOMAIN OF THE V1 LOOP.
AS I POINTED OUT EARLIER, HUMAN
TRIM 5 -- ONLY TWOFOLD
PROTECTION AGAINST HIV-1 IN
SIMILAR ASSAYS, WHEREAS RHESUS
TRIM 5 ENCODES NEARLY 100 FOLD
PROTECTION, WHICH OBVIOUS IS
BIOLOGICALLY PROTECTIVE.
INTERESTINGLY, HOWEVER, JUST
TAKING THE V1 LOOP, WHICH
REMEMBER IS THE MOST RAPIDLY
EVOLVING PART OF TRIM 5 AND
SWAPPING IT FROM RHESUS INTO
HUMAN IS ABLE TO CONFER NEARLY
COMPLETE PROTECTION IN THE
OTHERWISE INEFFECTIVE HUMAN TRIM
5 BACKBONE.
THIS IS ONE OF THOSE EXAMPLES WE
YOU CAN SEE HOW RAPID EVOLUTION
CAN GUIDE US TO THE INTERFACES
WHERE WE CAN ACTUALLY GRAFT
SPECIFICITY FROM ONE BACKBONE
INTO THE OTHER.
EVEN MORE SPECTACULARLY, JUST
ONE AMINO ACID CHANGE AT ONE OF
THESE CRITICAL POSITIONS, THIS
PROLINE AND SORT OF AN ARGININE
IS ABLE TO CONFER WHAT WOULD BE
BIOLOGICALLY MEANINGFUL
PROTECTION IN THE HUMAN TRIM
5 BACKBONE.
YES?
>> [INAUDIBLE]
>> THIS IS THE QUALIFIED UNIT OF
PROTECTION.
I'LL TALK MORE ABOUT THE ASSAY
IN A FEW MINUTES.
BUT THIS IS JUST A FULL
PROTECTION RELATIVE TO AN EMPTY
VECTOR CONTROL.
SO THIS JUST BASICALLY PROVIDES
THE EXAMPLE THAT YOU CAN HAVE
SINGLE AMINO ACID CHANGES THAT
COULD ACTUALLY BE MEANINGFULLY
PROTECTIVE.
I'LL JUST PREEMPT THE QUESTIONS
HERE THAT UNFORTUNATELY, WE DO
NOT HAVE ANY STANDING RADIATION
IN THE V1 LOOP THAT WOULD BE
PROTECTIVE AGAINST HIV-1 DUE TO
AN ADAPTIVE SWEEP THAT WENT
THROUGH TRIM 5 RIGHT OF THE OUT
OF AFRICA SORT OF MIGRATION OF
HUMANS, WE WIPED OUT ALL
POLYMORPHISMS AND WE'VE NOT
RECOVERED ENOUGH TO BE
PROTECTIVE LANDSCAPE HERE.
HOWEVER, TRIM 5 IS VERY
POLYMORPHIC IN ORWELL MONKEYS.
THIS PROJECT STARTED OFF WITH
THIS VERY OLD STUDY WHERE WE
SHOWED -- CAN BE RAPIDLY EVOLVE
AND CONFER PROTECTION, BUT A NEW
POSTDOC JOINED THE LAB AND SHE
WAS REREADING ALL THE OLD LAB
PAPERS AND SHE BASICALLY
REALIZED YOU CALL THIS RAPID
EVOLUTION BUT THERE'S NOT THAT
MANY CHANGES IF YOU THINK ABOUT
THEM, IT'S ALSO AS IF EVOLUTION
IS MOVING WITHIN A BOX WHERE
ONLY A FEW AMINO ACID -- IS THAT
CONSTRAINED OR AN INSTANCE WHERE
HELPFUL MUTATIONS ARE REALLY
RARE, OR THESE ARE THE MUTATIONS
THAT HAPPENED TO GET FIXED AND
MAYBE THERE'S A WIDE SPECTRUM OF
MUTATIONS TO CHOOSE FROM.
IRONICALLY, WE DON'T HAVE THE
MEANS TO DISTINGUISH BETWEEN
THOSE POSSIBILITIES BECAUSE WE
ARE LOOKING AT ALL OF THE
SUCCESSFUL TRIM 5 VARIANTS,
RIGHT?
THESE ARE ALL VARIANTS THAT
FOUGHT OFF SOMETHING AND HAVE
SURVIVED.
SO WE CAN SEPARATE MUTATION FROM
SELECTION.
SO SHE DECIDED THAT THAT'S THE
PROJECT SHE WANTED TO DO, MOSTLY
TO PROVE MY EARLIER DISCUSSION
TO BE WRONG.
SO THIS IS THE WORK I'M GOING TO
DESCRIBE TODAY.
SO SHE REALLY WANTED TO ASK THAT
IF I WERE TO LIKEN THE
EVOLUTIONARY LANDSCAPE OF TRIM S
THE SEQUENCE ACCESS OF TRIM
5 MUTATION, WHERE THE HEIGHT OF
THE PEAK DETERMINES HOW MUCH THE
HIV-1 RESTRICTION IS OCCURRING,
YOU CAN SEE THAT RHESUS HAPPENS
TO BE ON ONE OF THE PEAKS, AND
HUMAN TRIM 5 HAPPENS TO BE IN
ONE OF THE VALLEYS.
SO SHE WANTED TO ASK ARE --
SOLUTION JUST VERY RARE, AND IF
YOU OWE KUR ON A WINNING
SOLUTION, IS THAT VERY THA
MUTATIONALLY FRAGILE OR
RESILIENT?
SO TO DO THIS, SHE NEEDED TO
ACTUALLY DO AN EXPERIMENT WHERE
SHE WOULD INTRODUCE ALL POSSIBLE
MUTATIONS AND THEN SUBJECT THEM
TO SELECTION, AND WE CAN DO THAT
THANKS TO NICE ADVANCES IN DEEP
MUTATIONAL SCANNING METHODS
PIONEERED BY STAN FIELDS AND
OTHERS.
SO IN THIS CASE, WE DID NOT
ACTUALLY DO A SCAN OF THE ENTIRE
TRIM 5 BECAUSE WE WERE ONLY
INTERESTED IN THE V1 LOOP SO WE
INTRODUCED ALL POSSIBLE SINGLE
AMINO ACID CHANGES IN THE
V1 LOOP OF HUMAN TRIM 5 AND WE
INTRODUCED ALL OF THESE VARIANTS
INTO CAT CELLS.
THE CHOICE OF CAT CELLS IS
PARTLY HISTORICAL.
THAT'S THE CELL LINE -- USED TO
DO HIS INITIAL TRIM
5 EXPERIMENTS BUT THAT TURNS OUT
TO BE VERY FORTUITOUS THAT HE
DID THAT BECAUSE CATS AND DOGS
HAVE INDEPENDENTLY LOST THEIR
TRIM 5 FOR REASONS WE CANNOT
ACTUALLY DISCUSS IN THE Q & A
SECTION, WHICH MEANS THAT ALL OF
THESE CAT CELLS EACH HAS NOW AN
INDIVIDUAL HUMAN TRIM 5 VARIANT,
EACH SLIGHTLY DIFFERENT AT THE
V1 LOOP, AND THAT'S ALL THE TRIM
5 THAT THEY'RE CAPABLE OF
ENCODING.
SO WE SUBJECTED THIS POPULATION
OF CAT CELLS EACH ENCODING A
DIFFERENT HUMAN TRIM 5 TO
INFECTION WITH HIV-1, BUT THIS
HIV-1 HAS BEEN ENGINEERED TO
ONLY ENCODE GSP UPON
INTEGRATION, SO IT DOESN'T HAVE
THE HIV-1 GENOME, IT JUST HAS
THE ABILITY TO INTRODUCE GSP
UPON INTEGRATION.
FOR THE AFICIONADOS I SHOULD
POINT OUT THAT WE DO THIS AS A
HIGH MULTIPLICITY OF INFECTION
BECAUSE WE WANT TO MAXIMIZE
INFECTION RATES IN THIS ASSAY.
SO THIS ASSAY IS GOING TO RESULT
IN TWO CONSEQUENCES.
CONSEQUENCE NUMBER ONE IS YOU'RE
GOING TO TURN CELLS GREEN.
THIS MEANS YOU ARE NOT ABLE TO
PREVENT HIV-1 FROM INTEGRATING
THIS GENOME INTO THE CELL, THAT
MEANS YOU DO NOT HAVE A --
VARIANT.
WE'RE INTERESTED IN THE
NON-GREEN CELLS BECAUSE FOR SOME
REASON, THEY WERE PROTECTED
AGAINST HIV-1, WHICH IS WHY HIV
WAS NOT ABLE TO INTEGRATE INTO
THE GENOME.
SO THIS WOULD BE ENCODING
RESTRICTOR VARIANTS OF TRIM 5,
IT'S JUST WHAT WE ARE INTERESTED
IN, BUT WE ARE ALSO CONVINCED
THAT THERE ARE PROBABLY
UNINFECTED CELLS WHICH, DESPITE
OUR BEST EFFORTS, DID NOT GET
INFECTED WITH HIV WHEN THOSE
WOULD BE A SOURCE OF NOISE AND
WE WANT TO ELIMINATE THEM.
SO SHE SORTED ALL OF THE PF --
NEGATIVE CELLS, SUBJECTED THEM
TO INFECTION.
NOW IT'S VERY UNLIKELY THAT A
CELERY MAINS UNINFECTED IN BOTH
ROUNDS.
SIMPLY BY SEQUENCING THE
STARTING POOL AND ENDING TOOL,
SHE GETS ENRICHMENT SCORES FOR
HOW LIKELY IS IT THAT YOU HAVE A
PROTECTIVE TRIM 5 ALLELE
VARIANT, WE HAVE TESTED ALL
POSSIBLE -- AND I A SED THEM IN
TERMS OF THEIR ENRICHMENT
RELATIVE TO THE BACKGROUND.
THIS IS SORT OF JUST A SANITY
CHECK HERE, WE'VE DONE TWO
BIOLOGICAL REPLICATES OF THIS.
THE SCORES ARE BEAUTIFULLY
CORRELATED.
SO THAT'S OF COURSE VERY
REASSURING.
A SECOND SANITY CHECK CAME WHEN
WE BASICALLY LOOKED AT THE
ENRICHMENT SCORES ON THIS WATER
FOLLOW PLOT, WHERE THE LEFT --
THE MORE RESTRICTIVE YOU ARE.
YOU'LL NOTICE THAT THE WILD TYPE
HUMAN TRIM 5 IS HERE IN THE
CLUSTER.
THERE'S ACTUALLY MORE THAN ONE
TRIM 5 VARIANT THAT ARE
DIFFERENT AT SYNONYMOUS
POSITIONS.
THEY'RE BASICALLY IDENTICAL
BECAUSE THEY ENCODE THE SAME
PROTEIN, EVEN THOUGH I TOLD YOU
THAT HUMAN TRIM 5 IS VERY POOR
AGAINST HIV-1 YOU'LL NOTICE IT
IS STILL STATISTICALLY BETTER
THAN TRIM 5 VARIANTS THAT STOCK
CODONS -- THIS IS A VERY
REASSURING KIND OF STEP THAT WE
ARE MAKING A BIOLOGICALLY
MEANINGFUL PREDICTION ABOUT
RESTRICTION.
WHAT ABOUT TESTING INDIVIDUAL
VARIANTS?
THIS IS ACTUALLY IN RESPONSE TO
THE QUESTION ABOUT THE NATURE OF
THE ASSAY.
SO THE NATURE OF THE ASSAY IS WE
BASICALLY INFECT CELLS WITH
INCREASING VIRAL DOSES BECAUSE
ESSENTIALLY WE'RE ABLE TO
OVERCOME -- IN THE ABSENCE OF
ANY TRIM 5, IT TAKES ABOUT THIS
MUCH VIRUS TO INFECT 10% OF THE
CELLS.
IN THE PRESENCE OF HUMAN TRIM 5,
THIS SHIFT RIGHT WARD, BECAUSE
YOU NEED A LITTLE BIT MORE VIRUS
TO INFECT 10% OF THE CELLS.
WITH RHESUS, IT WOULD SHIFT EVEN
MORE RIGHT WARD.
SO THESE ARE UNITS OF SELECTION
FOR HOW MUCH RESTRICTIVE
POTENTIAL IS PROVIDED BY
INDIVIDUAL VARIANTS IN THE
COURSE OF THEIR HIV-1
PROTECTION.
WHAT'S REALLY NICE IS EVEN
THOUGH SHE TESTED ONLY A HANDFUL
OF THESE VARIANTS, SHE FOUND THE
RESTRICTION BEAUTIFULLY
CORRELATED WITH THE POOR
ENRICHMENT SCORE THEY FOUND IN
HER ONE PART EXPERIMENT WHICH
MEANT THAT THE ASSAY EVEN THOUGH
IT WAS DONE ALL TOGETHER WAS A
BEAUTIFUL KIND OF REFLECTION OF
WHAT THE ACTUAL CONSTRAINTS
MIGHT BE.
SO I WANT TO RETURN TO THIS
WATERFALL PLOT BECAUSE ONE OF
THE SURPRISING THINGS TO US WAS
WE MADE RANDOM MUTATIONS IN THE
V1 LOOP HOPING SOME OF THEM
WOULD BE BETTER, SOME WORSE.
WE FOUND THAT NEARLY 60% OF
THESE RANDOM VARIANTS WERE
ACTUALLY BETTER THAN WILD TYPE
HUMAN TRIM 5 WHICH MEANT THAT
FOR SOME SORT OF UNFORTUNATE
CIRCUMSTANCES WE WERE SITTING IN
THIS VALLEY BUT WE COULD EASILY
BE GO UP A PEAK WHERE WE WOULD
BE PROTECTED WITH JUST A SINGLE
AMINO ACID CHANGE.
SEVTHAT WOULD BE BIOLOGICALLY
MEANINGFUL PROTECTION SO IT WAS
NOT THE CASE THAT HELPFUL
MUTATIONS WERE RARE HERE.
SO WE BECAME VERY INTERESTED IN
UNDERSTANDING WH WHAT'S THE
BIOCHEMICAL BASIS FOR THIS?
BECAUSE THIS SEEMS LIKE, YOU
KNOW, HUMAN TRIM 5 SHOULD BE
DOING A LOT BETTER, AND SO TO DO
THAT, WE BASICALLY MODELED OUR
RESTRICTERS ON THIS KIND OF 2 BY
2 PLOT HERE WHERE THE ROWS
INDICATE EACH POSITION IN THE V.
THE ARROW HEADS JUST INDICATE
THOSE RAPIDLY EVOLVING POSITIONS
THAT I TOLD YOU ABOUT, AND EACH
COLUMN REFERS TO MUTATIONS TO A
PARTICULAR AMINO ACID WITH EACH
OF THE 20 AMINO ACIDS AND CODONS
BECAUSE WE COULD NOT -- THAT IN
OUR MUTATIONAL STRATEGY.
SO WHAT WOULD YOU EXPECT HERE?
THE BLUER YOU ARE, THE MORE
RESTRICTIVE YOU ARE.
OVER THE ENTIRE DIAGRAM IS
DECIDEDLY -- CERTAINLY NOT THAT
HE PROBABLY MADE ANY MONEY OFF.
THAT'S BECAUSE WE HAD THESE VERY
ROW-LIKE AND COLUMN-LIKE
STRUCTURES IN OUR GRAFTING.
SO THE TWO THAT ARE MOST OBVIOUS
IS THAT HUMAN TRIM 5 HAS THESE
DOUBLE ARGININES AT POSITION 33.
IT TURNS OUT THAT ANY AMINO ACID
OTHER THAN AN ARGININE OR A
LYSINE WOULD BE PROTECTIVE.
SO WE'RE SORT OF LIKE DOUBLY
UNFORTUNATE FOR THESE TWO
ARGININES AR WITH WE WOULD BE
BETTER WITH ANYTHING ELSE AT
THESE POSITIONS AND REALLY
UNDERSCORING THAT, YOU CAN ALSO
SEE THAT AT ANY POSITION,
MUTATION TO ACIDIC RESIDUE, A D
OR AN E, WOULD BE ACTUALLY
PROTECTIVE.
I DRAW YOUR ATTENTION TO THIS
ABSOLUTELY CONSERVED GLYCINE
RESIDUE ACROSS ALL MAMMALS AND
YET GLYCINE IS ONE OF THE WORST
RESIDUES TO HAVE AT THIS PO
POSITION IN TERMS OF PROTECTION.
THE REASON I'M NOT GOING TO
FOCUS ON GLYCINE TODAY IS
BECAUSE CHARGE CAN EXPLAIN 90%
OF WHAT WE SEE ON THIS PLOT.
SO TO HIGHLIGHT THAT IN
AGGREGATE TERMS, WILD TYPE
HAS -- OF PLUS TO.
IF YOU MAKE THE V1 LOOK MORE
POSITIVELY CHARGED YOU MAKE IT
WORSE, BUT AS YOU MAKE THE
V1 LOOP LESS AND LESS POSITIVELY
CHARGED, YOU GET MORE AND MORE
PROTECTIVE.
SO EVEN THOUGH WE'VE KNOWN ABOUT
TRIM 5 FOR NEARLY 20 YEARS NOW,
IT TURNS OUT THAT THE MUTATION
STRATEGY ALLOWED US TO ACTUALLY
PREDICT THAT IN FACT THE KEY
DETERMINANT ABOUT WHETHER TRIM R
NOT AGAINST HIV-1 IS THE
POSITIVE CHARGE -- AND A
PRESUMED POSITIVELY CHARGED --
CAPSID, AND JENNETTE IS NOW
DOING SIMILAR EXPERIMENTS ON THE
CAPSID SIDE IN HER OWN LAB TO
BASICALLY FULLY KIND OF
FORMULATE THIS OPINION.
SO AGAIN RETURNING TO THIS GLASS
HALF FULL, GLASS HALF EMPTY
HYPOTHESIS, 30% OF RANDOM
MUTATIONS CAN CONFER PROTECTION.
SO THIS IS AN INDICATION THAT
WINNING SOLUTIONS ARE NOT RARE.
SO WHAT WE SAW WHEN WE ASSESSED
PRIMATE TRIM 5s WAS A SMALL
TIP OF THE ICEBERG OF A GIANT
ICEBERG OF POSSIBLE RANDOM
MUTATIONS.
SO NEXT SHE TURNED HER ATTENTION
TO WHAT ABOUT TRIM 5 ALREADY IN
A WINNING POSITION, IS THAT A
MUTATIONALLY FRAGILE OR
RESILIENT SOLUTION?
IN OTHER WORDS, IF YOU GUYS ARE
SKIERS IN THE AUDIENCE, YOU ARE
ON A BLACK DIE MONDAY SKI SLOPE,
EVERY -- DOWN THE FITNESS LAND
LANDSCAPE.
OR ARE YOU ON ONE OF THOSE BUNNY
SLOPES WHERE YOU LEARN HOW TO
SKI, EVERYTHING IS QUITE GENERAL
10 AND UNLIKELY TO BREAK ANY
BONES HERE.
SO TO DO THIS, SHE DID A
RECIPROCAL VERSION OF THE
EXPERIMENT.
THIS TIME FOCUSING ON RHESUS --
AND NOW MUTATING EACH OF THE
V1 RESIDUES AND DOING THE SAME
INFECTION WITH HIV-1 GFP BUT
THIS TIME REMEMBER WE STARTED
OFF WITH SOMETHING THAT WAS
ALREADY PROTECTIVE AGAINST
HIV-1, SO NOW WE ARE INTERESTED
IN THE GREEN CELLS, BECAUSE
THESE ARE VARIANTS THAT HAVE
LOST THEIR PROTECTION, SO IT'S
THE OPPOSITE OF THE ASSAY THAT
WE DID EARLIER, WHERE WE DNT
WANT THE GREEN CELLS, NOW WE
WANT THE GREEN CELLS BECAUSE
THESE ARE THE LOSS OF FUNCTION
MUTATIONS THAT ARE INTRODUCED
INTO RHESUS.
SO AGAIN IN RHESUS IS
MUTATIONALLY FRAGILE WE WOULD
PREDICT MOST WE MADE IN THE
V1 LOOP WOULD BE LOSS OF
FUNCTION.
TO OUR SURPRISE, TH THERE WERE Y
FEW LOSS OF FUNCTION MUTATIONS.
IN FACT, THE ONLY MUTATIONS WE
FOUND THAT WERE IN THE LEFT-HAND
SIDE ENRICHED FOR THE
NON-RESTRICTORS, WERE THE TRIM
5 -- WE HAD MADE TRUNCATION
MUTATIONS IN THE TRIM 5.
MOST OF THE RHESUS TRIM
5 VARIANTS WERE JUST AS GOOD AS
THE WILD TYPE RHESUS.
IN FACT, 51% ARE NOT
DISTINGUISHLIABLE FROM WILD TYPE
RHESUS.
WHICH MEANS AMAZINGLY EVEN
THOUGH THIS IS A PRIMARY LIGAND
INTERACTION -- IT'S MUTATIONALLY
RESILIENT ENOUGH THAT IT CAN
TOLERATE MUTATIONS WITHOUT
LOSINGITY BASE INTERACTION WITH
THE HIV-1 CAPSID.
IS THIS A GENERALLY MUTATIONALLY
RESILIENT PHENOMENON OR IS THIS
SOMETHING THAT HAS AN
EVOLUTIONARY SELECTIVE PROPERTY
LIKE RHESUS ON TRIM 5?
WE THOUGHT WE COULD ASK THAT
QUESTION BY ASKING WHAT IF WE
WERE TO ENGINEER NEW RESTRICTION
AND REQUESTS WHETHER THAT IS
MUTATIONALLY RESILIENT.
WE CAN DO THAT BECAUSE REMEMBER
I SHOWED YOU A SINGLE AMINO ACID
CHANGED FROM RHESUS INTO HUMAN
TRIM 5 CAN PROVIDE YOU
SUBSTANTIAL RESTRICTION.
SO JENNETTE DID YET ANOTHER SCAN
OF THIS VERY HUMAN TRIM 5 WHERE
THIS PARTICULAR POSITION HAS
BEEN CHANGED FROM AN ARGININE TO
A PROLINE, REMOVING ONE OF THOSE
ARGININES THAT I TOLD YOU ARE SO
BAD FOR RESTRICTION.
AND WHEN SHE DID THAT, SHE
FOUND, ONCE AGAIN, THAT THIS
NEWLY ENGINEERED TRIM 5 IS JUST
AS RESILIENT.
SO ONCE YOU GO UP THE FITNESS
LANDSCAPE, IT'S NOT INEVITABLE
THAT THE NEXT IS GOING TO DRIVE
YOU -- THIS IS A PRETTY
INTERESTING PROPERTY THAT HAS
BEEN GRAFTED WE THINK BECAUSE OF
ITS INTERACTIONS WITH RAPIDLY
CHANGING VIRAL PATHOGENS SO THE
TWO SORT OF QUESTIONS THAT SHE
WANTED TO ASK, HOW RARE ARE
WINNING SOLUTIONS.
THAT WAS A SU SURPRISE BEING
THEY'RE NOT RARE AT ALL.
IN FACT EVOLUTION TURNED OUT TO
BE MISLEADING, AND MANY
SOLUTIONS TURN OUT TO BE QUITE
STABLE AND NOT AT ALL
MUTATIONALLY FRAGILE.
I JUST WANT TO CONTRAST WHAT SHE
HAS FOUND HERE WITH WHAT OTHERS
HAVE FOUND WHEN THEY DO SCANNING
WITH PROTEIN-PROTEIN INTERACTION
INTERFACES.
IN PARTICULAR WHEN YOU COME TO
LILIGAND BINDING INTERACTIONS,
WHAT PEOPLE HAVE FOUND IS AS YOU
GET CLOSER AND CLOSER TO THE
LIGAND BINDING DOMAIN, YOU GET
MORE AND MORE DEVASTATING
EFFECTS OF -- YOU HAVE ALMOST A
LOCK AND KEY-TYPE MECHANISM
WHERE ANY NEED THE LOCK YOU NEED
FOR THE LIGAND INTERACTION.
IN CONTRAST TO THIS, WE HAVE
THESE ROLLING HEADLINE -- WHICH
YOU CAN SEE WOULD BE VERY HUGELY
BENEFICIAL FOR ANTIVIRAL
PROTEINS TO ACTUALLY HAVE,
BECAUSE YOU'RE BASICALLY PLAYING
THESE AFFINITY-BASED ARMS RACES
WITH CHANGING VIRAL PATHOGENS.
SO THIS IS ONE OF THOSE REGIMES
WHERE IT EASY TO MAKE A
RESTRICTION HAPPEN BUT HOW TO
BREAK THE RESTRICTIONS ONCE YOU
GET MEANS YOU CAN GENERATE
STANDING VARIATION WITHOUT A
HUGE COST TO YOUR STANDING SORT
OF RESTRICTION PR PROFILE AND TE
STANDING VARIANTS ARE LIKELY TO
BE BENEFICIAL, WHEN YOU
ENCOUNTER A NOW VARIANT OR
PATHOGEN ENTIRELY.
SO WHAT I HAVEN'T TELL YOU SO
FAR BUT I WILL RIGHT NOW IS THAT
THERE'S A REASON WHY WE THINK
THAT ANTIVIRAL PROTEINS LIKE
TRIM 5 HAVE THIS VERY UNUSUAL
ROLLING HILL-LIKE LANDSCAPE,
WHICH IS THAT THE V1 LOOP EVEN
IN STRUCTURAL ANALYSIS IS
BASICALLY UNSTRUSTRUCTURED.
WHAT THAT MEANS IS THAT IT'S
REALLY AN ENSEMBLE OF MANY
DIFFERENT STRUCTURAL STATES
WHICH WE CANNOT REALLY CAPTURE
IN ONE PARTICULAR STRUCTURE.
UNLIKE THE REST OF TRIM 5 WHICH
IS QUITE NICELY STRUCTURED.
YOU CAN MABLG IN THIS SITUATIONS
SITUATION, IT'S ACTUALLY ONE OF
THE SUBSETS IS ACTUALLY CAPABLE
OF INTERACTING WITH THE HIV-1
CAPSID AND THAT'S WHERE WE GET
THE SPECIFICITY FROM.
HOWEVER, NO SINGLE MUTATION IS
GOING TO COMPLETELY BREAK THIS
ENSEMBLE OF STATES.
IN FACT, THE VERY FEW MUTATIONS
THAT DID BREAK THIS ENSEMBLE OF
STATES IN RHESUS TRIM 5 HAPPEN
TO BE THINGS WHERE WE HAD
INTRODUCED PROLINES, WHICH THOSE
OF YOU WHO ARE SORT OF
BIOCHEMICALLY INCLINED CAN
APPRECIATE WOULD BE A BAD THING
TO INTRODUCE INTO AN
UNSTRUCTURED LOOP IN ORDER TO
SORT OF IMPOSE STRUCTURE INTO
THAT.
THIS SHOULD BE VERY FAMILIAR TO
THOSE OF YOU WHO REMEMBER YOUR
IMEU KNOLL COURSES BECAUSE THIS
IS EXACTLY WHAT HAPPENS IN THE
COURSE OF SORT OF AFFINITY
MATURATION FOR ANTIBODIES, WHERE
ANTIGEN RECOGNITION SITES START
OFF IN THIS ENSEMBLE OF
UNSTRUCTURED STATES, AND AS YOU
GET MORE AND MORE MATURE AND YOU
SORT OF GET LESS AND LESS
DIVERSE IN YOUR EPITOPE
RECOGNITION, YOU GET MORE AND
MORE STRUCTURED AND DEFINED, BUT
REALLY YOU START OFF IN THESE
ENSEMBLE STATES WHICH GIVES YOU
THE MAXIMUM ABILITY TO SORT OF
DIVERGE INTO THESE MULTIPLE KIND
OF VALLEYS OF AFFINITY
MATURATION.
IN FACT, TRIM 5 IS NOT THE ONLY
PROTEIN THAT WE SEE THIS IN.
I'M GOING TO SORT OF SWITCH TO
OUR SECOND VIGNETTE NOW AND
FOCUS ON ANOTHER ANTIVIRAL GENE
CALLED MXA JUST TO MAKE A
DIFFERENT POINT, WHERE ONCE
AGAIN THE HERO OF THE STORY, IF
YOU WILL, HAPPENS TO BE THIS
UNSTRUCTURED LOOP CALLED LOOP
L4, WHICH IS THE PRIMARY
DETERMINANT FOR AFFINITY FOR A
COMPLETELY DIFFERENT CLASS OF
VIRUS AS WELL.
SO THE DISORDERED LOOPS REALLY
PROVIDE THE EVOLUTIONARY
FLEXIBILITY THAT ALLOWS THESE
ANTIVIRAL PROTEINS TO KEEP PACE
WITH CHANGING VIRAL PATHOGENS.
SO WHAT IS MXA?
IT'S AN INTERFERON TRIGGER GENE
WHICH IS ONE OF THOSE MANY ALARM
GENES THAT YOU TURN ON WHEN YOU
SENSE THAT SOME VIRAL PATHOGEN
IS TAKING OVER YOUR BODY.
MXA HAPPENS TO AB VERY CLOSE
RELATIVE IN EVOLUTIONARY TERMS
OF A PROTEIN CALLED DYNAMIN,
IMPORTANT FOR MEDIATING ENDO
CYTOSIS AS THEY RE-ENTER THE
CYTOPLASM.
MXA IS NOT ESSENTIAL AND IS
ACTUALLY DEDICATED TO A ROLE IN
ANTIVIRAL IMMUNITY.
PEOPLE IN THE IMMUNOLOGY WORLD
REALLY REVERE MXA BECAUSE IT'S
THE VERY FIRST INNATE ANTIVIRAL
PROTEIN TO BE DISCOVERED IN
MAMMALS.
IT WAS DISCOVERED BECAUSE OF A
HAPPY ACCIDENT, LET'S CALL IT
THAT, OF MOUSE BREEDING WHERE
MOST LABORATORY STRAINS OF MICE
HAPPEN TO BE DELETED FOR THE MX
LOCUS.
THIS WAS DISCOVERED MANY YEARS
LATER, WHICH MAKES THESE STRAINS
EXQUISITELY SUSCEPTIBLE TO BOTH
HUMAN AND BIRD INFLUENZA.
MOST STRAINS ARE DEAD WITHIN
FOUR DAYS OF INFECTION.
THIS IS A PRETTY SIGNIFICANT
INFECTION.
WHEREAS IF YOU WERE TO RESTORE
THE MOUSE MX1 GENE OR EVEN BRING
IN THE HUMAN MXA GENE, YOU CAN
COMPLETELY PROTECT THESE MICE
AND YOU CAN DO SO EVEN IN MICE
THAT ARE MUTATED FOR THE
INTERFERON RECEPTOR, WHICH MEANS
THAT NOW YOU DON'T HAVE ACCESS
TO THOSE 200 GENES THAT PROTECT
AGAINST VIRUSES.
YOU ONLY HAVE ACCESS TO ONE
GENE, MXA, AND THAT IS
SUFFICIENT TO PROTECT YOU LONG
ENOUGH FOR THE ADAPTIVE IMMUNE
SYSTEM TO KICK IN AND PROTECT
YOU.
SO REMEMBER LIKE THE JOB FOR
TRIM 5 AND MXA IS NOT TO CLEANSE
THE BODY OF THE PARTICULAR VIRAL
INFECTION.
IT'S JUST TO DELAY THE VIRAL
INFECTION LONG ENOUGH FOR THE
ADAPTIVE IMMUNE SYSTEM TO KICK
IN, EDUCATE ITSELF AND THEN
CLEANSE THE BODY OF THE
INFECTION.
THAT'S REALLY THE PRIMARY JOB OF
INNATE IMMUNITY IN VERTEBRATES.
SO WHAT IS MXA ACTING AGAINST?
I TOLD YOU IT ALREADY WORKS
AGAINST INFLUENZA AND SUBSEQUENT
STUDIES SHOW IT ACTUALLY WORKS
BY RECOGNIZING THE NUCLEAR
PROTEIN OF INFLUENZA, WHICH IS
THE PROTEIN THAT ACTS ALMOST
LIKE A HISTONE TO WRAP AROUND
THE RNA AND MEDIATE BOTH
TRANSCRIPTION AND REPLICATION OF
THIS RNA VIRUS.
SO I'M GOING TO SORT OF -- IT
WORKS A LOT LIKE TRIM 5 SO I'M
ACTUALLY GOING TO JUST GO
THROUGH THIS SORT OF SENSE OF
DEJA VU.
PATRICK MITCHELL, FORMER
GRADUATE STUDENT IN THE LAB
REALLY FOCUSED ON MXA BECAUSE
DESPITE DISCOVERY NEARLY FOUR
DECADES AGO, WE HAD NO IDEA WHAT
IT'S USING TO INTERACT WITH THE
MP PROTEIN.
THE
NP PROTEIN.
SO ONCE AGAIN, WE USED THIS
EVOLUTION-GUIDED APPROACH WHERE
WE REALIZE THAT THERE WERE
REALLY TWO HOT SPOTS FOR
POSITIVE SELECTION, AND ONE OF
THEM HAPPENED TO BE THIS
UNSTRUCTURED LOOP CALLED LOOP
L4, WHICH I'VE DRAWN IN HERE
WITH MY ARTIST'S RENDITION,
WHICH HAD THE MAJORITY OF
POSITIVELY SELECTED SITES IN
THIS, AND WE WONDERED WHETHER
THIS WAS, IN FACT, THE MISSING
SPECIFICITY DOMAIN THAT ALLOWED
MXA TO BIND THE NUCLEAR PROTEINS
OF INFLUENZA.
AND INDEED, PATRICK SHOWED THAT
WITH AN INFLUENCE -- WHERE IN
THE ABSENCE OF MXA YOU HAVE 100%
INFECTION, HUMAN MXA IS VERY,
VERY GOOD.
IN FACT, IT'S STILL THE VERY
BEST MXA VARIANT WE'VE FOUND
AGAINST -- VIRUS.
WE HAVE NO IDEA WHY BECAUSE THIS
IS A VIRUS THAT INFECTS MICE BUT
DOES NOT INFECT HUMANS.
HUMAN MXA, STRONG PROTECTION SO
IT'S THE OPPOSITE OF TRIM 5.
AFRICAN GREEN MONKEY IS NOT
PROTECTED.
THESE ARE 90% IDENTICAL AT THE
AMINO ACID LEVEL AND YET ONE IS
COMPLETELY PROTECTIVE, THE OTHER
IS COMPLETELY NOT PROTECTIVE.
JUST LIKE WHAT YOU CAN SEE FROM
THE TRIM 5 STORY, SWAPPING IN
THE MOST RAPIDLY EVOLVING PARTS
OF THE LOOP L4 REGION BETWEEN
THESE TWO BACKBONES IS ABLE TO
CONFER PROTECTION -- YET AGAIN A
SINGLE AMINO ACID RESIDUE OUT OF
THE -- IS ABLE TO CONVERT WHAT
WAS A LOSING SITUATION WITH
AFRICAN GREEN MONKEY MXA INTO
ESSENTIALLY A COMPLETELY
PROTECTIVE SITUATION, HUMAN
LIFE.
SO JUST TO REITERATE THAT WE ARE
ABLE TO USE EVOLUTION-GUIDED
APPROACH TO IDENTIFIED LOOP L4.
IT DOES MEDIATE THE TARGET
RECOGNITION AND SINGLE RESIDUE
CHANGES CAN DRASTICALLY ALTER
THE ANTIVIRAL ACTIVITY HERE.
SO UP TO THIS POINT, EVERYTHING
I'VE TOLD YOU HAS BEEN A
RETROSPECTIVE VIEW, WHICH IS
BASICALLY I'M LOOKING BACKWARDS
IN EVOLUTION, I'M TRYING TO
DECIPHER READING THE TEA LEAVES
OF WHETHER CHANGES HAVE OCCURRED
AND DESAI FER WHAT THE
INTERACTION INTERFACES MIGHT BE.
AND YOU KNOW, I WAS PERFECTLY
HAPPY TO DO THIS FOR THE
REMAINDER OF MY CAREER, BUT YOU
KNOW, GRADUATE STUDENTS COME IN
AND LIKE YEAH THAT'S BEEN DONE
BEFORE, I WANT TO DO SOMETHING
ELSE, WHICH IS WHAT ARE YOU
GOING TO USE THIS FOR?
I WAS LIKE WELL I'M GOING TO USE
THIS TO KEEP MY JOB.
THAT'S LIKE AN IMPORTANT, LIKE,
IMPORTANT TASK TO BEGIN.
SHE SAID NO, NO, BECAUSE YOU
LEARNED ALL OF THIS STUFF FROM
EVOLUTION, CAN YOU USE THIS TO
MAKE SORT OF A MORE SORT OF
DESIGNER SPECIFICITY INTO A
ANTIVIRAL PROTEIN THAT DOESN'T
HAVE IT.
THIS IS PRE-COVID SO WE WEREN'T
EVEN THINKING ABOUT SARS COV BUT
THAT WOULD BE, OF COURSE, VERY
USEFUL SO WE WONDERED CAN WE USE
THE SAME PLAY BOOK OF POSITIVE
SELECTION IDENTIFIED THROUGH
THIS RETROSPECTIVE ANALYSIS TO
NOW GO AFTER NEW SPECIFICITIES
IN THE LABORATORY TO ACTUALLY
ELICIT THAT AND WHAT IS THE
LANDSCAPE OF THAT NEW ADAPTIVE
LANDSCAPE WHICH MIGHT BE
REVEALED FROM THESE EXPERIMENTS.
SO THIS IS WHAT SHE DECIDED TO
DO.
SHE AGAIN CAME TO MY LAB
FOLLOWING UP ON PATRICK'S WORK
AND SHE SAID I'M REALLY
INTERESTED IN LOOP L4, BUT I'M
NOT INTERESTED IN THE 20 ODD
PRIMATE SEQUENCES THAT YOU'VE
SEQUENCED BECAUSE THAT'S A VERY
SMALL SUBSET OF THE POSSIBLE
COMBINATORIAL SPACE.
I WANT TO EXPLORE WHAT ARE THE
POSSIBLE STATES THAT MIGHT BE
REVEALED BY THIS APPROACH.
SO I SAID THIS SOUNDS LIKE A
GOOD IDEA, WE SHOULD DO THIS.
THIS WAS MANY YEARS AGO.
THIS WOULD NOW BE LIKE A PHONE
CALL TO TWIST BIOSCIENCES AWAY
BUT UNFORTUNATELY THAT WAS NOT
ROSSAN A'S GOOD LUCK, SO SHE
ASSESSED THEM INDIVIDUALLY IN
TRIPLICATE FOR FUNCTIONALITY AND
SHE DID ABOUT A THOUSAND
VARIANTS.
THIS WAS A LOT OF WORK.
I'M NOT GOING TO SORT OF DENY
THAT THIS WAS A LOT OF EFFORT
THAT SHE DID IN THIS PART.
AND IT WAS VERY DISAPPOINTING.
SO WHAT SHE FOUND WAS THAT 95%
OF THE VARIANTS WERE ACTUALLY
WORSE THAN WILD TYPE MXA.
SO EVEN THOUGH WE HAD STARTED
WITH HUMAN MXA, WHICH IS VERY
GOOD, AND WE HAD ONLY MESSED
WITH THE RAPIDLY EVOLVING
POSITIONS, IT TURNS OUT YOU CAN
STILL BREAK MXA QUITE EASILY.
AND ABOUT 5% WERE ABOUT AS GOOD
AS WILD TYPE MXA AND THEN WE HAD
A FEW VARIANTS THAT WERE
SLIGHTLY BETTER EVEN THAN WILD
TYPE MXA.
SO THIS WAS LIKE ONE OF THOSE
DEEPLY DEPRESSING SERIES OF LAB
MEETINGS WHERE YOU SORT OF GET
THESE ACCUSATORY LOOKS ABOUT
GRADUATE STUDENTS ABOUT HOW YOU
SHOULD HAVE REALLY TOLD ME
BETTER THINGS TO DO, AND THEN WE
SORT OF REALIZED AFTER THAT HAD
ENDED THAT WE SHOULD LOOK AT THE
VARIANTS THAT DID HAVE SOME
ACTIVITY, AND IT TURNS OUT THAT
ALL OF THOSE VARIANTS ACTUALLY
HAVE THIS RESIDUE RESTRICTION,
WHICH IS THAT THEY NEED TO HAVE
A PHENYL ALANINE, TYROSINE OR
TRYPTOPHAN -- THOSE OF YOU THAT
REMEMBER IT WAS EXACTLY THE SAME
RESIDUE THAT PATRICK HAD
ACTUALLY MUTATED FROM HUMAN INTO
AFER GANGRENE MONKEY TO CONFER
PROTECTION.
SO THE MORAL OF THE STORY IS WE
SHOULD DEFINITELY REREAD OUR OWN
PAPERS BEFORE EMBARKING ON THE
NEXT STUDY.
SO MY SUCCESSFUL CONTRIBUTION TO
THIS PROJECT WAS TO CONVINCE
ROSSANA TO NOT QUIT GRAD SCHOOL
AND REDO THE SCREEN AGAIN BUT
GUIDED BY THIS APPROACH WHERE
SHE ALREADY FOUND PHENYL ALANINE
IS PRETTY GOOD, TYROSINE IS EVEN
BETTER AND TRYPTOPHAN CAN WORK
BUT IT'S ACTUALLY NOT AS GOOD
COMPARED TO THE OTHER
HYDROPHOBIC RES KNEWS SO WE
DECIDED -- PHENYL ALANINE AND TO
SWEETEN THE POT A LITTLE BIT I
HAD A ROTATION STUDENT JOIN
ROSSANA AND THEY SAID WE'RE ONLY
GOING DO ABOUT 250 VARIANTS
BECAUSE WE'RE GOING TO TEST THEM
INDIVIDUALLY, AND THIS COULD
HAVE GONE REALLY BADLY BUT
LUCKILY FOR ME, IT DID NOT.
SO NOW 70% OF THE VARIANTS WERE
AS GOOD OR BETTER THAN HUMAN MXA
BECAUSE WE HAD NOW HONORED THE
REQUIREMENT OF A HYDROPHOBIC
AROMATIC RESIDUE AT THE CRITICAL
POSITION.
AND WE STILL HAVE ABOUT 30% OF
VARIANTS THAT ARE WORSE THAN
WHAT YOU'D EXPECT.
AND I WOULD BE REALLY INTERESTED
IN ANSWERING WHY THAT IS.
BUT OF COURSE WE NEVER DID THAT
BECAUSE WE REALIZED THAT WE HAD
NOW NEARLY TWO DOZEN VARIANTS
THAT WERE SIGNIFICANTLY BETTER
THAN WILD TYPE HUMAN MXA, WHICH
IS THE VERY BEST NATURAL
RESTRICTOR OF THE VIRUS WE HAVE
IN OUR ARSENAL.
THIS IS SHOWN IN TRIPLICATE
AGAIN.
WE USE THIS AS A CONTROL
NORMALIZED TO ONE.
WILD TYPE HUMAN MXA AT THIS
LEVEL OF PROTECTION IS TENFOLD
PROTECTION, REALLY, REALLY GOOD
PROTECTION.
HERE'S OUR INITIAL FIVE SITE
SCREEN AND HERE IS NOW OUR FOUR
SITE SCREEN WHERE YOU FIX THE
PHENYL ALANINE.
SO I JUST WANT TO YOU APPRECIATE
WHAT WE'VE DONE HERE.
WE ARRIVED AT LOOP L4 BY A
RETROSPECTIVE ANALYSIS THAT
IDENTIFIED THESE POSITIONS.
WE HAVE NOW JUST ALLOWED THOSE
POSITIONS, FOUR OF THOSE
POSITIONS TO MUTATE, AND WE NOW
HAVE 10 TO 20 FOLD BETTER
PROTECTIVE VERSIONS OF MXA JUST
BY TAKING THIS EVOLUTIONARY SORT
OF HANDBOOK AND JUST ALLOWING
MOLECULAR BIOLOGY IN THE LAB TO
TELL US ABOUT ALLOWING US TO
SCREEN MORE VARIANTS OF THAT
TIME.
I JUST WANT TO POINT OUT
FOCUSING ON THE THREE VERY BEST
SUPER RESTRICTORS WE HAVE ON THE
RIGHT HERE THAT THESE SUPER
RESTRICTIVE VARIANTS ARE BETTER
AT ALL LEVELS OF ECK PRETION TO
SHE OUTPERFORM WILD TYPE MXA
WHERE I'M GOING TO FOUND LIKE
VERY BEST NATURAL RESTRICTOR AND
THESE VARIANTS WE HAVE IN THE
LAB CAN OUTPERFORM THEM
SIGNIFICANTLY AT ALL LEVELS OF
EXPRESSION, AND UNSURPRISINGLY
BUT STILL REASSURINGLY, THE
REASON THEY OUTPERFORM THIS IS
BECAUSE OF HIGHER AVIDITY WITH
THE PROTEIN OF THE VIRUS.
SO HERE YOU CAN SEE THAT WILD
TYPE MXA HAS THE REALLY NICE
ABILITY TO -- DURING VIRALLY
INFECTED CELLS BUT EACH OF THE
SUPER RESTRICTORS IS MUCH MORE
AVID BINDER OF THE NP PROTEIN
AND THAT MAKES IT A MUCH MORE
AVID RESTRICTOR OF THE VIRUS
ITSELF.
SO LET'S RETURN BACK TO THIS
SORT OF DIAGRAM WHERE WE HAVE
NOW THE FIVE SITE AND FOUR SITE
MUTATIONS THAT ARE BENEFICIAL.
THIS IS THE POINT IN THE TALK
WHERE I OFTEN GET ASKED, NOW YOU
COULD USE DEEP LEARNING TO
ACTUALLY LIKE FIGURE OUT WHAT IS
THE SECRET HERE?
WHAT COULD YOU DO?
THAT'S THE POINT OF THE TALK
WHERE I TELL YOU THIS IS NOT A
REPRESENTATIVE SET.
THIS IS LITERALLY ALL THE
VARIANTS WE FOUND AT THE END WE
COULD CERTAINLY FIND MORE BUT
THAT WOULD REQUIRE HER TO DOM
BACK TO GRAD SCHOOL, WHICH IS
NOT HAPPENING.
SO WE USED MY VERSION OF SHALLOW
LEARNING, WHICH IS THE P.I.
STARES AT THESE VARIANTS FOR A
LONG TIME AND ALLOWS PATTERNS TO
EMERGE.
ACTUALLY THIS DID WORK IN THIS
CASE BECAUSE WE REALIZED THAT
THE Q RESIDUE THAT GLUTAMATED AT
THE FIRST POSITION WAS ACTUALLY
KIND OF OVERREPRESENT THE HERE,
IN SPITE OF THE FACT THAT
GLUTAMATE ITSELF IS NOT THAT
WELL REPRESENTED IN THE GENETIC
CODE, SO WE SAID THE GLUTAMATE
AT THIS POSITION 540 MUST BE
CRITICAL.
SO JUST TO REMIND YOU AGAIN,
WILD TYPE MXA WITH THE FIVE
RESIDUES, RAPIDLY EVOLVING
POSITIONS.
A G TO Q MUTATION IS A SMALL
BUMP IN RESTRICTION BUT ACTUALLY
IF YOU TAKE EACH OF THE SUPER
RESTRICTORS THAT ALREADY HAVE A
QUEUE, REVERTING TO A WILD TYPE
VERSION, YOU LOSE THE SUPER
RESTRICTION.
SO THIS IS OUR FIRST EVIDENCE
THAT RESIDUE 540, IN ADDITION TO
RESIDUE 561, IS PLAYING A VERY
CRITICAL ROLE.
THAT PROMPTED US TO ASK OKAY
WHAT ABOUT OTHER AMINO ACIDS AT
THIS POSITION?
MAYBE WE SHOULD MUTATE THEM TO
ALL THAT ARE PRESENT.
SO WE DID THAT AND IT ACTUALLY
TURNS OUT THAT PROLINE IS
TERRIBLE, WE'RE NOT GOING TO
KIND OF DWELL ON THAT, THAT
TURNS OUT TO BE A UNIVERSALLY
TRUE THING.
BUT YOU CAN SEE THAT YOU CAN GO
FROM TRYPTOPHAN ON THIS END TO
ALANINE ON THIS END AND THAT'S A
TENFOLD DIFFERENCE IN
RESTRICTION.
THAT'S THE DIFFERENCE BETWEEN
PROTECTION AND NO PROTECTION, IF
YOU WILL, AND THAT'S JUST
HAPPENING WITH A SINGLE AMINO
ACID RESIDUE.
AND WE STARR STARED AT THIS VERD
TO DETERMINE ARE THERE ANY
BIOLOGICAL PATTERNS, ET CETERA,
AND NOTHING BECAME OBVIOUS UNTIL
AGAIN SHALLOW LEARNING CAME TO
THE RESCUE BECAUSE IT TURNS OUT
THAT -- ARE REALLY BAD IN THE
WILD TYPE CONTEXT BUT I
REMEMBERED THAT VAI LEAN AND
THREONINE WERE ACTUALLY PRESENT
IN TWO OF OUR BEST SUPER
RESTRICTORS SO SUDDENLY VISIONS
OF CONVERTING THOSE TO ALANINE
AND GETTING A SUPERDOOPER
RESTRICTOR LIKE WOW, THAT WOULD
MAKE A REALLY GREAT FINAL FIGURE
FOR A PAPER, SO THAT'S WHAT I
ENCOURAGED ROSSANA TO DO, LET'S
TAKE THE WILD TYPE RESIDUE IN
WHICH THERE'S THIS DRAMATIC
DIFFERENCE IN RESTRICTION, LET'S
DO THE SAME FOR THE SUPER
RESTRICTOR THAT ALREADY HAD A
VAI LEAN AN MAYBE WE CAN BUMP UP
THE RESTRICTION EVEN MORE.
OF COURSE THE OPPOSITE HAPPENED.
SO THE SAME MUTATION IN A
DIFFERENT CONTEXT REMEMBER THESE
ARE OWL DIFFE ONLY DIFFERENT IN-
COMPARED TO THE 600 AMINO ACID
ASSAY PROTEIN HAVE COMPLETELY
OPPOSITE EFFECTS ON RESTRICTION
PRPROFILE, JUST AGAIN REFLECTING
THE UNPREDICTABLE NATURE OF THE
ENSEMBLE, ALSO REALLY
HIGHLIGHTING THAT THIS KIND OF
EPISTASIS MEANS WE WERE REALLY
LUCKY THAT WE DID IMI NIGH TORE
YAL -- RATHER THAN SINGLE
RESIDUE SCANNING.
BUT THAT LOSS OF FUNCTION
MUTATION ACTUALLY PERMITTED THE
GAIN OF FUNCTION SUBSEQUENT
MUTATIONS TO ACTUALLY OCCUR.
SO YOU HAD TO GO THROUGH THE
VALLEY TO GET TO THE PEAK THAT
WAS ACTUALLY AFFORDED.
JUST TO SUMMARIZE WHAT I TOLD
YOU SO FAR, WE STARTED WITH THIS
APPROACH WHERE WE LOOKED AT
EVOLUTION AND LOOKED AT LOOP
L4 -- TURNS OUT NOT JUST FOR TA
GOA DA VIRUS BUT INFLUENZA.
NOW ROSSANA HAS TAKEN THIS
APPROACH WHERE SHE'S MUTATED ALL
OF THE RESIDUES EXCEPT ONE AND
SHE'S NOW FOUND SUPER
RESTRICTORS.
I CAN GUARANTEE MOST OF YOU HAVE
NEVER HEARD OF THOGOTO VIRUS OR
WISH YOU WOULDN'T HAVE BUT YOU
ALL CARE ABOUT INFLUENZA VIRUS.
SO SUDDENLY VISIONS OF GRAND
FUNDING BEGAN DANCING IN MY HEAD
I SAID OH MY GOD, WHAT IF YOU'VE
DISCOVERED SUPER RESTRICTORS OF
INFLUENZA, WOULDN'T THAT BE
AMAZING.
AND MAYBE THAT'S WHAT THE WHOLE
STORY IS GOING TO BE ABOUT.
THAT'S WHEN ROSSANA TESTED MANY
OF HER SUPER RESTRICTORS AGAINST
THOGOTO VIRUS ON THE X AXIS
AGAINST THE -- HERE'S THE WILD
TYPE MMXA, AND YOU REALIZE WHAT
BECAME APPARENT TO US OVER LIKE
WEEKS OF DOING THIS THAT YOU
COULD GET REALLY GOOD AT THOGOTA
VIRUS BUT YOU'D GET WORSE AND
WORSE WITH INFLUENZA VIRUS.
SO EVEN THOUGH WE'RE MESSING
WITH THIS VERY SMALL LANDSCAPE
OF THE V1 LOOP, THERE IS THIS
SPECIFICITY TRADEOFF WHERE THE
WILD TYPE VERSION WE HAVE IN OUR
GENOME IS REALLY THE JACK OF ALL
TRADES VERSION AGAINST BOTH
VIRUSES.
YOU CAN GET BETTER BUT YOU OFTEN
DO SO AT THE EXPENSE OF THE
BREADTH OF RESTRICTION THAT MXA
CAN PROVIDE.
SO UNDAUNTED BY THIS, A NEW
GRADUATE STUDENT IN THE LAB, RI
CHRACHEL GEIGER, SAID I WANT TOO
THIS APPROACH BUT THIS TIME
AGAINST THE INFLUENZA VIRUSES
BECAUSE THAT'S THE ONES THAT I
CARE ABOUT.
LONG STORY SHORT, THIS WORKS
AGAINST INFLUENZA VIRUSES BUT WE
FIND COMPLETELY DIFFERENT
VARIANTS.
WE MADE THE JUDICIOUS CHOICE OF
ALLOWING ALL CRE HYDROPHOBIC
RESIDUES TO OCCUR IN THIS
LIBRARY AND THAT TURNED OUT TO
BE A VERY JUICE DI JUDICIOUS CHE
BECAUSE WHAT SHE DISCOVERED WAS
THE TRYPTOPHAN RESIDUE IS
OVERREPRESENTED AMONG THE
INFLUENZA SUPER RESTRICTORS, AND
THE TYROSINE RESIDUE IS
UNDERREPRESENTED AMONG THE
INFLUENCE IS A SUPER
RESTRICTORS.
IN FACT, THIS IS CLEARLY
APPARENT BY TAKING JUST ONE
VARIANT WHERE YOU'VE BASICALLY
MUTATED JUST THIS RESIDUE FROM A
TRYPTOPHAN, WHICH IS SUPER
RESTRICTIVE, AND A PHENYL AL
LEAN OR TYROSINE WHICH LOSES
THAT SUPER RESTRICTION, AND IF
YOU'LL RECALL, THIS IS EXACTLY
THE OPPOSITE OF WHAT SHE FOUND
WITH THE HYDROPHOBIC RESIDUE
PRESENCE.
SO IT TURNS OUT THIS TRADEOFF
OFTEN JUST COMES DOWN TO WHICH
HOE DREE PHOBIC RESIDUE DO YOU
HAVE FIXED OR NOT FIXED AT
RESIDUE 561?
IF IT'S A TRYPTOPHAN YOU'RE MUCH
MORE LIKELY TO BE AN INFLUENZA
VIRUS RESTRICT TORE, IF YOU'RE
PHENYL ALANINE, TURNS OUT YOU
STILL HAVE SOME PLAY, YOU CAN BE
BOTH, AND WE'RE HAPPY TO EXPLORE
THAT.
SO I JUST WANT TO TELL YOU THAT
THE JURY IS STILL OUT, WHETHER
WE CAN USE THIS APPROACH FOR
SUPER RESTRICTORS OF INFLEUN SHA
CYRUS.
WE'LL BE CONFIDENT WE'LL BE ABLE
TO DO THAT, AND THE NEXT STEP,
OF COURSE, IS TO GO AFTER
PANDEMIC STRAINS OF INFLUENZA
THAT BASICALLY HAVE ESCAPED
HUMAN MXA AND ASK, CAN WE USE
THE SAME APPROACH TO BRING THEM
BACK WITHIN THE REA STRICKTIVE
MANIFOLD OF MXA RESTRICTION?
SO HOPEFULLY I'VE CONVINCED YOU
TODAY THAT DISORDERED LOOPS EVEN
THOUGH THAT'S KIND OF MESSY FROM
A STRUCTURAL STANDPOINT ARE
REALLY AN IMPORTANT TOOL FOR
ANTIVIRAL PROTEINS THAT PROVIDE
BOTH THE EVOLUTIONARY
FLEXIBILITY AND THE ADAPTIVE
POTENTIAL THAT ALLOWS ANTIVIRAL
PROTEINS, EVEN SLOWLY EVOLVING,
MOST OF WHAT I TOLD YOU TODAY
ARE ONE ON ONE ARMS RACES, AND
THESE RED QUEEN CONFLICTS CAN
NEFORT THE LESS BE QUITE
SUCCESSFUL.
I JUST WANT TO CLOSE BY
ACKNOWLEDGING THE PEOPLE WHO DID
THE WORK.
ALL OF THIS WORK IS ACTUALLY THE
CONSEQUENCE OF A VERY INFLUENCE
SAL COFFEE THAT I HAD 20 YEARS
AGO WHEN I STARTED MY LAB WITH
MY SENIOR COULD LEAGUE, MICHAEL
EMERMAN.
I HOPE IT WAS NOT TOTALLY ON
VEES BUT I'M AN EVOLUTIONARY
BIOLOGIST BY TRAINING AND ALL OF
THIS IS A BEAUTIFUL SYMBIOSIS
BETWEEN A BIOLOGY DRIVEN LAB AND
EVOLUTION -- THERE ARE STUDENTS
IN MY LAB DOING EXPERIMENTS IN
WHICH I HAVE ABSOLUTELY NO
TRAINING IN DOING AND NO
BIOSAFETY ABILITY TO ACTUALLY
TRAIN THEM, BUT MICHAEL DOES,
AND THAT'S GOOD.
I ALSO WANT TO THANK JENNETTE
FOR LEADING ALL THE OF THE TRIM
5 WORK AND ACTUALLY RETRACING
OUR STEPS ON AN IMPORTANT STUDY
AND FINDING EVEN MORE INSIGHTS,
AND ROSANNA AND RACHEL FOR THE
SUPER RESTRICTOR.
THANK YOU VERY MUCH.
[APPLAUSE]
>> HELLO.
THANK YOU VERY MUCH FOR A VERY
BEAUTIFUL TALK.
I HAVE LIKE TWO QUICK QUESTIONS.
THE FIRST ONE ABOUT TRIM 5.
DO YOU THINK THE FACT THAT
HUMAN -- VERSION OF TRIM 5 IS
LIKELY A RESULT THAT IT'S BAD
FOR HIV BUT GOOD FOR OTHER RETRO
RETROVIRUSES?
>> EXCELLENT QUESTION.
I HAVE A WHOLE DIFFERENT PART OF
THE TALK WHERE IT TALKS ABOUT
WHY DID WE FIX THOSE DOUBLE
ARGININES.
THE SHORT ANSWER IS, DOUBLE
ARGININES SEEM TO BE BAD FOR HIV
AND FOR SIV, ALL THE SIVs
WE'VE TESTED.
SO WE ARE STILL ON THE HUNT
FOR -- THIS WAS CLEARLY DRIVEN
BY ADAPTIVE MUTATION, WHICH
MEANS THAT THIS WAS BENEFICIAL
WHEN IT OCCURRED THE DOUBLE
ARGININES, BUT WE STILL DON'T
HAVE WHAT WAS THE DRIVING FORCE
FOR THAT LEFT USING, WHETHER IT
WAS A VIRUS OR WHETHER IT WAS TO
DIMINISH SOME SORT OF AUTOIMMUNE
RESPONSE, WE DON'T KNOW.
>> THANK YOU.
AND THEN SECOND QUESTION RELATED
TO THAT, SO IF THERE SEEMS TO BE
SOME SORT OF LIKE HOW TO SAY
MUTUAL EXCLUSIVITY LIKE ONE IS
GOOD FOR ONE VIRUS, ANOTHER FOR
ANOTHER VIRUS, AS GENES LIKE
FREQUENTLY DUPLICATED IN THE
EVOLUTION BECAUSE THAT WOULD BE
VERY BENEFICIAL FOR THEM TO BE
EVOLVING SEPARATELY.
>> THIS IS AN EXCELLENT
QUESTION.
EVEN IF THERE WAS NO
SPECIFICITY, IT WOULD BE
MASSIVELY ADVANTAGEOUS TO HAVE
ALLELE IK -- AT EACH OF THESE.
THAT IS TRUE FOR TRIM 5, AND
IT'S ACTUALLY TRUE FOR MOST
SELF-RESPECTING ANTIVIRAL GENES
AMONG MAMMALS.
IT IS NOT TRUE FOR MXA, AND WE
DO NOT KNOW WHY.
I WOULD COMPLETELY AGREE WITH
YOUR PREDICTION THAT MXA SHOULD
DUPLICATE AND BASICALLY EACH
VERSION SHOULD SPECIALIZE FOR A
DIFFERENT VIRUS BECAUSE THEN IT
DOESN'T HAVE A TRADEOFF OR A
COMPROMISE.
WE DON'T UNDERSTAND THE NATURE
OF THE TRADEOFF.
THERE'S ANOTHER STUDENT IN THE
LAB WHO'S VERY INTERESTED IN WHY
THAT TRADEOFF IS OCCURRING.
EVERY MAMMAL, DESPITE MULTIPLE
ROUNDS OF DUPLICATION, HAS
2MXs, ONE THAT WORKS IN THE
CYTOPLASM AND ONE THAT WORKS IN
THE NUCLEUS.
THEY WORK ON COMPLETELY
DIFFERENT VIRUSES BUT THERE ARE
NOT CASE WHERE THERE ARE
MULTIPLE MX PARALOGS WITHIN THE
SAME.
SO WE ARE ACTIVELY CONSIDERING
THE PROSPECT THAT BECAUSE MX
ACTS AS AN OLIGOMER E HAVING --
IN THE SAME COMPARTMENT RUNS THE
RISK OF POISONING THE OL GER
MIEZATION BETWEEN THEM AND WE
MIGHT LOSE THE ACTIVITY AND
THAT'S SOMETHING WE'RE ACTIVELY
STUDYING RIGHT NOW.
>> THANK YOU.
>> THANK YOU SO MUCH.
VERY INTERESTING TALK.
MY QUESTION IS ABOUT THE KIND OF
SUPER RESTRICTORS AND THE
TRADEOFF YOU MENTIONED BETWEEN
THE THOGOTO VIRUS AND INFLUENZA.
WHEN YOU LOOK AT SPECIES -- HOST
SPECIES WHERE ONLY ONE OF THOSE
VIRUS FAMILIES HAVE CIRCULATED,
DO YOU SEE KIND OF A PREFERENCE
FOR ONE OR THE OTHER KIND OF
MUTATIONAL STATUS OF THE SUPER
RESTRICTORS?
>> THAT'S AN EXCELLENT QUESTION.
SO WE DON'T HAVE THE ABILITY TO
MAKE THAT JUDGMENT QUITE SO
WELL.
BECAUSE AS I POINTED OUT, THE
THOGOTOVIRUS IS NOT SOMETHING
THAT IS PRESENT IN HUMAN
POPULATIONS, IT'S SOMETHING THAT
IS ENDEMIC IN EUROPEAN
POPULATIONS, BUT THAT DOESN'T
MEAN THAT IT WASN'T ABLE TO
ACTUALLY PROVIDE SOME
PROTECTION, SO IT'S REALLY LIKE
IF YOU WERE SUCCESSFUL, YOU
PROBABLY DON'T HAVE THE VIRUS
BECAUSE YOU'RE PROTECTED FROM
ZOO KNOW CYS --SO THAT'S HARD FO
RECONCILE.
I WILL ALSO POINT OUT THAT
INFLUENZA VIRUSES, AT LEAST IN
THEIR NORMAL COURSE OF
INFECTION, PROBABLY DIDN'T DRIVE
THE MAJORITY OF THE EVOLUTION
WE'VE SEEN BECAUSE AGAIN, THAT
WAS IN THE AFRICAN DIASPORA.
SO THERE ARE VIRUSES THAT HAVE
DRIVEN THIS BUT WE'RE USING
THOGOTO AND INFLUENZA VIRUSES
FOR WHAT THIS MIGHT HAVE BEEN.
>> THANK YOU.
>> IT SEEMED LIKE FOR TRIM 5,
THERE WAS NO EVOLUTIONARY
PRESSURE WITH REGARD TO HIV-1,
PROBABLY BECAUSE THE HISTORY WAS
TOO SHORT.
FOR MXA, OBVIOUSLY, THERE WAS A
LOT OF EVOLUTIONARY PRESSURE AND
YOU HINTED AT THE POSSIBILITY
THAT THERE COULD BE VERSIONS OF
MXA THAT WOULD TREAT INFLUENZA
AND THE OTHER VIRUSES PERHAPS
EQUALLY WELL AND BETTER THAN THE
WILD TYPE VERSION.
HAVE YOU LOOKED AT WHETHER
PERHAPS ANY OF THOSE MUTATIONS
THAT WOULD BE BETTER FROM AN
ANTIVIRAL PERSPECTIVE WOULD LEAD
TO RESPONSES IN THE ORGANISM
THAT WOULD BE DISADVANTAGE --
>> SORT OF TOXIC EFFECT --
>> YEAH, TOXIC EFFECTS OR
TRIGGER IMMUNE RESPONSES OR
WHATEVER, BECAUSE THAT SEEMS TO
BE ANOTHER POINT.
>> SO I WILL COMPLETELY AGREE
WITH THAT.
THAT'S ACTUALLY, BY THE WAY,
TRUE OF TRIM 5 AS WELL, WHERE
TRIM 5 SAN E3 -- WITH A RAPIDLY
EVOLVING LIGAND SPECIFICITY
DOMAIN AND YOU CAN IMAGINE THAT
SOME OF THE ADAPTIVE LANDSCAPE
IS GOING TO BE RESTRICTIVE
BECAUSE OF THE CHANCES OF AN
INTERACTION WITH THE -- PROTEIN.
ALL OF OUR EXPERIMENTS ARE DONE
ON CELL LINES SO WE HAVE NOT
MANIPULATED -- EACH IS LIKE A
SIGNIFICANT INVESTMENT, AND
ASSESSING WHICH OF THE VARIANTS
THAT DIDN'T WORK, WE WANT TO
TEST, IS MORE PROBLEMATIC.
I WILL SAY, THOUGH, THAT
EVERYTHING IN OUR ASSAY, WE DID
NOT ACTUALLY SEE ANY
OVERTOXICITY WITH ANY OF THE
VARIANTS.
SO BOTH IN THE V1 LOOP OF TRIM E
ABLE TO ASSESS, BECAUSE WE
TESTED EACH OF THESE
INDIVIDUALLY, NEARLY 2,000
VARIANTS, IF THERE WAS A
TOXICITY VARIANT EFFECT, WE
WOULD HAVE NOTICED THAT.
THEY ALL APPEAR TO BE EXPRESSED
WELL AND ALL ARE CAPABLE OF
OLIGOMERIZATION.
THAT'S STILL ONLY ABOUT ONE MILL
YONTMILLIONTH.
I ALSO WANT TO MAKE A SMALL
CORRECTION ACTUALLY WITH MXA, WE
TONIGHT KNOW EXACTLY WHAT DROVE
IT BUT TRIM 5 WE HAVE A MUCH
BETTER SENSE OF WHAT DROVE IT
BECAUSE THE HUMAN TRIM 5 IS
VERY, VERY GOOD AT RESTRICTING
ANTEROPICK TROA PEEN -- LEUKEMIA
VIRUS, FOR MANY OF THE
ENDOGENOUS RETROVIRUSES WE HAVE
INTEGRATED INTO OUR GENOME.
ALMOST CERTAINLY, I DON'T THINK
THERE'S ANY AMBIGUITY, WERE
DRIVEN WITH THOSE ENDOGENOUS
RETROVIRUSES.
>> THANK YOU.
>> IT SEEMS TO ME THAT THE BEST
STRATEGY FOR A VIRUS WOULD BE TO
NOT ONLY EVOLVE TO AVOID THE
RECOGNITION BY THE INNATE
IMMUNITY PROTEIN, BUT TO ALSO
EVOLVE SUCH THAT IT BECAME MORE
SIMILAR TO A HOST PROTEIN TO
ALMOST TAKE A HOST PROTEIN
HOSTAGE SO THAT IF THAT INNATE
IMMUNITY PROTEIN EVOLVES TO
CHASE IT AND HIT IT IN THE
FUTURE, IT'S GOING TO HAVE TO
SOMEHOW, LIKE, DO A SECOND STEP
TO AVOID HURTING ANOTHER HOST
PROTEIN.
SO IS THERE ANY EVIDENCE THAT
SOMETHING LIKE THAT EXISTS OR --
>> I'M SMILING BECAUSE TOM IS IN
THE AUDIENCE AND HIS LAB HAS,
ALONG WITH MY LAB, STUDIED MANY
EXAMPLES OF VIRAL MIMIC RI,LY,
EXACTLY WHAT YOU'RE SAYING,
THEY'LL USE THEM AS DECOYS TO
ESSENTIALLY PREVENT ANTIVIRAL
PROTEINS, OFTEN THEY'LL STEAL
HOST GENES WITH JUST SUBSTRATES
OF ANTIVIRAL PROTEINS AND MAKE
VERSIONS THAT ARE CAPABLE OF
BEING BOUND BY THE ANTIVIRAL
PROTEINS BUT NOT CAPABLE OF
BEING CATALYZED, WHICH BASICALLY
TAKES THEM OUT OF THE PICTURE.
THIS HAS HAPPENED -- THIS IS A
VERY SEC SUS FELL STRATEGY THAT
MANY VIRUSES THAT HAVE THE
ABILITY TO HAVE LARGE GENOMES
LIKE DOUBLE SIDED DNA VIRUSES
HAVE EMPLOYED VERY, VERY
SUCCESSFULLY IN THE PAST.
THERE'S NO EVIDENCE FOR THAT FOR
MXA OR TRIM 5, BUT IN THEORY,
YOUR POINT IS WELL TAKEN THAT
MIMICRY IS A VERY, VERY GOOD
EVOLUTIONARY STRATEGY IN
GENERAL.
>> JUST SO INTERESTING BECAUSE
YOU FOCUS ON THE FLEXIBLE LOOPS
BECAUSE THOSE ARE VERY
IMPORTANT.
IT WAS HERE IN MY LAB, WE
SHOWED -- FLEXIBLE LOOPS IN
PROTEIN STRUCTURES.
NOW WHAT I WANT TO ASK, YOU HAVE
A GLYCINE RESIDUE AND THE
GLYCINE CONFORMATION, YOU MUST
ANALYZE THE CONFIRMATION AT THAT
REGION AND THE DYNAMICS OF THE
CONFORMATION.
THAT'S VERY IMPORTANT.
GLYCINE CHAINS --
>> GLYCINE AND PROLINE ARE
INTERESTING AT BOTH ENDS OF THE
SPECTRUM.
>> THAT STUDY IS VERY IMPORTANT.
>> I SHOULD POINT OUT THAT WE
ACTUALLY NOT HAVE DONE MUCH IN
TERMS OF THE CONFORMATIONAL
STATE IN PART BECAUSE WE THINK
ABOUT -- WITH MXA WE'VE BEEN
THINKING MORE THAT IT'S ALMOST
LIKE AN UNSTRUCTURED LOOP THAT
BECOMES -- WHEN IT BOUND AND
THERE ARE NO CO-STRUCTURES OF
THE MXA WITH THE NP PROTEIN IN
PART BECAUSE IT WAS NOT STRONG
ENOUGH FOR A CRYSTAL STRUCTURE.
WE ARE HOPING THAT OUR -- WILL
HELP THAT BECAUSE WE HAVE 24
MORE AVID STRUCTURES AND WE ARE
ACTIVELY WORKING WITH
COLLEAGUES.
WE'VE BEEN TOLD THAT THE
CONFORMATIONAL STATUS IS
SOMETHING THAT IS REALLY THE
PURVIEW OF NMR.
THIS IS FAR TOO BIG FOR THAT.
BUT WE'RE HOPING THAT EVEN
CRYO-EM STUDIES MIGHT ACTUALLY
REVEAL A LITTLE BIT MORE ABOUT
THIS.
THANKS FOR THE QUESTION.
>> IT'S PART 2 OF THIS ONE
QUESTION FROM OUR COLLEAGUE IN
THE LIBRARY.
NATIONAL LIBRARY OF MEDICINE.
COULD THE STORY ABOUT THE TWO
ARGININES, RATHER THAN BEING AN
UNFORTUNATE COINCIDENCE, REFLECT
ADAPTATION TO HIV TO HUMANS?
>> IT'S NOT ABOUT ADAPTATION TO
HIV OR REALLY TO ANY SIVs
WE'VE TESTED NEARLY A DOZEN
SIVs THAT COULD HAVE POSSIBLY
JUMPED INTO HUMANS BUT MAYBE
WERE PROTECTED BECAUSE WE HAD
THE DOUBLE ARGININES BUT IT WAS
CERTAINLY ADAPTIVE.
THIS IS AN UNFORTUNATE
COINCIDENCE ONLY WITH RESPECT TO
HIV SUSCEPTIBILITY BUT IT WAS
NOT AN UNFORTUNATE COINCIDENCE
BECAUSE BOTH OF THESE WERE
DELIBERATELY OCCURRED AND
DELIBERATELY SELECTED FOR IN THE
CONTEXT THAT THEY OCCURRED.
WE JUST DON'T KNOW SINCE THIS
HAPPENED IN THE COMMON ANCESTOR
OF CHIMPS, GUERILLAS AND HUMANS,
WE JUST DON'T KNOW WHAT THAT
AGENT WAS, WHETHER IT WAS A
VIRAL AGENT OR AUTOIMMUNE AGENT.
ONE OF THE PROBLEMS IS MAKING A
CONTEMPORANEOUS ARGUMENT FOR
WHAT DROVE THIS REQUIRES US TO
HAVE A PRETTY GOOD SINCE OF WHAT
THE VIRUSES WERE.
WITH RETROVIRUSES WE HAVE A
PRETTY GOOD SENSE OF THAT
BECAUSE MANY OF THEM INTEGRATED
INTO OUR GENOME BUT THINGS LIKE
HIV -- THEY'RE NOT GERMLINE
INTEGRATION EVENTS.
SO WE HAVE HALF THE MIRROR IMAGE
OF THE STORY.
WE KNOW ITS ADAPTATION BUT WE
DON'T KNOW WHAT CAUSED IT.
>> OKAY.
>> ALL RIGHT.
THANK YOU VERY MUCH FOR COMING.
[APPLAUSE]