Intrinsic host defenses against HIV-1

Intrinsic host defenses against HIV-1

January 21, 2020 0 By Jose Scott


>>WELCOME TO THE 2014 GEORGE KHOURY MEMORIAL LECTURE PAUL BIENIASZ TO GIVE THIS
LECTURE I AM INTERRESTED IN ASPECTS OF
REPLICATION, PARTICULARLY THE ABILITY OR OTHERWISE OF RETROVIRUSES TO COLONIZE CELLS OF A PARTICULAR TYPE OR SPECIES. MORE RECENTLY, IN THE PAST DECADE OR SO WE’VE COME TO REALIZE THAT THAT’S ONLY HALF THE STORY. AND EVOLUTION HAS ENDOWED HOST DEVELOPS WITH A RANGE OF DEFENSE MECHANISMS WHOSE SPECIFIC JOB IT IS TO ATTENUATE VIRUS, PARTICULAR RETROVIRUS REPLICATION. SO I’M GOING TO TALK TO YOU ABOUT THESE INTRINSIC ANTIVIRAL DEFENSES, THAT START WITH THE DEFINITION, DIFFERENT FROM THE CONVENTIONAL ADAPTIVE AND INNATE IMMUNE SYSTEMS. THESE ARE GERMLINE ENCODED PROTEINS WHOSE MAJOR OR ONLY FUNCTION IS TO DIRECTLY INHIBIT VIRUS REPLICATION OR TO REDUCE CELLULAR PERMISSIVENESS TO VIRUS REPLICATION. SOME OF THESE PROTEINS ARE INDUCED, SOME ARE CONSTITUTIVELY EXPRESS THE, SOME EVOLVED TO TARGET PARTICULAR TYPES OF VIRUSES. THIS IS A FIELD SOMEWHAT IN ITS INFANCY. IT’S BEEN REALLY EXPANDED OVER THE LAST DECADE OR SO, SO THE KEY QUESTIONS ARE QUITE FUNDAMENTAL. WHAT ARE THE MOLECULES INVOLVED, HOW DO THEY WORK, AND HOW DO VIRUSES, IN OUR CASE PARTICULARLY HOW RETROVIRUSES EVADE THEM? THERE ARE ALSO INTERESTING EVOLUTIONARY QUESTIONS I’LL TOUCH ON BRIEFLY TODAY, FROM WHERE DID THEY ORIGINATE, HOW HAVE THEY EVOLVED, AND FROM A PRACTICAL POINT OF VIEW CAN WE MAKE KNOWLEDGE OF THE USE OF THEM IN WAYS THAT ARE USEFUL, I’LL TALK ABOUT THAT TOWARDS THE END OF THE TALK. WE’RE ABLE TO IDENTIFY SUCH ANTIVIRAL ACTIVITIES IN PER MISSIVE CELLS, THAT DOESN’T OCCUR IN NONPERMISSIVE CELLS BECAUSE REPLICATION CAN BE BLOCKED ATTIC SETS IN THE RETROVIRAL LIFE CYCLE. THE DIFFERENCE CAN BE GOVERNED BY WHAT SPECIES THEY COME FROM, WHEN YOU DELETE ACCESSORY GENES. SO THE KEY EXPERIMENT IS TO TOGGLE BETWEEN THESE TWO PHENOTYPES, BY TRANSFERRING GENES FROM NONPERMISSIVE CELLS TO PERMISSIVE CELLS, MAKING A NONPERMISSIVE OR VIRUS VERSA, MAKING THEM PERMISSIVE. HOW DO YOU CHOOSE WHAT CANDIDATE GENES TO TARGET? THERE ARE A VARIETY OF WAYS WE CAN MAKE CDNA LIBRARIES FROM A PARTICULAR RESISTANT SPECIES, AND SCREEN FOR RESISTANT CELLS. AN APPROACH THAT’S BEEN USEFUL TO US AND SEVERAL OTHERS IN THE FIELD IS TO DO SIMPLE MICROARRAY EXPERIMENTS WHERE YOU LOOK FOR GENES THAT SELECTIVELY EXPRESS IN THE NONPERMISSIVE STATE. MORE RECENTLY WE’VE BUILT ARRAYED LIBRARIES OF INTERFERON STIMULATED GENES, PARTICULARLY RICH SOURCE OF ANTIVIRAL PROTEINS AS I’M SURE YOU KNOW, TREATMENT OF CELLS OF TYPE 1 INTERFERON INDUCES WHAT’S KNOWN AS THE RATHER POORLY UNDERSTOOD SO-CALLED ANTIVIRAL STATE, GENES INDUCED BY INTERFERON OBVIOUSLY RESPONSIBLE FOR THIS SOME HAVE ANTIVIRAL ACTIVITIES. THE FIRST STORY INVOLVES AN HIV-1 ACCESSORY PROTEIN Vpu, A SMALL PROTEIN, A MEMBRANE PROTEIN WITH A TRANSMEMBRANE DOMAIN, SHORT CYTO PLASTIC TALE. FOR A WHILE, A BACK WATER IN HIV RESEARCH BECAUSE IN MOST DEVELOPS Vpu IS NOT REQUIRED, YOU CAN DELETE IT FROM THE VIRAL GENOME, THE VIRUS IS QUITE HAPPY. AS FIRST SHOWN HERE BY CLAUS STRABEL AND OTHERS THERE ARE IS A SIGNIFICANT DEFECT, IT’S A PARTICULAR DEFECT AT THE TIME AT WHICH ONE WOULD EXPECT VIRUS PARTICLES TO BE RELEASED FROM CELLS. SO WILD TYPE HIV-1 STRAIN REPLICATION, PERMISSIVE AND NONPERMISSIVE, Vpu DELETED STRAIN REPLICATION THROUGH SO CALLED PERMISSIVE CELLS BUT FAILS TO RELEASE PARTICLES FROM INFECTED NONPERMISSIVE CELLS. AND OUR ENTRY INTO THIS FIELD INVOLVES THE FINDINGS THAT THE VIRIONS PRODUCED ARE FULLY FORMED, APPARENTLY FULLY FUNCTIONED, BUT RETAINED ON THE SURFACE OF CELLS BY PROTEASE SENSITIVE TETHERS. ANOTHER KEY OBSERVATION THAT REALLY HELPED US FIND WHAT THE MOLECULE INVOLVED WAS WAS THE NONPERMISSIVE STATE COULD BE INDUCED BY TREATING THE CELLS WITH TYPE 1 INTERFERON AND SO IN THE ABSENCE OF INTERFERON MOST CELLS WOULD RELEASE VIRUS IN THE ABSENCE OF Vpu AND Vpu BECOMES REQUIRED. BY TAKING SOME CELLS IN WHICH THE NONPERMISSIVE STATE WAS CONSTITUITIVEE COMPARING THE GENES EXPRESSED WITH THOSE EXPRESSED IN PERMISSIVE CELLS, WE COULD TRIANGULATE AND ISOLATE A CDNA THAT APPEARED TO BE A GOOD CANDIDATE FOR BEING RESPONSIBLE FOR THIS PHENOTYPE. THAT’S A GENE ENCODES A PROTEIN WE CALLED TETHERIN. IT’S A PROTEIN OF ABOUT 119 IMMUNOACIDS, IT’S A VERY UNUSUAL PROTEIN IN THAT IT HAS A TRANSMEMBRANE DOMAIN AND A GPI ANCHOR THAT ARE LINKED BY A ROD SHAPE THAT’S PREDOMINANTLY COILED-COIL. THIS WAS A PRIME CANDIDATE FOR THE INDUCTION OF THIS PHENOTYPE, LOOKING AT A CARTOON PICTURE OF THIS PROTEIN INSPIRES HYPOTHESES AS TO HOW IT MIGHT WORK, ASLY SHOW YOU IN A MOMENT. TO SHOW YOU THE PHENOTYPE ASSOCIATED WITH IT, IT IS A RATHER IMPORTANT SLIDE IN MY CAREER. THIS SHOWS TETHERIN IMPOSE AS REQUIREMENT FOR HIV-1 VPI, THIS IS A SINGLE CYCLE REPLICATION EXPERIMENT DONE IN A NORMALLY PERMISSIVE CELL, WILD-TYPE DELETES THE SAME IN A SINGLE REPLICATION, TO ENGINEER THAT CELL TO EXPRESS THIS ONE EXTRA GENE, THEN IT IMPOSES A STRONG REQUIREMENT FOR Vpu. AS ERIC SHOWED YOU, IF YOU EXPRESS THIS PROTEIN ON OTHERWISE PERMISSIVE CELLS YOU GET THIS VERY IMPRESSIVE ACCUMULATION OF VIRION PARTICLES THAT DOESN’T HAPPEN WHEN THE VIRUS IS WILD-TYPE OR THE CELLS DON’T HAVE THE TETHERIN PROTEIN. HOW DOES THIS PROTEIN WORK? ONE MAKES LOTS OF MUTANTS OF THE PROTEIN AND ASKS WHAT PARTS ARE REQUIRED TO FUNCTION. ONE THING WE QUICKLY AND FRUSTRATINGLY FOUND, IT WAS DIFFICULT TO MAKE MUTANTS OF THIS PROTEIN THAT LACK FUNCTION. ALTHOUGH THAT WAS INITIALLY DISPOINTED, IT INSPIRED AN EXPERIMENT ULTIMATELY QUITE SATISFYING, AND SO WHAT WE DID WAS IN EFFECT TAKE THE TETHERIN PROTEIN WE REBUILT PROTEIN FROM COMPONENTS THAT HAVE NO SEQUENCE TO TETHERIN BUT ARE EXPECTED TO HAVE A SIMILAR OVERALL CONFIGURATION SO WE TOOK A BIT OF THIS PROTEIN, A BIT OF THIS PROTEIN AND A BIT OF THAT PROTEIN AND PUT THEM TOGETHER TO MAKE A PROTEIN THAT SHOULD FIT IN THE MEMBRANE SOMETHING LIKE THIS. AND REMARKABLY ENOUGH IT WORKED. HERE IS THE SURFACE OF THE CELLS. YOU CAN SEE THAT THIS PROTEIN THAT HAS EFFECTIVELY NO SEQUENCE CAN RECAPITULATE THE BIOLOGICAL ACTIVITY OF THE NATIVE TETHERIN PROTEIN SUGGESTING IT WAS UNLIKELY THAT TETHERIN WAS DOING SOMETHING SOPHISTICATED OR COMPLICATED IN ORDER TO GENERATE THIS VIRION TETHERING PHENOTYPE. IF IT HAD TO SEND A SIGNAL TO CAUSE VIRION TETHERING, IT WOULD BE UNLIKELY TO RECAPITULATE THAT WITH THIS EXPERIMENT. IT WAS MORE LIKELY TETHERIN ACTS DIRECTLY TO TRAP VIRUSES ON THE SURFACE. WE DID AN EXPERIMENT THAT DEMONSTRATES THAT. WE TOOK ADVANTAGE OF THIS VERY DRAMATIC PERMISSIVEITY TO PUT PROTEASE CLEAVAGE SITES, A FACTOR 10 SITE HERE AND ASKED WHETHER TREATMENT OF THE SURFACE OF CELLS OF THAT PROTEASE WOULD RELEASE VIRIONS, INDEED THAT PROVED TO BE THE CASE. HERE WE HAVE CELLS THAT HAVE BEEN INFECTED WITH A Vpu MINUS VIRUS, THE VIRAL PROTEINS ACCUMULATE HERE, AND THEN AT THOSE VARIOUS TIME POINTS WE EITHER INCUBATE THEM WITH BUFFER OR BRIEFLY WITH FACTOR 10A PROTEASE, AND THE CLEAVAGE OF THE TETHERIN PROTEIN RELEASES THE VIRAL PARTICLE, AND THIS ONLY WORKS IF YOU PROGRAM THE TETHERIN PROTEIN TO HAVE THE CLEAVAGE SITE. ALSO QUITE INTERESTINGLY IS THE FRAGMENTS OF THE TETHERIN PROTEIN ARE RELEASED WITH THE VIRUS. BY PLAYING GAMES WITH WHERE YOU POSITION THE TAGS, YOU CAN DEMONSTRATE THAT MOST OF THE TIMES ABOUT 3/4 OF THE TIME VIRIONS ARE HELD BY TETHERIN PROTEIN IN THIS CONFIGURATION. THE GPI ANCHOR INFILTRATING THE VIRUS PARTICLE, AND THE TRANSMEMBRANE DOMAIN RETAINED IN THE CELL. AND SO THIS IS A SIMPLE AND I WOULD ARGUE ELEGANT MECHANISM BY WHICH CELLS EXPRESS A PROGRAM WITH BROAD SPECIFICITY, TRAP SIRONS ON — VIRIONS ON THE
CELL SURFACE, ONLY WITHOUT A Vpu, THE ANSWER TO TETHERIN. Vpu BINDS TO THE TETHERIN PROTEIN, MANY GROUPS HAVE SHOWN USING CO-IP ASSAYS, I’M SHOWING A CROSS-LINKING ASSAY WHETHER WE’VE PLACED THEM AND SO YOU EXPRESS THE SINGLE IMMUNOACID SUBSTITUTED PROTEINS IN VARIOUS CELLS, AND THEN IF YOU THROUGH IMMUNOPRECIPITATION ASSAY PULL ON THE H-A TAG AFTER DENATURING THE SAMPLE AND RUN A WESTERN BLOG FOR THE FLAG TAG YOU CAN FIND A COMPLEX THAT IS CONSISTENT CAN A DIMER OF TETHERIN CROSS LINKED TO MOLECULES OF Vpu, YOU GET THE CYSTEINE IN THE RIGHT PLACE AND FORM THESE COMPLEXES INDICATING THESE DOMAINS ARE IN ROUGHLY MOLECULAR PROXIMITITY TO EACH OTHER IN THE MEMBRANE. WE CAN GET GENETIC EVIDENCE IN SUPPORT OF THAT CLAIM, BASED ON EXPERIMENTS SUCH AS THIS, SO THE HIV-1 Vpu PROTEIN IS AN EFFICIENT ANTAGONIST OF THE TETHERIN PROTEIN FOUND IN HUMANS, SO THAT ASSAY IS SHOWN HERE, SO HERE WE JUST EXPRESS INCREASING AMOUNTS OF THE TETHERIN PROTEIN AND ASKING IT TO INHIBIT THE RELEASE OF INFECTIOUS HIV-1 PARTICLES AS MEASURED BY AN INDICATOR CELL, AND YOU CAN SEE THAT TETHERIN DOES THAT VERY NICELY TO A Vpu MINUS VIRUS WHEREAS Vpu COMPETENT VIRUS ANTAGONIZES TETHERIN AND IS RESISTANT. NOW, IMPORTANTLY, REMEMBER THIS FOR WHAT FOLLOWS, IF YOU TAKE A MONKEY TETHERIN PROTEIN, OR EVEN IN THIS CASE SIMPLY REPLACE THE TRANSMEMBRANE DOMAIN OF HUMAN TETHERIN WITH THAT FROM A MONKEY TETHERIN PROTEIN, THEN THIS INTERACTION FAILS, ALTHOUGH THE CHIMERIC TETHERRER INIS ACTIVE IT’S RESISTANT TO THE Vpu PROTEIN CEMENTING THE IDEA OF INTERACTION BETWEEN THE PROTEINS IS BIOCHEMICALLY AND FUNCTIONALLY MEDIATED BY THE TRANSMEMBRANE DOMAIN. OKAY. SO THIS IS AN INTERESTING MECHANISM, NOT PREVIOUSLY DESCRIBED IN THE CONTEXT OF VIROLOGY, AND NOT AS FAR AS I’M AWARE ASSOCIATED WITH ANY PARTICULAR CELLULAR ACTIVITY. UNLIKE OTHER ANTIVIRAL PROTEINS THAT HAVE BEEN DISCOVERED, TETHERIN DOESN’T HAVE OBVIOUS ACTIVITIES FROM WHICH IT’S DERIVED. IT’S NOT RELATED TO ANY OTHER PROTEINS FOUND IN THE HUMAN GENOME. AND SO WE BECAME INTERESTED IN HOW DID THIS ACTIVITY EVOLVE? WHERE DID IT COME FROM? IT’S A RATHER STRANGE THING FOR CELLS TO DO OTHER THAN IN THE CONTEXT OF THE INHIBITION OF VIRAL PARTICLES, SO HOW MECHANICKISTICALLY DID THIS ACTIVE ARRIVE? WE LOOKED TO SEE HOW OLD THE TETHERIN GENE WAS, WHAT WAS DESCENDED FROM? WHEN WE LOOK AT GENOMES, YOU CAN FIND PROTEINS THAT HAVE THE CONFIGURATION OF TETHERIN, AND I SHOULD POINT OUT THAT OTHER THAN TETHERIN THERE’S NO OTHER PROTEIN WHICH WE’RE AWARE THAT HAS THIS CONFIGURATION, TRANSMEMBRANE DOMAIN AND EXTRACELLULAR COILED-COIL AND GPI ANCHOR. YOU CAN FIND PROTEINS THAT HAVE THAT CONFIGURATION, IN ROUGHLY SENTINOUS POSITION OF ANYTHING FROM ANCESTRAL — REPTILES HAVE IT, MARSUPIALS HAVE IT, ANYTHING THAT’S MORE DISTANT, FOR EXAMPLE, AMPHIBIANS DON’T APPEAR TO HAVE A TETHERIN PROTEIN. THE EQUIVALENT POSITION, AND WE LOOK AT MODERN GENOMES, THAT POSITION IS UNOCCUPIED IN AMPHIBIANS. SO IT LOOK AS IF TETHERIN CAME INTO EXISTENCE AT SOME POINT GREATER THAN 300 MILLION YEARS AGO BUT LESS THAN 370 MILLION YEARS AGO, BETWEEN THE TIME THAT AMPHIBIANS DIVERGED. SO IF YOU LOOK AT THE SEQUENCES OF THESE TETHERIN PROTEINS THAT DIVERGED 300 MILLION YEARS AGO YOU’LL SEE THAT THEY ARE DISTANTLY RELATED. EITHER THERE HASN’T BEEN A STRONG PRESSURE TO MAINTAIN SEQUENCE OR THERE’S BEEN PRESSURE TO DIVERSE SEQUENCE OR BOTH. IF YOU LINE UP TASMANIAN DEVIL, HUMAN AND ALLIGATOR TETHERIN YOU’LL FIND VERY FEW POSITIONS IN ALIGNMENT. IN FACT, SOME OF YOU MIGHT THINK THIS ALIGNMENT IS CONTRIVED, AND IT IS. IT’S VERY DIFFICULT TO ALIGN THESE PROTEINS TOGETHER. THEY ARE VERY DIVERSE. OBVIOUSLY THAT MAKES TRACING ITS ORIGINS QUITE CHALLENGING. DESPITE THIS VERY LARGE DEGREE OF SEQUENCE DIVERGENCE, ALL OF THESE PROTEINS, HUMAN, TASMANIAN DEVIL AND ALLIGATOR, ARE POTENT INHIBITORS OF HIV-1 PARTICLE RELEASE, THE HIV-1 PROTEIN HAS LEARNED TO ANTAGONIZE HUMAN TETHERIN, DOESN’T DO ANYTHING AGAINST THESE MORE DISTANT TETHERINS. AND SO HOW DOES ONE GO ABOUT FINDING THE ORIGINS OF A PROTEIN THAT HAS THIS LEVEL AM OF SEQUENCE DIVERSITY? AND IN FACT, BY JUST INEXPECTING THE GENES IN CHROMOSOMES AROUND WHERE TETHERIN IS LOCALIZED, WE FOUND THIS RATHER INTERESTING PROTEIN, PV-1, THAT IS POSITIONED NEXT TO THE AT THE TIMERRING GENES. A COMMON MECHANISM FOR HOW GENES ARISE IS DUPLICATION OF NEIGHBORING GENES. THERE’S NO SEQUENCE HOMOLOGY AS FAR AS WE CAN TELL BETWEEN MODERN VERSIONS THE TETHERIN AND PV-1 BUT THERE’S STRUCTURAL HOMOLOGY AND JUST LIKE TETHERIN, IT’S QUITE A BIT LONGER BUT THE ONLY MAJOR DIFFERENCE IS THAT TETHERIN HAS A GPI ANCHOR, AND PV 1 — THE FUNCTION OF PV 1 IS TO MAKE THINGS LIKE THIS, A DIAPHRAGM ON CAVIOLI, FOR EXAMPLE. AND SO WE THOUGHT, WELL, PERHAPS IT SEEMED AN OUTLANDISH HYPOTHESIS BUT NOT TO RIDICULOUS TO SUPPOSE PERHAPS TETHERIN MIGHT HAVE DESCEND THE FROM THE DUPLICATE COPY OF PV-1. IF ONE RECONSTRUCTS WHAT MIGHT PUTATIVI, IF YOU TAKE THE GPI ANCHOR FROM TETHERIN AND PUT IT ON THE C-TERMINUS PV-1 YOU WITH CHANGE IT FROM A PROTEIN INACTIVE IN TERMS OF INHIBITING VIRUS PARTICLE RELEASE TO ONE THAT IS ALMOST AS ACTIVE AS WILD-TYPE TETHERIN, AT LEAST AS ACTIVE AS ARTIFICIAL TETHERIN I TOLD YOU ABOUT A FEW MOMENTS AGO. PERHAPS EVEN MORE COMPELLINGLY, WHEN WE LOOK VERY CLOSELY, AND IN FACT DEDUCE THE ANCESTRAL SEQUENCE OF PV-1 USING MAXIMUM LIKELIHOOD METHODS TO DETERMINE WHAT THE APPROXIMATE SEQUENCE WAS OF PV-1, 300 MILLION YEARS AGO, BEFORE IT PUTATIVEY DUPLICATED TO MAKE TETHERIN, YOU SEE STATISTICALLY LOW LEVELS, BUT STATISTICALLY SIGNIFICANT HOMOLOGY. THIS IS A FRAGMENT OF THE MODERN VERSION OF TETHERIN FOUND IN PRIMATES ALIGNED TO THE ANCESTRAL PV 1, NOT THE FRAGMENT OF HOMOLOGY AND MAY HAVE BEEN DESCENDED FROM THE PV-1 PROTEIN. I DIDN’T MENTION THIS BUT IT HAS VERY BROAD SPECIFICITY, THE FACT THAT IT WORKS SIMPLY BY INFILTRATING LIPID ENVELOPES MAKES ESSENTIALLY ANY ENVELOPE VIRUS FAIR GAME, AND MANY ENVELOPE VIRUSES ARE SUSCEPTIBLE TO THEIR RELEASE BEING INHIBITED BY TETHERIN, BECAUSE IN EFFECT IT TARGETS AN INDISPENSABLE INVARIANT HOST COMPONENT OF THE VIRUS AND THERE’S NO SPECIFIC INTERACTION WITH VIRAL PROTEINS THAT’S REQUIRED. OF COURSE, THIS HAS SOME IMPORTANT BIOLOGICAL CONSEQUENCES, IT VERY MUCH REDUCES THE OPPORTUNITY FOR VIRUSES TO EVOLVE RESISTANCE. YOU CAN’T BECOME RESISTANT BY A SIMPLE CHANGE IN AMINO ACID SEQUENCE, RATHER IT PROVIDES THE IMPETUS FOR THE ACQUISITION OF NEW ANTAGONIST ACTIVITY AS EXEMPLIFIED BY Vpu, BUT AS IT TURNS OUT THERE ARE MANY OTHER WAYS IN WHICH TETHERIN IS ANTAGONIZED BY OTHER VIRUSES, IN THE SIVs, THEY CAN TAKE ON THIS ROLE, SOMETIMES THE ENVELOPE PROTEIN OF SIVs AND OTHER VIRUSES CAN TAKE ON THE ROLE OF TETHERIN ANTAGONIST, BUT THIS SPECIALIZATION TO TETHERIN PROTEINS THAT ARE VERY VARIABLE IN SEQUENCE BETWEEN SPECIES IS A THEME THAT CAN REOCCUR AND BE A BARRIER TO CROSS-SPECIES TRANSMISSION. SO LET ME MOVE ON NOW TO TALK ABOUT A SECOND ANTIVIRAL MECHANISM THAT WE’VE DISCOVERED, WHICH WE’VE FOUND USING SIMILAR APPROACHES THAT ACTUALLY WORKS IN A COMPLETELY DIFFERENT WAY, AND ACTUALLY WORKS TO PROTECT TARGET CELLS FROM INFECTION. AND THIS ARISES FROM A SET OF EXPERIMENTS WHERE WE — THAT WE DID IN THE LAB WHERE WE COMPARED THE ABILITY OF VARIOUS CELL LINES TO RESIST HIV-1 INFECTION, INCOMING HIV-1 INFECTION WHEN TWEETED WITH TYPE 1 INTERFERON. HERE IS THAT EXPERIMENT DONE WITH THREE MONOCYTE CELL LINES. YOU CAN SEE IN THIS CELL LINE INTERFERON HAS A RATHER DRAMATIC EFFECT ON HIV INFECTION AND VERY LITTLE EFFECT ON INFECTION IN THESE TWO CELL LINES. IF YOU SIMPLY COMPARE THE GENES THAT ARE INDUCED BY INTERFERON HERE, WITH THOSE THAT ARE INDUCED BY INTERFERON HERE, AND SELECT THOSE THAT ARE ONLY UPREGULATED HERE, YOU GET A LIST OF CANDIDATE GENES AND THEN IF YOU EXPRESS THOSE INDIVIDUAL GENES IN PERMISSIVE CELLS AND INFECT THEM WITH AN HIV GFP REPORT YOU FIND AMONG THE CANDIDATES, ONE OF THEM, Mx2 PROTEIN CAN INHIBIT HIV-1 INFECTION, IN THE HUMAN AND MONKEY Mx2 PROTEINS. A MODEST BUT CLEARLY SIGNIFICANT INHIBITION OF HIV INFECTION. SO HERE YOU CAN SEE HOW THE Mx2 PROTEIN IS INDUCED BY INTERFERON IN THP-1 CELLS, WHEN THAT HAPPENS INTERFERON INHIBITS IN HIV-1 INFECTIONS, BUT IF THE CELLS ARE EXPRESSED IN SHORT HAIRPIN mRNA, IT’S BLUNTED. SO THIS PROTEIN IS SUFFICIENT TO INHIBIT HIV-1 INFECTION, AND AT LEAST IN THIS PARTICULAR CELL CONTEXT, NOT IN ALL CELL CONTEXTS BUT IN THIS PARTICULAR CELL CONTEXT IS REQUIRED FOR THE FULL ANTI-VIRAL ACTIVITY OF INTERFERON ALPHA. SO WHAT IS Mx2? IT’S A MEMBER OF A FAMILY OF PROTEINS, THE MX PROTEINS HAVE A PRETTY RICH HISTORY AS ANTIVIRAL PROTEINS, GTP-ASES, A CRYSTAL STRUCTURE OF THE MX-1 PROTEIN WHICH HAVE LONG BEEN KNOWN TO HAVE ANTIVIRAL ACTIVITY AGAINST A RANGE OF RNA VIRUSES, THE Mx2 PROTEIN, THIS PART OF THE TREE HERE, THIS IS A TREE OF MX PROTEIN SEQUENCES. WHAT I CALL MX 1 AND 2 IS NOT NECESSARILY THE SAME AS WHAT YOU’LL READ IN THE LITERATURE SO THAT YOU’LL FIND SOME LITERATURE MX 1 AND 2S IN HERE AND EVEN MX 3s. BUT PHYLO GENETICALLY Mx2s ARE HERE. THEY WERE THOUGHT TO HAVE OTHER FUNCTIONS. SO THESE PROTEINS ARE FOUND IN MAMMALS, OTHER VERTEBRATES, ALL THE WAY DOWN TO LAMP RAYS. IT APPEARS WITH ANTIHIV-1 ACTIVITY, A RATHER MORE RECENTLY EVOLVED ACTIVITY IN MX PROTEINS COMPARED TO TETHERIN PROTEINS. IF YOU TAKE PROTEINS IN THIS PLACE FROM PRIMATES THEY ALMOST UNIVERSALLY HAVE NPH 1 ACTIVITY. SHEEP AND DOGS, NO ACTIVITY. IN ANY EXPERIMENT WE’VE PUT Mx2 UNDER A DOCKS OH SIGH CLEAN, YOU GET THIS. WHAT’S DIFFERENT ABOUT THE PRIMATE Mx2 PROTEINS AS COMPARED TO THE NONPRIMATE THEY LOCALIZE TO NUCLEAR PORES. I DON’T KNOW IF YOU CAN SEE THE RED STAINING. YOU CAN SEE IT QUITE CLEARLY HERE, A STAIN IN RED, DNA IN BLUE, AND THIS IS Mx2, THE CLOSER YOU LOOK THE MORE CONVINCING THE CO-LOCAL IDESSIZATION BECOMES. THIS IS A PROMINENT FEATURE OF ALL OF THE PRIMATE Mx2s THAT WE’VE ANALYZED, MUCH LESS EVIDENT IN THE NONPRIMATE Mx2. ANOTHER THING AS COMPARED TO MX-1 AND OTHER PROTEINS, IT HAS THIS AMINO TERMINAL EXTENSION THAT APPEARS TO BE THE INTERESTING PART OF THE PROTEIN, IT HAS THAT NUCLEAR PORE LOCALIZATION SIGNAL CONTAINED WITHIN THE AMINO TERMINAL 25 AMINO ACIDS, AND THEY ARE ABSOLUTELY REQUIRED TO ANTIVIRAL ACTIVITY. I’LL SPARE YOU DETAILS BUT WE’VE DONE IN COLLABORATION WITH MY FORMER POSTDOC SAM WILSON, A NUMBER OF EXPERIMENTS WITH VARIOUS HIV MUTANTS THAT INDICATE THE NEXT PORTION OF THE PROTEIN IS 25 THROUGH 90 CONTAINS SPECIFICITY DETERMINANTS, WHICH GOVERN WHICH PARTICULAR VIRUSES ARE INHIBITED BY Mx2. IF YOU LOOK AT THE SEQUENCE OF VARIOUS Mx2 PROTEINS YOU CAN SEE THIS AMINO TERMINUS LOOKING AT NDS OR PROBABILITY OF DNDA BEING GREATER THAN 1, THIS IS THE — OTHER PARTS APPEAR RAPIDLY EVOLVING TOO. SO THIS AMINO TERMINUS APPEARS TO BE WHAT GOVERNS THE ANTIRETROVIRAL ACTIVITY OF THE Mx2 PROTEINS AND APPEARS TO HAVE BEEN QUITE, QUITE RECENTLY ACQUIRED. SO IT’S THE CAPSID OF THE HIV-1 PROTEINS THAT DETERMINES WHETHER IT’S SENSITIVE TO Mx2 OR NOT. HERE IS A WILD TYPE STRAIN, IN THE ABSENCE OR PRESENCE OF Mx2 YOU’LL SEE IT’S NOT A VERY POTENT INHIBITOR BUT IT CLEARLY DOES INHIBIT INFECTION, BUT YOU CAN FIND MUTANTS. THIS IS INTERESTING, IT’S A MUTANT THAT CONVERTS HIV-1 FROM AN INDEPENDENT VIRUS TO ONE THAT CAN ONLY INFECT DIVIDING CELLS. THAT MUTATION MAKES HIV-1 COMPLETELY RESISTANT TO Mx2. THIS MUTATION, WHICH AMONG OTHER THINGS, IN BLOCKS FINDING TO A HOST PROTEIN AND CHANGES THE NUCLEAR PORE REQUIREMENTS FOR HIV-1 INFECTION. THERE ARE A NUMBER OF MUTATIONS ONE CAN IDENTIFY THROUGHOUT THE VIRAL CAPSID THAT CONFER PARTIAL OR COMPLETE RESISTANCE TO Mx2. SO YOU MIGHT ASK WHY DON’T THE VIRUS MAKE THESE CHANGES? EVERY ONE OF THESE CHANGES COMES AT A SIGNIFICANT FITNESS COST TO THE VIRUS. EVEN IF YOU GROW VIRUS IN CELLS EXPRESSING Mx2, YOU CAN GET RESISTANT VIRUSES, BUT THOSE VIRUSES INVARIABLY HAVE A FITNESS PENALTY ASSOCIATED WITH THEM. MUTATING CAPSID IS SOMETHING THE VIRUS CAN DO BUT IT’S ONE OF THE MOST GENETICALLY — THE MOST GENETICALLY FRAGILE PROTEIN THAT’S EVER BEEN ANALYZED, MOST MUTATIONS IN CAPSID ARE IN FACT LETHAL. SO THIS FINDING THAT Mx2 LOCALIZES QUITE SPECIFICALLY AT NUCLEAR PORES, AND ITS SENSITIVITY TO VIRUS IS MANIPULATED WHEN YOU CHANGE THE ROUTE BY WHICH HIV GAINS ACCESS TO THE NUCLEUS MADE US CONSIDER IT MAY PATROL NUCLEAR PORES AND INTERCEPT HIV-1 PARTICLES AS THEY ATTEMPT TO INFECT CELLS. AND CONSISTENT WITH THAT IDEA, IF YOU EXPRESS Mx2 IN THIS PARTICULAR CELL LINE, AND AS YOU CAN SEE HERE, IN THIS CELL LINE Mx2 IS CAUSING A FIVE-FOLD DECREASE IN HIV INFECTIVITY ON THIS CELL WHEN IT’S DIVIDING, IF YOU GROW THE CELL, IT DOESN’T AFFECT HIV-1 INFECTION, HIV-1 INFECTION DOESN’T REQUIRE CELL DIVISION BUT INCREASES THE POEANCY OF Mx2, CONSISTENT WITH THE NOTION THAT WHAT Mx2 LIKELY DOES IS TO, AS I MENTIONED, FIT ON THE NUCLEAR PORE AND BASICALLY INTERCEPT VIRUSES AS THEY TRY TO ENTER THE NUCLEUS. I DIDN’T SHOW THIS BUT IT’S TRUE THAT Mx2 INHIBITS INFECTION BY AN ARRAY OF PRIMATE LENTIS HAVE VIRUSES, IT DOESN’T AFFECT REVERSE TRANSCRIPTION IN ANY WAY AT ALL. THE VIRUS SYNTHESIZES NORMAL LEVELS OF DNA,IT JUST APPEARS NOT TO GET IN THE NUCLEUS. AS HIV-1 CAPSID IS THE MAJOR DETERMINANT THAT GOVERNS NUCLEAR ENTRY, IT STANDS TO REASON, IT’S TRUE, THAT THE HIV-1 CAPSID DETERMINES WHETHER A VIRUS IS SENSITIVE OR RESISTANT TO THIS PARTICULAR HOST INHIBITOR. OKAY. SO I’VE GONE OVER IN SOME DETAIL A COUPLE OF THE ANTIVIRAL MECHANISMS THAT WE’VE BEEN INVOLVED IN STUDYING. I SHOULD MENTION AT THIS POINT THAT BOTH OF THOSE MECHANISMS WERE ALSO DISCOVERED IN OTHER LABS, SO JOHN GOTELLI WAS CO-DISCOVERER OF TETHERIN, AND MICHAEL AND CHANG’S GROUP IN TORONTO CO-DISCOVERED THIS Mx2 AS AN INHIBITOR OF HIV-1 INFECTION. WHAT I’M GOING TO DO NOW IS TALK VERY BRIEFLY ABOUT TWO OTHER ANTIVIRAL MECHANISMS THAT TARGET HIV-1, BECAUSE THEY ARE IMPORTANT FOR THOSE, THE LAST PART OF WHAT I’LL TALK TO YOU ABOUT IN A COUPLE OF MINUTES. AND SO TWO OTHER ANTIVIRAL MECHANISMS ARE INDUCED BY THE SO-CALLED APOBEC3 AND TRIM5 ANTIVIRAL PROTEINS, THE APOBEC3 PROTEIN DISCOVERED TO BE ANTIVIRAL IN MIKE’S LAB INFILL INFILTRATE THE PARTICLES AND DURING REVERSE TRANSCRIPTION THESE ARE SINGLE STRAND SPECIFIC CYTO DEAMINASE, AND A VERY LARGE FRACTION OF THE CYTODEAMINASE, CAUSING CODING POTENTIAL. IN A CONCEPTUALLY SIMILAR WAY THAT HIV DEVELOPS AN ANTAGONIST OF TETHERIN IN THE Vpu PROTEINS, SO IT HAS ALSO DEVELOPED AN ANTAGONIST THAT DENUDES INFECTED CELLS OF THE APOBEC3 PROTEIN, AND SO LENTIVIRAL PROTEIN BINDS DIRECTLY TO APOBEC3 PROTEIN AND UBIQUITIN LIGATE COMPLEX. IT’S REMOVED FROM INFECTED CELLS. AND AGAIN IN CONCEPTUALLY MUCH THE SAME WAY THAT Vpu EVOLVED TO BE RATHER SPECIFIC TO THE PARTICULAR VARIANTS OF TETHERIN IT FINDS IN ITS NATURAL TARGET CELLS, SO THE SAME IS TRUE OF THIS PROTEIN. HIV-1 IS A KILLER OF APOBEC3 BUT DON’T TOUCH MONKEY APOBEC3 PROTEIN. AND THE CONVERSE IS TRUE OF SIV PROTEINS, IN PROTEINS OF MONKEY APOBEC3 PROTEINS BUT DO NOT TOUCH THE HUMAN PROTEIN. ANOTHER ANTIVIRAL MECHANISM DISCOVERED IN JOE’S LAB WORKS IN A CONCEPTUALLY SIMILAR WAY TO Mx2, I HAVE PROTEIN BINDS DIRECTLY, AND IT’S BEEN DEMONSTRATED TO BIND DIRECTLY TO INCOMING HIV CAPSIDS, AND BLOCKS PROGRESSION OF THE VIRAL LIFE CYCLE, THE DIFFERENCE FROM Mx2 IS THIS HAPPENS BEFORE REVERSE TRANSCRIPTION RATHER THAN AFTER. AND THERE’S AGAIN A SPECIES-SPECIFIC COMPONENT TO THIS ACTIVITY, SO THE HUMAN TRIM5 PROTEIN DOESN’T TOUCH HIV-1 CAPSIDS, HIV-1 HAS EITHER EVOLVED OR WAS ALREADY RESISTANT TO THE HUMAN TRIM5 PROTEINS, BUT THE TRIM5 PROTEINS IN MONKEYS ARE EFFECTIVE INHIBITORS OF HIV-1 INFECTION. TOGETHER THESE TWO PROTEINS IN PARTICULAR CAN CREATE A PROFOUND BARRIER TO HIV-1 REPLICATION, IN MONKEY CELLS. AT THE TIME WE STARTED THE PROJECT THAT I’M ABOUT TO TELL YOU ABOUT, THESE WERE THE ONLY TWO DEFENSE MECHANISMS THAT WE KNEW ABOUT THAT DIFFERED SUFFICIENTLY BETWEEN HUMAN AND MONKEY CELLS TO CREATE THIS SPECIES-DEPENDENT BARRIER TO HIV-1 REPLICATION. AND THIS IS NOT JUST OF ACADEMIC INTEREST. AS I’M SURE YOU WILL HAVE DEDUCED, IF YOU WEREN’T ALREADY AWARE, HIV-1 DOESN’T INFECT ANY SPECIES OTHER THAN HUMANS AND CHIMPANZEES. THIS CREATES A BARRIER TO THE GENERATION OF ANIMAL MODELS OF HIV-1 INFECTION. THERE HAVE VARIOUS TYPES OF ANIMAL MODELS, FOR EXAMPLE IMMUNODEFICIENT MICE THAT IN WHICH YOU CAN GROW HUMAN IMMUNE CELLS, THE MICE THEMSELVES DON’T DEVELOP AN IMMUNE SYSTEM, THEY DEVELOP A PARTIAL IMMUNE SYSTEM AND HIV CAN GROW IN THEM BUT THEY ARE NOT IMMUNOCOMPETENT MICE. THE MAINSTAY OF AIDS RESEARCH IS IMMUNOCOMPETENT HOSTS, SIMIAN EFFICIENCY IN MACAQUES, WHILE THESE ARE VERY USEFUL, ONE WOULD ALSO LIKE TO HAVE A MODEL THAT INVOLVED A NEARLY INTACT HIV-1 STRAIN, BECAUSE THE SIV COMPONENTS ARE DIVERGENT, 50% OF AMINO ACIDS ARE DIFFERENT FROM HIV-1. AND SO THIS COMPLICATES THE PRE-CLINICAL EVALUATION OF VACCINES AND SOMETIMES EVEN DRUG CANDIDATES THAT ARE EFFECTIVE AGAINST HIV-1 BUT DON’T WORK ON SIV. SO WHAT WE HAVE BEEN DOING RECENTLY IS TO ASK THE QUESTION, CAN WE USE ENGINEERING AND ADAPTATION OF PROTEASE TO DEVELOP HIV-1 STRAINS THAT CAN REPLICATE IN NONHUMAN, SPECIFICALLY IN MACAQUES, AS A BETTER ANIMAL MODEL OF HUMAN AIDS? AND SO ONE THING THAT ENCOURAGED US IN THIS ENDEAVOR WAS A FINDING WE MADE SOME YEARS AGO THAT THE TRIM5 PROTEIN IS POTENT INHIBITOR OF HIV-1 REPLICATION IS KNOCKED OUT IN PIG-TAIL MACAQUES, THE PART OF THE PROTEIN THAT RECOGNIZES THE HIV-1 CAPSID IN OUR MONKEYS, AND IN PIGTAIL MACAQUES HAS BEEN REPLACED AS THE RESULT OF A RETRO TRANSPOSITION EVENT. IN THE PARTICULAR CASE OF PIGTAIL MACAQUES, THIS PORTION EVOLVE AND NO LONGER RECOGNIZES HIV-1. PRESUMABLY, IT EVOLVED TO CHASE SOME OTHER RETROVIRUS BUT THE MAIN POINT IS THAT PIGTAIL MACAQUES LACK THIS VERY POTENT BLOCK TO HIV-1 REPLICATION IN THAT SPECIES. AND IF YOU TAKE CELLS FROM PIGTAIL MACAQUES AND ASK HIV TO GROW IN IT YOU CAN GET A LITTLE BIT OF REPLICATION, NOT AS MUCH AS WITH A SIV STRAIN THAT’S PATHOGENIC AND GROWS NICELY IN PIGTAIL MACAQUES, BUT IF WE MAKE ONE CHANGE IN THE HIV GENOME, TO SUBSTITUTE THE GENE THAT’S RESPONSIBLE FOR KILLING THE APOBEC3 PROTEINS, IF WE CHANGE THE HIV-1 VERSION FROM SIMEON IMMUNODEFICIENCY TO A VIRAL GENOME THAT’S 93% HIV-1 AND HAS A SINGLE GENE FROM SIV, THAT VIRUS THEN GROWS VERY NICELY IN THESE PIGTAIL MACAQUE LYMPHOCYTES. SO WE TEAMED UP WITH JEFF LISTEN’S GROUP AT NCI FREDERICK, WE GAVE IT RELEVANT ENVELOPES FROM PROTOTYPES, RF STRAINS AND INFECTED PIGTAIL MACAQUES. OVER THE NEXT FEW SLIDES, WHAT YOU’LL SEE ON THE BOTTOM ARE TWO CHARTS, ONE MEASURES THE AMOUNT OF VIRUS IN BLOOD OF MACAQUES WEEKS AFTER INFECTION, AND ANOTHER TRACKS A PATHOGENIC SEQUENCE, THE NUMBER OF CD-4 POSITIVE T-CELLS IN THE BLOOD OVER THE SAME TIME FRAME. WHAT WE FOUND IN THESE PASSAGE ONE MACAQUES IS A REASONABLE TAKE OF VIRUS UP TO ABOUT 100,000 COPIES OF RNA PER MILLILITER OF PLASMA, BETWEEN ONE AND TEN PER CENT OF THE LEVEL EXPECTED IN HUMANS AND HOPES TO FIND IN ANIMAL MODEL. THE VIRUS REPLICATION WAS DETAILED IN ONE ANIMAL, CONTROLLED TO LOW LEVEL IN ANOTHER ANIMAL, AND THEN AFTER ABOUT 30 OR SO WEEKS OF INFECTION, WE GAVE THE ANIMALS AN INJECTION OF AN ANTICD 8 ANTIBODY TO RELIEVE SOME OF THE IMMUNE PRESSURE AND ESSENTIALLY BOOST THE POPULATION SIZE AND I’M JUST GOING TO SHOW YOU THIS FOR ONE ANIMAL. WHERE YOU SEE ONE OF THESE TRIANGLES, AN ANTICD-8 ANTIBODY INFUSION, AND THE CONSEQUENCE IS A TEMPORARY REMOVAL OF THE CDA CELLS FROM THE PERIPHERAL CIRCULATION SO THE CD-8 CELLS ARE DEFLEETED FROM BLOOD AND LYMPH NODES, ALMOST UNTOUCHED IN THE GUT, LASTING 2 TO 8 WEEKS AND THEN THE CD-8 CELLS RETURN TO PRE-DELETION LEVELS, WE GET A SPIKE OF VIRUS WITH YOU WE USE TO INITIATE PASSAGE. THERE’S NO SIGN OF PATHOGENIC CONSEQUENCE WITH THIS INFECTION. PASSAGE TWO, WE GOT ANOTHER REASONABLE TAKE OF VIRUS, AND IN PASSAGE TWO AND EVERY PASSAGE AFTERWARDS UNLESS I SPECIFIC OTHERWISE WE GIVE THE ANIMALS AN ANTICD-8 ANTIBODY JUST DEPLETING CD-8 CELLS FOR A FEW WEEKS TO GIVE THE VIRUS AN ASSIST FOR WANT OF A BETTER WORD. WE GOT, AGAIN, A NICE TAKE OF VIRUS AND THEN VIRUS PERSISTED AT 10,000 COPIES FOR MILL. 20 WEEKS TOOK VIRUS FROM THE ANIMAL, INITIATED PASSAGE THREE AND FOUR AND SO ON. I’LL JUMP TO PASSAGE FOUR BECAUSE THREE WAS UNEVENTFUL BUT SOMETHING CHANGED QUITE DRAMATICALLY AND IMPORTANTLY AT PASSAGE FOUR. SO THE FIRST THING I’LL SHOW YOU ARE THE NUMBERS OF CD-4 CELLS IN THE GUT ASSOCIATED LYMPHOID TISSUE, A SOMEWHAT MORE SENSITIVE INDICATOR OF THE ABILITY OF A VIRUS TO DEPLETE CD-4 CELLS IN AN ANIMAL AND IN ALL THREE OF THESE ANIMALS THE NUMBER OF CD-4 CELLS WAS VERY LOW LEVELS QUITE SOON AFTER INFECTION, RETAINED AT LOW LEVELS THEREAFTER. JAKE HESTER, WHO DID THESE IMMUNOSTAINING NOTED PATHOLOGICAL CHANGES. YOU CAN SEE ARCHITECTURE IN THIS PARTICULAR ANIMAL WHERE THE CHANGES WERE ESPECIALLY EVIDENT AND COLLAGEN SCARRING THE LYMPH NODES, COMMONLY SEEN IN PROGRESSIVE HIV-1 AND SIV IN MACAQUES, VIRUS REPLICATION IN ONE PARTICULAR ANIMAL, P4C WAS NOT CONTROLLED, CONTINUING AT EXTREMELY HIGH LEVELS, CD-4 CELLS WERE EFFECTIVELY REMOVED FROM THE ANIMAL AND WERE NEARLY UNDETECTABLE AT 28 WEEKS AFTER INFECTION, AT WHICH TIME THE ANIMAL HAD TO BE EUTHANIZED WITH A RATHER CLASSIC CASE OF AIDS, FIRST TIME AIDS HAS BEEN INDUCED BY HIV-1 IN A NONHOMINUS SPECIES. THE ANIMAL WAS SACRIFIESED BECAUSE IT HAD TUMORS, WIDELY DISSEMINATED, COMMONLY ASSOCIATED WITH HERPES IN PRIMATE SPECIES. ONE CLASSICAL AIDS DEFINING CONDITION, A SECOND IN THE SAME ANIMAL, THESE BLACK SPOTS, REVEALED BY THIS SAME, NOT FOUND IN CONTROL ANIMALS. OF COURSE, ONE DEAD MONKEY DOES NOT A COMPELLING STORY MAKE. SO WE TOOK BLOOD FROM THIS ANIMAL AND PASSED IT TO FOUR MORE MACAQUES, AND IN THIS PASSAGE WE LEFT THE ANIMALS OTHERWISE UNTREATED, SO JUST INFECTED AND THEN TWO ANIMALS WERE ALSO CD-8 DEPLETED AT THE TIME OF INFECTION. THE CD-8 DEPLETED ANIMALS HE RECAPITULATED THE FINDING WITH PEOPLE, VERY HIGH LEVELS OF VIRUS REPLICATION AND HAD TO BE EUTHANIZED, EITHER VERY QUICKLY OR SOMETIME LATER FOR CLINICAL CAUSE AND PROFOUND LOSS OF CD-4 POSITIVE T-CELLS. THE OTHER TWO ANIMALS, THOSE NOT CD-8 DEPLETED, THERE WAS A GOOD TAKE OF VIRUS BUT IT WAS A VERY RAPIDLY CONTROLLED, AND THOSE ANIMALS HAD A COURSE OF INFECTION THAT ONE — SUPERFICIALLY RESEMBLES THAT ASSOCIATED WITH A SMALL MINORITY OF HUMAN INFECTIONS, SO-CALLED LONG TERM NONPROGRESSION. WE DID A CD-8 DEPLETION IN THOSE ANIMALS AT SOME POINT AND GOT A SPIKE BUT IT RETURNED TO ITS PRE-DEPLETION LEVELS. IN NO WAY RECAPITULATED THE EFFECT OF CD-8 DEPLETION DONE EARLY DURING INFECTION. AND THIS SUGGESTS THAT IN THIS MODEL IN PARTICULAR, AND PERHAPS MORE GENERALLY, THE BATTLE BETWEEN HIV AND THE IMMUNE SYSTEM DURING THIS EARLY WINDOW OF INFECTION CAN BE REALLY DETERMINISTIC OF CLINICAL CAUSE AND WE CAN TOGGLE BETWEEN RAPID PROGRESSION AND ELITE CONTROL BY INCLUSION OR LACK OF INCLUSION OF THIS SIMPLE IMMUNOLOGICAL MANIPULATION DURING THE FIRST WEEKS OF INFECTION. WE COULD AGAIN RECAPITULATE BY TAKING LOAN FROM P5C AND INFECTING TWO HORMONES, ONE WAS CD-8 DEPLETED, RAPID PROGRESSION AND DEATH, IN THE OTHER ANIMAL NOT CD-8 DEPLETED EFFECTIVE CONTROL. SO OBVIOUSLY SOMETHING WAS DIFFERENT HERE TO HERE. WHAT, IF ANYTHING, INTERESTING HAPPENED TO THE VIRUS DURING THIS PASSAGE SERIES IN MACAQUES AND HOW DID IT ADAPT TO BECOME MORE VIRULENT IN ITS NEW HOST? WELL, AS YOU’LL REMEMBER FROM THE FIRST PART OF THE TALK, AND I DELIBERATELY NEGLECTED TO MENTION WHEN I TALKED ABOUT ADAPTATION TO THE MACAQUES, YOU’LL RECALL HIV-1 HAS AN ANTAGONIST OF TETHERIN PROTEIN, Vpu THAT INHIBITS THIS ANTIVIRAL PROTEIN AND I TOLD YOU IT DOESN’T WORK IN MONKEYS AND ACTUALLY DOESN’T WORK IN PIGTAIL MACAQUES EITHER. SO WE INFECTED ANIMALS WITH A VIRUS THAT WAS INCAPABLE MUCH DEALING WITH THIS NEW ANTIVIRAL PROTEIN. BUT WHAT HAPPENED OVER THE COURSE OF PASSAGE WAS THAT MUTATIONS OCCURRED IN THE TRANSMEMBRANE DOMAIN OF Vpu, THE VERY DOMAIN RESPONSIBLE FOR INTERACTION WITH TETHERIN. AND SO WHILE THE WILD-TYPE Vpu CANNOT ANTAGONIZE THE PIGTAIL MACAQUE TETHERIN PROTEIN, SO PIGTAIL MACAQUE TETHERIN INHIBITS AN HIV-1 STRAIN THAT HAS THE WILD-TYPE Vpu, THE MUTATIONINGS IN THE TRANSMEMBRANE DOMAIN CONFERRED ON Vpu THE ABILITY TO ANTAGONIZE THE PIGTAIL MACAQUE TETHERIN AND GAINED FUNCTION, I.E. THE ABILITY TO ANTAGONIZE MA TAG TETHERIN IN THE ADAPTATION PROCESS. ANOTHER THING HAPPENED THAT WE WEREN’T EXPECTING. WE FOUND UNUSUAL MUTATIONS IN CAPSID, MUTATIONS THAT ARE RARELY AND IN SOME CASES NEVER FOUND IN HIV-1 SEQUENCES THAT ARE FOUND IN HUMANS, HERE THEY ARE DEPICTED ON THE HIV-1 CAPSID PROTEIN. WHAT THESE MUTATIONS APPEAR TO DO IS TO GIVE A SUBTLE BUT REAL RESISTANCE TO THE Mx2 PROTEIN, AND SPECIFICALLY THE Mx2 PROTEIN THAT’S FOUND IN PIGTAIL MACAQUES. SO HERE ARE THESE VARIOUS MUTANTS. THESE ARE THE POSITIONINGS THAT WERE MUTATED. THE ACTUAL COMBINATIONS OF MUTATIONS OCCURRED IN VARIOUS FORMS, LISTED HERE, BUT THE VARIANTS WE FOUND FROM THE PIGTAIL MACAQUES WITH ONE EXCEPTION WITH A PROFOUND FITNESS DEFECT ASSOCIATED WITH IT REALLY DIDN’T DO MUCH IN TERMS OF SENSITIVITY TO HUMANS BUT CAUSED CHANGED THE PIGTAIL MACAQUE EMICS FROM TEN-FOLD TO THREE-FOLD, A SMALL BUT NEVERTHELESS SIGNIFICANT CHANGE IN SENSITIVITY TO — SPECIFICALLY TO THE MONKEYS, A FORM OF THIS INHIBITOR. LET ME SUMMARIZE THIS LAST PART OF THE TALK. FIRST, AN HIV-1 THAT’S BEEN ADAPTED AND ENGINEERED CAN CAUSE AIDS IN PIGTAIL MACAQUES. THE DISEASE MIMICS KEY FEATURES OF THE DISEASE IN HUMANS. AND CRUCIALLY, SORT OF TIEING EVERYTHING TOGETHER, ADAPTATION OF HIV-1 TO MACAQUES IS ACCOMPANIED BY ACQUISITION EFFECTIVELY OF RESISTANCE TO ANTIVIRAL PROTEINS SO Vpu GAINS FUNCTION TO ANTAGONIZE PICK TAIL MACAQUE TETHERIN WITH A REDUCTION IN SENSITIVITY TO Mx2. AN IMPORTANT FEATURE OF THIS MODEL, THERE ARE STARK DIFFERENCES IN OUTCOMES THAT CAN BE DETERMINED BY EVENTS INFECTION, I MENTION A PAPER THAT ADAPTED INTERFERON RESPONSE, EARLY IN INFECTION HAS GREATER THAN EXPECTED LONG-TERM EFFECTS ON INFECTION, IN AN SIV MODEL, ARGUES THAT THERE ARE VERY IMPORTANT EVENTS THAT HAPPEN EARLY DURING THE INFECTION PROCESS. OBVIOUSLY ADAPTATIONS IN THE NEW HOST IS NOT COMPLETE. WE DO STILL HAVE TO INJECT THE ANIMALS WITH AN ANTICD-8 ANTIBODY TO GIVE THE VIRUS BREATHING SPACE IN ORDER FOR IT TO HAVE ITS FULL VIER LENS. V I R ULENCE. LET ME THANK PEOPLE ASSOCIATED WITH THE WORK. MANY PEOPLE IN MY LAB OVER THE YEARS HAVE WORKED ON THIS TETHERIN PROTEIN, INCLUDING ZHANG, BILERATORE STEWART NEIL WAS RESPONSIBLE FOR THE DISCOVERY OF TETHERIN AND DID SOME MECHANISTIC STUDY, SAM WILSON, AND PARTICULARLY MELISSA KANE, RESPONSIBLE FOR THE WORK ON Mx2 THAT I SHOWED YOU. WORK WITH TRYING TO ADAPT HIV-1 TO GROW IN MACAQUE HOST IS VERY MUCH A FOUR-WAY COLLABORATION THAT INVOLVES MY GROUP, MY WIFE, THEODOREA, AND HER TEAM DOWN THE HALL FROM MY LABORATORY, AND IN PARTICULAR I’D LIKE TO ACKNOWLEDGE OUR COLLABORATORS HERE, AT NCI-FREDERICK, PARTICULARLY JEFF LISTEN, AND HIS TEAM, AND ESPECIALLY NOTE THE CONTRIBUTIONS OF JAKE AND BRANDON AND GREGORY, AND MICHAEL WHO SADLY PASSED AWAY RECENTLY, AND THESE OTHER COLLABORATORS CONTRIBUTED IN OUR EARLIER WORK ON THE TETHERIN PROTEIN. THANK YOU VERY MUCH FOR LISTENING. [APPLAUSE]>>HI, PAUL. WONDERFUL TALK, AS USUAL.>>THANK YOU.>>I WANTED TO ASK YOU SINCE THE PATROL, THE Mx2 PATROL IS NOT WORKING TO STOP EVERYTHING, AND I KNOW EARLY EVENTS ARE VERY IMPORTANT, WHAT ARE OR IS THE DETERMINANT EVENT THAT MAKES THE VIRUS HIV ESCAPE Mx2 COMPLETELY, PROVIDED THAT IT’S ACTUALLY AT THE ENTRY AT THE NUCLEAR PORT? BECAUSE THE CAPSID MUTATIONS ARE MAJOR, YOU KNOW, PROBLEMS FOR THE VIRUS, SO WHAT MAKES IT OR WHAT ARE THE EVENTS, NOT ONE EVENT I’M GUESSING?>>SO I WAS PERHAPS VAGUE ABOUT HOW WE THINK Mx2 WORKS, AND THAT’S BECAUSE WE DON’T REALLY KNOW. SO A FAVORITE HYPOTHESIS IS THE Mx2 PROTEIN ACTUALLY DIRECTLY BINDS TO THE CAPSID, AS IT ENTERS OR APPROXIMATE TO THE TIME AT WHICH IT WOULD BE EXPECTED TO ENTER THE NUCLEAR PORE, GENETICS ARGUES IN FAVOR OF THAT TYPE OF MODEL. AND IN THAT INSTANCE, THEN THE MUTATIONS IN CAPSID COULD BE LOST OF BINDINGS EFFECT. I DO HAVE TO SAY THOUGH IT’S BEEN VERY CHALLENGING, AND IMPOSSIBLE IN FACT IN MY LAB TO SHOW SPECIFIC INTERACTIONS BETWEEN THE HIV-1 CAPSID AND Mx2. WE CAN SHOW INTERACTIONS BUT NONE OF THE MUTANTS THAT CONFER RESISTANCE ABOLISH THAT ACTION SO WE’RE SUSPICIOUS. ANOTHER POSSIBILITY IS WHAT Mx2 MIGHT DO IS SIMPLY COMPETE FOR FACTORS THAT HIV NEEDS TO ENTER THE NUCLEUS. IT’S LOCALIZATION, ITS LOCALIZATION INDICATES IT’S IN THE RIGHT PLACE TO DO THAT. AND THE FACT THAT IF YOU GROW THE CELLS SO INFECTION HAS TO GO THROUGH A NUCLEO PORE, THAT INCREASES POTENCY, AND IT INFECTS CELLS — ONLY ENTERS THE NUCLEAR DURING MITOSIS WHEN THERE ISN’T A NUCLEUS, IT’S COMPLETELY RESISTANT, COMPATIBLE WITH THAT SECOND NOTION. BUT DISTINGUISHING BETWEEN THE TWO IS VERY DIFFICULT AT PRESENT. WE’RE GATHERING LOTS OF DATA, DOING LOTS OF NUCLEAR PORE KNOCKDOWNS BUT NOT READY TO TAKE A POSITION ON HOW WE BETWEEN Mx2 IS WORKING YET.>>PAUL, ONE MORE QUESTION. AS INTRIGUED ABOUT THE STRUCTURE OF TETHERIN AND GPI TAIL, THAT DOESN’T STAY IN THE CELL MEMBRANE?>>HOW — NO, IT’S NOT CLEAR. THAT IT HAPPENS IS PRETTY CLEAR, BUT WHY THOSE SELECTIVITIES FOR THE GPI ANCHOR TO BE INCORPORATED, WE DON’T KNOW. IT MIGHT BE THAT, AS I’M SURE YOU’RE AWARE, THERE’S A LONG HISTORY ABOUT HOW HIV FAVORS PARTICULAR MEMBRANE DOMAINS, LIPID, WHATEVER NAME YOU LIKE TO GIVE THEM, FOR ITS ASSEMBLY, IT’S ALSO TRUE THAT GPI ANCHORS LIKE TO PARTITION INTO LIPID RATS, OR THE ALTERNATIVE MODEL IS TO — IT ACTUALLY MAKES ITS OWN LIPID ENVIRONMENT AS IT ASSEMBLES AND THAT COULD FAVOR INCORPORATION OF A GPI ANCHORED PROTEIN OVER A TRANSMEMBRANE DOMAIN WITH A CYTOPLASTIC TAIL. TRANSMEMBRANE DOMAIN CONTAINING PROTEINS CAN BE INCORPORATED INTO VIRAL PARTICLES, BUT THE RULES GOVERNING THAT ARE POORLY UNDERSTOOD. BUT WE DON’T KNOW THE ANSWER, BUT I’M SORT OF COMFORTABLE WITH THE NOTION THE ANSWER IS SOMEWHERE IN THE HAND-WAVING THAT I JUST GAVE YOU. [ LAUGHTER ]>>OKAY. IF THERE NO FURTHER QUESTIONS, LET’S THANK PAUL FOR AN EXCELLENT TALK, AND PLEASE JOIN US AT THE RECEPTION. [APPLAUSE]