>> HAPPY NEW YEAR TO EVERYONE. WE START A NEW CALENDAR YEAR WITH A WONDERFUL SPEAKER, SONJA BEST. SONJA COMES TO US INITIALLY FROM AUSTRALIA WHERE HE GOD A UNIVERSITY DEGREE AT UNIVERSITY OF ADELAIDE AND Ph.D. IN MOLECULAR BEOLOGY FROM THE AUSTRALIA NATIONAL UNIVERSITY. AND THEN SHE DECIDED THAT THAT WAS A LITTLE DOOR FROM MOTHER NIH SO SHE MOVED TO ROCKY MOUNTAIN LAB AS VISITING FELLOW. INITIALLY WORKING IN MINK PARVOVIRUS AND HER INTEREST IS INTERACTION WITH VIRUSES IN THE IMMUNE SYSTEM. FOR WHILE A RESEARCH FELLOW AND BRIEF STINT AS STAFF SCIENTIST AND BECAME A TENURE TRACK INVESTIGATOR IN LABORATORY OF VIROLOGY AT RML IN 2009 AND LAST YEAR WAS TENURED AS A SENIOR INVESTIGATOR AND CHIEF OF THE INNATE IMMUNITY AND PATHOGENESIS SECTION OF THE LABORATORY OF VIROLOGY AT RML. NOW, SHE'S MOST OF HER CAREER SO FAR IS INTERESTED IN IN STUDYING FLAVIVIRUSES AND INTERACTION WITH THE INNATE IMMUNE SYSTEM. JUST TO REMIND YOU FLAVIVIRUSES INCLUDE A LOT OF VIRUSES IN THE NEWS H WEST NILE, ZIKA, YELLOW FEVER, DENGUE VIRUS. A LOT OF IMMUNITY TO THIS IS INNATE AND THEY MANAGE TO DEAL WITH TYPE 1 INTERFERON SYSTEM. AND I THINK THE TALK TODAY WILL FOCUS ON THAT INTERACTION. AND THE TITLE OF SONJA'S TALK IS NEW INSIGHT TO THE HUMAN TYPE 1 INTERFERON RESPONSE REVEALED BY FLAVIVIRUS HOST INTERACTION. WITHOUT FURTHER ADIEU, LET YOU SPEAK FOR YOURSELF. >> THANK YOU VERY MUCH. HI TO THE RML FOLKS AND EVERYBODY HERE AND JUST WANT TO SAY WHAT A PLEASURE AND HONOR IT IS TO BE HERE ON CAMPUS GIVING THIS TALK. TODAY I WANT TO TELL YOU TWO STORIES THAT WE HAVE BEEN WORKING ON OVER THE PERIOD OF MY TENURE TRACK TIME. THE FIRST IS TO ILLUSTRATE THE POWER OF STUDYING THESE VIRUS AND THE ENTERAS WITH THE INNATE IMMUNE -- INTERACTIONS WITH INNATE IMMUNE RESPONSE IN ORDER TO LEARN MORE ABOUT OUR OWN IMMUNE RESPONSE AND THE INTERFERON RESPONSE IN PARTICULAR. AND THEN THE SECOND STORY IS A NEWER STORY IN THE LAB THAT IS CURRENTLY UNFOLDING. WE REALLY I THINK ARE STARTING TO LEARN ABOUT THE VIRUSES THEMSELVES DUE TO THESE INTERACTIONS WITH THE HOST. SO I WORK PREDOMINANTLY ON FLAVIVIRUS, WE ALSO HAVE A SMALL PROGRAM NOW EBOLA VIRUS FOLLOWING THE OUTBREAK, MOST RECENT OUTBREAK BUT TODAY I WILL CONCENTRATE ON THESE VIRUSES WHICH REALLY CAPTURE MY IMAGINATION EXTENSIVELY IN THE TIME IN THESE SLIDES. SO THESE VIRUSES, THERE'S ABOUT 53 RECOGNIZED SPECIES OF FLAVIVIRUSES, SO THEY HIGHLY DIVERSE AND APPROXIMATELY 40 ARE KNOWN TO CAUSE DISEASE IN HUMANS. THE MOST WELL KNOWN OF THESE, I DON'T KNOW IF I HAVE A -- THE ONES YOU'RE FAMILIAR WITH AND HAVE BEEN IN THE NEWS, ZIKA VIRUS EMERGED QUITE DRAMATICALLY STARTING IN 2014, 15 IN SOUTH AMERICA. FROMSOME OF THESE OTHER VIRUSES WE HAVE ISSUES WITH SUCH A DENGUE VIRUS WHICH CAUSING 90 MILLION CASES ON AN ANNUAL BASIS AND FOUR TIMES AS MANY INFECTIONS ARE ESTIMATED TO OCCUR. WEST NILE VIRUS SUCCESSFULLY EMERGED TO NORTH AMERICA IN '99 AND BECOME ENDIME AND IS STILL A PROBLEM WITH TWO TO THREE THOUSAND CASES OF WEST NILE DISEASE ANNUALLY. AND THEN SOME VIRUSES THAT YOU PROBABLY LESS FAMILIAR WITH, IN EUROPE AND ASIA THERE'S QUITE VIRULENT VIRUS KNOWN AS TICK BORNE ENCEPHALITIS VIRUS. THIS VIRUS IS A COMPLEX OF VIRUSES THAT HAD HIGH CASE FATALITY RATES OF PROTEIN, 30, 40% DEPENDING ON VIRUS. SO IN THE U.S. WE WORK ON THAT BSL 4 WHICH IS WHY WE WORK AT ROCKY MOUNTAIN LABORATORIES. BECAUSE I'M GOING THE TALK MORE ABOUT THESE VIRUSES VERY UNDERSTUDIED I WANT TO TELL YOU THE NAMES OF SOME OF THESE VIRUSES. YOU DON'T HAVE TO REMEMBER BUT TO BE FAMILIAR WITH THEM. SO WE HAVE TICK BORNE ENCEPHALITIS VIRUS ITSELF AS WELL AS COUSINS FOREST DISEASE VIRUS ENDEMIC AND POISON VIRUS EMERGING HERE IN THE NORTHEAST UNITED STATES, TRANSMITTED BY THE SAME TICKS THAT TRANSMIT LYME DISEASE. THEN WE HAVE A NATURALLY ATTENUATED MEMBER THESE VIRUSES CALLED LENGAT VIRUS ISOLATED OUT OF TICKS IN MAY LA SHAH AND THAILAND BUT A NICE MODEL TO STUDY SOME OF THESE VIRUSES AT A LOWER BIOSAFETY LEVEL. SO I WILL MENTION QUITE A FEW OF THESE. THE SUCCESS OF THESE VIRUSES IS PATHOGENS IS BECAUSE THEY'RE TRANSMITTED BY TICKS AND MOSQUITOES AND FOLLOWING INFECTION OF THE SKIN, PROBABLY THE MOST IMPORTANT EARLY CELL TYPES THAT ARE INFECTED INCLUDE DENDRITIC CELLS AND MACROPHAGES AND YOU HAVE REPLICATION IN LIMB NODES AND THEN ESTABLISHMENT OF A PRIMARY VIREMIA. FOR MOST PEOPLE THAT MIGHT BE ASSOCIATED WITH FEVER BUT THEN THAT INFECTION IS RESOLVED. AND ONLY IN A FEW PEOPLE, MAYBE 25% OR SO, THE VIRUS IS THEN ABLE TO CROSS CRITICAL BARRIERS AND CAUSE DISEASE. SO WE HAVE VIRUSES LIKE WEST NILE AND TICK BORNE ENCEPHALITIS VIRUS THAT ESTABLISH INFECTION IN CENTRAL NERVOUS SYSTEM AND CAUSE DAMAGE TO DIRECT KILLING OF CELLS AS WELL AS INFLAMMATORY RESPONSE. THE ZIKA VIRUS IS ABLE TO CROSS THE PLACENTAL BARRIER AND IT'S STILL NEUROTROPIC BUT HAS THIS UNUSUAL TROPISM FOR THE DEVELOPING FETUS AND EXPECTTANT MOTHERS. OTHER VISCERAL TROPIC VIRUSES DENGUE AND YELLOW FEVER, THEY HAVE PRIMARY TARGET FOR REPLICATION IS THE LIVER AND COMPLICATIONS ASSOCIATED WITH VARIOUS INFLAMMATORY RESPONSES TO THOSE VIRUSES. SO THESE VIRUSES ARE REPEATEDLY EMERGING AND RE-EMERGING AND FOR ANY VIRUS TO BE ABLE TO DO THIS IT HAS TO PARTICULARLY TO ESTABLISH INFECTIONS LIKE IN HUMANS, IT HAS TO OVERCOME A NUMBER OF BARRIERS TO INFECTION. THE FIRST IS OF COURSE THE RECEPTOR IF THE VIRUS CAN USE RECEPTOR, THEY'RE QUITE PROMISE SKEW WOWS IN THIS MANNER HOE PROMY SKEW -- CELLULAR RESPONSE TO INFECTION OR ABILITY OF CELLS TO RECOGNIZE THEY'RE INFECTED AND MOUNT AN INTERFERON RESPONSE WHICH IS MEDIATED THROUGH THREE GROUPS OF INTERFERENCE TYPE 1, 2 AND 3, WE WILL CONCENTRATE ON TYPE 1 FOR TODAY BUT THESE INTERFERONS ARE EXTREMELY POWERFUL BECAUSE OF THEIR ABILITY TO UPREGULATE HUNDREDS OF INTERFERON STILL LATED GENES. THESE GENES ARE RESPONSIBLE FOR THE BIOLOGICAL EFFECTS OF INTERFERON THAT INCLUDE DIRECT RESTRICTION OF VIRUS INFECTIONS SO SUPPRESSION OF VIRUS REPLICATION IN THOSE INDIVIDUAL INFECTED CELLS. AS WELL AS PROMOTION OF INNATE AND ADAPTIVE IMMUNE RESPONSE TO PROVIDE CLEARANCE AND ESTABLISHMENT OF IMMUNE MEMBRANE. THIS SYSTEM IS SO POWERFUL THAT IT REALLY, ANY VIRUS THAT IS GOING TO CAUSE, EVEN BE ABLE TO REPLICATE IN HOST AND BE ABLE TO CAUSE DISEASE HAS TO BE ABLE TO OVERCOME THAT RESPONSE THROUGH DIRECT EVASION AND ANTAGONISM FOR MAYBE SIMPLY AVOIDANCE. BUT THAT RELATIVE ABILITY OF VIRUS TO DO THAT CAN DETERMINE HOST TROPISM AND HOST SUSCEPTIBILITY. SO WE WILL EXPLORE THOSE CONCEPTS A LITTLE BIT IN THIS TALK SO WHAT IS THE INTERFERON RESPONSE I'M TALKING ABOUT? WE DON'T -- THE FLAVIVIRUSES SEEM TO BE ABLE TO AVOID TRIGGERING THE INITIAL PRODUCTION OF INTERFERON WHICH OCCURS THROUGH FOLLOWING RECOGNITION OF VIRUS NUCLEIC ACIDS THROUGH THESE RNA LIKE HELOCASES, MDA 5. THESE HELOCASES SIGNAL THROUGH ADAPTOR MOLECULE MITOCHONDRIA TO ACTIVATE CANONICAL TRANSCRIPTION FACTORS AND DRIVE PRODUCTION OF INTERFERON. THE VIRUSES DEPENDING ON THE VIRUS THEY DON'T TEND TO SPEND A LOT OF GENETIC INFORMATION IN SUPPRESSING THIS RESPONSE EXCEPT FOR PERHAPS DENGUE. BUT THEY DO SPEND A LOT OF GENETIC INFLAMMATION IN TERMS OF SUPPRESSING SIGNALING ONCE INTERFERON IS BEING MADE. AND HAS BEEN MADE. SO ONCE INTERFERON IS MADE, IT'S SECRETED AND THEN LIGATING TWO RECEPTOR SUBUNITS, NF 1 AND 2, TO ACTIVATE THE CASCADE AND UP REGULATE THE STIMULATOR NEURON GENES. SO ALL THE FLAVIVIRUSES HAVE POTENT WAYS TO SUPPRESS THE SIGNALING PATHWAY. BUT THEY DO IT AT SLIGHTLY DIFFERENT STEPS. SO THIS WORK HAS BEEN MAINLY DONE BY MY LAB AND AT MOUNT SINAI, THIS SUMMARIZES WHAT WE KNOW ABOUT THIS PART OF ANTAGONISM. SO WHERE IS THESE VIRUSES DENGUE, ZIKA AND YELLOW FEVER CONCENTRATE ON THE ACTUAL STATS THEMSELVES? THESE TWO ENCEPHLYTIC VIRUSES, TBB AND WEST NILE, SUPPRESS SIGNALING BY SUPPRESSING ACTIVATION OF THE GENUS KINASES. WE HAVE KNOWN THIS FOR A LONG TIME, PROBABLY CLOSE TO 15 YEARS ACTUALLY. BUT THIS IS THE POINT IN THE PATHWAY WHERE THESE VIRUSES SUPPRESS SIGNALING. HOWEVER FOR THE LONGEST TIME THE ACTUAL MECHANISM OF INTERFERON ANTAGONISM BY THESE TWO VIRUSES HAS REMAINED QUITE ELUSIVE. YOU WILL UNDERSTAND WHY WHEN I TELL YOU THE ANSWER. THIS IS JUST AN EXAMPLE OF THESE VIRUSES. HERE IS HUMAN DENDRITIC CELLS THAT WE HAVE INFECTED WITH LENGAT VIRUS, A ATTENUATED MEMBER OF TICK 1 COMPLEX AND STIMULATED WITH TYPE 1 INTERFERON AND STAINED FOR PHOSPHORYLATED STAT #. IF ALL SIGNALING CASCADE IS WORKING, STAT 1 WILL MOVE TO THE NUCLEUS AND YOU CAN SEE NOW THREE INFECTED CELLS HERE THAT IT'S COMPLETELY SHUT DOWN FOR SIGNALING. IT'S A REMARKABLE ABILITY TO ANTAGONIZE SIGNALING PATHWAY. SO THEN WE WANTEDDED TO KNOW WHAT VIRAL PROTEIN WAS RESPONSIBLE. THE FLAVIVIRUSES ARE GENETICALLY SIMPLE, A SINGLE STRANDING MOLECULE OF RNA IN A POSITIVE SENSE. ONCE RELEASEDDED IN TO THE CELLS IT ACTS AS MESSENGER RNA THAT'S TRANSLATED INTO A LONG OPEN READING FRAME THAT SUBSEQUENTLY CLEAVED INTO THE INDIVIDUAL VIRAL COMPONENTS. THESE COMPONENTS INCLUDE THREE STRUCTURAL PROTEINS HERE THAT MAKE THE VIRION AND SEVEN NON-STRUCTURAL PROTEINS. THE TWO PROTEINS FOR THE PURPOSE OF THIS TALK WE'RE GOING TO CONCENTRATE ON IS NS 5 AND NS 3. NOTABLE BECAUSE THEY ENCODE FUNCTIONS FOR REALLYCATION THESE VIRUSES REPLICATE UNMODIFIED ER MEMBERS OF THE JURY BRAINS YOU CAN SEE THROUGH THIS TWO SCALES SCHEMATIC, JUST KIDDING WE HAVE COMPLEXING OF NS 5 WHICH IS RNA DEPENDENT POLYMERASE AS WELL AS NS 3 VIRAL PROTEASE RESPONSIBLE FOR A LOT OF CLEAVAGE EVENTS, ALSO RNA HELOCASE AND THAT NS 3 PROTEIN IS ANCHORED TO THE ER MEMBRANE ANOTHER STRUCTURAL PROTEIN. THESE GUYS ARE PRESENT TOGETHER IN REPLICATION COMPLEX AND WITHOUT THAT, YOU WOULDN'T HAVE RNA REPLICATION. SO WE LOOK AT THE ABILITY OF ALL THOSE INDIVIDUAL PROTEINS TO SUPPRESS THIS INTERFERON RESPONSE SO THIS IS A SIMPLE IMMUNOFLUORESCENCE ASSAY IN VIRAL CELLS TREATED WITH INTERFERON, YOU HAVE THAT LIGHTING UP OF NUCLEUS BY FLOW CYTOMETRY H SHIFT IN PHOSPHORYLATED PROTEIN. NOW IF WE EXPRESS NS 5 PROTEIN ALONE YOU CAN SEE THAT BOTH CELLS ARE REALLY NICELY SUPPRESSED FOR INTERFERON SIGNALING, ESPECIALLY COMPARED TO OTHER VIRAL PROTEINS. FROM THESE TICK BORNE VIRUSES YOU CAN SEE THEY MORE THAN 90% ARE SUPPRESSED FOR THAT INTERFERON RESPONSE. WE HAVE KNOWN THIS LEVEL OF INFORMATION FOR A LONG TIME. WHEN WE FINALLY STARTED TO LOOK AT THE ACTUAL RECEPTOR ITSELF WHICH IS NOT EASY WITH THE INTERFERON RECEPTOR, WE STARTED TO LOOK AT THE INTERFERON RECEPTOR AND WE FOUND IT PROBABLY WASN'T THE GENUS KINASES THEMSELVES BEING TARGETED, BUT MORE MORE RECEPTOR. IF YOU LOOK AT INFECTED CELLS IN EACH CASE WITH THESE FLAVIVIRUSES THAT SUPPRESS GENUS KINASE SIGNALING AT THE JACKS, THEY'RE DEPLETED FOR THAT TYPE 1 INTERFERON RECEPTOR SUBUNIT. WE WANT TO KNOW WHETHER OR NOT NS 5 WAS RESPONSIBLE FOR THIS DEPLETION. WE MADE STABLE 293 CELLS EXPRESSING EITHER NS 5, THIS TIME FROM THAT LANGET VIRUS AGAIN OR NS 5 WITH SINGLE AMINO ACID MUTATION UNABLE TO SUPPRESS SIGNALING. HERE WE HAVE NS 5 EXPRESSION LEVELS IF WE TREAT WITH INTERFERON YOU CAN SEE THAT CELLS ARE PHOSPHORYLATED FOR STAT 1 IN CELLS EXPRESSING GFP AND MUTANT BUT NOT WILD TYPE NS 5. NOW IF WE CO-EXPRESS THESE INTERFERON RECEPTOR SUBUNITS IFNAR 1 OR 2 HERE YOU CAN SEE IN CELLS EXPRESSING THIS WILD TYPE NS 5 THOSE CELLS ALSO DEPLETED OF INFAR 1 LEVELS BUT MUTANTS IS UNABLE DO IT LINKING NS 5 FUNCTION WITH THIS PARTICULAR ROLE IN INTERFERON ANTAGONISM. SO THE NEXT QUESTION THOUGH BECAME WHAT WAS THE CELLULAR TARGET OF NS 5. WE COMPLETED A YEAST 2 HYBRID STUDY THAT REVEALED A POTENTIAL INTERACTION BETWEEN NS 5 OF LENGET VIRUS AS MODEL VIRUS AND HOST PROTEIN, DIPEPTIDEASE KNOWN AS PROLIDASE, IT'S INVOLVED IN ME TAB LIMB, CLEAVES DIPEPTIDES CONTAIN PROLINE FOR RECYCLING OF DIETARY ENDOGENOUS PROTEIN SUCH AS COLLAGEN. BUT OF COURSE AS YOU MIGHT EXPECT ROLE FOR PRO DAYS IN IMMUNITY IS NOT PREVIOUSLY ESTABLISHED. WE DID A NUMBER OF ASSAYS SO SHOW THE NS 5 PROTEIN OF VIRUSES INCLUDING TICK BORNE ENCEPHALITIS VIRUS AND INTERACTS WITH PAROLE DAYS. THIS IS AN -- PAROLADASE. YOU CAN SEE THE INTERACTIONS APPEARS LOCALIZED TOWARDS ER CONSISTENT WITH REPLICATION THESE VIRUSES. SO THESE PROTEINS THAT ARE INTERACTING BUT WE CERTAINLY HAD LIMITED INFORMATION THAT PROLIDASE MIGHT BE INVOLVED IN INTERFERON RESPONSE. TO LOOK AT THIS WE MADE STABLE CELL LINES THAT WERE KNOCKED DOUBT BACK WHEN SHORT HEAVY RNA WAS THE BEST WAY TO KNOCK OUT GENES. NOW WITH CRISPER, BUT TO KNOCK OUT OR KNOCK DOWN PROLIDASE EXPRESSION, WE USE HIF 1 AS CONTROL FOR REASONS I WON'T GO INTO. WHEN YOU LOOK AT THE LEVEL OF I HAVE FAR 1 EXPRESSION US SEE IN ABSENCE OF PROLIDASE YOU LOSE A LOT OF EXPRESSION OF THAT PROTEIN AND ABILITY OF CELLS TO RESPOND TO INTERFERON IS REDUCED BY ABOUT 85% AS INDICATED BY IS GENE EXPRESSION ESSENTIALLY IN THESE REPORTER ASSAYS. IF WE NOW TAKE THESE PROLIDASE DEFICIENT CELLS AND OVEREXPRESS IT WE RECOVER INTERFERON RESPONSIVENESS OF CELLS, TO THE EQUIVALENT LEVELS AS IF WE OVEREXPRESS I HAVE FAR 1, AND THEN INTERESTINGLY PROLIDASE IS A DIPEPTIDEASE BUT IF WE OVEREXPRESS THE MUTANT OF PROLIDASE THAT IS INACTIVE WE ALSO GET THAT EFFECT SUGGESTING THIS IS POTENTIALLY A SECOND FUNCTION OF PROLIDASE INDEPENDENT OF NORMAL ROLES IN PEPTIDE CATABOLISM. WHAT DOES THIS MEAN FOR THE VIRUS? IT LOOK A LONG TIME TO WORK OUT BUT WE FINALLY WERE ABLE TO REVERSE ENGINEER INTO MUTATIONS INTO FLAVIVIRUSES SO THEY CAN NO LONGER BIND TO PROLIDASE. WHEN WE DID THAT THIS IS IN THE CONTEXT OF TICK BORN ENCEPHALITIS VIRUS, IF I HAVE FAR 1 PROPHENES IN CELLS INFECTED WITH WILD TYPE VIRUS COMPARED TO THE SINGLE MUTATION IN NS 5 PROTEIN WHICH MEANS THIS VIRUS CAN NO LONGER BIND PROLIDASE AND IT RECOVERS NOT ONLY THE INTERFERON RECEPTOR EXPRESSION BUT PHOSPHORYLATION OF STAT 1 IN THOSE INFECTED CELLS AS COMPARED TO THE WILD TYPE HERE. IMPORTANTLY, THIS IS ONE OF MY FAVORITE SETS OF DATA BECAUSE IT TOOK SO LONG TO COME ABOUT BUT IMPORTANTLY IF WE INFECT JUST CONTROL CELLS HERE WITH OUR WILD TYPE TICK BORNE ENCEPHALITIS VIRUS AND TREAT WITH TYPE 1 INTERFERON AT 24 HOURS POST INFECTION SO YOU HAVE ALREADY GOT A LOT OF VIRUS HERE IN THE SYSTEM. THIS VIRUS REALLY DOESN'T CARE AT ALL BUT MUTANT VIRUS IS REDUCED BY A LOG OF 90% REDUCTION IN VIRUS REPLICATION OVER TIME. IF YOU DEPLETE OUT THE INTERFERON RECEPTOR, THAT COMPENSATES FOR THIS MUTATION IN TVV AS EXPECTED. SIMILARLY IF YOU DEPLETE PROLIDASE FROM THESE CELLS THAT COMPENSATES FOR THIS MUTATION. AND SO IT ESSENTIALLY COMPLIMENT S THE SENSITIVITY OF THIS VIRUS IN THE PRESENCE OF TYPE 1 INTERFERON. SUGGESTING PROLIDASE IS WORKING IN THE SAME SIGNALING PATHWAY. IMPORTANTLY IN MOUSE MODEL OF TBV, HERE WE HAVE SET IT UP WITH ABOUT 50% SUSCEPTIBILITY TO WILD TYPE VIRUS. IF YOU INDIRECT WITH ATTENUATED -- WE DON'T KNOW IT'S ATTENUATED YET. IF WE INFECT WITH VIRUS THAT NO LONGER BINDS PROLIDASE OR DOWN REGULAR PLATE THE RECEPTOR THE VIRUS IS ATTENUATED ASSOCIATED WITH REDUCED VIREMIA, ALMOST UNDETECTABLE LEVELS. AND DELAYED ENTRANCE INTO THE CENTRAL NERVOUS SYSTEM AND REPLICATION IN THE BRAIN. SO THIS SUGGESTS AT LEAST PROVIDES SOME PROOF OF CONCEPT YOU CAN RATIONALLY DESIGN OR TARGET SOME OF THESE INTERFERON ANTAGONISM STRATEGIES FOR REVERSE ENGINEERING THESE VIRUSES AS LIVE ATTENUATED VACCINE CANDIDATES. BUT OF COURSE WE CERTAINLY HAVEN'T ATTENUATED THIS VIRUS ENOUGH IN THIS SENSE BUT AT LEAST IT SAYS WE CAN DO IT. SO WHAT IS THE FUNCTION OF PROLIDASE THEN? IT HASN'T REALLY HAD A ROLE IN TERMS OF INTERFERON REGULATION, AND THEM TOOK US A LONG TIME TO UNDERSTAND BECAUSE THE INTERFERON RECEPTOR IS GENERALLY CONTROLLED IN ABUNDANCE THROUGH DEGRADATION OF THE RECEPTOR AND A LOT OF VIRUSES DO TARGET THE INTERFERON RECEPTOR AS MIGHT BE EXPECTED BUT THEY DO SO BY ACCELERATING DEGRADATION OF THE PROTEIN. WHEN WE LOOK AT THIS IN CONTEXT OF CELLS KNOCKED DOWN FOR PROLIDASE EXPRESSION WE FOUND IF WE TREATED CELLS WITH CYCLE EXPRESSION OVER TIME WE FOUND NO DIFFERENCE IN THE HALF LIFE OF THAT PROTEIN. SO THERE'S NO INCREASE TURN OVER OF INTERFERON RECEPTOR. HOWEVER, THE INTERFERON RECEPTOR IS HIGHLY GLYCOSYLATED AS YOU CAN SEE HERE, SO THESE TWO BANDS OF THE RECEPTOR ARE ACTUALLY THE PARTIALLY GLYCOSYLATED AND FULLY GLYCOSYLATED FORM. IF YOU TREAT TMGA YOU GET RID OF AND GO DOWN TO EXPECTED MOLECULAR MESS OF THAT PROTEIN. AND WHEN WE ENUMERATED OR SEMIQUANTIFIED THE RATIO OF THIS UPPER BAND TO LOWER BAND YOU CAN SEE -- YOU CAN ALMOST WATCH THIS PROTEIN MATURE AS IT MOVES THROUGH GLYCOSYLATION PROCESS AND THIS IS LARGELY ABSENT IN THE ABSENCE OF PROLIDASE. SO THIS LEADS TO A CONCLUSION THAT PROLIDASE IS HERE PROBABLY RESIDENT SOMEWHERE IN THIS PROTEIN QUALITY CONTROL PATHWAY WHERE YOU HAVE THE RECEPTOR GOING FROM THE ER THROUGH THE TP EXPRESSION ON SURFACE MATURING AND BEING GLYCOSYLATED AS IT GOES. THE CONTEXT FLAVIVIRUSES TBB SHOPE AS WELL, YOU HAVE SUPPRESSION OF THIS ACTIVE GNAR MATURATION THROUGH SUPPRESSION OF GLYCOSYLATION AND EVENTUALLY DEGRADED. IN SUBSEQUENT WORK WHICH I DON'T REALLY HAVE TIME TO TELL YOU ABOUT IS THAT WE THINK THAT PROLIDASE IS ACTING IN A PROTEIN COMPLEX TO PROTECT THE INTERFERON RECEPTOR FROM UBIQUITINATION AND ALLOW ITS MATURATION. SO IN HUMANS THERE'S A RARE GENETIC DISORDER ASSOCIATED WITH MUTATIONS KNOWN AS PROLIDASE DEFICIENCY. THIS IS ONE OF THE PATIENTS THAT WAS SEEN HERE AT THE NIH CLINICAL CENTER AND YOU CAN SEE FROM THESE IMAGES COURTESY OF MAN FRED BALM, THESE PATIENTPS ALTHOUGH CLINICALLY VERY VARIABLE, SOME PATIENTS AREN'T DIAGNOSED UNTIL THEY'RE IN THEIR 20s OR LATER. BUT IN THE MOST SEVERELY AFFECTED PATIENTS YOU HAVE THESE CHRONIC INTRACTABLE ULCER USUALLY THE LOWER EXTREMITIES, RECURRENTS INFECTIONS THOUGH INTERESTINGLY GENERALLY NOT VIRAL. USUALLY BACTERIAL. FACIAL FEATURES, COGNITIVE IMPAIRMENT, PULMONARY INFECTIONS HYPER IG AND MOST SERERLY AFFECTED PATIENTS DEATH BY ABOUT AGE FOUR OR FIVE YEARS OLD. WE WERE LUCKY ENOUGH OR I SHOULD SAY THAT AT LEAST IN THE LITERATURE IT'S BEEN HYPOTHESIZED THAT THERE MUST BE OTHER FUNCTIONS OR ROLES OF PROLIDASE BECAUSE COLLAGEN METABOLISM IN PARTICULAR ARE NOT SUFFICIENT TO EXPLAIN DISEASE SO WE WERE FORTUNATE ENOUGH TO BE PUT IN CONTACT FIRST WITH ALEXANDER FREEMAN AND MANFRED BAUM AND THEN ANOTHER LAB IN ITALY AND WE RECEIVED SOME PRY MAY RECALL FIBROBLASTS FROM THESE PATIENTS. SO THIS STAINING HERE IS JUST STAINING FOR THE INTERFERON RECEPTOR IN CONTROL FIBROBLASTS NOT TREATED WITH INTERFERON. OR TREATED WITH TYPE 1 INTERFERON YOU CAN SEE RECEPTOR GOES TO THE NUCLEUS WHICH IS UNKNOWN BUT NOT WELL UNDERSTOOD PHENOMENON. IF WE NOW LOOK IN THE CELLS OF THESE DIFFERENT PATIENTS YOU CAN SEE THAT INTERFERON RECEPTOR ACCUMULATING ABNORMALLY INTRACELLULARLY. AND THAT SO HERE ARE CLOSE UPS IN THE SECOND PATIENT YOU CAN SEE WE SEE THIS IN ABOUT 10 TO 15% OF CELLS. AT THE POPULATION LEVEL THIS IS ASSOCIATED WITH AN OVERALL LOSS OF INTERFERON RECEPTOR AND FAILURE TO SIGNAL. IF WE ADD INTERFERON TO FIBROBLASTS AND LOOK FOR ACTIVATION OF SIGNALING INTERMEDIATES, YOU CAN SEE PHOSPHORYLATED STAT 1 HERE IS GREATLY DIMINISHED IN THESE PATIENTS. THIS IS A HIGHER EXPOSURE. SO YOU CAN SEE THE EXTENT TO WHICH THAT IS. AND THEN YOU DON'T GET UPREGULATION OF ASSOCIATED CANONICAL INTERFERON STIMULATED GENES SO THIS STUDY STARTED AS A QUESTION HOW THESE VIRUSES ANTAGONIZE INTERFERON RESPONSES, AND HAS LED TO AN UNDERSTANDING OF A NEW MECHANISM THAT CONTROLS CELLULAR RESPONSIVENESS TO TYPE 1 INTERFERON AND LINKED PROLIDASE DEFICIENCY AS A PRIMARY IMMUNE DEFICIENCY IN PEOPLE. DESPITE THIS UNDERSTANDING AND THE ABILITY OF THESE VIRUSES TO EFFICIENTLY ANTAGONIZE THE HOST INTERFERON RESPONSE, TYPE 1 INTERFERON REMAINS ABSOLUTELY CRITICAL FOR HOST RECOVERY FROM INFECTION. THIS MEANS THERE'S ISGs ABLE TO PROTECT FROM INFECTION, WHAT THE VIRUSES ARE TRYING TO SHUT DOWN. BUT WE DON'T UNDERSTAND THE FULLY HOW THIS SYSTEM WORKS. SO IN THE SECOND PART OF THIS TALK I WANT TO MOVE TO THIS NEWER STUDY, TRYING TO PROBE ISGs THAT PROTECTED EFFICACY AGAINST THESE FLAVIVIRUSES. THERE'S HUNDREDS OF ISGs UPREGULATED IN RESPONSE TO INTERFERON STIMULUS. MOST OF THESE ISGs GENERALLY ANTIVIRAL. SO THEIR FUNCTIONS TO SHUT DOWN TRANSCRIPTION TRANSLATION INCREASE APOPTOSIS, DETECT MEMBRANE CURVATURE, THEY CAN REALLY GENERALLY CREATE INHOSPITABLE ENVIRONMENT FOR VIRUS REPLICATION AS A WHOLE. AS AN RNA VIRUS YOU DEFINITELY WANT TO AVOID THIS. BUT THE MORE INTERESTINGLY I THINK ARE A NUMBER OF VERY VIRUS SPECIFIC INTERFERON STIMULATED GENES THAT CONTRIBUTE TO HOST SUSCEPTIBILITY. IF WE CAN UNDERSTAND THESE RELATIONSHIPS HAVE OCCURRED OVER MILLENNIA OF VIRUS HOST EVOLUTIONS SO IF WE CAN UNDERSTAND THOSE PARTICULAR INTERACTIONS, I THINK IT TELLSES US A LOT ABOUT VIRUS HOST EVOLUTION AND VIRAL EMERGENCE POTENTIALLY. SO THE LARGEST CLASS OF PROTEINS THAT ACT IN A HOST SPECIFIC MANNER ARE CALLED THE TRIM PROTEINS OR TRY PAR TIDE MOTIF PROTEINS SO NAMED BECAUSE OF CHARACTERISTIC DOMAIN STRUCTURE. THE END TERMINUS HERE THEY GENERALLY HAVE A RANGE DOMAIN AN FUNCTION IS E 3 UBIQUITIN LIGASE. THE MOST FAMOUS OF THESE TRIM PROTEINS IS TRIM 5 WHICH MANY OF YOU MAY KNOW. I KNOW SOME OF YOU KNOW VERY WELL. I WANT -- I WANT TELL YOU COUPLE OF INTERESTING FACTS ABOUT TRIM 5, IT'S A TRY STRICKS FACTOR IN PRIMATES THAT RECOGNIZE LATTICE STRUCTURE OF RETRO VIRUSES THIS EVENT IS THOUGHT EXQUISITELY SPECIFIC TOWARDS RETRO VIRUSES, IT FORMS THIS AMAZING HEXAGONAL CAPSID BASED ON CRYO-EM STUDIES AND STRUCTURAL STUDIES. BUT DOES SO IN A VERY SLICER SPECIFIC HOST SPECIFIC MANNER. SO THE TRIM 5 PROTEIN FROM OLD WORLD MONKEYS IS VERY ABLE TO RECOGNIZE TESTIMONY CAPSID LATTICE PROTEIN FROM HIV-1 AND IT THEN CAUSES EITHER PREMATURE UNPROTEIN WHICH IS THOUGHT TO COMPROMISE VIRUS INFECTIVITY OR THERE'S A SECOND BLOCK THAT HOWEVER HUMAN HIV-1 TRIM 5 IS NOT THOUGHT TO BE FUNCTIONAL HIV ACQUISITION OR DISEASE PROGRESSION. WE NAIVELY IN SOME WAYS DECIDESSED TO LOOK AT WHETHER OR NOT TRIM 5 COULD HAVE ANTIVIRAL EFFECTS ANTI-FLY SRI VIRUSES. HI I'M NOT TELLING YOU THE BACK STORY WHY YOU THOUGHT THIS MIGHT BE INTERESTING BUT IN PART IT WAS BECAUSE HE HAD VIRUSES LIKE DENGUE YELLOW FEVER ZIKA, ALL EMERGING FROM TRANSMISSION CYCLES THAT INVOLVE NON-HUMAN PRIMATES. WHEREAS OTHER VIRUSES OF THIS HUGE GROUP UTILIZING OTHER RODENTS BIRDS, SWINE, ET CETERA. SO WE WONDERED WHETHER THERE WAS SOME HOST SPECIFIC RESTRICTION FACTORS AND TRIM 5 WAS THEREFORE A CANDIDATE. WE KNOW IT HAS HIGH CONSEQUENCE FOR HUMAN SUSCEPTIBILITY TO VIRUS INFECTION. TO DO THIS WE MADE STABLE CELL LINES AGAIN, ABOUT TOOL KIT, WE EXPRESSED VARIOUS TRIM 5 CONSTRUCTS, FOR PURPOSES OF THIS TALK JUST CONCENTRATE ON THE HUMAN TRIM 5 RHESUS TRIM 5 OR HUMAN TRIM 22 AS CONTROL. WE TEST IN CELLS TO SUSCEPTIBILITY FOR THESE PSEUDOTYPE HIV-1. HUMAN TRIM 5 LOOKS LIKELY NON-FUNCTIONAL, RHESUS TRIM 5 IMPACTS A VERY STRONG RESTRICTION AND SO THESE CELLS ARE ACTING IN WAY THEY SHOULD IN THE CONTEXT OF RETRO VIRUS INFECTION. WE THEN INFECTED WITH VARIOUS FLAVIVIRUS. THIS IS TICK BORNE ENCEPHALITIS VIRUS, IT MAY SEEM LITTLE IRRELEVANT BUT RELEVANCE WILL COME LATER. WE INFECTED WITH VARIOUS FLAVIVIRUS AND YOU CAN SEE THAT TICK 1 ENCEPHALITIS VIRUS IN PARTICULAR IS SUSCEPTIBLE TO TRIM 5 MEDIATED SUPPRESSION. IN CELLS EXPRESSING HUMAN TRIM 5 YOU HAVE REDUCTION OF REPLICATION BY ABOUT A LOG. IN THE CONTEXT OF RHESUS, THIS IS UP TO THREE LOGS OR A THOUSAND FOLD REDUCTON IN VIRUS REPLICATION DURING THE EXPONENTIAL STAGE OF VIRUS. IT DOES EVENTUALLY CATCH UP BUT CONSISTENT WITH VIRUSES SATURATING RESTRICTION FACTORS. THIS IS NOT A PROBLEM. WE LOOKED AT VARIOUS OTHER FLAVIVIRUSES. HERE IS THE PANEL. YOU CAN SEE IF TICK BORN TBB LANGETT VIRUS YOU HAVE QUITE GOOD SUPPRESSION OF VIRUS REPLICATION PARTICULARLY BY RHESUS TRIM 5. HOWEVER WEST NILE DENGUE ZIKA AND YELLOW FEVER ARE NOT SUSCEPTIBLE TO RESTRICTION. THE INTERESTING ONE HERE IS POISON VIRUS, SUSCEPTIBLE NOT TICK BORNE, I'LL TRY TO COME BACK AT THE END BUT WE HAVE THIS CLEAR -- BESIDES THAT VIRUS WE HAVE CLEAR DEMARCATION BETWEEN THE TICK BORNE VIRUS AND MOSQUITO BORNE VIRUS IN SUSCEPTIBILITY TO TRIM 5. WHERE ARE WE GOING? SO AS I SAID, HUMAN TRIM 5 HAS NOT REALLY BEEN SHOWN TO BE FUNCTIONAL IN THE CONTEXT OF THESE RETRO VIRUS -- RETRO VIRUSES, AT LEAST CURRENTLY EXISTING RETRO VIRUSES SO WE WANTED TO LOOK FURTHER WHETHER OR NOT HUMAN TRIM 5 IS REALLY FUNCTIONAL AND TO DO THIS WE KNOCKED OUT TRIM 5 EXPRESSION USING CRISPER CAS IN HAP 1 CELLS. YOU CAN SEE WITH THIS EXAMPLE, SUSCEPTIBLE VIRUS, THIS VIRUS IS REPLICATES QUITE A LOT BETTER ABOUT A LOG IN THE ABSENCE OF TRIM 5. IMPORTANTLY IF WE TREAT CELLS WITH INTERFERON AND NOW YOU -- THIS IS PRETREATING CELLS WITH INTERFERON FOR SIX HOURS PRIOR TO INFECTION YOU CAN REDUCE VIRUS REPLICATION AS EXPECTED. IN THE ABSENCE OF TRIM 5 INTERFERON DOESN'T WORK SO THIS IS A POWERFUL RESTRICTION FACTOR IN CONTEXT OF SUS IS HE WANTABLE VIRUSES. BUT ZIKA IS AN EXAMPLE OF A VIRUS RESISTANT WITH NO EFFECT. AND WE FINISHED THIS NOW, THANK YOU, WITH OUR FULL PANEL OF VIRUSES AND WE FIND THIS AFFECT TO BE CONSISTENT ACROSS THOSE VIRUSES THAT I WAS TALKING ABOUT EARLIER. SO TRIM FIVE IN THE CONTEXT OF RETRO VIRUSES, WORKS IN THE POST ENTRY EVENT SO WE WANTED TO LOOK AT WHERE THIS WAS OCCURRING IN THE CONTEXT OF FLAVIVIRUSES. THE REPLICATION COMPLEX OF FLAVIVIRUS CAN LOOSELY BE DEFINED AT THIS RESOLUTION OF FLUORESCENCE BY CO-STAININGSTAINING WITH MS 3, THE NON-STRUCTURAL PROTEIN, THE VIRAL PROTEASE AS WELL AS DOUBLE STRANDED RNA SO HERE YOU CAN SEE THESE PERINUCLEAR FOCI. IF WE NOW TAKE CELLS STABLY EXPRESSING RHESUS TRIM 5 AND INFECT WITH A SENSITIVE VIRUS, THIS IS LANGETT VIRUS AGAIN AND STAIN FOR MS 3 YOU CAN SEE TRIM 5 WHICH HAS THIS GRANULAR STAINING IN UNINFECTED CELLS IS RECRUITED TO THOSE REPLICATION SITES, HERE IS ANOTHER NON-STRUCTURAL PROTEIN NS 5, EXPRESSED MORE WIDELY THROUGHOUT THE CELL, NOT ONLY SITES OF REPLICATION AND DOESN'T SEEM TO HAVE THAT SAME SPATIAL DISTRIBUTION OR RELATIONSHIP WITH TRIM 5, YOU HAVE THIS INTERACTION BUT LIKELY TO BE OCCURRING WHERE NS 5 AND 3 INTERACT IN THE REPLICATION COMPLEX. THIS IS SUPPORTED BY THIS HERE, SO THIS IS JUST -- THESE ARE ALL CONTROLS, YOU JUST HAVE CONCENTRATE ON LAST THREE LANES SO WE HAVE TAKEN EITHER OUR EMPTY VECTOR VELLS, CELLS EXPRESSING HUMAN TRIM 5 OR RHESUS TRIM 5 AND AFFECTED WITHIN LANGET VIRUS, YOU CAN SEE BY STAINING OF THE NS 3 PROTEIN THE VIRUS IS RESTRICTED. IF WE PRECIPITATE OUT NS 3 SPECIFICALLY CO-PRECIPITATE OUT NS 5 AND TRIM EITHER HUMAN OF RHESUS SUGGEST SUGGESTING THESE INTERACTIONS ARE LIKELY TO BE OCCURRING AT THAT REPLICATION COMPLEX. SO IS IT NS 5 INTERACTING WITH FAVORITE PROTEIN? UNFORTUNATELY NOT. YOU CAN SEE IF YOU CO-EXPRESS JUST THE NS 5 PROTEIN FROM ONE OF THESE SENSITIVE VIRUSES, YOU HAVE NO CO-LOCALIZATION WITH TRIM 5. BUT NOW IF YOU EXPRESS THE NS 3 PROTEIN HERE WE HAVE MADE IN THE CONTEXT OF NS 2B 3 FUSION, THIS IS A FUNCTIONAL PROTEASE THAT LOCALIZES TO THE ER. SO IT HAS THAT LOVELY RE RETICULAR PATTERN. IF YOU CO-EXPRESS WITH RHESUS TRIM 5 YOU CAN SEE THAT NS 3 IS NICELY COLOCALLIZING WITH TRIM 5 IN THESE TRIM BODIES, IN THIS -- IT IS QUITE SURPRISING. JUST AS CONTROLS HERE IS Z DA NS 3 BY ITSELF AND WHEN WE CO-EXPRESS WITH RHESUS TRIM YOU CAN SEE THAT THOSE PROTEINS ESSENTIALLY IGNORE EACH OTHER AND IF WE LOOK AT DOUBLE STRANDED RNA STAINING TO LOOK AT SITES OF REPLICATION IN INFECTED CELLS, ZIKA INFECTED CELLS WITH RHESUS TRIM 35, -- 5, RHESUS TRIM 5 DOESN'T RECOGNIZE THOSE CELLS ARE INFECTED. FINALLY THE MECHANISM OF RESTRICTION FOR TRIM 5 ONE OF THESE SENSITIVE VIRUSES, HERE WE HAVE THE NS 2B 3 PROTEIN, THERE'S TWO BANDS BECAUSE THIS IS THE IMMATURE PRECURSOR, THIS IS THE AUTOCLEAVED MATURE PROTEIN. IF YOU EXPRESS THIS WITH INCREASING AMOUNTS OF TRIM 5, YOU SEE A LOSS OF NS 3 AND THERE'S TWO PATHWAYS THAT THIS COULD BE OCCURRING THROUGH IN TERMS OF PROTEIN DEGRADATION IN THE PROTEOSOME OR LYSOSOME. IF WE INHIBIT CELLS WITH USING THAT INHIBIT LYSOSOME WHICH IS IMPLICATED THE TRIM 5 MEDIATED RESTRICTION OF RETRO VIRUSES YOU RECOVER A LOT OF NS 3 PROTEINS BUT THE RATIO OF LOSS OF NS 3 STILL REMAIN IN THE PRESENCE OF TRIM 5. BUT WHEN YOU INHIBIT THE PROTEASOME USING NG 132 YOU RECOVER THE VAST MAJORITY OF THAT PROTEIN SUGGESTING THAT THE TRIM 5 IS UTILIZING THE PROTEASOME. AND THEN FINALLY, WHAT IS TRIM 5 SEEING? HERE IS A REPRESENTATION OF THE DOMAIN STRUCTURE OF THIS CRITICAL PROTEIN FOR VIRUS REPLICATION. NS 2B IS GREEN, IT'S REQUIRED THIS CENTRAL DOMAIN FORMS AN INTEGRAL PART OF THE PROTEASE. IT PERFORMANCE PART OF THE ACTIVE SITE OF PROTEASE SO WITHOUT NS 2B -- SORRY, WITHOUT NS 2B YOU DON'T HAVE A FUNCTIONAL ENZYME HERE. HERE IS THE PROTEASE DOMAIN, THERE'S A SHORT LINKER AND THEN YOU HAVE HELOCASE DOMAIN IF YOU EXPRESS NS 3 BY ITSELF YOU DON'T SEE DEGRADATION. IF WE EXPRESS THIS LITTLE FUNCTIONAL PROTEASE, WE ALSO, I DON'T KNOW -- WE DON'T SEE DEGRADATION SO IS IT'S NOT NS 2 MEDIATING AND IT'S NOT NS 3 MEDIATING IT. BUT IF YOU GET LUCKY AND MAKE A MISTAKE IN THE LAB, AND MAKE THIS CONSTRUCT THAT HAS JUST PROTEASE DOMAIN, THE -- SORRY, THE -- YEAH, THE PROTEASE DOMAIN FROM NS 2B, PROTEASE DOMAIN FROM NS 3 AND SHORT LINKER DOMAIN HERE, NOW YOU GET DEGRADATION. SO IT'S LIKELY THAT TRIM 5 IS ACTUALLY RECOGNIZING THE VIRAL PROTEASE AS CONFIRMATIONALLY DEPENDENT STRUCTURE. WHICH IS INTERESTING. THIS IS MORE FOR THE TRIM 5 A FISHNA DOES. IF U YOU -- AFICIONADOS. THIS IS THE PARENT STATE OF WHAT IS KNOWN ABOUT RESTRICTION OF TRIM 5 IN TERMS OF THE RETRO VIRUSES. THE ONLY THING THAT I WANT TO SUMMARIZE HERE IS THAT IN CONTRAST TO WHAT IS CURRENTLY THOUGHT OF AS TRIM 5, IT'S EXQUISITELY SPECIFIC FOR THESE MOLECULAR PATTERNS OF RETROVIRAL CAPSID LATTICES, IT SEEMS INSTEAD THIS EXAMPLE ILLUSTRATES TRIM 5 TO HAVE REMARKABLE PLASTICITY IN RECOGNITION OF DIVERSE VIRAL PATTERNS. IT'S HARD TO SAY THAT WITHOUT THE STRUCTURAL INFORMATION OF WHAT TRIM 5 IS SEEING BUT AT LEAST I CAN SAY IT'S STRUCTURALLY AND NON-STRUCTURAL. IMPORTANTLY HUMAN TRIM 5 IS FUNCTIONAL IN THE CONTEXT OF THESE VIRUSES. WHY IS THIS SO IMPORTANT AND MORE THAN JUST AN ACADEMIC QUESTION? SO THIS IS MY LAST SLIDE, MY SUMMARY SLIDE H THE RELATIVE HOST USAGE OF THESE FLAVIVIRUSES AND THEIR RELATIVE ABILITY TO EMERGE TO HUMANS AND START TO ESTABLISH OVER CYCLES OF REPLICATION. SO THE TICK 1 VIRUSES YOU DON'T HAVE TO OBVIOUSLY SEE THESE, BUT HERE IS THE TICK BORNE VIRUSES IN BLUE. THESE ARE THE MOST ANCIENT OF THE VECTOR BORNE FLAVIVIRUS AND THEY'RE ALSO EVOLVING AT APPROXIMATELY HALF THE RATE OF MOSQUITO BORNE VIRUS. THIS IS GIVING MORE ANCIENT SNAP SHOT INTO WHAT THESE -- THE ANCESTRAL FLAVIVIRUS LOOKS LIKE. THESE ARE THE VIRUSESNA ARE BEING SUPPRESSED BY TRIM 5, REPLICATION. I'M SORRY TRIM 5 ANTIVIRAL AFFECTS. HOWEVER MOSQUITOES THAT HAVE BEEN DIVERGE FROM THESE MOSQUITO FROM THE TICK BORNE VIRUSES WHICH YELLOW FEVER IS OLDEST ONE WE HAVE TESTED IN AT LEAST IN EVOLUTIONARY TERMS, ARE ABLE TO AVOID THE ANTIVIRAL VEX OF TRIM 5. THIS ALLOWS THEM IN PART AT LEAST TO UTILIZE PRIMATES AS HOSTS AND POTENTIALLY ESTABLISH A SUFFICIENT VIREMIA TO ENABLE MOSQUITO BORNE TRANSMISSION. IT POTENTIALLY INCREASES HUMAN SUSCEPTIBILITY TO THESE VIRUSES IN PARTICULAR. IN GOING FORWARD WHAT WE NEED TO KNOW IS HOW SIGNIFICANT IS TRIM 5 IN LIMITING FLAVIVIRUS EMERGENCE. IT'S CERTAINLY I THINK HAS BEEN INVOLVED IN IN DRIVING FLAVIVIRUS EVOLUTION. THAT'S A BIG STATEMENT THAT WE'LL KEEP WORKING ON TRYING TO APPROVE THAT BUT WE NEED TO NOW KNOW HOW SIGNIFICANT TRIM 5 IS AND WHETHER OR NOT THERE ARE HUMAN POLYMORPHISMS IN TRIM 5 THAT AFFECT SUSCEPTIBILITY TO THESE VIRUSES IN PARTICULAR. I BET WE CAN FIND THEM WITH TVB. SO WHILE THIS SUMMARIZES THERE IN TERMS OF EVOLUTION, YOU MIGHT ASK WHY WEST NILE DOESN'T CIRCULATE IN NON-HUMAN PRIMATES IS ALSO RESISTANT BECAUSE IN TERMS OF EVOLUTION IT WAS THE '80s SPECIES VIRUSES THAT SPLIT OFF FIRST AND THE KULEX VIRUSES SPLIT OFF LATER AND I THINK WEST NILE IS RETAINED THAT RESISTANCE. THAT ESTABLISHED FOR THE '80s GROUP. WITH THAT, I HOPE THAT WASN'T TOO AMBITIOUS TRYING TO TELL TWO STORIES. BUT I JUST WANT TO LET YOU KNOW WE'RE OUT HERE AT THIS ROCKY MOUNTAIN LABORATORIES CAMPUS THAT'S OUR LITTLE SLICE OF HEAVEN MOST DAYS. IN ROCKY MOUNTAINS HERE. I CAN'T POSSIBLY THANK EVERYBODY ON A SLIDE BUT THIS IS MY CURRENT LAB HERE. THERE'S BEEN LOTS OF PEOPLE THAT HAVE BEEN INVOLVED IN THE NS 5 STORY OVER THE YEARS. ALEX CRO MIXERC IN QUEENSLAND, TRIM 5 COULDN'T HAVE BEEN DONE WITHOUT EFFORTS OF SARA SAWYER IN COLORADO. ALONG WITH A FEW STUDENTS WHO WERE SUPPORTED THROUGH THE IN ROAD PROGRAM, OMYRA AND VANESSA. OUR EARLY START WOULDN'T HAVE HAPPENED WITHOUT SASHA PUTNAM AND STEVE WHITEHEAD. AND I HAVE ENJOYED IMMENSE SUPPORT FROM MY CHIEF HEINZFELDMAN, THE FORMER SD OF NIAID KATHERINE ZOON AND STEVEN HOLLAND AND I'M ETERNALLY GRATEFUL TO BE HERE AMONGST YOU. THANK YOU. [APPLAUSE] >> SO THERE'S TIME FOR QUESTIONS. I'LL ASK YOU TO GO TO THE MICROPHONE SINCE WE ARE TRANSMITTING TO POINTS FAR AWAY. INCLUDING ARAMELLA. FROM >> VERY NICE TALK, SONJA, YOU MAY IMAGINE THE PROLIDASE IS INVOLVED IN MORE THAN JUST IFNAR 1. ARE YOU LOOKING FOR OTHER RECEPTORS THAT COULD CONTRIBUTE TO THE FACT OF THE BLOCK PREGNANT OF THE PROLIDASE? >> WE HAVEN'T BUT I THINK THAT THIS IS NOW POSSIBLE WITH ALL THE PROTEOMIC APPROACHES THAT ARE AVAILABLE. I THINK WE SHOULD GO BACK AND REVISIT THAT. WE WERE MOST CONCERNED WITH FINDING RECEPTORS THAT WEREN'T AFFECTED SO WE CAN SAY THERE'S SOME SPECIFICITY TO IT BUT WE HAVEN'T GONE BACK AND LOOKED. THERE'S GOT TO BE MORE. >> SECOND QUESTION, I WAS INTRIGUED THE PROLIDASE DEFICIENT PATIENTS SHOW MORE SUSCEPTIBILITY TO -- THAN VIRAL. CAN YOU SPECULATE WHAT'S GOING ON THERE? >> THERE'S TWO THINGS THAT I CAN SAY TOWARDS THAT. SO EVEN THOUGH THESE PATIENTS ARE UNABLE TO RESPOND VERY WELL TO TYPE 1 INTERFERENCE, WHEN YOU LOOK AT BASAL LEVELS OF ISGs, THEY'RE THROUGH THE ROOF. SO ACTUALLY I WOULD -- I QUESTIONED WHETHER THESE PATIENTS ACTUALLY HAVE SOME KIND OF INTERFERONOPATHY GOING ON. SO I REACHED OUT TO YANNICK PROVOST AND HE HAD SOME PATIENTS WHO HAD A MILDER PHENOTYPE THAT WAS CONSISTENT WITH AN INTERFERONOPATHY AND HE DID IT FOR PROLIDASE AND THEY WERE DEFICIENT IN PROLIDASE. SO NOT THE CLASSIC INTERFERONOPATHY WHERE YOU HAVE A LOT OF INTERFERON BEING MADE. IN FACT WHEN WE LOOK AT BETA EXPRESSION OR INTERFERON ALPHA SUBTYPE EXPRESSION BY REAL TIME WE DON'T SEE A LOT OF UPREGULATION. BUT THERE ARE INTERFERON INDEPENDENT WAYS THAT YOU CAN GET ISG UPREGULATION. SO THEY ACTUALLY HAVE THIS INFLAMMATORY PHENOTYPE ASSOCIATED WITH ELEVATED ISGs. WHEN YOU LOOK AT BACTERIAL INFECTIONINGS OCCURRING FOLLOWING SOMETHING LIKE INFLUENZA INFECTION, SOME OF THOSE COMPLICATIONS ARE -- HAVE BEEN ATTRIBUTED EVEN TO INDIVIDUAL ISGs. SO IT MAYBE MORE OF THAT KIND OF PHENOTYPE GOING ON. I THINK THAT'S IT, TWO POINTS. BUT THAT WAS THE MAIN. THEY'RE HIGH IN ISGs. >> SONJA, THAT WAS REALLY ELEGANT AND ENJOYED HEARING MORE ABOUT THAT. TYPE 1 INTERFERONS, COME ON, THEY' EAR JUST YOU START AT ONE BECAUSE YOU HAVE TO GO HIGHER. SO YOU SHOWED ELEGANTLY THAT THE TYPE 1 INTERFERONS ARE INHIBITED BY SOME OF THESE VIRAL PROTEINS THROUGH THE INH INHIBITION OF IFNAR OR DEGRADATION OF IFNAR 1. IF WE THINK THAT STAT ACTIVATION IS A CRITICAL PART OF THE INTERFERON PATHWAY, WHAT HAPPENS IF YOU GO TO SOME OF THOSE LIKE MORE INTERESTING INTERFERONS, WHAT HAPPENS WITH TYPE 2 OR TYPE 3 INTERFERONS THAT CAN ALSO ACTIVATE STAT 1 IN SOME OF THOSE CASES? ARE THOSE ALSO AFFECTED BY NS 5 AND SO ON? >> SURPRISINGLY NOT SO MUCH. WHEN WE FIRST DID THIS WORK, IT DOES SEEM MORE SPECIFIC TO TYPE 1 INTERFERON FOR THESE VIRUSES. WHEN WE FIRST DID THIS WORK WE LOOK AT TYPE 2 INTERFERON AND WE SAW A SUPPRESSION. IT IS A WEAKER SUPPRESSION. THESE VIROS ALSO ENCODE NON-STRUCTURAL PROTEINS AND INDUCE ER STRESS AND OTHER THINGS AND I THINK THAT THAT IMPACTS ON SOME OF THOSE SIGNALING PATHWAYS. WHY DON'T THEY CARE ABOUT INTERFERON GAMMA, FOR EXAMPLE? TYPE 3 INTERFERONS AT LEAST THE VIRUSES THAT ARE SUPPRESSING THE SIGNALING PATHWAY AT THE STAT, ZIKA, DENGUE, YELLOW FEVER , THEY'RE AFFECTING TYPE 3 INTERFERES TOO BY DOING THAT BUT WEST NILE AND TVB AREN'T DOING THAT. SO IT SEEMS TO BE QUITE EXCLUSIVE. I DON'T KNOW WHEY DON'T -- MAYBE THEY WANT SOME OF THOSE RESPONSES AROUND ACTUALLY. >> ONE OF THE LESSONS WE LEARNED FROM STUDYING PEOPLE WHO WERE GETTING VIRAL ILLNESSES IS THATMENT SO PEOPLE GET VERY SHEEVE SOME PEOPLE GET SEVERE DISEASE SOME NOT SO MUCH. OBVIOUSLY A LOT OF THESE FLAVIVIRUS INFECTIONS ARE SEVERE IN MOST OF THE POPULATION BUT WEST NILE IS NOT NECESSARILY, RIGHT? THERE'S A PRETTY LOW PERCENTAGE OF SEVERE DISEASE AND MOST PEOPLE HAVE SUBCLINICAL OR MILD DISEASE. >> CORRECT. >> HAS ANYONE LOOKED IN THOSE PATIENTS WHO GET SEVERE DISEASE THE DEFECTS IN ANY OF THE PATHWAYS YOU'RE INTERESTED IN? >> IN TERMS OF LOOKING FOR POLYMORPHISMS THAT MIGHT BE ASSOCIATED WITH DISEASE SEVERITY, THE ONES THAT HAVE COME UP FOR ALL THESE FLAVIVIRUSES ARE ASSOCIATED WITH INNATE IMMUNE PATHWAYS SO TLR 3 IS AFFECTED, CCR 5 INTERESTINGLY IS PROBABLY ONE OF THE MAIN ONES FOR WEST NILE AND TBV BOTH. SO THE POLYMORPHISMS ARE THERE. BUT I THINK SOME OF THESE OTHER PATIENTS LIKE ZIKA WOULD BE A FANTASTIC ONE, I KNOW PEOPLE ARE LOOKING BUT THAT WE DON'T KNOW ENOUGH ABOUT. THOSE TYPES OF THINGS YET. YEAH. >> DR. BEST, GREAT TALK. [APPLAUSE] >> THANK YOU.