Articles, Blog

The Structural Basis of Ebola Viral Pathogenesis

December 30, 2019


>>GOOD AFTERNOON, EVERYONE. THIS IS A SPECIAL DAY BECAUSE WE ARE IN THE FIRST DAY OF THE NIH RESEARCH FESTIVAL AND A SPECIAL DAY BECAUSE WE HAVE A REMARKABLE LECTURER AS PART OF OUR REGULAR WEDNESDAY AFTERNOON SERIES WHO IS HERE TO TEACH US SOMETHING PRETTY INTERESTING ABOUT VIRAL HEMORRHAGIC FEVER, SPECIFICALLY
EBOLA VIRUS. ERICA OLLMANN SAPHIRE HAS AN INTERESTING AND VERY PRODUCTIVE CAREER BRINGING HER TO WHERE SHE IS A PROFESSOR IN IMMUNOLOGY AND MICROBIAL SCIENCE AT THE SCRIPPS RESEARCH INSTITUTE. WE FOUND A PROFILE OF HER IN THE SAN DIEGO UNION TRIBUNE WHERE SHE WAS CALLED, THE VIRUS HUNTER. AND VARIOUS COMMENTS WERE MADE ABOUT HER CONTRIBUTIONS, WHICH ARE OBVIOUSLY SUBSTANTIAL. I WON’T COMMENT UPON WHAT THEY CALLED HER, ALIAS, STEEL MAGNOLIA. I THOUGHT THAT WAS ODD TO BE PUTTING IN A PROFILE OF A SCIENTIST BUT YOU CAN DECIDE FOR YOURSELF. SHE GOT HER UNDERGRADUATE DEGREE AT RICE WITH A DOUBLE MAJOR IN BIOCHEM AND CELL BIOLOGY AND ECOLOGIY AND EVOLUTIONARY BIOLOGY AND PH.D. AT THE SCRIPPS IN THE YEAR 2000. AND HAS BEEN THERE IN THIS REMARKABLE PRODUCTIVE ENTERPRISE FOCUSED ON TRYING TO UNDERSTAND HOW PATHOGENS EVADE AND USURP THE INNATE AND ADAPTIVE IMMUNE RESPONSES. SHE HAS QUITE A DIVERSITY OF PROJECTS GOING ON IN THE LAB INCLUDING LASSA AND MARRER AND EBOLA FEVER AND SHE IS AN EXPERT IN INCORPORATING DIFFERENT APPROACHES TO INTERESTING THIS INCLUDING IMFROG NOLOGY AND EXTRA CRYSTALLOGRAPHY — IMMUNOLOGY — AND I WANT TO POINT OUT AT THE END OF THE LECTURE, WE WILL HAVE TIME FOR QUESTIONS AND THE MICROPHONES ARE IN THE AISLE AND WELCOME TO THOSE OF YOU WHO ARE WATCHING ON THE WEB. WE’LL TRY TO BE SURE THAT QUESTIONS ARE POSED FROM THE MICROPHONE SO YOU CAN HEAR THEM AND THEN AT 4:00, WE’LL ADJOURN DOWN THE HALL FOR CONTINUATION OF INFORMAL CONVERSATIONS WITH OUR SPEAKER BUT ALSO THE ACTUAL FORMAL UNVEILING OF THE NEW FAES CENTER, WHICH I THINK YOU’LL WANT TO COME AND HAVE A LOOK AT BECAUSE IT IS REALLY QUITE BEAUTIFUL FACILITY AND WE’LL HAVE A RIBBON CUTTING AND A FEW HOPEFULLY SHORT SPEECHES AND THAT WILL MORPH INTO A POSTER SESSION WHERE THE SCIENTIFIC DIRECTORS WHO ARE THEMSELVES STANDING BY THEIR POSTERS TALKING ABOUT THEIR SCIENCE GIVING YOU A CHANCE TO HAD THE THEM UP WITH REALLY HARD QUESTIONS. SO IT WILL BE QUITE AN AFTERNOON. BUT, TO GET US GOING HERE, IN MA SUR, LET ME ASK YOU PLEASE TO, GIVE A WARM WELCOME TO ERICA OLLMANN SAPHIRE. [ APPLAUSE ]>>THANK YOU, DR. COLLINS. IT’S A REAL PLEASURE TO BE HERE. MY LABORATORY WORKS ON A LOT OF DIFFERENT VIRUSES. TODAY I’M GOING SHOW YOU CHAMPS FROM TWO OF THEM. THE FIRST ONE IS EBOLA VIRUS, A LONG VIRUS AND THE SECOND ONE IS A SMALLER ROUNDER PARTICLE AND IT BELONGS TO THE ARENA VIRUS FAMILY. WHAT THEY HAVE IN COMMON IS A SIMILAR DISEASE. THEY BOTH CAUSE HEMORRHAGIC FEVER AND THE SYMPTOMS LOOK SIMILAR ESPECIALLY AT FIRST. WHEREAS EBOLA IS QUITE RARE, LASSA IS UNFORTUNATELY EXTREMELY COMMON. THERE ARE HUNDREDS OF THOUSANDS OF CASES EVERY YEAR IN WESTERN AFRICA AND THE FEVER IS MOST FREQUENTLY IS IMPORTED TO THE UNITED STATES AND EUROPE. NOW WHAT ELSE THESE VIRUS VS. IN COMMON IS A VERY SMALL GENOME. EBOLA ENCODES SEVEN GENES LASSA ONLY 4. SO WHERE YOU HAVE 25,000 GENES AND YOU CAN MAKE 25,000 PROTEINS, THESE VIRUSES MAKE ONLY A FEW. SO, USING THIS VERY LIMITED PROTEIN TOOLKIT, HOW DOES A VIRUS ACHIEVE ALL THE DIFFERENT FUNCTIONS OF THE VIRUS LIFE CYCLE FROM ATTACH WANT TO A NEW HOST CELL, FUSION AND ENTRY AND ENCODING AND TRANSCRIPTIONS AND ASSEMBLY AND EXIT AND SOME OF THE MORE SOPHISTICATED FUNCTIONS FOR LOTS OF DIFFERENT PATHWAYS. HOW DO THEY DO THAT? ONLY A VERY FEW PROTEINS AT THEIR DISPOSAL. THIS IS THE GENOME OF LASSA VIRUS. THOSE ARE — THAT WAS EBOLA AND THIS IS LASSA. SO HOW DOES A HANDFUL OF PROTEINS CONSPIRE TO CREATE SUCH EXTRAORDINARY PATHOGENESIS IN HEMORRHAGIC FEVER? THE ANSWER IS THAT EACH PROTEIN THESE VIRUSES DO ENCODE IS ESSENTIAL. THESE VIRUS VS. NO JUNK. MANY OF THESE PROTEINS ARE MULTI-FUNCTIONAL AND SOME ARE EXTREMELY ADAPTABLE. BY STUDYING THE PROTEINS THESE VIRUSES MAKE, WE SEE THE VULNERABILITIES OF THE VIRUS, THE ACHILLES HEEL, THE PLACE TO TARGET A DRUG OR VACCINE OR ANTIBODY. BUT PERHAPS MORE IMPORTANTLY, WE CAN UNDERSTAND SOMETHING MORE ABOUT PROTEINS THEMSELVES. BECAUSE EVOLUTION HAS COMPELLED THESE PROTEINS TO BE REMARKABLE, TO DO MORE WITH LESS THAN OTHER PROTEINS BY STUDYING WHAT THESE PROTEINS ARE CAPABLE OF, WE LEARN ABOUT THE CAPABILITIES OF PROTEINS IN MOLECULAR BIOLOGY. SO I’LL SHOW YOU A FEW EXAMPLES. THE FIRST ONE COMES FROM THE FIRST STEP OF THE VIRUS LIFE CYCLE. SO THE FIRST STEP, THE VIRUS HAS TO FIND AND ATTACH TO A NEW HOST CELL. THIS IS ACHIEVED BY THE GLYCOPROTEIN CALLED GP. BOTH VIRUSES EXPRESS ONLY ONE PROTEIN ON THE SURFACE CALLED GP AND IT IS SOLELY RESPONSIBLE FOR ATTACHING WITH THAT CELL. SO EBOLUS VIRUS FILAMENT US. THIS HAS A MEMBRANE OF GREEN SURROUNDING A NUCLEO CAP SID. AND THERE ARE SPIKES. THOSE ARE FORMING 450 KILLADALTON TRIMERS AND THEY ARE QUITE HEAVILY GLYCOSYLATED. SO THE QUESTION YOU MIGHT ASK IS, IF THIS SPIKE IS IMPORTANT FOR ATTACHMENT AND ENTRY, WHAT DOES IT LOOK LIKE AND HOW DOES IT WORK? WE HAD TO MAKE ABOUT 140 VERSIONS OF THIS GP TO GET ONE THAT WOULD CRYSTALLIZE WELAND WE HAD TO THROW BACK 150. BEFORE WE HAVE A STRUCTURE, WE THINK OF A PROTEIN WITH AN END TERMINUS AND C TERMINUS. THIS IS CLEAVED IN THE PRODUCER CELL, WITH 2 SUB UNITS. A GP1 WHICH MEDIATES THE RECEPTOR BINDING AND GP2 WHICH MEDIATES FUSION. SO THE BP1 HAS RECEPTOR BINDING DOMAINS AND THE GP2 HAS TO UNDERGO A CHANGE. ALSO IN GP1 IS THIS UNUSUAL MUSE IN-LIKE DOMAIN IT’S VERY HEAVILY GLYCOSYLATED. THERE IS A LOT OF UNSTRUCTURED PROTEIN HERE. SO THIS IS THE CRYSTAL STRUCTURE OF THE EBOLA VIRUS GP. YOU CAN SEE THE 3GP1 SUBUNITS IN BLUE AND GREEN. THESE RECEPTOR BINDING ARE TIED TOGETHER AT THE BOTTOM BY THE GP2 FUSION SUBUNITS. NOW THERE IS SOMETHING INTERESTING HERE. WHEN YOU THINK ABOUT A FUSION PEPTIDE OR FLU OR HIV, IT’S A HYDROPHOBIC PEPTIDE TUCKED UP INSIDE THE STRUCTURE. HERE ARE THE FUSION LOOP IT IS TACKED ON TO THE OUTSIDE. THIS REACHES ALONG THE OUTSIDE AND BINDS INTO THE NEXT ONE. IN ORDER TO GET THIS TO CRYSTALLIZE, WE HAD TO EXIZE AND WE WANT TO UNDERSTAND WHAT THE REAL GP LOOKS LIKE ON THE SURFACE. IT HAS HEAVILY GLYCOSYLATED DOMAINS ATTACHED AT THE TOP. NOTE GP ONE TAINING THAT DOMAIN CRYSTALLIZES AND WE HAD TO USE A DIFFERENT TECHNIQUE. A SMALL SCATTERING, TINY X-RAYS AND PROTEIN MOLECULES TUMBLING AROUND IN SOLUTION GET A LOW RESOLUTION VIEW, MAYBE 10 RESOLUTION. AND THEN THIS, TURNS OUT THIS IS THE SOLUTION SCATTERING ENVELOPE OF THE GLYCOSYLATED EBOLA VIRUS GP. SO THE CRYSTAL SHUCK STRUCTURE IS IN THE RIBBON CENTER. SO THESE ARE THE DOMAINS ATTACHED. SO THE EFFECTIVELY TRIPLE THE SIZE OF THE MOLECULE. AND THIS IS A HELL OF A GLYCAN SHE WOULD. THEY REACH ABOUT 100 FROM THE CORE OF THE G. AND THEY ARE QUITE FLEXIBLE SO I EXPECT THE ACTUAL WILT OF THIS DOMAIN TO BE HALF THAT. I THINK VISUALIZING THE FLEXIBILITY AS WELL. THE SALIENT FEATURE OF THIS IS THAT THESE MUSE IN-LIKE DOMAINS ARE MASSIVE AND THEY DOMINATE THE STRUCTURE. S OKAY? SO THIS IS WHAT IS ON THE BIOSURFACE. HOW DOES IT WORK? HOW DOES IT FIND AND GET INTO A NEW CELL? WELL, THIS I’M SHOWING AGAIN THE CRYSTAL STRUCTURE AND COLORING THE SURFACE WHITE. PATCHES THAT ARE COLORED PINK ARE AREAS THAT MUTAGENESIS TELLS US ARE IMPORTANT FOR INFECTIVITY. THEY ARE SEQUESTERED INSIDE THE BOWL SHIP THAT IT MAKES. THE RESIDUES MOST IMPORTANT TO A SEPARATE BINDING ARE VERY SEQUESTERED. INSIDE STRUCTURE UNDER THIS DOMAIN. SO THAT IS SORT OF A REPRESENTATION OF WHERE THE DOMAINS ARE. THE PARTS THAT ARE IMPORTANT FOR THE RECEPTOR BINDING ARE PINK AND THEY ARE UNDER THESE DOMAINS CALLED THE GLYCAN CAP. SO, DOES THIS MAKE INNOCENCE HOW ON EARTH IS THIS AFFECTED UNDER THIS ENTIRE CANOPY OF PROTEIN CARBOHYDRATES? THE ANSWER IS THAT IT IS KNOWN FROM BIOLOGY THAT GP NEEDS TO BE CLEAVED BY HOST CAPSAICIN ENZYMES FOR THIS TO OCCUR. THIS IS ESPECIALLY IMPORTANT FOR EBOLA VIRUS. SO, WHY? WELL, IN SOLVING THE CRYSTAL STRUCTURE, WE SEE THAT ALL OF THIS STRUCTURE, THE GLYCAN CAP AND THE WHOLE MUSE IN LIKE DOMAIN ARE ATTACHED BY A SINGLE POLYPEPTIDE TETH THEY’RE CONNECTS RESIDUE 189 TO 213. AND THAT PIECE OF POLYPEPTIDE IS DISORDERS. SO SOMETHING THAT IS DISORDER IN A CRYSTAL STRUCTURE AND FLEXIBLE AND MOVING AROUND. SO THIS LOOKS LIKE A PRETTY ATTRACTIVE CLEAVAGE SITE. IF PROTEASES WERE TO CLEAVE ON THAT YELLOW LOOP, THIS WOULD BE THE EFFECT. A MUCH BETTER EXPOSURE. NOW WE ARE NOT MAKING THAT UP. THIS IS ACTUALLY THE CRYSTAL STRUCTURE NOW CLEAVED GP AND ANOTHER LAP SHOWS THAT YES, CLEAVAGE STRIPS OFF 85% OF THE MASS OF GP1 LEAVING THE RECEPTOR BINDING SITES EXPOSED. SO IN IS WHAT THE PROTEIN LOOKS LIKE ON THE VIRAL SURFACE. WHAT DO WE LEARN FROM THIS? RECEPTOR BINDING PROBABLY DOESN’T HAPPEN AT THE VIRAL SURFACE. BY LOOKING AT THE STRUCTURE, YOU CAN SEE SPOTS NEEDED TO BIND THAT RECEPTOR ARE NOT ACCESSIBLE. THEY ARE NOT WELL EXPOSED IN THIS KIND OF PROTEIN. INSTEAD, THE VIRUS THAT BEARS THIS SURFACE ENTERS CELLS BY MACRO 15AL CYTOSIS. ONCE IN THE ENDOSOME, THIS IS CLEAVED TO STRIP OFF ALL THAT SURFACE SUGAR IN THE MEW SIN LIKE DOMAINS LEAVING THE RECEPTOR BINDING SITE EXPOSED AND ALLOWING BINDING BY THE RECEPTOR AND THIS BINDING SITE IS RIGHT THERE WHERE THE GLYCAN CAP USED TO BE. SO WHAT WE SEE HERE IS ONE POLYPEPTIDE AND ALSO TWO DIFFERENT BIOLOGICALLY RELEVANT MANIFESTATIONS. THIS IS THE MOLECULE SUBJECT TO ANTIBODY SURVEILLANCE AND THIS IS THE MOLECULE FUNCTIONAL FOR RECEPTOR BINDING. SO WHAT DOES THAT MEAN TO THE IMMUNE RESPONSE? WELL, NOTHING GOOD. MANY CAN BE CLIPPED OFF. A LOT OF VACCINATION STUDIES, THESE SITES CAN BE IMMUNODOMINANT. YOU CAN SEE THAT ANY ANTIBODY THAT BINDS TO THESE EPITOPES WILL BE CUTTING RIGHT OFF IN THE END ZOME LEAVING A RECEPTOR BINDING CORE THAT IS NOW ANTIBODY FREE. THOSE KINDS OF ANTIBODIES DON’T NEUTRALIZE. THE ESSENTIAL CONSERVED SITES ARE NOT WELL EXPOSED. SO FOR EXAMPLE, ALL OF THESE VIRUSES SHARE THE SAME RECEPTOR SO THAT’S A CONSERVED BINDING SITE, AN ESSENTIAL SITE FOR THE MOLECULE. WE WOULD LIVE TO TARGET THAT WITH ABET BODY. IT’S PARTIALLY HIDDEN UNDER THE CAP AND VIRAL SURFACE SO THE ANTIBODY MIGHT NOT SEE IT UNLESS YOU FOUND A WAY TO ENGINEER THE ANTIBODY. BECAUSE OF THIS CAN NUN DRUM, WE ARE LEFT A PUZZLE THAT NEUTRALIZATION AND PROTECTION DON’T ALWAYS CORRELATE EBOLA VIRUS. SO NEUTRALIZATION IS YOUR ABILITY TO INACTIVATE THE VIRUS IN VITRO. PRO SECTION YOUR ABILITY TO SAVE THE ANIMAL IN VIVO. SO FOR EXAMPLE, ANTIBODIES LIKE THIS, THIS IS THE HUMAN KZ52 FROM THE SURVIVOR. OUTBREAK NEUTRALIZES BRILLIANTLY AND DOESN’T PROTECT. ANTIBODIES LIKE THESE, INCLUDING TWO THAT BIND THE MUSE IN LIKE DOMAINS, DON’T NEUTRALIZE BUT THEY DO PROTECT THE PRIMATE. SO THIS DOESN’T MAKE A LOT OF SENSE. LEAVING YOU WONDERING WHAT WORKS HERE. WE HAD THIS RESULT YEARS BEFORE AND IT REALLY COOLED EVERYBODY’S OPINION ON ANTIBODIES AGAINST EBOLA VIRUS THINKING WHETHER IT WOULD BE POSSIBLE TO PROTECT ANIMALS T TURNS OUT THAT YOU CAN. THESE ARE QUITE PROTECTED. EVEN IF YOU WAIT LONG ENOUGH FOR HEMORRHAGIC FEVER TO DEVELOP. THE DIFFERENCE MIGHT BE THAT THESE ARE GIVEN IN A COCKTAIL AS THIS WAS GIVEN ALONE. SO DOES THAT MEAN WE HAVE TO HAVE A COCKTAIL? IS THE LENGTH AND THE NUMBER OF EBOWL VIRUS SUCH THAT WE NEED TO HAVE MULTIPLE ANTIBODIES AGAINST MULTIPLE ESTIMATES IF SO, WHICH ONES DO WE PUT TOGETHER? TWO-THIRDS OF THIS COCKTAIL SELL MUSE IN. DOES THAT MEAN THAT IT WORKS? OR IS THIS ONE THE CHAMP THAT BINDS THE TOP? WE DON’T KNOW. NOW IN THE FIELD WE HAVE ABOUT 200 DIFFERENT MONOCLONAL ANTIBODIES IDENTIFIED IN THIS VIRUS. WHAT DO YOU PUT TOGETHER IN A COCKTAIL. NOW I’M GOING DIVERT A LITTLE BIT FROM MY THEME WHEN THE PROTEINS OF THE VIRUS AND THEN TELL YOU HOW TO USE THE STRUCTURE TO GET AT THAT PROBLEM. THIS IS THE WEBSITE THAT THE VIRAL HEMORRHAGIC FEVER -UE CAN FIND THIS LINK THROUGH SCRIPPS VERY SOON. THIS IS MORE THAN 20PIs AND 7 DIFFERENT COUNTRIES HAVE GOTTEN ON THE SAME PAGE. WE PUT ALMOST ALL THE ANTIBODIES KNOWN AGAINST THESE VIRUSES TOGETHER IN ONE POOL. WE BLINDED THEM AND THEN COMPARE THEM SIDE-BY-SIDE TO SEE WHAT IS MORE EFFECTIVE. IN OTHER WORDS HOW TO PUT TOGETHER THE RIGHT COCKTAIL. RIGHT NOW WE HAVE THREE FROM THE ARMY IN A COOK TAIL THAT NEUTRALIZE AND WE HAVE THREE FROM CANADA IN A COCKTAIL THAT NEUTRALIZES. WHAT IF THE MOST EFFECTIVE IS ONE FROM JAPAN AND ONE FROM THE ARMY AND ONE FROM HAMILTON? WE WON’T KNOW UNTIL WE PUT THEM ALL TOGETHER IT’S NICE THAT EVERYONE IS ON THE SAME PAGE IN THE SAME STUDY. SO UNTILE WOO MAKE THAT COCKTAIL, LET’S ASSUME THAT VIRAL INFECTION WILL PIQUE. SO THE NEXT VIRAL INFECTION AFTER THE VIRAL MEMBRANE IS FUSED TO THE HOST ENDOSOME MEMBRANE AND GENETIC MATERIAL EXCERPTS THE VIRUS STARTS TO REPLICATE. NOW, SOMETHING IMPORTANT HAPPENS HERE. MOST PEOPLE DIE FROM EBOLA VIRUS INFECTION. 50-90%. SOME PEOPLE LIVE. WHAT SILENT DIFFERENCE? THE DIFFERENCE SEEMS TO BE THAT THOSE PEOPLE THAT SURVIVE THE EBOLA VIRUS INFECTION TEND TO GENERATE AN EARLY AND STRONG IMMUNE RESPONSE AGAINST THE VIRUS AND THE VIRAL TITER STARTS TO DROP BY AROUND DAY 4. THOSE PEOPLE THAT ULTIMATELY SUCCUMB TO THE VIRUS INFECTION ARE MORE LIKELY TO BE CHARACTERIZED BY A VERY POOR IMMUNE RESPONSE AND THEIR VIRAL TITERS GET QUITE HIGH. 10 TO THE 9 TO 10 TO THE 10. SO FOR THIS DECISION POINT TO OCCUR BY AROUND DAY 4, THAT MEANS THAT THE INNATE IMMUNE SYSTEM IS QUITE IMPORTANT IN MAKING THIS DECISION OF SURVIVAL OR NOT SURVIVAL. SO WHAT IS THE VIRAL FACTOR AT PLAY IN THIS AMAZING DECISION POINT? ONE OF THEM IS A PROTEIN CALLED VP35. VIRAL PROTEIN 35 KILL DALTONS. IT’S A COMPONENT OF THE NUCLEO CAPS IN REPLICATION COMPLEX. IT ALSO HAS ANOTHER JOB, INTERFERON ANTAGONIST. WHAT IT DOES IS BIND DOUBLE-STRANDED RNA. NOW YOU TYPICALLY WOULD ONLY HAVE DOUBLE-STRANDED RNA IN THE CONTEXT OF A VIRAL INFECTION. SO IT IS A PATHOGENESIS MOLECULAR POWDER. YOUR INNATE IMMUNE SYSTEM THAT HAS SENSORS LOOKING FOR DOUBLE-STRANDED RNA AND THEY MOUNT A ANTIVIRAL RESPONSE. SO HOW DOES THIS WORK? THIS IS A CRYSTAL STRUCTURE VP35 BOUND TO DOUBLE-STRANDED RNA. SO THE DOUBLE-STRANDED RNA APPEARS IN GREEN. WE HAVE FOUR COPIES OF VP35 BOUND TO IT. NOW THIS HALF IS IDENTICAL TO THIS HALF IN THE STRUCTURE. SO YOU CAN REALLY ONLY LOOK THAT THE HALF IF YOU WANT. THIS IS NOT THE MODE OF GLYCAN BINDING YOU LEARNED ON YOUR MOTHER’S KNEE AS A BIOCHEMIST. WHAT YOU TIICALLY THINK OF WHEN YOU THINK OF A PROTEIN BINDING A LIGAND, IT HAS ONE BINDING SITE. THIS LASER POINTER IS A LIGAND. MY HAND IS THE PROTEIN, IT BINDS IN THE PALM AND THAT IS THE BINDING SITE. PERFECTLY SHAPED. WHAT WE HAVE HERE IS THE SAME PROTEIN BINDING IN TWO DIFFERENT WAYS. TWO COPIES BIND THE BACKBONE, TWO COPIES CAP THE END OF THE THESE ARE THE IDENTICAL PROTEINS. YOU CAN PULL OFF THE END CAP AND ROLE IT AROUND AND ATTACH IT BY THE BACKBONE. THEY USE DIFFERENT BINDING SITES TO DO THIS. THE END CAPPING HUGHES SYSTEM A HYDROPHOBIC PATCH AND THE BACKBONE USES A HYDROPHILIC PATCH. SO INSTEAD OF IT BINDING IN ONE SITE, YOU HAVE TWO IDENTICAL COPIES OF THE PROTEIN AND ONE BINDS THIS WAY AND ONE BINDS THIS WAY. IT TURNS THOUGHT DIMERIZATION IS ESSENTIAL. POINT MUTATION THAT IS BROCK THAT INTERFACE ATTENUATE THE EBOLA VIRUS. AFTER YOU FORM THIS DIMER ON THE END, IT SPIRALS AROUND THE RNA. IT IS INTERESTING THAT IT HAS REPURPOSED ITSELF FROM NUCLEO CAPSID PROTEIN TO HAVE THIS ADDITIONAL FUNCTION AND USED DIFFERENT SIDES OF ITSELF IN ORDER TO MAKE TWO DIFFERENT BANDING STATES. BINDING SITES. HERE IS THIS PROTEIN. I’M GOING TO SHOW YOU THIS NEXT WITH A DIFFERENT STRATEGY FOR MANAGING DOUBLE-STRANDED RNA. THIS IS THE NUCLEAR PROTEIN OF LASSA VIRUS. SO THE DAY JOB OF THE NUKE LA PROTEIN IS TO BIND AND PLAY A ROLE IN REPLICATING THE VIRAL GENOME. RNA VIRUS THAT IS PROTECT OUR GENOME BY HAVING IT CONTINUALLY BOUND BY A NUCLEO PROTEIN. LASSA HAS FOUR GENES. THIS PROTEIN HAS ANOTHER FUNCTION THAT IS ALSO INTERFERON ANTAGONIST. BUT IT WAS KNOWN THAT IT WAS IMMUNOSUPPRESSIVE BUT IT WASN’T KNOWN HOW. SO HOW DOES THIS GENOME BINDING PROTEIN SUPPRESS IMMUNE SIGNALING? WE DIDN’T KNOW. SO WE SOLVED THE STRUCTURE. HERE IS THE STRUCTURE. IT HAS STRANDS, HELIXES AND LOOPY BITS AND BOUND ZINC. THAT DEPENDENT TELL US ANYTHING. — THAT DIDN’T TELL US ANYTHING. THIS LOOKS LIKE ANOTHER NUCLEO VIRUS. SO WE HAVEN’T LEARNED ANYTHING FROM THE SEQUENCE. SO WE ASKED OURSELVES IS IT STRUCTURE LIKE ANYTHING WE SEEN BEFORE? EACH THOUGH THE SEQUENCE ISN’T. SO WE DID A DOLLY SEARCH FOR THINGS OF SIMILAR FOLD AND WE FOUND ONE. SO IN GREEN, SILENT LASSA VIRUS NUCLEO PROTEIN. I’M GOING DO OVERLAY OTHER PROTEINS WHICH ARE ALL NUCLEASES OF THE DEDDH SUPER FAMILY. THIS IS ISG20. DNA POLYMERASE SUBUNITS. THE FOLDS ARE SIMILAR. THEY HAVE THE SECONDARY STRUCTURAL ELEMENTS IN THE SAME PLACES. THIS THE SILENT SUPER FAMILY OF NUCLEASES — IT HAS A SIMILAR FOLD EVEN RIGHT DOWN TO THE NUCLEOAISE ACTIVE SITE. SO ALL OF THESE ENZYMES ARE CHARACTERIZED BY THE DEDDH. THESE CATALYTIC RESIDUES. THE LASSA NUCLEAR PROTEIN IS COLORED GREEN. IT HAS THE SAME RESIDUES IN THE SAME PLACE. IF YOU LOOKED IN THE SEQUENCE, YOU COULD SEE THEY WERE THERE AND THEY ARE ACROSS THE IMMUNEOY VIRUSES BUT THE SPACING WASN’T ANYTHING YOU COULD APPRECIATE THAT WOULD WIND UP BEING AN EXNUCLEASE UNTIL WE SAW THE STRUCTURE. SO IT LOOKS LIKE A EXNUCLEASE DOES. IT FUNCTION LIKE ONE? SO TO ANSWER THAT, WE GIVE IT DNA, RNA, SINGLE STRANDED AND DOUBLE-STRANDED AND IT DIGESTED SOME OF THEM. SO THIS DIGESTS NUCLEIC ACID AND DOUBLE-STRANDED RNA. SO THE OTHER EXNUCLEASES IN THE SUPER FAMILY CAN BE MORE CATHOLIC IN THEIR SPECIFICITY. THIS ONE ONLY DIEGISTS DOUBLE-STRANDED RNA. THE PATHOGENESIS MOLECULAR PALTERERN. WE THINK THAT ENZYMATIC ACTIVITY IS LINKED TO THE IMMUNOSUPPRESSION. BECAUSE WHEN YOU MAKE POINT MUTE APPOINTMENTS AROUND THE ACTIVE SITE, THE WILDTYPE PROTEIN DIEGISTS DOUBLE-STRANDEDS RNA. THE MUTANTS DON’T. IF YOU LOOK AT A REPORTER ACTIVITY, THE WILDTYPE POE TEEN SUPRESS IT IS AND THE MUTANTS DON’T. SO IF YOU KNOCKOUT THE EXNUCLEASE ACTIVITY, YOU KNOCKOUT THE IMMUNOSUPPRESSION. HERE IS A STRUCTURE OF THE NUCLEASE COMPLEX DOUBLE-STRANDED RNA. THE YELLY FEEDS INTO THE ACTIVE SITE. THE PAIRED PURPLE STRAND ARCHES UP. AND WE CAN LOOK IN HERE AND COMPARE THIS TO OTHER EXNUCLEASES AND SEE THERE ARE ONLY TWO AMINO ASATOIDS GIVE THE UNIQUE IMMUNOSUPPRESSIVE SPECIFICITY. SO WHAT IT IS DOING IS MAYBE RAPIDLY ERASING THE THING THE IMMUNE SYSTEM IS LOOKING FOR. DOUBLE-STRANDED RNA IS A REPLICATION INTERMEDIATE OF A SINGLE STRANDED RNA VIRUS MAYBE AS ONE DOMAIN BINDS, THE OTHER COMES ALONG AND RAISES IT. WE ARE STILL TRYING TO FIGURE OUT HOW THAT WORKS. WE SEE THAT THIS STRUCTURE AND THAT MOTIF SEEMS TO BE SHARED AMONG THE ARENA VIRUS FAMILY. THIS IS A FAMILY OF 50 DIFFERENT VIRUSES THAT ARE EXISTING NEARLY EVERY CONTINENT. SO, AN ENZYME WITH A NUMBER OF HUMAN PATHOGENS LOOKS LIKE IT COULD BE AN EFFECTIVE TARGET FOR BROAD SPECTRUM ANTIVIRAL AND I’M LOOKING FOR SOMEONE TO WORK WITH ME ON THAT. SO WHAT WE SEE HERE AND THIS EXAMPLE IS A POLYTEP TIED WITH MULTIPLE ACTIVITIES. IT’S DAY JOB IS TO ERASE A KEY SIGNATURE TO SPARK INNATE IMMUNE SIGNALING. SO THE VIRUS HAS ENTER THE THE CELLS, SUPPRESSED IMMUNE SIGNALING AND REPLICATED AND ITS NEXT JOB IS TO ASSEMBLE NEW VARIANTS AND BUD OUT. THAT OCCURS BY PROTEIN CALLED MATRIX FOR EBOLA VIRUS IT’S CALLED VP40. SO THE MATRIX IS THE LAYER RIGHT UNDER THE MEMBRANE BETWEEN THE MEMBRANE AND THE NUCLEO CAPS IN AND IT GIVES THE VIRUS ITS SHAPE. SO IF YOU TRANSFECT CELLS OF VP40 ALONE IT WILL ASSEMBLE BUT OUT VIRUS LIKE PARTICLES THAT LOOK LIKE EBOLA VIRALS. SO ALL THE INFORMATION YOU NEED TO BUILD AND BUD A ENVELOPE PARTICLE IS CONTAINED IN VP40. SO, HOW DOES IT DO THAT? WHAT DOES THIS PROTEIN LOOK LIKE? THE FIRST CRYSTAL STRUCTURE WAS SOLVED 13 YEARS AGO NOW. HERE IT IS. AS AN N-TERMINAL DEMAIN AND A C TERMINAL DOMAIN. IT STILLS HAS TO BE A MONOMER. WHAT IS INTERESTING ABOUT A MATRIX PROTEIN IS NOT WHAT IT LOOKS LIKE AS A MONOMER BUT HOW IT ASSEMBLES, HOW TO BUILD A MATRIX? SO THEY KNEW THAT IF THEY TINKERED WITH THE VP40, CUTTING OFF C TERMINAL TO RENALONS OR OTHERS, THEY COULD GET TO THE TO FORM RINGS. SO HERE IS EM OF A HEX MERIC RING AND A CRYSTAL STRUCTURE OF THIS RING. SO THEY EXPRESSED THE N-TERMINAL DOMAIN WITH USE WITHOUT THE ORANGE C TERMINAL DOMAIN. EIGHT OF THEM MAKE THIS RING AND UNEXPECTEDLY, IT PULLED OUT RNA FROM THE E.COLI SYSTEM. THERE IS A LITTLE ORANGE BOUND TO EACH ONE OF THE EIGHT COPIES OF VP40 IN THIS RING. SO FOR THE LAST DECADE, THAT HAS BEEN OUR ONLY MODEL FOR HOW VP40 COULD ASSEMBLE. THIS IS A LOT OF EFFORT THAT HAS GONE INTO DESIGNING DRUGS TO INHIBIT RING FORMALIZE INHIBIT MATRIX FORMATION. A LOT OF MODELS GENERATED BY TAKING THIS CHEERIO AND MAKING LINOLEUM PATTERN AND WRAPPING IT AROUND THE FILLA VIRUS. BUT THERE ARE A NUMBER OF PROBLEMS WITH THIS SLIDE. THE FIRST ONE IS THE RINGS ARE NOT FOUND IN PURIFIED VARIETIES. SO IF THEY ARE NOT IN THE VARIANT, ARE THEY A COM PONE INNOCENT THEY ARE FOUND IN INFECTED CELLS. JUST NOT THE ACTUAL VIRUS. THE SECOND PROBLEM IS THAT THERE IS NO RNA IN THE VIRUS MATRIX LAYER. WHAT THAT WAS WASN’T ENTIRELY CLEAR. THE RN SAMPLE BOUND TO THE NUKE LID CAPSID AT THE CENTER. THE THIRD PROBLEM IS MUTATION THAT IS PREVENT RING FORMATION GAVE PERFECTLY NORMAL LOOKING VIRAL PARTICLES. SO IF YOU ABOLISH THE RING YOU CAN BUD OUT A NORMAL LOOKING VIRUS. THE CRYSTALLING ONFERS DIDN’T THINK THIS IS HOW THE MATRIX WAS ASSEMBLED BECAUSE THEY DID ALL THIS WORK BUT THE FIELD PROCEEDED AS IF VP40 MADE THESE RINGS, SOMETHING WAS HELD AS SIMPLE. HOW DOES IT ASSEMBLE? WE DIDN’T INTEND DO DO ANY OF THE WORK. I’M GOING TO SHOW YOU THIS NEXT. WE WERE MAKING VP40 FOR SOME OTHER REASON. AND WHAT WE NOTICE IS WHEN WE PURIFY VP40, IT CAME OUT AS A DIMER, NOT A MONOMER. SO WE ARE USING SIZE EXCLUSION ANGLE LIGHT SCATTERING. SO IT’S A MORE SENSITIVE METHOD IN TOMORROWING SOMETHING THAT WASN’T WIDE LIVEABLE A DECADE AGO. SO VP40 WAS ALWAYS A DIMER. DOES THAT MATTER? WE WERE LOOKING FOR A DIFFERENT WAY. WE HAD ALL THIS PROTEIN, WE HAD ROBOTS SO WOE GREW CRYSTALS AND WE SOLVED 9 STRUCTURE. HERE IS THE STRUCTURE. WE SEE THE END TERMINAL AND C TERMINAL. SO THE STRUCTURE FROM DIMER IS COLORED. HERE IS THE STRUCTURE FROM THE MONOMER. NO CHANGE. SO, THE REVELATION THAT IT WAS A DIMER INSTEAD OF A MONOMER HASN’T TOLD US ANYTHING ABOUT THE FOLD OF THE PROTEIN. BUT IT WAS THE PIECE OF INFORMATION THAT WE NEEDED TO GO LOOKING IN THE CRYSTAL PACKING. BECAUSE WE KNEW THAT IT WAS A DIMER IN SOLUTION. SO SOMEHOW THOSE PROTEINS ASSEMBLED IN THE CRYSTALS WE ARE GOING TO SEE THE DIMER INTERVASE. THIS SILENT CRYSTAL PACKING. — THIS IS THE CRYSTAL PACKING. C ARE BLUE, PROTEINS ARE ORIENTED LIKE THIS DOWN A FILAMENT SO THEY MAKE THIS NNCCNNCC FILAMENT. SOMEWHERE IN THIS IS THE DIMER THAT FLOATS AROUND THE SOLUTION. SO THE DIMER MADE BY THE BLUE-BLUE OR THE ORANGE ORANGE INTERACTION? WELL, THE BLUE BLUE VARIES MORE MOLECULAR SURFACE BUT THE PROOF CAME FROM A POINT MUTATION WE MADE, LEU117. SO THE DIMER INTERNATION IS PROBABLY THE BLUE ONE EXTHIS IS THE DIME THEY’RE FLOATS AROUND THE SOLUTION THAT LOOKS LIKE A BUTTERFLY. INCIDENTALLY THAT LEUCIN 117 IS ON THE OUTSIDE OF THE RING. IT’S NOT INVOLVED IN ANY RING ASSEMBLING INTERFACESES. SO LET’S HAVE ANOTHER LOOK AT THAT FILL MEANT. HERE THIS BELONGS TO EBOLA VIRUS. THIS IS THE SIDE VIEW. ROLE IT AROUND AND THERE IS THE TOP VIEW. THIS IS HOW THE CRYSTALS ASSEMBLE. ALL THOSE FILAMENTS LINE UP SIDE-BY-SIDE. WELL, WE WONDERED IF THAT WAS INTERESTING. IS THIS ASSEMBLY PHYSICAL LOGICALLY RELEVANT OR A ARTIFACT? THE ODD THING WE NOTICED IS THAT NO MATTER HOW WE TRIED TO CRYSTALLIZE VP40, WE ALWAYS GOT THE SAME FILAMENT. THIS GROUP C2, BASE GROUP EXPLORE — THIS IS THE ORIGINAL STRUCTURE. NO MATTER WHAT SPECIES WE WORKED WITH OR WHICH CRYSTAL SYMMETRY WE GOT, WE ALWAYS GOT THE SAME FILAMENT ORGANIZED THE SAME WAY. HERE, THE RIGID AND THEY LINE UP NEXT TO EACH OTHER AND DEFRACT WELL. HERE THEY FORM FOUR TWISTED AROUND EACH OTHER. HERE THEY MAKE A 10-STRANDED CONDUIT TUBE AND DON’T REFRACT TOO WELL. BUT THEY ARE ALWAYS ASSEMBLED BY THE SAME FIRST FACES. SO, THAT’S STARTING TO GET UNCANNY. CRYSTALLIZE THE PROTEIN FOUR TIMES AND MAYBE IF MAKES IT IS SOMETHING IT WANTS TO DO. SO IT FORMS A DIMER IN SOLUTION AND EVERY TIME YOU CRYSTALLIZE A FULL-LENGTH PROTEIN WE GET THE SAME FILAMENT. UNDER OTHER CIRCUMSTANCES, CUT OFF THE C TERMINAL DOMAIN AND FORM A RING AND THERE IS ONE CRYSTAL STRUCTURE OF THAT. SO, WHICH ONE OF THESE ASSEMBLIES MAKE THE VIRAL MATRIX OR DO NEITHER ONE OF THEM? TO ANSWER THAT QUESTION WE MADE MUTATIONS IN EACH INTERFACES BECAUSE THIS ASSEMBLY AND THE RING ASSEMBLY ARE BUILT BY DIFFERENT SURFACES. AMINO ACIDS THAT MAKE THIS FILAMENT ON ARE THE OUTSIDE OF THE RING AND AMINO BINDING RING FORMATIONS ARE NOT WHAT ASSEMBLED THIS FILAMENT. SO LET ME SHOW YOU THE MUTATIONS. SO THIS IS THE DIMER INTERFACE. YOU’RE LOOKING TOP DOWN AT THE BUTTERFLY. BLUE-BLUE. LEUCIN 117 AND 112 ARE IMPORTANT TO THE DIMER. IF YOU MUTATE THEM, YOU GET THIS. SO THE WILDTYPE PROTEIN IS THE DIMER. MUTATE THE DIMER INTERFACE, YOU GET MONOMER AND RING. CAN YOU SEE THAT OR DO WE NEED TO DIM THE LIGHTS MORE? SO MUTATE THE DIMER INTERFACE AND GET MONOMER AND RING. IF YOU TRANSFECT CELLS AND NOW WE ARE STANDING IN GREEN. THE WILDTYPE PROTEIN TRAFFIC TO THE CELL MEMBRANE AND BUDS OUT THE FILAMENTOUS VIRUS LIKE PARTS. SO SOME LENGTH WISE IN MANY OF THE CROSS SECTION. THE MONOMER AND RING MUTATIONS DON’T TRAFFIC AS WELL AS THE MEW TAIN AND DON’T BUD ANYTHING AT ALL. SO THAT END-TO-END DIMER INTERFACE IS IMPORTANT FOR MATRIX ASSEMBLY AND BUDDING. EVEN IF THE MUTANTS MAKE A RING. HOW ABOUT THE FILAMENT WE KEEP SEEING MADE BY PACKING SIDEWAYS OF THE DIMERS? THAT IS MADE BY THE C-C INTERACTION. SO IF THAT INTERFACE IS 214 AND LOU SIN 307. LET’S MUTATE THOSE. WILDTYPE PROTEIN IS A DIMER. THIS FIRST MEW SUBSTANT A DIMER. WE EXPECTED THAT. SO WE STILL HAVE THE DIMER. THE WILDTYPE PROTEIN TRAFFIC TO THE MEMBRANE AND BUDS OUT THE VIRUS PARTICLES AND MAKES THESE FUNNY RUFFLES. WE DON’T KNOW WHAT THEY ARE. THE WILDTYPE PROTEIN DOES THAT. THIS MUTANT PROTEIN DOESN’T TRAFFIC QUITE AS WELL BUT IT HAS A CRAZY RUFFLING MORPHOLOGY. IT DOESN’T BUD ANY PARTICLES BUT MAKES A MEMBRANE WITH A FUNNY RUFFLING EFFECT. SO WE SAW THE STRUCTURE OF THE MUTANT TO FIND OUT WHAT WAS HAPPENING. YOU HAVE THE SAME DIMER, THE GREEN BUTTERFLY AND THE BLUE BUTTERFLY. BUT INSTEAD OF BEING PACKED SIDE-BY-SIDE LIKE EVERY OTHER CRYSTAL STRUCTURE, WE MUTATED THAT INTERDAYS AND THEY ARE TWISTED RELATIVE TO EACH OTHER. SO IT MIGHT BE WHEN THEY TRAFFIC TO THE MEMBRANE, THEY ARE MAKING A FUNNY TWISTED FILAMENT MAKING THAT RUFFLED MORPHOLOGY THATICANT QUITE GET-TOGETHER AND RELEASE THE VIRUS. THIS OTHER MUTANT IS DIFFERENT. IT DOESN’T MAKE DIMER. IT ONLY MAKES RINGS. AND THESE RINGS BIND RNA. THE VP40 DIMER DOESN’T BIND RNA. ONLY THE RING BIND RNA. AND WE LOOKED AS THESE BY EM AND THE SAME SIZE AND SHAPE BY THE OTHER RINGS MADE BY DELETING THE C TERMINUS. THE RNA BINDING RINGS DO NOT TRAFFIC TO THE MEMBRANE. INSTEAD THEY HUG THE NUCLEUS AND DON’T BUD ANYTHING AT ALL. SO WHAT WE SEE FROM THOSE EXPERIMENTS IS THAT DISRUPTING THE INTERACTION THAT IS BUILD THAT FILAMENT PREVENTS VIRUS ASSEMBLY AND BUDDING EVEN IF YOU’RE MAKING ONLY RINGS. NOW LET’S BREAK THE RING. THIS MUTATION WAS PREVIOUSLY KNOWN AND PREVENTS RNA BINDING AND RING FORMATION IT’S A DIMER. IT TRAFFIC TO THE MEMBRANE. IT BUDS OUT VIRUS-LIKE PARTICLES AND THE SAME KIND OF MEMBRANE RUFFLES AND LOOKS IDENTICAL TO WILDTYPE AND ASSEMBLED AND BUDS. SAME MOREOVERROLOGY AND NUMBER OF PARTICLES. YOU CAN’T TELL IT APART FROM WILDTYPE. WE CONCLUDE THAT SOMETHING ABOUT THAT DIMER AND FILAMENT IS INVOLVED IN VIRUS ASSEMBLY NOT THE RING. SO HOW DIFFERENT YOU THE DIMER THAN THE RING? THEY ARE PRETTY DIFFERENT. SO EASY TO SEE HOW YOU’RE MAKING THIS FILAMENT BY LENGTH WISE ASSEMBLY OF THE DIMERS. TO MAKE THE RING, YOU HAVE TO SEPARATE THE NNC TERMINAL DOMAINS FROM EACH OTHER, SPLIT THEM APART, UNRALPH THE 70 AMINO ACETATE MAKE THE INTERFACE AND ROTATE THE DIMER FROM PARALLEL TO ANTIPARALLEL AND BACKWARDS. SO THEY ARE GOING TO REASSEMBLE NOW BY THE GREEN INTERFACE THAT USED TO BE HIDDEN BY THE C TERMINAL DOMAIN. AND THEN FOUR OF THESE ANTIPARALLEL BACKWARDS DIMERS MAKE THE RING AND THIS RING THEN HAS AN RNA BINDING SITE IN THE CENTER THAT WASN’T AVAILABLE FOR THE FILAMENT. SO WE THINK THIS IS SOMETHING TO DO WITH THE VIRUS ASSEMBLY. HOW? THERE ARE THREE QUESTIONS YOU MIGHT ASK YOURSELF. THE FIRST ONE WOULD BE WHAT SIDE OF THE THING INTERACTS WITH MEMBRANE? WELL, WE KNEW THAT THE INTERACTION WITH MEMBRANE WAS ELECTROSTATIC BECAUSE YOU COULD SALT IT OFF. SO, IF YOU LOOK FOR A BASIC PATCH IN VP40, THERE IS REALLY ONLY ONE AND THEY ARE ON THE SAME SIDE OF THE FILAMENT. IN THAT BASIC PATCH ARE FIVE LYSINES CONSERVED ACROSS THE EBOLA VIRUSES. 4 OF THE 5 ARE ESSENTIAL FOR MEMBRANE INTERACTION AND BUDDING VIRUSES. SO PROBABLY THIS SURFACE OF THE FILAMENT IS THE ONE THAT INTERACTS WITH MEMBRANE. NEXT QUESTION YOU MIGHT ASK YOURSELF IS, IS THIS IT? IS THIS FILAMENT HOW YOU BUILD THE FILAMENT VIRUS? THERE ARE A LOT OF SATISFYING THINGS ABOUT THIS MODEL. ALL THE AIRPORT FACES WE THINK ARE ESSENTIAL BECAUSE ANY TIME YOU MUTATE THEM YOU NO LONGER BUILD AND BUD A VIRUS. BUT, THERE ARE TWO OTHER PIECES OF INFORMATION THAT THIS MODEL DOESN’T ADDRESS. THE FIRST ONE IS THAT INTERACTION WITH MEMBRANE INDUCES A LIGMERRIZATION OF VP40 IN HEXAMERS AND BY OUR MODEL, WE SEE 2, 4, 6, 8, 10, WITH NO JUMP TO HEXAMER. THE SECOND THING IS THAT INTERACTION MEMBRANE SEEMS TO DO SOME KIND OF CONFIRMATIONAL CHANGE BETWEEN THE DOMAINS AND THIS DIDN’T ANSWER THAT EITHER. SO THE THIRD QUESTION WE ASKED OURSELVES AND YOU MIGHT BE ASKING YOURSELF RIGHT NOW, THERE IS SOMETHING DIFFERENT HAPPENING TO THIS STRUCTURE WHEN IT MAKES IT ELECTROSTATIC INTERACTION ACTION WITH MEMBRANE. SO WE WENT THROUGH A SERIES OF ATTEMPTS TO TRY TO SATISFY THE POSITIVE CHARGE WITH THAT BASIC PATCH. NOW IT IS KNOWN THAT THIS IS A NATURAL LIGAND OF VP40 IN THE MEMBRANE AND A MOLECULE WILL COMPETE. SO WE SOAKED A LOT OF CRYSTALS IN PHOSPHOR SEREIN. WE TRIED TO CO-CRYSTALLIZE WITH IT. WE FOUND SUCCESS WITH DEXTRAN SULPHATE. IF WE INCUBATE AND GROW CRYSTALS IN THE STRUCTURE, WHEN WE GET IS THE A VP40 THAT IS NOW HEX MERIC WITH N AND C SEPARATION. SO LET ME WALK YOU THROUGH THIS STRUCTURE. THE N-TERMINAL DOMAINS ARE BLUE. C ARE ORANGE. THIS IS ONE MONOMER WITH THE N-TERMINAL AND C TERMINAL DOMAIN. HERE ARE TWO MORE. SO MOLECULES ONE-6 AND THE HEXAMER. THESE C TERMINAL DOMAINS ARE STILL ATTACHED. IF YOU RUN THE CRYSTAL AND JELLY, THEY ARE THERE BUT WE DON’T SEE THEM. THEY HAVE SOMEHOW SPRUNG INTO SOLVENT CHANNELS WHERE THEY OCCUPY A LOT OF POSITIONS. WE KNOW THEY ARE ATTACHED BUT WE DON’T SEE THEM AND THEY BELONG TO THESE AND WE CAN SEE WHICH DIRECTION THEY ARE GOING FROM THE POLYPEPTIDE CHAIN THAT EXTEND. THIS HEXAMERRIC BUILDING BLOCK FORMS THIS FILAMENT IN THESE CRYSTALS AND THIS IS ASSEMBLED 3 INTERFACES. THE SAME DIMER INTERFACE MUTATING BEFORE WITH THE SAME LEUCIN 117 AND THE SAME C-C INTERFACE WITH THE ORANGE ORANGE THAT HAD THE SAME LEWISSINE AND SOMETHING ELSE WE ARE CALLING OLIGOMERIZATION INTERFACE EXPOSED BOY THE RELEASE OF THE C TERMINAL DOMAIN. SO WE KNOW BY MUTAGENESIS THAT EVERY INTERFACE, THIS REARRANGED ZIGZAG FILAMENT IS ESSENTIAL FOR BUDDING. DOES THIS FILAMENT NOW FIT WHAT WE KNOW ABOUT THE VIRUS? THE EBOLA VIRUS LOOKS LIKE THIS IN CROSS SECONDS. WE HAVE A NUCLEO CAPSID AT THE CENTER AND MEMBRANE ON THE OUTSIDE AND THEN THE TOPOGRAPHY TELLS US MULTIPLE PROTEIN LAYERS BETWEEN THE NUCLEO CAPSID AND THE MEMBRANE. SO MATRIX HAS A LOT OF PROTEIN LAYERS IN IT. IF YOU LOOK AT THE RADIAL DENSITY OF THE VIRUS INSIDE TO OUT, YOU SEE A BIG PEEK FOR THE NUCLEAR CAPSID AND A PEEK FOR MEMBRANE. WE CALL THIS INTERPEEK CENTRAL PEEK AND OUTER PEEK IN THE MEMBRANE. HERE IS OUR ZIGZAG FILAMENT. TURN IT ON ITS SIDE AND ROLE IT OVER ONCE MORE, THERE ARE 3 PROTEIN LAYERS, INNER AND CENTRAL AND OUTER LAYER. THEY FIT TO SCALE WHAT WE KNOW ABOUT THE WIDTH OF THE VEER YON, ALWAYS FIXED, THE WIDTH OF THE NUCLEO CAPSULE AND THE SPACE IN BETWEEN AND THE DIMENSIONS OF THE C TERMINAL AND N-TERMINAL CORE AND THE REACH OF THESE. THIS FITS THE BIOLOGY AS WELL. WE KNOW FROM BIOLOGY THAT AS THE C TERMINAL DOMAIN THAT BINDS MEMBRANE. WE ALSO KNOW THAT IT IS THE C TERMINAL THAT BINDS NUCLEO CAPS IN. HOW CAN THIS HAPPEN UNLESS SOME GO THIS WAY AND SOME GO THAT WAY? SO IT FITS WHAT WE UNDERSTAND ABOUT THE VIRUS ASSEMBLY. IT ALSO FITS THE SHAPE OF THE VIRUS TOO. SO HERE PROTEIN IS WHITE. NOT PROTEIN IS BLACK. WE ARE SHOO THETH INTO THE SIDE OF THE EBOLA VIRUS AND THE ZIGZAGGING FILAMENT SEEMS TO FOLLOW THE CHECKER BOARD PATTERN AND SCALE REPEATING DISTANCES. SO WHAT WE HAVE EXPRESSED AS A DIMER MAKES THIS FILAMENT INTERMEDIATE AT THE MEMBRANE IT SEEMS TO BE A REARRANGEMENT THAT MAKES THIS BUILDING BLOCK. THIS IS OUR CURRENT BEST MODEL FOR HOW TO MAKE THIS ASSEMBLE. UNDER SOME OTHER CIRCUMSTANCES, SPLIT THE THING APART AND ROTATE IT AND MAKE ANOTHER RING THAT BINDS RNA. WHAT IS THIS RING? IS IT REAL? REMEMBER THIS MUTATION. PREVENT RNA BINDING AND RING FORMATION? IT RESULT IN NORMAL LOOKING VIRUSES AND PERFECT SHAPES AND PERFECT NUMBER. THIS IS A LETHAL MUTATION. YOU CAN NOT PROPAGATE AN EBOLA VIRUS WITH THIS MUTATION. WHY NOT? YOU CAN STILL BUILD AND BUD A VIRUS. VIRUS CAN STILL ATTACHE CELL. WHY IS THIS LETHAL? THE RNA BINDING RING MUST DO SOMETHING. THAT’S WHAT WE CONCLUDE. IT MUST DO SOMETHING ESSENTIAL IN THE VIRUS LIFE CYCLE. WHAT WOULD THAT BE? THE RECENT DISCOVERY VP40 HAS A SECOND FUNDS IN ADDITION TO VIRUS ASSEMBLY AND BUDDING, VP40 EF CONTROLS VIRUS TRANSCRIPTION INSIDE THE INFECTED CELLS. MAYBE THIS IS WHAT THE RING IS FOR. IT’S THE OHMY STRUCTURE THAT BIND RNA. WE ONLIY SEE IT IN INFECTED CELLS AND NEVER SEE THE RING AND THE VIRUS. NOW WE HAVE NEW TOOLS TO BRING TO BEAR IN SITUATIONS. WE FOUND POINT MUTATIONS THAT MAKE ONLY RINGS AND THOSE THAT NEVER MAKE RINGS. SO WE PUT INTO A MINIGENOME ASSAY. WE HAVE THESE IN THE MIDDLE AND THE WILDTYPE VP40 EXHIBITS CONTROL FUNCTION AND THE VP40 WE LOCKED INTO THAT RING CONTROLS IT BETTER. SO IF YOU ANCHOR IT INTO THE RING YOU GET THE SAME FUNCTION. IF YOU PREVENT VP40 FROM FORMING THE RING, YOU GET LESS IT’S NOT A TOTAL KNOCK OUT. MAYBE TO BE IS MAKING PARTIAL STRUCTURE. SO THAT RING DOES SEEM TO HAVE SOME KIND OF FUNCTION INSIDE THE INFECTED CELL OF TRANSCRIPTIONAL CONTROL. SO THE WILDTYPE UNMODIFIED HERE MAKES A DIMER. THE DIMER IS CRITICAL FOR TRAFFICKING TO THE MEMBRANE T MAKES A FILAMENT TO BUILD AND BUD A VIRUS AND MAKES AN RNA BINDING TRICYCLE CONTROL THE CELLS. SO VP40 IS BOTH A STRUCTURAL AND A NONSTRUCTURAL PROTEIN. WE ARE DOING THIS ALL IN BACTERIA SO WE DON’T NEED A POSTTRANSLATIONAL MODIFICATION TO DO IT. MAYBE THERE IS ONE IN INFECTION. THERE IS NO MUTATION AND THE SAME POLYPEPTIDE MAKING DIFFERENT FUNCTIONS FOR DIFFERENT TIMES. WHAT DO YOU CALL A PROTEIN THAT DOES THAT? WELL, WEATOID AROUND WITH DIFFERENT NAMES BISTRUCTURAL, AMBEE FORM, FINALLY WE DECIDED THE TRANSFORMER WAS THE RIGHT ANALOGY. SO TRANSFORMERS ARE THESE TOY THAT IS REFOLD FROM A ROBOT INTO A VEHICLE. TRUCK, CAR, IT’S NEVER A CUP OF COFFEE OR AUGRAT BUT THEY REFOLD FROM ONE TO ANOTHER. WHAT I LIKE ABOUT THIS ANALOGY IS YOU SEE THE SAME SECONDARY STRUCTURAL ELEMENTS ACHIEVING DIFFERENT ROLES IN THE DIFFERENT MANIFESTATIONS. SO FOR EXAMPLE, THE TIRES ARE THE SEAT OF HIS PANTS AND HIS ANKLES AND THE TIRES ARE OF COURSE TIRES ON THE TRUCK. IF YOU DID NOT KNOW THAT THIS TRUCK EXISTED, I’M GOING BLOCK IT OUT. ALL YOU KNEW IS THE ROBOT AND YOU KNEW THAT SOMETIMES THIS PROTEIN COULD WALK AND TALK AND SHOOT AND SOMETIMES THIS ROBOT COULD CARRY A LOT OF CARGO AND DRIVE FAST AND YOU NEEDED TO FIND OUT WHY. IT’S EASY TO SEE HOW THE TIRES WOULD FLATTEN THE CARGO CARRYING CAPACITY BUT IF YOU WERE JUST LOOKING AT THIS, YOU WOULD CONCLUDE THIS ROBOT HAD ROCKET POWERED PANTS BECAUSE THEY ARE ESSENTIAL FOR CARRYING A LOT OF CARGO. THE HEAD HERE IS THE HYDROPHOBIC CORE ABOUT WHICH THE TRUCK IS FOLDED. SO IF YOU MUTATEED HEAD, YOU KNOCKOUT EVERYTHING. SO YOU SAY, THE HEAD IS THE THINKING CENTER. WE WERE NOT ALLOWED TO USE THAT ANALOGY BECAUSE THE TOY COMPANY WOULDN’T LET US AND SO INSTEAD, WE CALLED IT MOLECULAR ORIGAMI. AND SO, WHAT YOU CAN THINK ABOUT IN THIS SITUATION IS THE PROTEIN AS A BLANK SHEET OF THEY WERE FOLDS INTO DIFFERENT STRUCTURES ACCORDING TO DIFFERENT NEEDS AND THE VIRUS LIFE CYCLE. AND SO WE JUST SHOWED YOU HOW WE THINK THE DIMER MAKES RNA BINDING RING AND THE SAME ONE REARRANGES TO MAKE THE HEXAMER 2345 BUILDS AND BUDS THE VIRUS. VIRUSES ARE COMPELLED BY EVOLUTION TO BE SMALL. ESPECIALLY RNA VIRUSES. THEY DON’T VEY PROOF READING MACHINERY. YOU HAVE TO KEEP BELOW THAT THRESHOLD. HOW DO THEY KEEP THE GENOMES LEAN AND MEAN? HOW DO THEY DO MORE WITH LESS? THEY CAN HIJACK HOST PROTEINS FOR CENTRAL FUNCTIONS? THEY CAN OVERLAP READING FRAMES. THE SAME IN NUCLEIC ACID THAT MAKES DIFFERENT PROTEINS. THEY CAN HAVE MOONLIGHTING PROTEINS FOR THE SAME PROTEIN DOES DIFFERENT FUNCTIONS SO THE NUCLEO CAPSID SUPRESSES INTERFEAR ON SIGNALING. THIS IS A FOURTH WHERE A VP40 CRYSTAL STRUCTURE AND HERE IS ONE AND HERE IS ONE. THE POLYPEPTIDE THAT THE GENE ENCODES REARRANGE INTUSE DIFFERENT STRUCTURES FOR DIFFERENT FUNCTIONS AT DIFFERENT TIMES TO GET MORE FUNCTION FROM LESS GENE. SO THIS IS WHAT WE BRING TO GO TO THE VIRUS’S TERRITORY. THIS IS WHAT IT BRINGS. IT TRAVELS LIGHT. BECAUSE THIS ACTUALLY, THE FEW PROTEINS IT DOES MAKE, ACHIEVE A MULTITUDE OF FUNCTIONS. THIS IS MY LAB AT SCRIPPS. WE COLLABORATE WITH A NUMBER OF WONDERFUL LABS AND VERY SUPPORTIVE. ONE LAB DID THE VP40 AND THESE GROUPS ARE WORKING WITH US TO DEVELOP ANTIBODIES AGAINST THE EBOLA VIRUS AND UNDERSTAND VIRAL ENTRY AND I’D LIKE TO THANK THE NIH FOR FUNDING ALSO THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY AND I’D LIKE TO THANK YOU FOR YOUR ATTENTION. [ APPLAUSE ]>>WHAT GREAT STORIES. PLEASE, IF PEOPLE HAVE QUESTIONS, THERE ARE MICROPHONES IN THE AISLES. FEEL FREE TO COME FORWARD AND ASK WHAT IS ON YOUR MIND. WHILE PEOPLE ARE THINKING, I HAVE TO COME BACK TO SOMETHING YOU SAID EARLY IN THE TALK ABOUT THE DIFFERENCE IN WHO SURVIVES EBOLA AND WHO DOESN’T IN TERMS OF WHO IS ABLE TO MOUNT SOME KIND OF AN IMMUNE RESPONSE IN FOUR DAYS. DO YOU HAVE ANY IDEA WHAT THAT IS ABOUT? WHAT DETERMINES WHETHER YOU’RE IN THE SURVIVAL CATEGORY OR NOT?>>WE DO NOT. THERE ARE SOME OBVIOUS ANSWERS THAT YOU CAN RULE OUT. HEALTH CARE STATUS, NUTRITIONAL STATUS. IF YOU RULED THAT OUT, I THINK WE STILL NEED TO DO THE WORK TO UNDERSTAND. I THINK THAT WE KNOW THAT THE VIRAL FACTORS AT PLAY BUT WE DON’T THE HUMAN GENETIC FACT AUTHORITIES CONTROL EXISTENCE. WE KNOW THESE FOR LASSA VIRUS. EBOLA IS QUITE NEW. LASSA IS QUITE OLD. FOR EXAMPLE, LASSA VIRUS HAS BEEN IN NIGERIA FOR THOUSANDS OF YEARS AND SIERRA LEON 150 YEARS AGO. PEOPLE EVOLVED MUTATIONS IN THEIR RECEPTOR LIKE A SICKLE CELL ANEMIA SO THEY ARE LESS SUSCEPTIBLE TO THE VIRUS. EBOLA WE DON’T KNOW. WE NEED PEOPLE ON THE GROUND TO LOOK AT THE HUMAN GENETICS OF THE SURVIVORS.>>VERY INTERESTING TALK. AND IN A DIFFERENT VERSION OF QUESTION THAT DR. COLLINS ASKED, MY QUESTION IS, DO WE KNOW — YOU MENTIONED YOU DON’T KNOW BUT THE THOUGHT IS, ARE YOU IMPACTING DIFFERENT COMPOSITION OF CELLS PERHAPS THE MUTANT CELLS THAT CAN ACTUALLY NEUTRALIZE? OR IS THERE ANYTHING KNOWN ABOUT THE PROFILE OF IMMUNOGLOBULIN SYNTHESIS AND THE EFFECTED CELLS THAT COULD EITHER NEUTRALIZE AND PRODUCE IMMUNE RESPONSES, OR THEY ARE NOT AFFECTED IMMEDIATELY.>>I DON’T THINK WE REALLY KNOW THE ANSWERS TO THAT QUESTION YET BECAUSE WE HAVEN’T DONE AS MUCH IN-DEPTH ANALYSIS OF THE HUMAN SURVIVORS AS WE NEED TO HAVE DONE. SO A LOT OF THE STUDIES ARE ONGOING. IT DOESN’T INFECT MOST CELL TYPES, MONOCYTES AND MACROPHAGES TO BEGIN. SO IT’S QUITE A POLICE OFFICERRIC VIRUS BUT WE HAVEN’T LOOKED ENOUGH AT THE SURVIVORS TO FIND OUT WHAT THE DIFFERENCE IS IN THE CELL TYPES.>>THE RING AND THE FILAMENT THEY COEXIST. SO, BASICALLY SOMETHING IS DETERMINING THE FOLDING PATTERN. DO YOU KNOW WHAT THAT IS? IS IT A CHAPTER?>>NO, AND IT’S KILLING ME. WHAT IS THE TRIGGER? WHAT MAKES IF DO ONE THING AND — SO I DON’T KNOW. WE CAN SPECULATE. SO, MAYBE AT SOME STAGE OF THE VIRUS LIFE CYCLE THERE IS A LOT OF VIRAL RNA AND WHAT I HAVE DRAWN IS RNA BINDING AND DRAWING A WEDGE BETWEEN THE DOMAINS AND KICKING OFF THE C TERMINAL AND OPENING UP. IF IT’S NOT RNA, MAYBE IT’S A PROTEIN COMPLEX LIKE A POLYMERASE OR SOMETHING LIKE THAT. IS THERE A CHAPERON? COULD BE. WHAT WE ARE TRYING TO DO NOW IS USE THESE POINT MUTATION THAT IS WE HAVE THAT LOCK IT INTO ONLY RING OR NEVER RING OR EITHER AND SEE WHAT THEY PULL DOWN AND BASED ON WHAT IT PULLS DOWN DOES IT TELL US IF IT NEEDS A HOST FACTOR TO DRIVE THOSE OR PURELY A VIRAL FACTOR? IS IT RNA? AND RNA, WHICH RNA. THE RN. THAT PICKED OUT FROM THE CRYSTAL STRUCTURE OF THE UGA, UGA, UGA. SO DOES IT RECOGNIZE STOP CODONS? IS THAT WHAT IT IS TRYING TO FIND? DOES IT LOOK FOR THE END OF THE GENE? WE DON’T KNOW. AND THOSE ARE EXACTLY THE KIND OF QUESTIONS WE ARE TRYING TO ASK. ALSO WHAT IS THE THERMODYNAMICS? ARE THESE ALL EQUALLY STABLE OR DO YOU NEED THE INPUT OF ENERGY OR CHAPERON IN ORDER TO GET FROM ONE TO THE OTHER?>>THERE IS A KINETICS DIFFERENCE IN THE SYNTH US?>>WE DON’T KNOW. WE HAVE TRAFFICKED WHERE DP40 IS AT DIFFERENT STAGES OF THE VIRUS LIFE PSYCHE E8. EARLY IT HUGS THE NUCLEUS AND NEIGHBOR IS IN THE RING FORMATION AND MAKING COPIES. LATER IN THE VIRUS LIFE CYCLE, IT CATCHES A RIDE ON MICROTUBULES UP TO THE SURFACE AND MAKES FILL COMMENTS BUDS OUT. SO THE LOCATION TRAFFIC MAY BE WITH FUNCTION. WHAT CAUSES IT TO MAKE THE DIFFERENT STRUCTURES WE STILL NEED TO FIGURE OUT. BUT WHAT IS INTERESTING ABOUT THAT IS IT IS A DIFFERENT PERSPECTIVE ON THE PROTEIN FOLDING PROBLEM, RIGHT 1234 INSTEAD OF UNKNOWN, UNFOLDED THING TO ONE SINGLE FOLDED STRUCTURE, WE HAVE A FOLDED AND A FOLD ED AND THEY CONVERGE AND ARE THESE EQUAL OR DIFFERENT OR WHAT CAN WE LEARN ABOUT THE PROTEIN FOLDING PROBLEM GETTING FROM ONE TO THE OTHER? CAN WE LEARN SOMETHING ABOUT INFORMATION AND CODING THAT WE HAVE MULTIPLE FUNCTIONS ENCODED IN THIS ONE PIECE OF CODE.>>SO, THE EXON NUCLEASE YOU TALK ABOUT, ONE WOULD ENVISION THAT IT MIGHT ACTUALLY DO HARM TO THE REPLICATION OF THE VIRUS TOO IF IT GET ACTIVATED IN AN OPPORTUNE TIME. IS THERE A TIMING INTERIM OF WHEN THIS VIRAL ENCODED NUCLEUS GET ACTIVATEED DURING THE LIFE CYCLE SO IT ALLOWS IT TO EVADE IMMUNE DETECTION BUT DOESN’T HURT ITS OWN PRODUCTION?>>THAT’S A FANTASTIC QUESTION. THAT’S SOMETHING I WANTED TO ASK EARLIER TODAY. WE DON’T KNOW. WE THAN ONE DOMAIN BIND GENOME AND THE OTHER DOUBLE-STRANDED RNA. FOR THE PROTEIN TO FUNCTION THEY MUST BE GENETICALLY LINKED. SO THEY HAVE TO BE TETHERED TOGETHER. THE MP DOESN’T EXIST AS A MONOMER, IT’S A LIGMER. SO SOMEHOW, THIS INTERACTS WITH THE FRIENDS. SO, WE ALSO KNOW THAT THE LINKER IS QUITE PATROLAISE SENSITIVE AND IN INFECTION A LOT OF C TERMINAL DOMAINS GO FREE. IS THE THE C TERMINAL DOMAIN GOING FREE THAT IS SCRUBBING OUT DOUBLE-STRANDED NA OR IS THAT A ACCIDENT OF CONTAMINATION? OR THIS FUNCTION PHYSICALLY TETHERED TO THE REP CALLS SIDE OR TO ERASE SOME INTERMEDIATE AS IT IS BEING MADE OR DOES IT HAVE SOME OTHER FUNCTION THAT WE NEVER THOUGHT ABOUT?>>DOES IT DO ANY EDITING FOR THE VIRAL GENOME ITSELF?>>THAT IS ANOTHER QUESTION. WE WANTED TO KEEP CYCLES OF REPLICATION GOING TO SEE IF IT HAD A PROOF READING FUNCTION. WE DON’T KNOW. WE LOVE TO DO THOSE KIND OF STUDIES.>>SO I HAVE A SECOND QUESTION ABOUT THIS RNA BINDING TOWARDS BY THE PROTEIN AT THE END AND ALSO ON THE SIDE. SO, I GUESS YOU MENTIONED THAT CHEMICAL NATURE TOTALLY DIFFERENT, SON HYDROPHOBIC AND ONE IS ELECTROSTATIC. WHICH DOMINANTS? IF YOU ANALYZE THE BINDING AFFINITY — AUTOMOBILEY THE CAP END BINDING HAS A DISADVANTAGE BECAUSE IT NEEDS EACH STRAND TO BIND TWO. IF IT BIND ON THE SIDE YOU CAN BIND MULTIPLE COPIES. SO THERE IS ADVANTAGE AND DISADVANTAGE IN THERE.>>SO WE HAVEN’T — WHAT WE HAVE HAVE DONE AND HAVEN’T DONE IS HE HOW IT BIND TO A CIRCULAR DOUBLE-STRANDED RN. THAT HEADS NO END. WE SEE WHAT HAPPENS IS IT MAKES THE CAP FIRST AND THEN THIS BACKBONE BINDER DOES THE SAME INTERACTION ALL THE WAY DOWN THE REST. SO IF YOU KEEP POLYMERIZING ONCE IT HAS THIS CAP ON THERE. MAR BERG VIRUS DOESN’T NEED THE CAP AND IT IS HAPPY TO JUST POLYMERASE. SO, ANOTHER GROUP HAS BEEN DOING SIMILAR INSTRUCT US AND THEY SAY THE SAME THING. STRUCTURES. SO, BASED ON THAT, I WOULD SAY IT IS THE BACKBONE BIND THEY’RE MIGHT BE DOMINANT BUT WE HAVEN’T BEEN ABLE TO TEASE THEM APART YET. YOU LOSE BINDING WHEN YOU HAVE AN OVER HANG THAT PROJECTS INTO THE SPACE THAT NEEDS TO BE OCCUPIED BY THE END CAP. SO, I DON’T KNOW WHAT THAT SAYS ABOUT RELATIVE INFINITY BUT IT SEEMS YOU NEED THE END CAP TO GET IT GOING. INFINITY IS NOT HIGH. IT’S MAYBE 10 OR MAYBE A MICROMOLAR.>>ONE MORE QUESTION AND THEN WE WILL ADJOURN TO THE RIBBON CUTTING PLEASE.>>IN THE CRUSTAL STRUCTURES OF VP40, YOU SHOWED A HEX MERIC RING I DIDN’T SEE LATER. IS THAT INVOLVED IN THE CELL DURING THE FASHION –>>HEXAMER VERSUS OPT MER? WE DON’T EVEN KNOW IF INFECTED CELLS IT IS EVEN A COMPLETE RING OR IF IT’S THE SAME INTERFACE BUT SPLIT OPEN AND SPIRALING AROUND THE NUCLEO CAPSID. WE DON’T KNOW. WE JUST KNOW THAT YOU CAN GET IT TO FORM BOTH KIND OF RINGS AND ONE CRYSTALIZED AND THE OTHER DIDN’T.>>ALL RIGHT.>>WE WILL NOW INVITE EVERYBODY TO WALK UP THE HALL TO THE RIBBON CUTTING. BEFORE YOU DO SO, PLEASE JOIN ME IN THANKING ERICA FOR A REALLY FASCINATING SEMINAR. [ APPLAUSE ]

12 Comments

  • Reply Nasiruddin84 August 3, 2014 at 2:10 pm

    Excellent lecture. Thank you.

  • Reply Chris Brisson August 6, 2014 at 4:41 pm

    Prediction:  Nobel Prize

  • Reply alz123alz September 16, 2014 at 12:07 am

    THANK YOU FOR THE EXCELLENT LECTURE.  WHERE ARE THE BRILLIANT BLACK COLABORATORS? THEY CAN ANSWER THE QUESTIONS THAT YOU NEED ANSWERS. 

  • Reply keine031 September 24, 2014 at 12:44 pm

    Smart confident women are really hot.

  • Reply Jai Cilento I Love That Guy ʕ•ᴥ•ʔߛ ̋ October 1, 2014 at 8:42 pm

    Awesome

  • Reply Meekseek October 14, 2014 at 8:20 pm

    little cheerios, oh my

  • Reply Patrick Mwaura October 16, 2014 at 3:06 pm

    Wow what an inspiration!

  • Reply Tim Kasey November 1, 2014 at 6:50 am

    If I were to write 200,000 pages on roads, and not explain anything about driving, would my information be of value to a new driver?  If I were to write 2 paragraphs, giving both: 1) Blood Ph level needed to stop Ebola duplication; 2) Temperature needed to kill the virus;  would my information be less 'vain' than yours? 

  • Reply offmeds2nite January 29, 2015 at 5:46 am

    Dr. Saphire rocks my socks 🙂

  • Reply Mathavan Muthaiyan April 14, 2015 at 7:18 am

    Valuable lecture thank you!

  • Reply U Alam November 16, 2015 at 12:47 pm

    would you be able to identify receptor binding molecule that Ebola gp binds to on host cell, like influenza virus gp binds to sialic acid.

  • Reply U Alam November 16, 2015 at 12:48 pm

    I have been trying to search for the answer but have not come up with anything specific.

  • Leave a Reply