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>> 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.