>> 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 ]