>> MY NAME'S ERIC FREED PART OF
THE NCI PROGRAM AT FREDERICK AND
A PLEASURE TO INTRODUCE THE
LECTURE.
SO A FEW WORDS ABOUT GEORGE.
HE WAS A GRADUATE OF PRINCETON
UNIVERSITY AND HARVARD MEDICAL
SCHOOL AND BECAME HEAD OF THE
LABORATORY OF MOLECULAR VIROLOGY
AT THE NCI AND BECAME CHIEF IN
1980 OF THAT LAB AND IN 1981 HE
RECEIVE THE ARTHUR S. FLEMING
AWARD FOR OUTSTANDING GOVERNMENT
SERVICE AND IN 1987 THE YEAR OF
HIS IN TIMELY DEATH WAS ELECTED
TO THE NATIONAL ACADEMY OF
SCIENCES.
THIS IS A PICTURE OF HIM AND
RECEIVING THE AWARD.
GEORGE PUBLISHED OVER 140 PAPERS
DURING HIS SHORT CAREER
DECIPHERIDECIPHER
DECIPHERING MANY MECHANISMS.
THE GEORGE KHOURY LECTURES BEGAN
IN 1995 AND FEATURED A SIMILARLY
DISTINGUISHED GROUP OF SPEAKERS
SOME OF WHOM RECEIVE THE NOBEL
PRIZE AND THESE ARE THE MOST
RECENT SPEAKERS.
IT'S A PLEASURE TO INTRODUCE THE
LECTURER.
A NATIVE OF ARIZONA AND RECEIVED
HER BACHELOR'S DEGREE FROM
UNIVERSITY OF ARIZONA AND DID A
POSTDOC AT UCSF AND IN 2006 WAS
RECRUITED TO AN ASSISTANT
PROFESSOR POSITION AT BERKELEY
IN THE DEPARTMENT OF PLANT AND
MICROBIOLOGY AND RECEIVED TENURE
IN 2012 AND BECAME AN HHI
INVESTIGATOR AND PULL PROFESSOR
IN 2017 AND SINCE 2021 HAS BEEN
ASSOCIATE CHAIR OF THAT
DEPARTMENT AT BERKELEY.
SHE RECEIVED A NUMBER OF HONORS
AND RECOGNIZES.
TOO MANY TO LIST HERE BUT IF YOU
KNOW THE ONES THE KECK
DISTINGUISHED YOUNG SCHOLAR
AWARD.
THE U.C. BERKELEY EXCELLENCE IN
GRADUATE MENTORSHIP AND 2019
ELECTED FELLOW OF THE AMERICAN
ACADEMY OF MICROBIOLOGY.
HER RESEARCH FOCUSSED ON SEVERAL
P.O.s INCLUDING MECHANISMS AND
CONSEQUENCES OF mRNA
DEGRADATION AND TRANSCRIPTION
REGULATION DURING LATE STAGES OF
IN STAGES AND NON-CODING RNA.
THE SEMINAR IS ENTITLED
CONTROLLING THE MESSAGE HOW
HERPES VIRUSES MANIPULATE THE
GENE EXPRESSION LANDSCAPE.
BEFORE BRITT DOES HER SEMINAR WE
DISCOVERED AT DINNER LAST NIGHT
SHE NEVER HEARD OF OLD BAY SO
FOR A TOKEN OF APPRECIATION I'D
LIKE TO PRESENT HER WITH THIS
SMALL TIN OF GENUINE MARYLAND
OLD BAY.
IT'S A REAL PLEASURE TO BE HERE.
THE FLOWERS ARE IN BLOOM.
IT'S A BEAUTIFUL SUNNY DAY
COMPARED TO THE RAIN STORM I
LEFT IN CALIFORNIA YESTERDAY AND
AS ERIC ALLUDED TO I SPENT
PRETTY MUCH MY ENTIRE CAREER
STUDYING VIRUS INTERACTION AND
IT'S AN HONOR TO GIVE THIS
LECTURE FOR GEORGE KHOURY AND
I'M FAMILIAR WITH THE RESEARCH
HE CARRIED OUT RELATED TO USING
DNA VIRUSES IN PARTICULAR TO
DISSECT FUNDAMENTAL PRINCIPLES
OF GENE REGULATION.
MUCH OF HIS RESEARCH PROVIDED
THE FOUNDATION OF KNOWLEDGE THAT
UNDER LIES MANY OF THE PROJECTS
IN MY OWN LAB NOW.
I FEEL LIKE IT'S A REAL DELIGHT
TO BE ABLE TO PARTICIPATE IN
THIS LECTURE SAYS.
-- SERIES.
WHEN I THINK OF VIRAL INFECTION,
REALLY GENE REGULATION AND THE
ABILITY TO MANIPULATE GENE
REGULATION IS AT THE CRUX OF
WHETHER OR NOT OR NOT AN
INFECTION IS GOING IT TAKE OVER
OR PETER OUT.
AND THIS IS BECAUSE FROM THE
HOST SIDE THE ABILITY TO ELICIT
A PARTICULAR GENE EXPRESSION
PROGRAM IN RESPONSE TO INFECTION
IS GOING TO DETERMINE WHETHER OR
NOT THE HOST CELL CAN COUNTER
ACT AND FIGHT BACK AGAINST THE
VIRAL INFECTION AND CONVERSELY
THE ABILITY OF THE VARIOUS TO
COMMANDEER THOSE GENE EXPRESSION
MACHINERY AND SHUT DOWN THE ATS
ASPECTS IN CONTROLLING VIRAL
INFECTION WILL DETERMINE WHETHER
THE VIRAL INFECTION WILL TAKE
OUT.
REHOSTING THE ENVIRONMENT IS
CRITICAL IMPORTANCE FOR VIRUSES
AND SOMETHING I'VE BEEN
INTERESTED IN FOR A LONG TIME.
BECAUSE IT'S CENTRAL IMPORTANCE
TO VIROLOGY MANY CODE PROTEINS
WITH A DEDICATED JOB OF SHUTTING
THEM DOWN AND THEY CAPTIVATED ME
FOR YEARS.
THE IDEA HERE SOME OF WHICH ARE
SHOWN IN THE DIAGRAM EXPRESSION
OF THE PROTEINS, MANY OF WHICH
TARGET RNA DIRECTLY LEADS TO A
LARGE-SCALE DOWN REGULATION OF
HOST GENE EXPRESSION.
FIRST, IT HELPS THE VIRUS SHUNT
TRANSLATION RESOURCES AWAY FROM
THE HOST AND TOWARDS THE VIRUS
BECAUSE NO VIRUS OF COURSE CAN
ENCODE THE TRANSLATION MACHINERY
ALL VIRUSES NEED.
SECOND, BY CASTING A WIDE NET
AND TARGETING MOST OF THE
MESSENGER RNA THE VIRUS IS ABLE
TO CAPTURE MANY OF THE IMMUNE
STIMULATORY FACTORS IN THE
ATTEMPT TO STOP THE VIRUS.
IN THIS REGARD THE VIRUSES BY
GLOBALLY SHUTTING DOWN GENE
EXPRESSION CAN HELP COUNTER
ACTED RESPONSE.
WE STUDIED THE SHUT OFF FACTORS
BUT THE MAIN VIRUS IS FOCUSSED
ON THE HERPES VIRUS AND THAT
WILL BE THE TOPIC OF THE VIRUS
TODAY.
HERPES VIRUS I WOULD ARGUE AND
MANY WOULD AGREE ARE AMONG THE
MOST SUCCESSFUL PATHOGENS IN THE
PLANET IN PART BECAUSE THEY'RE
UBIQUITIOIOUIOUIOUS -- UBIQUITOUS AND
IT'S NOT JUST HUMANS, HERPES
VIRUSES ARE INCREDIBLY
WIDESPREAD ON THE PLANET.
MANY VERTEBRATES HAVE THIS VIRUS
ASSOCIATED WITH THEN AND OYSTERS
HAVE THEM AND THEY'RE AROUND 200
AND 400 MILLION YEARS OLD AND
IT'S AN EXTENSIVE
CO-EVOLUTIONARY HISTORY WITH US
AND OTHER HOSTS AND ONE OF THE
REASONS WE THINK THEY MAKE
EXCELLENT MODEL TO DISSECT GENE
REGULATION IN MAMMALIAN CELLS
UNDER STRESSFUL CONDITIONS LIKE
PATHOGEN INVASION.
MY LAB IS FOCUSSED ON THE GAMMA
HERPES VIRUSES THAT HAVE TWO
HUMAN PATHOGENS, EPSTEIN BARR
VARIETY WHICH CAUSES A NUMBER OF
B CELL LYMPHOMAS AND IDENTIFIED
AS A MAIN CAUSE OF MULTIPLE AND
KAPOSI'S SARCOMA HERPES VIRUS IN
AIDS PATIENTS AND WE WORKED ON
MOUSE HERPES VIRUS MHV68
COMMONLY USED IN THE FIELD FOR
IN VIVO AND STUDYING THE
WHITTAKER CYCLE WE'RE INTERESTED
IN.
DURING LYTIC AMPLIFICATION THEY
HAVE A SHUT OFF FACTOR AND SHUT
OFF THE NUCLEASE OUR LABS AND
OTHER LABS HAVE SHOWN THE
ABILITY OF THIS NUCLEASE TO
ELICIT WIDESPREAD RNA DECAY AND
PLAYS A VARIETY OF ROLES IN THE
GAMMA HERPES LIFE CYCLE FROM
PROMOTING IMMUNE EVASION TO
ENABLING THE VIRUS TO TRAFFIC
WHERE IT NEEDS TO GO IN THE IN
VIVO MODEL IN A MOUSE.
ESTABLISHING LATENCY BEING ABLE
TO REPLICATE IN B CELLS THE MAIN
NICHE FOR GAMMA HERPES VIRUSES
AND BEYOND CONTROLLING THE HOST
GENE EXPRESSION ENVIRONMENT
SETTING THE HOST GENES VERSUS
VIRAL GENES INDUCES VIRAL GENE
EXPRESSION AND PROTEIN
PRODUCTION AND WHAT GETS
PACKAGED TO NASCENT VIRAL
PARTICLES INFLUENCING NEW ROUNDS
OF INFECTION.
VARIOUS STAGES ARE INFLUENCED BY
THE ABILITY TO CONTROL THE GENE
EXPRESSION CASCADE AND WE
STUDIED THIS PROCESS FOR A
NUMBER OF YEARS.
IN PART STUDYING THE RNA
MECHANISMS OF THE ENZYMES THE
SOCK PROTEIN CAN BROADLY TARGET
CELLULAR MESSENGER RNA AND
TARGETS ONLY TRANSCRIBED RNA AND
THOUGH IT CAN BROADLY TARGET RNA
IT HAS SPECIFICITY BUILT IN AND
CLEAVES THROUGH A COMBINATION OF
SEQUENCE AND STRUCTURE CAUSING
AN INTERNAL CLEAVAGE EVENT
BASICALLY IT CREATES UNPROTECTED
FREE ENDS OF THE RNA THAT CAN
THEN BE ATTACKED BY THE RESIDENT
EXISTING NUCLEASES OUR OWN CELLS
NORMALLY USE TO CONTROL RNA
TURNOVER.
THE TWO-STEP PROCESS OF THE
VIRUS INDUCING AN INTERNAL
CLEAVE CATHAGE AND CLEARING THE RNA IS
IMPORTANT AND HOLD THAT THOUGHT
IN YOUR MIND I'LL TOUCH ON IT
LATER IN THE LECTURE.
SO BEYOND STUDYING HOW THESE
ENZYMES WORK AND TARGETING RNA
AND WHAT SORT OF RNA FEATURES
ARE REQUIRED FOR TARGETING OR
ESCAPE, IN THE LAST SEVERAL
YEARS WE'VE SHIFTED OUR FOCUS
SOMEWHAT TO SAYING CAN WE USE
THESE AS TOOLS TO PROBE HOW
CELLS SENSE AND RESPOND TO LARGE
CHANGES IN MESSENGER RNA
ABUNDANCE.
BECAUSE THESE ARE FEATURES
CHARACTERISTIC OF A NUMBER OF
PATHOGENIC SITUATIONS.
SO I'M GOING IT TELL YOU TWO
RELATED STORIES TODAY.
THE FIRST STORY FOCUSES ON THE
HOST SIDE AND JUST THAT QUESTION
THAT I OUTLINED OF USING THE
VIRUSES AS A TOOL TO EXPLORE
CONNECTIONS BETWEEN SEEMINGLY
VERY DISTAL STAGES OF THE VIRAL
LIFE CYCLE AND HOW THOSE ARE
INTERCONNECTED AND THE CELL USES
THOSE IN RESPONSE TO STRESS AND
IN THE SECOND PART OF MY TALK
I'LL FOCUS ON THE VIRUS ITSELF
AND HOW IT BENEFITS FROM THIS
AND IS ABLE TO ORCHESTRATE AN
UNUSUAL GENE EXPRESSION CASCADE
OR REGULATION FOR THE VIRAL
GENES.
SO THIS FIRST PART OF THE STORY
BEGINS WITH THE IDEA THAT CELLS
AND THEY'RE UNINFECTED STATE
BASICALLY ARE TRYING TO ACHIEVE
HOMEOSTASIS.
THEY WANT AN APPROPRIATE OVER
ALL BALANCE OF MESSENGER RNA.
THE IDEA OF CELLS TRYING TO
ACHIEVE HOMEOSTASIS BY
CONTROLLING THEIR MESSENGER
LEVELS IS WELL ESTABLISHED IN
YEAST AND MAMMALIAN CELLS.
IT'S BEEN SHOWN FOR EXAMPLE IF
YOU DECREASE THE OVER ALL RATE
OF MESSENGER RNA SYNTHESIS, FOR
EXAMPLE USING SLOW RNA
POLYMERASE 2 THE CELL WILL
RESPOND AND COMPENSATE BY
GENERALLY STABLIZING THE
MESSAGES THAT EXIST.
FEWER BEING PRODUCED AND YOU
NEED THOSE THERE STICKING AROUND
LONGER.
CONVERSELY FURTHER SLOW DOWN THE
DEGRADATION BY GETTING RID OF
KEY DECAY FACTORS, FOR EXAMPLE,
THE CELL WILL COMPENSATE BY
SLOWING THE RATE OF
TRANSCRIPTION BECAUSE YOU HAVE
THOSE STICKING AROUND LONGER AND
DON'T NEED TO PRODUCE AS MANY TO
ACHIEVE YOUR OPTIMAL BALANCE.
BUT WE WONDERED WHAT ABOUT IF
YOU WERE LOOKING IN THE OTHER
DIRECTION INSTEAD OF STABLIZING
RNA A MUCH MORE COMMON SITUATION
IN VIRAL INFECTION IS GLOBAL
DESTABILIZATION OF RNA.
AND SO IN THIS REGARD WITH THE
CELL ALSO TRY AND MAINTAIN
HOMEOSTASIS WE THOUGHT NOT.
CELLS GENERALLY WHEN FAZED TRY
TO SLOW THING DOWN WITH PRK
ACTIVATION AND THINGS LIKE THAT.
WHAT WE FOUND IN THE CASE OF
CELLULAR THREAT THE HERPES VIRUS
INFECTION THAT ELICIT RNA DECAY
THE BALANCE IT TIPPED FROM
HOMEOSTASIS AND THE CELL INSTEAD
OF AMPLIFYING TRANSCRIPTION
RESPONDS BY SHUTTING DOWN
TRANSCRIPTION ESSENTIALLY MAG
MAGNIFYING THE SCREEN AND WE
HYPOTHESIZE IS BECAUSE MANY
VIRUSES LIKE DNA VIRUSES ARE
RELIANT ON THE MACHINERY.
WHEN CELLS SENSE THEY'RE
INFECTED ARE TRYING TO FIND WAYS
TO SHUT DOWN CORE CAN CELLULAR
PROCESSES AS I SAID TRANSLATION
BEING VERY WELL CHARACTERIZED
ONE BUT WE THINK TRANSCRIPTION
IS ANOTHER ONE PARTICULARLY FOR
DNA VIRUSES.
ALL RIGHT.
SO LET ME SHOW YOU WHAT I MEAN.
WE OBSERVE THE CONNECTION
BETWEEN RNA DECAY AND A --
TRANSSCRIPTION.
WE WERE USING A WILD TYPE VIRUS
USING A FUNCTIONAL SOX PROTEIN.
WHEN YOU INFECT CELLS AND WE'RE
USING MOUSE FIBROBLAST CELLS AND
IT INDUCES CYTO-MICK --
CYTOPLASMIC EFFECT AND IT
TARGETS mRNA s FOR DECAY.
THE VIRUSES REPLICATE SIMILARLY
BUT THE SOX MUTANT DOES NOT
ACCELERATE RNA DECAY IN THE
CYTOPLASM AND THE WILD TYPE
DOES.
HOW DOES THE CELL RESPOND TO
ACCELERATED RNA DECAY OR NORMAL
RNA DECAY AT THE TRANSCRIPTIONAL
LEVEL?
WE LOOK AT NASCENT RNA
DESCRIPTION AND IN THE
UNINFECTED CELL WHEN LOOK AT THE
PILE UP OF POLYMERASE IT'S
CENTERED NEAR THE TRANSCRIPTION
START SITE AS YOU'D EXPECT
DURING INFECTION WITH WILD TYPE
THERE'S A LARGE SCALE CLEARANCE
OF THE POLYMERASE FROM THE HOST
GENOME AND LINKED TO CYTOPLASMIC
RNA DECRAY -- DECAY AND THERE
IS SOMETHING ABOUT RNA DECAY IN
THE CYTOPLASM THAT IS CAUSING A
REDUCTION IN RNA SYNTHESIS IN
THE NUCLEUS.
SO HOW MIGHT THAT HAPPEN?
THERE'S A COUPLE POSSIBILITIES.
ONE, SOMETHING ABOUT TARGETING
TRANSCRIPTS INDIVIDUALLY THAT
MATTER.
FOR EXAMPLE, IF YOU HAVE FACTORS
THAT ENCODE TRANSCRIPTIONAL
REGULATORS AND YOUR GETTING RID
OF THEIR TRANSCRIPTS AND THE
PROTEINS ARE LABEL YOU MAY
CHANGE THE PROTEINS IN THE CELL
RESPONSIBLE FOR TRANSCRIPTION
AND DAMP ENNING TRANSCRIPTION
THAT WAY.
IN ADDITION, YOU CAN HAVE A
SITUATION WHERE THE CELL IS
SOMEHOW SENSING THE DEGRADATION
OF THE CLEAVED FRAGMENTS THE
ACTIVE RNA DECAY AND RESPONDING
TO THAT.
WHAT I'LL TELL YOU IS BOTH OF
THESE TURN OUT TO BE TRUE AND WE
DISCOVERED THEM IN REVERSE
ORDER.
THE ONE WE HIT ON FIRST WAS THE
IDEA THAT CELLS COULD ACTUALLY
SENSE ACCELERATED RNA DECAY.
THE REASON WE KNEW IT WAS THE
CASE WE COULD RECAPITULATE THE
RESULTS IN UNINFECTED CELLS IF
WE EXPRESSED THE VIRAL NUCLEASE
WHICH CAUSED RNA DECAY IN THE
OUTSIDE OF INFECTION AND IF WE
PREVENTED DEGRADATION MUCH THESE
CLEAVED FRAGMENTS YOU NO LONGER
HAVE THE TRANSCRIPTIONAL
REGRESSION.
IT'S POST TRANSLATIONAL IN
ACTIVATION BUT INVOLVING THE
DETECTION SOMEHOW OF THE
DEGRADING RNA.
WE SAT OUT TO FIGURE OUT HOW
DOES THAT HAPPEN?
IN THINKING ABOUT THIS WE
CONSIDERED WHAT HAPPENS TO AN
RNA AS IT'S BEING DEGRADED IT
MAY BE ABLE TO SENSE?
ONE OF THE HYPOTHESES WE ENDED
UP TESTING WAS THE IDEA THAT
CELLS THE RNA IN A CELL IS NOT
NAKED.
IT'S CODED BY SUITES OF RNA
BINDING PROTEIN.
AS THE RNAs ARE BEING CLEAVED
AND THE SUBSEQUENT CLEAVAGE
PRODUCTS BEING DESTROYED THE PRO
PROTEINS AND THEY'RE BEING
EVICTED AND YOU CAN ENVISION IF
YOU'RE RAMPING UP THE RATE WITH
WHICH RNAs ARE BEING DESTROYED
YOU HAVE MORE PROTEINS BEING
RELEASED FROM THE RNAs.
WE KNOW RNA BINDING PROTEINS
MANY OF THEM ARE SHUTTLING
PROTEINS THAT MOVE IN AND OUT OF
THE NUCLEUS DEPENDING AS
MESSENGER RNA ITSELF STARTS IN
ONE COMPARTMENT AND ENDS IN
ANOTHER.
WE CONSIDERED THE POSSIBILITY
PERHAPS RELEASE OF RNA BINDING
PROTEINS WAS BEING SENSED BY THE
CELL AND LEADING TO THE
TRANSCRIPTIONAL RESPONSE PERHAPS
BECAUSE THEY'RE MOVING WITHIN
THE CELL DIFFERENTIALLY
DEPENDING IF THEY'RE BOUND OR
RELEASED FROM RNA.
WE TEST THE HYPOTHESIS WITH A
LAB BY ANALYZING THE PROTEIN
DISTRIBUTION AND REDISTRIBUTION
THAT HAPPENS IN THE CELL IN
RESPONSE TO RNA DECAY.
THE FIRST TIME WE DID THIS WE
DID IT IN THE MINIMALIST SYSTEM
WE'RE JUST TRYING TO ISOLATE THE
RNA DECAY PHENOTYPE AND TAKING
H
HEXTILES OUT OF THE CONTEXT OF
INFECTION AND WE EITHER HAD A
WILD TYPE VERSION OF THE
NUCLEASE, WE HAD A CATALYTICALLY
VERSION AND WE RELEASED THE
BINDING PROTEINS AND THAT WAS
CARRIED OUT BY THE CELLULAR
NUCLEASE EXERON 1 TO SLOW THE
RATE OF DECAY OF THE ALREADY
CLEAVED TRANSCRIPT.
EACH OF THESE CELLS WE
FRACTIONATED AND EXTRACTED TOLD
PROTEIN LATED WITH TAGS AND
IDENTIFIED BY MASS SPECTOMETRY
AND LOOKING FOR THE SIGNATURE
AND THAT WOULD BE THE ABUNDANCE
OF A PROTEIN UP IN ONE
COMPARTMENT AND DOWN IN A
COMPENSATORY WAY IN ANOTHER
COMPARTMENT.
IF WE LOOK FOR THEM MOVING FROM
THE CYTOPLASM TO NUCLEUS YOU SEE
A DECREASE IN THE CYTOPLASMIC
SIGNAL AND THAT WAS THE DOMINANT
PHENOTYPE.
WE SAW A CLEAR SIGNATURE IN
CELLS EXPRESSING THE WILD TYPE
NUCLEASE OF PROTEINS THAT WERE
SHIFTING THEIR DISTRIBUTION FROM
THE CYTOPLASM OF THE CELL TO THE
NUCLEUS OF THE CELL.
AND SO WHAT I'M SHOWING YOU HERE
ARE THESE RESULTS IN A HEAT MAP
IN WHICH YOU CAN SEE THAT
COMPARED TO THE CATALYTICALLY
INACTIVE VERSION OF THE NUCLEASE
THERE'S A SET OF PROTEINS 30 OR
SO THAT REPRODUCIBLY ACCUMULATE
IN THE NUCLEUS IN THE CELLS AND
70% ARE NO LONGER IN CELLS
LACKING EXERON 1.
WHEN THEY SHIFT WE DID AN
ANALYSIS AND THE ONLY SIGNATURE
THAT POPS OUT ARE THINGS RELATED
TO MESSENGER RNA BINDING.
AGAIN, RNA BINDING PROTEIN
SIGNATURES LIKE POLY A AND U
BINDING AND MESSENGER RNA, UTR
BINDING THE SORTS OF PROTEINS
YOU EXPECT TO BE RELEASED FROM A
DEGRADING RNA ONCE RELEASED
TRANSITING TO THE NUCLEUS.
WHAT THIS TELLS US IS INDEED RNA
DESTRUCTION IN THE CYTOPLASM IF
YOU RAMP THAT UP CAUSES RELEASE
OF BINDING PROTEINS ANDS WITH
THE REDUCTION IN POL 2 OCCUPANCY
IN THE HOST GENOME AND
TRANSCRIPTION BUT DOESN'T TELL
US IF THE REDISTRIBUTION OF THE
RNA BINDING PROTEINS IS
NECESSARY TO CONVEY THE SIGNAL
OF CYTOPLASMIC TRANSCRIPTION.
TO TEST THAT A FORMER STUDENT
CHRIS DONE CAN LEWIS USED A DRUG
THAT TARGETS THE NUCLEAR IMPORT
MACHINERY.
THE IMPORTANT ALPHA BETA COMPLEX
IMPORTANT FOR RELOCALIZING
PROTEINS TO THE NUCLEUS.
WHAT HE COULD SEE IS IN CELLS
WHERE HE TREATED WITH MOX A DMSO
TREATMENT THAT EXPRESSED THE SOX
PROTEIN WE'RE LOOKING AT POL 2
OCCUPANCY HERE AT A COUPLE
REPRESENTATIVE HOST PROMOTERS IN
CELLS EXPRESSING THIS YOU HAVE
THE DECREASE IN POL 2 OCCUPANCY
BECAUSE OF CYTOPLASMIC RNA DECAY
AND IF THE BLOCKS THE TRANSPORT
BY TREATING THE CELLS BIG THIS
THE CELL STILL UNDER GO THE SAME
RNA DECAY IN THE CYTOPLASM BUT
NO LONGER CAUSE THE
TRANSCRIPTIONAL REPRESSION IT'S
THE SIGNAL READ OUT TO THE CELL
TO TRIGGER THIS TRANSCRIPTIONAL
REPRESSION.
SO ONE OF THE THINGS WE'RE
INTERESTED IN NOW IS THIS IS
KIND OF A LONG LIST OF PROTEINS
HERE SHUTTLING AND IS IT THAT
INDIVIDUAL PROTEINS WITHIN THE
LIST ARE KEY PLAYERS EITHER
CONTACTING OR SIGNALLING THINGS
IN THE NUCLEUS TO ELICIT THE
TRANSCRIPTIONAL REGRESSION
RESPONSE OR THE MASS ACTION OF
GENERALLY REDISTRIBUTING THE RNA
BINDING PROTEINS THAT'S CAUSING
A STRESS SIGNAL IN THE CELL THAT
LEADS TO TRANSCRIPTIONAL
REPRESSION?
WE FAVOR THE KEY HYPOTHESIS AND
DOING KNOCKOUT AND
OVEREXPRESSION FOR THE
INDIVIDUAL RNA BINDING PROTEINS
AND FOUND A COUPLE OF EXAMPLES
WHERE INDIVIDUAL PROTEINS LUKE
POLY A SEEM TO PLAY IMPORTANT
ROLES AND OTHERS I WON'T SHOW
THE DATA FOR BECAUSE THEY SHOWED
NO PHENOTYPE WHEN WE DID THESE
EXPERIMENTS.
AND CYTOPLASMIC POLY A BINDING
PROTEINS ARE STUDIED FOR A LONG
TIME AND THEY BIND TO THE POLY A
TAIL OF MESSENGER RNAs AND PLAY
IMPORTANT ROLES INSTABILITY AND
TRANSLATIONAL COMPETENCE IN THE
CYTOPLASM AND THEY TEND TO GET
RELOCALIZED AND ACCUMULATE IN
THE NUCLEUS IN RESPONSE TO LARGE
RNA CHANGES IN THE CYTOPLASM
THAT OCCUR DURING INFECTION.
IT'S NOT A PHENOTYPE WE SEE ONLY
IN HERPES VIRUS INFECTION OR IN
GAMMA AND SEEN IN ALPHA HERPES
AND OTHER VIRUSES AS WELL IN THE
NORMAL LEVELS, EXPRESSION OF THE
SOX PROTEIN CAUSE THE STRONG
TRANSCRIPTIONAL REPRESSION
HOWEVER, IF WE DEPLETE THE POLY
A BINDING PROTEINS FROM CELLS
YOU SEE THE PHENOTYPE IS
REVERSED SUGGESTING THEY'RE
PLAYING A ROLE IN THE REPRESSION
PHENOTYPE.
WE CAN ALSO ASK WHAT HAPPENS IF
YOU JUST DRIVE THE PROTEINS INTO
THE NUCLEUS IN THE ABSENCE OF
RNA DECAY IN THE CYTOPLASM.
WE CAN DO THIS THROUGH A COUPLE
OF STRATEGIES.
ONE OF WHICH IS A NUMBER OF
YEARS AGO WE HAD SHOWN THE
REASON POLY A BINDING PROTEINS
THE REASON THEY'RE RETAINED IN
THECYTE
THECYTEOPLASM THEY HAVE A SIGNAL
HIDDEN IN THE RECOGNITION MOTIFS
AND THE SIGNAL IS MASKED AND
KEPT IN THE CYTOPLASM AS THE RNA
IS DESTROYED AND THEY'RE
RELEASED, THE NUCLEAR
LOCALIZATION SIGNAL IS EXPOSED,
BINDS IMPORTANT ALPHAAN AND
TRANSPORTED TO THE NUCLEUS.
THE LEVELS ARE CAREFULLY TRI
TRITATED AND IT GOES NOT NUCLEUS
BECAUSE IT'S EXPOSED.
WHEN WE DO THIS IN THE ABSENCE
OF CYTOPLASMIC mRNA DECAY AND
INCREASING THE CONCENTRATION IN
THE NUCLEUS CAUSES A DECREASE IN
POL 2 OCCUPANCY AT THE CELLULAR
PROMOTERS.
FOR THIS REASON WE THINK THE
PROTEINS AS WELL AS WE SUSPECT A
COUPLE OTHERS MAY BE KEY PLAYERS
IN REMEDIATING THE
TRANSCRIPTIONAL EXPRESSION.
NOW THE QUESTION IS HOW ARE THEY
DEGREE THAT?
THIS IS AN ONGOING PIECE OF
WORK.
I'LL SHARE SHARE FAVORITE
HYPOTHESIS GENERATED BY A
CURRENT GRADUATE STUDENT SAM
RYDER WHO CAME UP WITH THE IDEA
THAT PERHAPS WHAT HAPPENS WHEN
YOU HAVE AN INFLUX OF THE RNA
BINDING PROTEINS IS THAT THEY
MAY COMPETE WITH RESIDENT
NUCLEAR BINDING PROTEINS WHICH
OF WHICH PLAY IMPORTANT ROLES IN
CO-TRANSCRIPTIONAL RNA
PROCESSING ACTIVITY HAPPEN
DURING THE PROCESS OF
TRANSCRIPTION.
AND INDEED EVEN THE PROCESS OF
TRANSCRIPTION HELPS COMPETE OFF
CHROMATIN MODIFIERIERS MAKE LOCUS
COMPETENT.
AND SHE HYPOTHESIZES AS THEY GO
IN THE NUCLEUS THEY MAY
RE-OCCUPY THE SPACE THE NORMAL
mRNA BINDING PROTEINS OCCUPY ON
NASCENT RNA AND THEREFORE
DISRUPT THE RNA EARLY ON IN THE
TRANSCRIPTION PROCESS AND ALTER
TRANSCRIPTION.
THESE IN THE PROCESS OF PURIFY
PURIFYING NASCENT WITH THE
PROTEOME ASSOCIATED WITH CELLS
UNDERGOING ACCELERATEDD DECAY AND
LOOKING AT WHAT IS THERE BY MASS
SPEC
SPECTOMETRY AND SEEING THE RVPs
TO THOSE THAT MOVED IN THE
CYTOPLASM.
I'LL CLOSE THIS SECTION BY
SAYING WE ASSUME THAT THIS
PATHWAY WE THINK IS RELATED TO
CELL STRESS SENSED BY REMOVAL OF
RNA BY THE CYTOPLASM IS BEING
USED PROBABLY DURING A NUMBER OF
VIRAL INFECTIONS AND PATHWAYS
TRIGGERED BY VIRUSES WE THINK
ARE THERE FOR OTHER REASONS AS
WELL.
SO WE ASSUMED THAT SURELY THERE
ARE CASES IF THIS IS A NORMAL
CELLULAR RESPONSE TO REMOVAL OF
RNA WHERE NON-VIRAL STRESSES MAY
TRIGGER THE ACTIVATION.
INDEED CHRIS DUNCAN LEWIS AND AS
DISCOVERED BY THE FREEDMAN LAB A
FORMER LECTURER AS A SIGNATURE
FEATURE OF APOPTOSIS BEFORE
THERE'S GROSS CHANGES AND A NEWS
NUCLEUS THAT IS RELEASED AND
CHRIS FOUND LIKE VIRAL SITUATION
THIS CAUSES RNA BINDING PROTEIN
RELOCALIZATION TO THE NUCLEUS IN
THE CELLS AND A SELECTIVE PRE
INVESTIGATION OF POL 2 ACTIVITY
BUT NOT POL 3 ACTIVITY WHICH
AGAIN IS SOMETHING THAT WE HAD
SEEN IN THE VIRAL CONTEXT AS
WELL.
MESSENGER RNA DECAY TRIGGERING
POL 2 TRANSCRIPTIONAL
REPRESSION.
OKAY.
SO THAT'S THE FIRST PATHWAY.
ONE IN WHICH RNA DESTRUCTION
ITSELF IS DETECTED.
BUT I TOLD YOU THAT THERE WERE
TWO PATHWAYS INVOLVED.
ALSO A CONTROL OF THE GENERAL
PROTEIN ABUNDANCE OF
TRANSCRIPTIONAL REGULATORS.
THIS PATHWAY WE FOUND WHEN WE
WENT BACK AND REPEATED THESE TMT
BASED MASS SPECT EXPERIMENTS BUT
IN THE CONTEXT OF INFECTION.
THIS PATHWAY ONLY MANIFESTED IN
INFECTED CELLS AND EXPRESSION OF
VIRAL NUCLEASE WAS NOT ENOUGH TO
TRIGGER IT AND WHEN REPEAT THIS
USING MICE FIBROBLASTS INFECTED
WITH THE SOX VERSION OR ANOTHER
VERSION WE SAW THE SAME BINDING
PHENOTYPE WE SAW IN THE SOX
EXPRESSING CELLS AND SAW
SOMETHING ELSE.
IT WAS A GENERALIZED DECREASE IN
MANY OF THE SUBUNITS OF mRNA
POLYMERASE AND POL 2 IS AN
ENZYME AND 7 OF 12 SUB UNITS OF
RNA POLYMERASE ARE DEPLETE WITH
THE WILD TYPE BUT NOT THE SOX
MUTANT AND NOT JUST RELOCALIZED
BUT SELECTIVELY DELETED FROM THE
NUCLEUS AND NOT CYTOPLASM.
ONE INTERESTING THING IS IT HAS
SEVEN SUB UNITS AND FIVE SHARED
WITH THE OTHER POLYMERASES POL 1
AND POL 3.
THE SHARED UNITS WAS NOT
CHANGING.
SOMETHING ABOUT INFECTION WAS
SPECIFICALLY DELETING THE POL 2
SPECIFIC SUB UNITS.
WOE THOUGHT THAT WAS
INTERESTING.
A NUMBER OF VIRUS KNOWN TO
TARGET POL II.
GENERAL MY PEOPLE LOOK AT THE
LARGE CATALYTIC SUBUNIT AND THIS
IS THE FIRST DEMONSTRATION THAT
VIRAL INFECTION COULD IN FACT
LEAD TO DEPLETION OF MULTIPLE
BUT NOT ALL POL II SUB UNITS.
THIS SUBUNIT DEPLETION WE SEE IN
KSHV INFECTED CELLS HAPPENS
THROUGH TURN OVER.
WE'RE TAKING B CELL, BCBL CELL
LINE INFECTED WITH KSHV AND
LOOKING AT WHAT THE HALF LIFE OF
THE PROTEINS ARE IN THE CELLS IN
THEIR LATENT STATE WITH NO HOST
SHUT OFF HAPPENING VERSUS WHEN
WE ENTER THEM IN THE LYTIC
STATE.
I'M SHOWING ONE EXAMPLE THE RPB2
SUB UNIT AND A POL II SPECIFIC
SUB UNIT IF WE TREAT CELLS WITH
CYCLOHEXIMIDE YOU CAN SEE THE
PROTEIN IN THE REACTIVATED CELLS
IS TURNING OVER MORE RAPIDLY
THAN IN THE UNREACTIVATED CELLS
AND ACCELERATED PROTEIN TURNOVER
AND SEE IT FOR THE POL II SUB
UNITS AND WE THINK SOMETHING IS
ACTIVATING A PROTEIN TURNOVER OF
THE POL II SPECIFIC SUB UNITS
AND INFECTED CELLS.
I MENTIONED THERE'S SEVEN
DIFFERENT POL II SUB UNITS.
IS IT THE VIRUS HAVING A
STRATEGY TO SOMEHOW PROMOTE
TURNOVER OF ALL OF THESE SEVEN
SUBUNITS SEPARATELY?
OR ALTERNATIVELY, IS IT POSSIBLE
IT IS TARGET SINGLE SUB UNIT AND
WHEN HAVE YOU PROTEIN COMPLEXES
THEIR STABILITY IS DEPENDENT ON
THE FORMATION OF THAT COMPLEX
ITSELF.
IF YOU REMOVE A COMPLEX SUBUNIT
FROM A PROTEIN COMPLEX IT CAN
LEAD TO DESTABILIZATION OF THE
OTHER MEMBERS OF THIS COMPLEX.
AND PERHAPS IT'S A SINGLE SUB
UNIT AND CO-DEPLETION OF A
PROTEINS ALL THE POL II SPECIFIC
SUB COMPLEX OF THAT POLYMERASE.
WE THINK THIS IS PROBABLY THE
CASE.
WE COULD DO THIS BY SELECTIVELY
DEPLETING RPB1 IN UNINFECTED
CELLS AND WHEN WE DEPLETE THE
SUB UNIT OF POL II WE SEE
CO-DEPLETION OF OTHER SUB UNITS
SUBJECT TO POLYMERASE II AND
LESS DEPLETION OF RPB5.
OUR HYPOTHESIS IS THE VIRUS IS
TARGETING A PARTICULAR SUB UNIT
OR ELICITING A STRESS TARGETING
A PARTICULAR SUB UNIT OF POL 2
FOR DESTRUCTION.
IT'S LEADING TO CO-DEPLETION OF
SUB UNITS CONTRIBUTING TO THE
TRANSCRIPTIONAL SHUT DOWN WE SEE
IN CELLS.
TO SUMMARIZE WHAT I'VE SHOWN YOU
IN THE FIRST PART IS THAT DURING
GAMMA HERPES VIRUS INFECTION
THERE'S A LARGE SCALE DEPLETION
OF MESSENGER RNA FROM THE
CYTOPLA
CYTOPLASM INITIATED FROM THE
SPECIFIC NUCLEASE CALLED SOX.
THIS RNA DECAY PHENOTYPE SIGNALS
IN AT LEAST TWO DIFFERENT WAYS
FROM THE CYTOPLASM TO CAUSE
REPRESSION BY POLYMERASE II AND
THE BINDING PROTEINS FROM THE
DEGRADING RNA FRAGMENTS AND THE
SECOND MECHANISM WE THINK I
DIDN'T SHOW THE DATA BUT A
PROTEOSOME UNIT SPECIFIC TO POL
II NOT SHARED WITH THE OTHER
POLYMERASES.
SO YOU MIGHT BE WONDERING, WELL,
THESE VIRUSES ARE
DOUBLE-STRANDED DNA VIRUSES.
THEY'RE TRANSCRIPTION DEPENDS ON
RNA POLYMERASE II LIKE THE HOST
DOES.
HOW ARE THEY ABLE TO GET AROUND
THE PHENOTYPE?
OBVIOUSLY THEY DO SINCE THEY'RE
SUCCESSFUL VIRUSES.
SO I'M GOING IT TURN MY
ATTENTION NOW TO THE SECOND
QUESTION OF VIRAL GENE
REGULATION.
SO OF COURSE WHILE HOST
TRANSCRIPTION IS BEING REPRESSED
DURING LYTIC INFECTION
PARTICULARLY LATER, VIRAL
EXPRESSION AND OTHER PROCESS
HUMMING ALONG NICELY IN THE
NUCLEUS IN VIRAL REPLICATION
COMPARTMENTS.
THESE ARE SUBCOMPARTMENTS OF THE
NUCLEUS NOT MEMBRANE BOUND BUT
CONCENTRATE REPLICATING VIRAL
DNA BUT EXCLUDE HOST DNA.
THE VIRAL DNA IN THE REPLICATION
COMPARTMENTS IS LARGELY THOUGHT
TO BE UNCHROMATINIZED AND
HUNDREDS OF COPIES AND THINK OF
IT AS A CHAOTIC PLACE BECAUSE IT
LACKS THE ORGANIZATIONAL
PRINCIPLES USED BY THE HOST DNA
AND YET ON THAT CHAOTIC
REPLICATING DNA MILIEU THERE'S
SOPHISTICATED GENE REGULATORY
PROCESSES AND JUST DNA PROCESSES
THAT ARE HAPPENING INCLUDING
VIRAL DNA REPLICATION AND THE
DNA IS GETTING PACKAGED.
THE LOCALIZATION OF THE VIRAL
GENOME WITHIN THE COMPARTMENTS
IS WHAT WE THINK PROTECTS THE
VIRAL GENOME FROM THE
TRANSCRIPTIONAL REPRESSION THAT
IS LEVIED ON THE HOST GENOME.
THIS PROVIDES A PROTECTIVE
ENVIRONMENT WHERE THE REMAINING
POL II FROM THE CELL IS
RECRUITED STRONGLY TO VIRAL
REPLICATION COMPARTMENTS
PROBABLY BECAUSE OF THE OPEN
NATURE OF THE VIRAL DNA.
NON CHROMATINIZED DNA IS LIKE A
SPONGE AND HELPS RECRUIT FACTORS
FROM THE REST OF THE NUCLEOPLASM
IT NEEDS TO COORDINATE ITS OWN
GENE EXPRESSION.
WHAT I'LL TELL YOU ABOUT TODAY
IS HOW IN THAT RECRUITMENT
PROCESS THERE IS LATENT
INFECTION, SPECIALIZED VIRAL
REGULATORS TO HELP COORDINATE ON
PROMOTERS AND WE FOUND CONNECT
EACH OF THESE PROCESSES IN SOME
SURPRISING WAYS.
LET ME FIRST REMIND YOU THAT FOR
ALL DNA VIRUSES IT DOESN'T
MATTER IF BACTERIA, PHAGE OR
HERPES VIRUS YOU HAVE SETS OF
GENES EXPRESSED PRIOR TO THE
VIRAL DNA REPLICATION CALLED THE
EARLY GENES.
THEY BY AND LARGE LOOK LIKE HOST
PROMOTERS.
MANY WERE USED AS MODEL SYSTEMS
TO UNDERSTAND THE BASICS OF HOW
EUKARYOTIC FACTORS WORK LIKE OUR
OWN PROMOTERS DO.
LATE GENES ARE THE SET OF GENES
WHOSE EXPRESSION INITIATES ONLY
AFTER THE VIRUS STARTS
REPLICATING ITS DNA AND THIS
MAKES SENSE.
LIKE CAPSID PROTEINS, ETCETERA,
THE THING THE VIRUS DOESN'T NEED
UNTIL THERE'S REPLICATED DNA TO
PACKAGE INTO NEW CAPSIDS.
THESE GENES ARE THE ONES THAT
ARE BEING EXPRESSED AT THIS
PARTICULARLY CHAOTIC TIME IN THE
VIRAL REPLICATION COMPARTMENTS
WHERE THERE'S A LOT GOING ON IN
THE DNA AND THEY'RE EXPRESSION
STRATEGIES ARE UNIQUE COMPARED
TO VIRAL EARLY GENES AND HOST
GENES.
FIRST OF ALL THEY HAVE
SIMPLISTIC PROMOTERS THAT HAVE A
LITTLE MORE THAN A MODIFIED BOX
AND THEIR TRANSCRIPTION IS
COORDINATED BY A COMPLEX OF
VIRALLY ENCODED PROTEINS OR SIX
IN THE BETA AND GAMMA HERPES
VIRUS CALLED THE VIRAL
TRANSCRIPTION ACTIVATORS THAT
DIRECTS TRANSCRIPTION OFF THE
LATE GENES.
WE STUDIED THEM 10 YEARS AND
MAPPED THE GENERAL ORGANIZATION.
IT'S A FASCINATING COMPLEX AND A
TOPIC FOR ANOTHER SEMINAR.
ONE IS A MIMIC OF CELLULAR
PROTEIN NOT AT THE SEQUENCE
LEVEL BUT STRUCTURALLY AND
MIMICS TVP AND DOES SOMETHING IT
DOESN'T DO WHICH ACTS LIKE A
BACTERIAL SIGNAL FACTOR IT CAN
BIND DIRECTLY IT POL II AND
BRING IT TO VIRAL PROMOTERS.
SO TVP DOES NOT MAKE CONTACT
LIKE THE VIRAL VERSION DOES.
SO WE WONDERED DESPITE THE FACT
WE HAVE A DECENT IDEA OF THE
ORGANIZATION OF THIS COMPLEX AND
THE PROMOTER ELEMENTS INVOLVED
WE DON'T REALLY KNOW HOW IT'S
REGULATE
REGULATED AND WHAT TRIGGERS THE
LATE TIME POST INFECTION AND
THIS IS WORK FROM A GRAD STUDENT
FIGURING OUT WHAT ARE THE SETS
OF THINGS THERE AT THE SCENE OF
THE CRIME.
THE CELLULAR AND VIRAL FACTORS
LOCALIZED TO THESE LATE GENE
TRANSCRIPTION COMPLEXES TO THE
LATE PROMOTERS AT THE POINT IN
TIME WHERE LATE GENE EXPRESSION
IS HAPPENING.
THIS SOUNDS SIMPLE BUT IT'S NOT
BECAUSE TRANSCRIPTION COMPLEXES
ARE INHERENTLY DYNAMIC.
YOU GENERALLY CAN'T STUDY THEM
USING STANDARD IP ASSAYS.
YOU'RE NOT GOING TO BE ABLE TO
SEE THE INTERACTION.
THEY'RE JUST HAPPENING IN A
SNAPSHOT OF TIME MOVING SHAPE
SHIFTING AS THEY GO ALONG.
SHE WANTED TO USE A PROXIMITY
LABELLING APPROACH TO CAPTURE
TRANSIENT INTERACTIONS HAPPENING
AT A PARTICULAR SNAPSHOT IN TIME
AND PLACE.
AND SO FOR THIS SHE USED
PROMISCUOUS LIGASE CALLED TURBO
I.D. AND FUSES ENDOGENOUSLY IN
THE VIRAL GENOME TO THE LATE
TRANSCRIPTION ACTIVATORS.
IF YOU BLOW BIOTIN IT WILL GO TO
WHERE LATE GENE TRANSCRIPTION IS
HAPPENING AND YOU CAN PULL OUT
THE BIOTIN PROTEINS AND IDENTIFY
THEM BY MASS SPECTOMETRY AND IT
TOOK A TON OF WORK PROBABLY A
YEAR OR MORE OF TRYING DIFFERENT
LOCATIONS FOR THE TAG AND
EXPRESSION AND CONTROLS TO LAYER
IN.
BE AWARE.
IT WAS A TON OF WORK.
NONETHELESS, CHLOE LOOK FOR
CELLS IN ASSOCIATION WITH THE
COMPLEX BUT NOT ANY OF HER
NEGATIVE CONTROLS AND CAME UP
WITH A LIST OF 45 PROTEINS AND
THESE ENCOMPASSED THE KNOWN
COMPONENTS OF THE COMPLEX IN POL
II.
EVERYTHING WE KNEW SHOULD BE
THERE WAS PICKED UP BY THE
SCREEN.
THE 45 LIST OF PROTEINS DOESN'T
TELL YOU WHETHER THEY'RE
INVOLVED IN LATE GENE PROTEINS
AND RAN THEM THROUGH FUNCTIONAL
ASSAYS TO TEST IF THEY WERE
IMPORTANT FOR THE TRANSCRIPTION
PROCESS.
FOR THIS SHE NEEDED ADDITIONAL
TOOL BUILDING AND ENGINEERED ON
THE KSHG GENOME ON THE BACKBONE
A REPORTER THAT WAS DRIVEN
EITHER BY AN EARLY GENE PROMOTER
OR A LATE GENE PROMOTER.
SHE DEPLETED EACH OF THESE THEN
USING A CRISPR SCREEN CAN ASK
WHETHER THE DELETION SELECTIVELY
IMPACTED LATE GENE EXPRESSION
AND WHETHER THEY WERE
FUNCTIONALLY INVOLVED AND RAN
THROUGH A SECOND ROUND OF
SCREENING TO AN ORTHOGONAL PRECH
APPROACH TO GET ACCURATE HITS.
SHE DID ALL THIS AND PICKED UP
EVERY KNOWN FACTOR THAT WAS
REQUIRED FOR LATE GENE
TRANSCRIPTION.
THAT WAS GREAT.
AND ONE NEW HIT.
SHE WAS VERY NERVOUS.
THIS IS A MEETING IN THE OFFICE
WHERE YOU'RE SPENDING A LOT OF
TIME SAYING IT'S GOING TO BE
OKAY BECAUSE WHEN YOU DO SCREENS
YOU HOPE TO GET ONE TRUE HIT AND
YOU HAVE ONE HIT AND SHE'S LIKE
WHAT IF SOMETHING ELSE TELLS ME
THIS HIT ISN'T RIGHT AND I'VE
DONE ALL THIS WORK FOR YEARS AND
NOTHING COMES FROM IT.
THOUGH THE ONE HIT WAS NOT A
PROTEIN WE EXPECTED QUITE
INTERESTING.
THIS IS TO SHOW YOU THE DATA FOR
THE HIT BUT THE HIT IS A PROTEIN
CALLED ORPH29 THE LATE GENE
DEPLETER AND IT RESIDES WITH THE
OTHER LATE GENE ACTIVATORS AND
HAVE A REDUCTION IN LATE GENE
SIGNAL.
IF WE DO IT WITH THEY WILL EARLY
GENE REPORTER LIKE ORPH30
THERE'S NO REPRESSION AND IT'S
THE PHENOTYPE THAT WE'RE LOOKING
FOR.
BUT THE PROTEIN WAS CONFUSING TO
US BECAUSE IT HAS NOTHING TO DO
WITH TRANSCRIPTION AS FAR AS WE
CAN TELL IT'S THE CATALYTIC PART
OF THE TERMINASE AND ESTABLISHED
ROLES IN PACKAGING DNA.
SO SHE TRIPLE CHECKED HERSELF
AND MADE THE MUTANT IN KSHV AND
IF YOU DON'T HAVE IT YOU CANT
HAVE PRODUCTION OF NEW
INFECTIOUS VIRON AND THE VIRUS
IS COMPLETELY DEAD.
INDEED GETTING RID OF THE
TERMINASE GOES TO PRODUCTION AND
WE LOOKED AT LATE OR EARLY
PRODUCED PROTEINS AND COULD
RECAPITULATE LATE PROTEINS LIKE
K.1 WERE SIGNIFICANTLY REDUCED
IN THE ORF VIRUS AND OTHERS WERE
NOT REDUCED AT ALL.
WHAT IS IT GETTING RID OF THE
TE
TERM
TERMINASE WILL DO.
HAVE YOU A PORTAL THROUGH WHICH
THE VIRAL DNA REPLICATED IN A
ROLLING CIRCLE MECHANISM TO HAVE
THESE PUNCTUATED BY TERMINAL
REPEAT AND THE COMPLEX BINDS
THESE AND IN AN ATP DEPENDENT
WAY PUMPS THEM IN THE CAPSIS AND
AS THE NEARLY LIQUID CRYSTAL IN
DENSITY.
IT'S A POWERFUL MOTOR TO BE ABLE
TO GET THESE IN AND THEN IT HAS
A NUCLEASE ACTIVITY THAT
SUBSEQUENTLY CLEAVES AT A
TERMINAL REPEAT WITH A FULL
GENOME PACKAGE TO THEN ALLOW THE
PORTAL TO SEAL AND THE VIRAL
PARTICLE TO UNDER GO THE REST OF
ITS MATURATION PROCESS.
SO ONE OF THE THINGS WE THOUGHT
WAS PERHAPS IF YOU'RE PREVENTING
THIS PROCESS YOU'RE MESSING UP
THE PACKAGING REACTION AND GOING
TO A CHAOTIC ENVIRONMENT AND
CREATING TOO MUCH SOMETHING IN
THE REPLICATION COMPARTMENT AND
IT'S NOT A DIRECT EFFECT BUT
INDIRECT EFFECT OF FAILURE TO
PACKAGE YOUR DNA.
WE COULD TEST THIS HYPOTHESIS
BECAUSE A COUPLE OF YEARS
EARLIER A FORMER POST DOCK IN
THE LAB IDENTIFIED ANOTHER
CRITICAL PACKAGING FACTOR CALLED
ORF68 WHOSE ACTIVELY IS
ESSENTIAL FOR PACKAGING BUT
UNRELATED TO THE TERMINASE
PACKAGING AND WE CAN BLOCK THIS
AND SEE IF WE GET THE SAME
REACTION AND YOU GET RID OF ORF6
YOU DON'T PACKAGE YOUR DNA.
IF YOU GET RID OF ORF68 YOU
EXPRESS K.1 JUST FINE.
STO STOPPING PACKAGES DOES NOT
HAVE THE BYPRODUCT OF REDUCING
GENE EXPRESSION AT LATE TIMES.
THERE'S SOMETHING SPECIFIC ABOUT
THE VIRAL TERMINASE IS DOING.
THIS IS MY LAST DATA SLIDE.
WHAT CHLOE WANTED TO DETERMINE
IS IT THE CATALYTIC PROTEIN
NECESSARY FOR THIS FUNCTION AND
MADE POINT MUTATIONS IN THE
WALKER A OR WALKER B MOTIFS
IMPORTANT FOR ATP BINDING AND
HYDROLYSIS FOR THE PACKAGING
REACTION AND IMPORTANT FOR
CLIPPING THE DOMAIN FOR FINISH
THE REACTION AND ASK CAN THE
MUTANTS COMPLEMENT WHEN SHE
TRANSDUCES THESE INTO CELLS A
FIRST WITH LAX ORF29 AND
COMPARED THE MUTANTS TO WILD
TYPE.
I'M SUMMARIZE THE DATA BECAUSE
THE MUTANTS ALL LOOK THE SAME
AND IF YOU MUTATE ANY ASPECT OF
THE CATALYTIC UNLIKE THE WILD
TYPE PROTEIN YOU'RE UNABLE TO
COMPLETE THE DETECT AND IT'S
CRITICAL FOR POTENTIATING GENE
EXPRESSION LATE IN INFECTION.
LET ME BRING THIS TOGETHER FOR
YOU.
THIS IS WHY THIS IS INTERESTING
AND IMPORTANT.
FIRST, FOR A LONG TIME IT'S BEEN
KNOWN FOR ALL DNA VIRUSES THAT
THIS ACTIVE DNA REPLICATION IS
REQUIRED IN SOME WAY TO LICENSE
TRANSCRIPTION OF GENES LATE IN
INFECTION BUT WHAT WE'RE SHOWING
NOW IS THAT INDEED IT IS NOT
JUST THIS CRITICAL ACTIVITY BUT
DOWN STREAM EFFECT THE PACKAGING
REACTION ALSO SOMEHOW FEEDS BACK
TO POTENTIATE GENE EXPRESSION AS
WELL.
AND HOW MIGHT THIS BE HAPPENING?
WE HAVE A COUPLE OF IDEAS.
BECAUSE THE ATP ACTIVITY IS
REQUIRED ONE POSSIBILITY IS THE
TERMINASE IS FUNCTIONING IN --
WE DON'T THINK IT'S JUST A
SCAFFOLDING FACTOR BUT MAYBE
SCANNING AND CLEARING THE DNA TO
MAKE IT EASIER TO TRANSCRIBE.
ALTERNATIVELY WE THINK THE
PACKAGING REACTION ITSELF IS
YOUR TWISTING THE DNA IN THE
CAPSID AND CREATING TORSIONAL
STRAIN ON DNA.
THE SAME THING WHEN OUR DNA IS
BEING REPLICATED YOU NEED TO
RELEASE THE STRAIN OTHERWISE IT
WILL CAUSE TRANSCRIPTIONAL
INHIBITION.
WE FAVOR THE IDEA AT PRESENT
PERHAPS THE TERMINASE THROUGH
PACKING IS RELIEVING THE STRAIN
DURING THE PACKAGING REACTION
NECESSARY TO ALLOW TRANSCRIPTION
TO CONTINUE TO HAPPEN ON THE
NEWLY REPLICATED GENOMES.
IT'S ALSO INTERESTING BECAUSE IT
SUGGESTS IF INHIBITERS CAN BE
DEVELOPED AGAINST THE PACKAGING
COMPLEX THE INHIBITORS MAY BE
POTENT NOT JUST BECAUSE THEY
BLOCK PACKAGING BUT ALSO POISON
LATE GENE TRANSCRIPTION CRITICAL
FOR COMPLETION OF THE VIRAL LIFE
TIME.
SO I'LL END THERE.
I'LL MENTION THE FIRST PART OF
MY TALK WAS CARRIED OUT LARGELY
BY THE FORMER Ph.D. STUDENTS WHO
START THE WORK.
THE POL II PROTEIN SUB TURNOVER
WORK IS CARRIED OUT BY A CURRENT
Ph.D. STUDENT AND THE LATE GENE
WORK THE STORY WAS LARGELY
CARRIED OUT BY CHLOE McCALLUM
AND WAS HELPED TO GET THE TOOLS
AND SYSTEM SET UP.
ALWAYS GRATEFUL FOR OUR FUNDING
AND I'M MORE THAN HAPPY TO TAKE
QUESTIONS I'LL LEAVE UP OUR
MODEL TO HELP YOU THINK OF ANY
IF THERE'S TIME.
THANKS.
>>WE DO HAVE A QUESTION
ONLINE.
I'LL TAKE THAT FIRST BEFORE WE
GO.
SO OUR FRIEND HOWARD YOUNG UP AT
NCI FREDERICK ASKED DO
INTERFERONS PLAY ANY ROLE IN
YOUR DATA?
>> I GUESS I WONDER -- I'M
GUESSING HE'S TALKING ABOUT THE
FIRST PART.
>> THE QUESTION CAME IN PRETTY
EARLY.
>> AN IDEA IS THAT ONE OF THE
ROLES OF HOST SHUT OFF IS TO
RESTRICT PRODUCTION OF
INTERFERON STIMULATED GENES.
THAT'S BEEN PRETTY WELL
ESTABLISHED FOR A NUMBER OF HOST
SHUTOFF FACTORS IN THESE AND
OTHER VIRUS.
SO COUNTER ACTING THE INTERFERON
RESPONSE IS AN IMPORTANT ROLE OF
HOST SHUTOFF THOUGH WE DON'T
HAVE EVIDENCE THAT INTERFERONS
ARE PREFERENTIALLY TAR GUEST GETED FOR
DISRUPTION.
>> GREAT SCIENCE AND GREAT
PRESENTATION AS WELL.
I HAVE A QUESTION ABOUT ON YOUR
MODEL SLIDE THERE OF POLY
BINDING PROTEINS BEING IMPORTED
INTO THE NUCLEUS.
COULD THAT POTENTIALLY INHIBIT
TRANSLATION OF VIRAL GENES OR
COULD THAT BENEFIT TRANSLATION
OF VIRAL GENES BECAUSE I'M NOT
REMEMBERING WHETHER THE BINDING
PROTEINS RELATE TO THE NUCLEUS
OR THE CYTOPLASM.
>> YOU'RE NOT REMEMBERING
BECAUSE IT'S NOT BEEN WELL
ESTABLISHED.
THE THINKI INING IS IT PROBABLY
BINDS THEM AND SHUTTLING PROTEIN
AND PROBABLY BINDS IN THE
NUCLEUS AND ESCORTED IN THE
CYTOPLASM AND CYCLES BACK TO THE
NUCLEUS.
YES, I WOULD THINK PAB IS
IMPORTANT FOR VIRAL TRANSLATION
AND LOOKED AT WHERE IT IS IN THE
NUCLEUS.
YOU CAN SEE IT IN VIRAL
REPLICATION COMPARTMENTS AS
WELL.
SO WE ORIGINALLY THOUGHT IT'S
EXCLUDED AND THIS IS MAYBE THE
WAY VIRUSES ESCAPE BUT IT'S
POSSIBLE IT'S USED BY THE VIRUS
IN THE REPLICATION COMPARTMENTS
AN PROCESSING THE RNAs SO THEY
CAN BE EXPORTED EFFICIENTLY AND
TRANSLATED EFFICIENTLY AS WELL.
THE VIRUS NEEDS IT TOO.
>> GREAT.
>> THAT WAS LOVELY.
THANK YOU.
I COULD ASK THE SAME THING.
IN THE LAST BIT THE TORSIONAL
RELEASE, DO YOU THINK YOU NEED
THE PACKAGE AT THE END?
IS THAT WHAT IT'S FIGHTING
AGAINST?
OR DID YOU DIDN'T HAVE SOMETHING
GOING ON THERE --
>> THERE WAS NO PACKAGING WOULD
YOU STILL NEED -- THAT IS AN
EXCELLENT QUESTION AND THE WAY
WE COULD ADDRESS THAT WOULD BE
TO MAKE THE DOUBLE MUTANT OF AN
ORF68 TO BLOCK PACKAGING AND
ORF28 IN THE BACKGROUND OF
PACKAGING DO YOU STILL NEED TO
RELIEF THE TORSIONAL STRAIN?
I'LL BRING IT UP TO MY STUDENT.
THANK YOU.
SHE'S PROBABLY ALREADY THOUGHT
OF IT.
>> THERE WAS A PROTEIN IN YOUR
THIRD.
WHAT IS THAT AND WHAT DO YOU
THINK IS GOING ON WITH THAT?
>> SO IT TURNS OUT THERE'S A
NUMBER OF FACTORS INVOLVED IN
RNA DECAY AND THAT'S ONE OF THEM
THAT TEND TO SHUTTLE INTO THE
NUCLEUS AND PARTICULARLY THINGS
IN DECAPPING.
WE DELVED INTO THAT WHEN WE WERE
FIRST WORKING INTO THE DATA.
AND I'LL SAY I DON'T HAVE
ANYTHING SATISFYING TO TELL YOU
THE OUTCOME EXCEPT WE NOTICED A
NUMBER OF THINGS INVOLVED IN
MESSENGER RNA DE CAPPING AND
OTHER REGULATORS OF RNA DECAY
THAT HAPPEN TO SHIFT
LOCALIZATION IN THE FACE OF RNA
DECAY.
YEAH.
>> DO YOU HAVE A QUESTION?
>> IS THERE ANY MODIFICATION
AFTER THIS INFECTION?
WHY I'M ASKING THE QUESTION --
>> GET CLOSER TO THE MICROPHONE.
>> AND BACK IN 1967 OR '68 THE D
FOR ATTACHES TO THE CELL THE
POLYMERASE GETS ENACTIVATED
BECAUSE OF THE ISOLATION OF THE
SIGMA FACTOR AND THAT MODIFIED
ONLY TRANSCRIBES THE D4 DNA.
I THINK IT MUST BE VERY
IMPORTANT SOME MODIFICATION
TAKES PLACE THAT THEY'RE ACTIVE
WITH RESPECT TO THE ALTERNATIVE.
>> YOU'RE ASKING WHETHER THERE'S
MODIFICATION OF THE POL II SUB
UNITS THAT UNDER GO DECAY?
>> YES.
>> GREAT QUESTION.
I DON'T KNOW BUT WE SUSPECT
THERE'S UBIQUITINATION AND SOME
TARGETS THESE FOR TURN OVER AND
THE OTHER HYPOTHESIS IS IT'S
LINKED TO CELL STRESS.
THERE'S OTHER STRESSORS, DNA
DAMAGE AND COLLISION THAT WILL
TRIGGER REMOVAL OF POLE --
POLYMERASE AND TYPICALLY THROUGH
MODIFICATION IS THE TURNOVER.
>> I HAVE A QUESTION ON THE
CLOSE.
YOU SHOWED DATA IN THE MOUSE AND
WE KNOW THE CODE ENDS.
>> IT'S THE IDEA SO YOU HAVE
THIS HYPER ABUNDANT NUCLEAR RNA
AND THE INFECTED CELLS IT'S
BINDING THE PAB C FROM THE
CYTOPLASM.
THE QUESTION IS IN THE CONTEXT
OF KSAB IS MAYBE HELPING RETAIN
PAD IN THE NUCLEUS.
CERTAINLY IT'S INFLUENCING
PRESUMABLY PAN STABILITY THERE.
WE DON'T THINK THAT'S WHAT'S
BRINGING PAP INTO THE NUCLEUS.
WE THINK IT'S SERVING AS A
SPONGE IN THE NUCLEASE AND I
THINK IT'S INTERESTING
POSSIBILITY I DON'T KNOW THE
ANSWER TO.
YEAH.
>> THANK YOU.
>> BEAUTIFUL TALK.
SO YOU SPOKE TO THE CHAOS OF
REPLICATION AND I THINK
PACKAGING'S BEEN MORE OF A BLACK
BOX.
YOU HAVE REPLICATION, LATE GENE
EXPRESSION AND PACKAGING WHICH
SEEM TO NOW BE LINKED.
ONE QUESTION I HAD MAYBE A
SIMPLE HYPOTHESIS FOR THE DEFECT
IS IF YOUR NOT PACKAGING THE
GENOMES DO YOU JUST HAVE MORE
GENOMES OVER ALL AND A
DISTRIBUTION OF POL II WITH THE
LATE COMPLEXES AND WHY YOU'RE
GETTING LATE GENE EXPRESSION?
>> I THINK THAT WAS A
POSSIBILITY THAT WE CONSIDERED
BUT THAT I THINK IS DISPROVEN WE
CAN BLOCK PACKAGING AND NOT
IMPAIR EXPRESSION OF LATE GENES
WHICH SUGGESTS JUST HAVING MORE
DNA AROUND IS NOT THAT YOU'VE
GOT A LIMITING AMOUNT OF POL II
AND SAMPLING THE DNA ALL OVER
THE PLACE WITH OTHER NUCLEIC
ACID PROTEINS.
>> ERIC, ANY CLOSING THOUGHTS.
>> THANK YOU VERY MUCH.