Retroviruses:Retroviruses: Endogenousendogenous MLVMLV Andand XMRVXMRV
Retroviruses:Retroviruses: EndogenousEndogenous MLVMLV andand XMRVXMRV
JohnJohn M.M. CoffinCoffin TuftsTufts UniversityUniversity
Tufts University
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA The Retrovirus Family Tree
Virus Genus HFV Spumaretrovirinae bel1, bel2 MLV GammaretrovirusGammaretroviru FeLV HERV-C WDS Epsilonretrovirus orfA, orfB, orfC HIV-1 tat, rev HIV-2 Lentivirus EIAV VMV
dut MPMV
sag new env MMTV Betaretrovirus HERV-K IAP
ASLV Alpharetrovirus
BLV HTLV-1 Deltaretrovirus tax, rex HTLV-2
New Genes Presented at the 1st Intl. Workshop on XMRV At least 30 million years ago! 7-8 September, Bethesda USA EndogenousEndogenous RetrovirusesRetroviruses
1.1. Remnants Remnants ofof germgerm lineline infectionsinfections byby exogenousexogenous (infectious)(infectious) retroviruses.retroviruses.
2.2. BecameBecame fixedfixed inin thethe hosthost speciespecies.Somes.Some conferconfer protectionprotection againstagainst futurefuture infectionsinfections byby thethe samesame oror similarsimilar viviruses.ruses. AA fewfew othersothers havehave salutarysalutary effects.effects.
3.3. InheritedInherited likelike normalnormal genes.genes.
4.4. PresentPresent inin everyevery vertebratevertebrate andand manymany invertebrates.invertebrates.
5.5. CompriseComprise 6-8%6-8% ofof thethe humanhuman genome.genome. (More (More virusesviruses thanthan us).us).
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA EndogenousEndogenous RetrovirusesRetroviruses
6. Provide a fossil record of pathogen-host interactioninteraction unavailableunavailable inin anyany other system.
7. Can participateparticipate in evolutionary processescesses asas wellwell asas informinform usus aboutabout them.
8. Involved in disease in some animals. Humans?
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA XMRVXMRV
1.1. First First describeddescribed aboutabout 55 yearsyears agoago inin aa fewfew patientspatients withwith prostateprostate cancer.cancer. 2.2. Within Within thethe lastlast year:year: •• Reported Reported inin 25%25% ofof prostateprostate cancercancer biopsies.biopsies. •• Reported Reported inin 67%67% ofof chronicchronic fatiguefatigue syndromesyndrome patients.patients. •• Reported Reported inin 4%4% ofof normalnormal controlscontrols inin bothboth studies.studies. •• Not Not alwaysalways foundfound inin studiesstudies fromfrom otherother labslabs onon samplessamples fromfrom eithereither disease.disease. 3.3. Isolates Isolates fromfrom thethe 22 diseasesdiseases atat didifferentfferent timestimes andand locationslocations areare almostalmost identicalidentical toto oneone another.another. 4.4. Causality, Causality, prevalence,prevalence, andand distridistributionbution remainremain toto bebe established.established. 5.5. Very Very closelyclosely relatedrelated toto endogenousendogenous xenotropicxenotropic (X)(X) MLV.MLV. 6.6. Recently, Recently, LoLo etet alal (PNAS)(PNAS) reportereportedd alsoalso findingfinding MLV-likeMLV-like (PMV(PMV andand MPMVMPMV related)related) virusvirus sequencessequences associatedassociated withwith CFS.CFS.
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA GammaretrovirusesGammaretroviruses
1.1. Simple Simple retrovirusesretroviruses (only(only gag,gag, polpol,, andand envenv genes).genes). 2.2. Widespread Widespread inin nature,nature, withwith isolatesisolates fromfrom mammalsmammals (mice,(mice, cats,cats, primates,primates, koalaskoalas andand others),others), birds,birds, andand reptiles.reptiles. 3.3. Readily Readily givegive riserise toto endogenousendogenous proviruses.proviruses. 4.4. Most Most commonlycommonly transmittedtransmitted verticallyvertically (mother(mother toto offspring).offspring). 5.5. Cause Cause aa widewide varietyvariety ofof diseasesdiseases (cancer,(cancer, neurological,neurological, degenerative,degenerative, immunodeficiency).immunodeficiency). 6.6. Cancer Cancer isis usuallyusually thethe resultresult ofof insinsertionalertional inactivationinactivation ofof protooncogenes.protooncogenes. 7.7. Infection Infection isis lifelong,lifelong, withwith persistencepersistence ofof infectedinfected cellscells establishedestablished early,early, and-and- unlikeunlike HIV,HIV, veryvery fewfew replreplicationication cyclescycles perper host.host.
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA XenotropicXenotropic MLVMLV
1.1. InheritedInherited asas endogenousendogenous provirprovirusesuses (ca(ca 10-2010-20 copies)copies) in all inbred mice. 2.2. Many Many moremore provirusesproviruses inin somesome wildwild MusMus MusculusMusculus subsubspecies.species. 3.3. SomeSome provirusesproviruses (e.g., Bxv1-XMV43)MV43) areare intactintact andand infectious.infectious. 4.4. Can Can infectinfect virtuallyvirtually allall mammals,mammals, exceptexcept somesome speciesspecies ofof MusMus,, duedue toto receptorreceptor (Xpr1)(Xpr1) polymorphism.polymorphism. 5.5. Not Not directlydirectly pathogenicpathogenic inin micemice (due(due toto lacklack ofof receptor),receptor), butbut LTRLTR isis oftenoften foundfound inin oncogeniconcogenic recombinantrecombinant MLVs.MLVs. 6.6. Pathogenicity Pathogenicity inin otherother speciesspecies isis unknown.unknown. 7.7. Common Common contaminantcontaminant ofof humanhuman tumortumor cellcell lineslines duedue toto passagepassage throughthrough nudenude micemice (which(which havehave Bxv1).Bxv1). 8.8. Closely Closely relatedrelated toto XMRV,XMRV, butbut nonenone isis identical,identical, perhapsperhaps excludingexcluding inbredinbred micemice asas thethe origin.origin. 9.9. Related Related MLVsMLVs causecause aa widewide varietyvariety ofof diseasesdiseases (malignant,(malignant, immunodeficiency,immunodeficiency, neurological)neurological) inin mice.mice. Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA PolytropicPolytropic andand ModifiedModified PolytropicPolytropic MLVMLV
1.1. InheritedInherited asas endogenousendogenous provirprovirusesuses (ca(ca 10-2010-20 copiescopies each)each) inin allall inbredinbred mice.mice. 2.2. Also Also foundfound inin wildwild MusMus MusculusMusculus subsubspecies.species. 3.3. No No infectiousinfectious virusvirus hashas beenbeen foundfound toto date.date. 4.4. Can Can infectinfect virtuallyvirtually allall mammals,mammals, ususinging bothboth forms of the Xpr1 Receptor. 5.5. Not Not directlydirectly pathogenicpathogenic inin micemice ((duedue toto lacklack ofof infectivity?),infectivity?), butbut envenv genegene isis oftenoften foundfound inin oncogeniconcogenic recombinantrecombinant MLVs.MLVs. 6.6. Pathogenicity Pathogenicity inin otherother speciesspecies isis unknown.unknown. 7.7. Both Both typestypes derivedderived independentlyindependently fromfrom XMV,XMV, probablyprobably inin responseresponse toto Xpr1Xpr1 mutation.mutation. 8.8. Unlike Unlike XMV,XMV, PMVPMV andand MPMVMPMV provprovirusesiruses havehave sufferedsuffered significantsignificant mutationsmutations mediatedmediated byby mousemouse APOBEC3.APOBEC3.
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA Formation of Endogenous Proviruses
Long terminal repeats (LTR’s) provirus Identical sequence
st Endogenous provirus Presented at the 1 Intl. Workshop on XMRV 7-8 September, Bethesda USA Host-Retrovirus Evolution
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA Effects of Endogenous Proviruses (Good and Bad)
Syncytin env
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA Four Classes of Endogenous MLV
BHBB B Ecotropic
pEco BBBE Xenotropic
js6/10 BB B E Polytropic
js5 BB H B E Modified polytropic js4
Selective hybridization probe Insert in LTR
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA Detection of Specific Proviruses
Specific restriction sites
Probe Specific hybridization probe A B
Size of fragments detected depends on location of cleavage site in flanking DNA, one band per provirus.
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA Distribution of Endogenous Proviruses in Inbred Mice B C Ak D H A L B H Ak D A L C B C Ak D H A L
PmvMpmv Xmv Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA Distribution of Endogenous MLV’s on Mouse Chromosomes
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=10cM =centromere =Emv =Pmv =Mpmv =Xmv =Mtv =Pltr =Mltr =Xltr
11 12 13 14 15 16 17 18 19 X Y
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA OncogenesisOncogenesis byby EndogenousEndogenous MLVMLV
1.1. In In AKRAKR andand somesome otherother inbredinbred strains,strains, mostmost micemice diedie withinwithin aa yearyear ofof thymicthymic lymphoma.lymphoma. 2.2. The The proximalproximal causecause isis integrationintegration adjacentadjacent toto oneone oror moremore protooncogenesprotooncogenes ((c-mycc-myc,, pim-1pim-1,, andand others)others) ofof aa provirusprovirus derivedderived fromfrom endogenousendogenous MLV.MLV. 3.3. Around Around thethe timetime ofof birthbirth allall micemice expressexpress aa replicatingreplicating ecotropicecotropic virusvirus (AKV).(AKV). 4.4. A A complex,complex, butbut highlyhighly reproduciblereproducible seriserieses ofof eventsevents leadsleads toto thethe formation formation ofof aa recombinantrecombinant virusvirus containingcontaining aa polytropicpolytropic envenv genegene andand anan LTRLTR derivedderived fromfrom bxv1bxv1 andand modifiedmodified byby enhancerenhancer duplicationduplication..
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA Generation and selection of Recombinant Proviruses in AKR Leukemia
Akv
gag pro pol env
Bxv1
gagpro pol env
Pmv
gag pro pol env
Viruses generated during leukemogenesis Recombinant 1
gagpro pol env
Recombinant 2
gagpro pol env MCF
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA gagpro pol env Endogenous MLVs in Lab and Wild Mice
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Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA X-IV (KT-59/60) Xltr Coevolution of Endogenous MLV’s and Mice
HEM
M. Spicelegus (formerly M. hortulanus)
M. Spretus
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Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA Distribution of Endogenous Proviruses in C57BL/6JInbred Mice
Pmv Mpmv Xmv Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA Relationship of Proviruses in the Sequenced Genome
MLV-like (Lo et al., PNAS Two weeks ago)
XMRV (Lombardi et al., Science 2009)
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA Relationship of XMRV to Endogenous MLVs Exogenous MLV
CFS pt 1010 Endogenization CFS pt 1042 PC VP 62 Endogenous MLV PC VP 42 PC VP 35 XMV13 Provirus NZB Xenotropic MLV Receptor Mutation BALB/c Xenotropic MLV AKR MLV Xenotropic MLV Moloney MLV
2%
XMRV Virus Mutation
MLV-like Polytropic MLV Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA XMRVXMRV vsvs MLV-Like MLV-Like
1.1. XMRV XMRV (Urisman(Urisman et et al,al, LombardiLombardi etet al,al, others):others): 1.1. Isolated Isolated asas infectiousinfectious virusvirus fromfrom ProstateProstate Cancer,Cancer, CFSCFS patients.patients. 2.2. Isolates Isolates areare veryvery similarsimilar inin sequence.sequence. 3.3. They They formform aa distinctdistinct subcladesubclade among among XMVXMV proviruses.proviruses.
2.2. MLV-like MLV-like sequencessequences (Lo(Lo etet al)al) 1.1. PCR PCR amplifiedamplified usingusing MLVMLV gaggag andand envenv primers.primers. 2.2. Found Found preferentiallypreferentially inin CFSCFS plasma.plasma. 3.3. No No infectiousinfectious virusvirus yetyet identified.identified. 4.4. Only Only fragmentary,fragmentary, bulk,bulk, sequencessequences availableavailable atat present.present.
3.3. Until Until thethe relationshiprelationship betweenbetween thethe twotwo isis furtherfurther clarified,clarified, itit isis importantimportant toto considerconsider thesethese toto bebe distinct,distinct, unrelatedunrelated phenomena.phenomena.
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA WhyWhy II Don’tDon’t SleepSleep WellWell atat NightNight
1.1. We We cancan bebe optimisticoptimistic thatthat XMRVXMRV wwillill turnturn outout toto bebe importantimportant inin thethe etiologyetiology ofof CFSCFS andand prostateprostate cancer,cancer, butbut thethe casecase isis notnot yetyet proven:proven: 1.1. Conflicting Conflicting reportsreports onon thethe associationassociation ofof eithereither diseasedisease withwith thethe virusvirus 2.2. Lack Lack ofof insightinsight intointo pathogenicpathogenic mechanismsmechanisms
2.2. Studies Studies cancan bebe complicatedcomplicated byby minuteminute tracestraces ofof mousemouse DNADNA oror byby virusesviruses derivedderived fromfrom lab strain mice.
3.3. One One mousemouse cellcell containscontains overover 100100 PMPMVV andand XMVXMV proviruses.proviruses. SensitiveSensitive PCRPCR assaysassays can detect the provirus in 10 fg of mouse DNA.
4.4. It’s It’s wellwell knownknown thatthat xenotropicxenotropic MLVsMLVs derivedderived fromfrom nudenude micemice havehave inadvertentlyinadvertently spreadspread throughthrough manymany labs.labs.
5.5. While While thethe majormajor XMRV studiesstudies publispublishedhed toto datedate appearappear toto bebe wellwell controlledcontrolled forfor thesethese problems,problems, extraordinaryextraordinary cautioncaution isis necessary.necessary.
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA WhyWhy II Don’tDon’t SleepSleep WellWell atat NightNight
6.6. In In subsequentsubsequent talks,talks, mymy colleaguescolleagues fromfrom TuftsTufts andand thethe NCINCI willwill presentpresent ourour effortsefforts toto dealdeal withwith thisthis problem:problem:
1.1. Mouse Mouse originorigin ofof XMRVXMRV andand aa sensitivesensitive assayassay forfor contaminationcontamination (Cing(Cingööz)z) 2.2. Environmental Environmental contaminationcontamination withwith mousemouse DNADNA (Huber)(Huber) 3.3. Sensitive Sensitive assaysassays forfor XMRVXMRV andand endogenousendogenous MLVsMLVs (Kearney)(Kearney)
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA MiceMice areare Everywhere!Everywhere! XMRVXMRV
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Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA One drop of mouse blood in my swimming pool…
Equals One MLV provirus per ml.
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Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA XMRV,XMRV, CFS,CFS, andand AntiviralsAntivirals
1.1. Like Like otherother MLVs,MLVs, XMRVXMRV isis sensitivesensitive toto inhibitioninhibition byby aa fewfew antiretroviralsantiretrovirals licensedlicensed toto treattreat HIVHIV infection.infection.
2.2. A A surveysurvey ofof thethe blogsblogs indicates indicates thatthat therethere maymay bebe significantsignificant off-labeloff-label useuse ofof somesome ofof thesethese compoundscompounds inin CFSCFS patipatients,ents, withwith anecdotalanecdotal resultsresults posted.posted.
3.3. While While therethere couldcould bebe valuevalue inin well-controlledwell-controlled randomizedrandomized trialstrials ofof thesethese agents,agents, wewe shouldshould allall opposeoppose uncontrolleuncontrolledd dosingdosing ofof patientspatients withwith antivirals:antivirals: 1.1. We We dodo notnot knowknow forfor suresure thatthat thethe virusvirus isis causal.causal. 2.2. The The drugsdrugs can can onlyonly workwork byby blockingblocking replicationreplication cycles,cycles, notnot reparingreparing damagedamage alreadyalready cousedcoused by by virusvirus replication.replication. 3.3. The The diseasedisease lackslacks objectiveobjective biomarkersbiomarkers toto monitormonitor treatment.treatment. 4.4. There There isis nono goodgood assayassay toto monitormonitor effectseffects onon virusvirus replication.replication. 5.5. Results Results ofof suchsuch studiesstudies willwill nevernever bebe acceptedaccepted byby third-partythird-party payerspayers oror regulators,regulators, keepingkeeping thethe treatmenttreatment outout ofof reachreach ofof thethe largelarge majoritymajority ofof thossethosse suffering suffering fromfrom CFS.CFS.
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA AcknowledgementsAcknowledgements -- Jonathan Jonathan StoyeStoye -Wayne-Wayne FrankelFrankel -Patric-Patric JernJern -Oya-Oya Cing Cingöözz -Brigitte-Brigitte HuberHuber
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Tufts University
Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA AcknowledgementsAcknowledgements
-Mary Kearney -Ann Wiegand -Jon Spindler -Wei Shao
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HIVHIV DrugDrug ResistanceResistance ProgramProgram
NationalNational CancerCancer InstituteInstitute atat FrederickFrederick Presented at the 1st Intl. Workshop on XMRV 7-8 September, Bethesda USA