Identification of Novel Positive-Strand RNA Viruses by Metagenomic Analysis of Archaea ...

12 Pages·2012·7.3 MB·English

Identification of Novel Positive-Strand RNA Viruses by Metagenomic
Analysis of Archaea-Dominated Yellowstone Hot Springs
BenjaminBolduc,a,dDanielP.Shaughnessy,a,cYuriI.Wolf,eEugeneV.Koonin,eFranciscoF.Roberto,fandMarkYounga,b,c
ThermalBiologyInstituteaandDepartmentsofMicrobiology,bPlantSciencesandPlantPathology,candChemistryandBiochemistry,dMontanaStateUniversity,Bozeman,
Montana,USA;NationalCenterforBiotechnologyInformation,NationalLibraryofMedicine,NationalInstitutesofHealth,Bethesda,Maryland,USAe;andIdahoNational
Laboratory,IdahoFalls,Idaho,USAf
TherearenoknownRNAvirusesthatinfectArchaea.Fillingthisgapinourknowledgeofviruseswillenhanceourunderstand-
ingoftherelationshipsbetweenRNAvirusesfromthethreedomainsofcellularlifeand,inparticular,couldshedlightonthe
originoftheenormousdiversityofRNAvirusesinfectingeukaryotes.WedescribeheretheidentificationofnovelRNAviralge-
nomesegmentsfromhigh-temperatureacidichotspringsinYellowstoneNationalParkintheUnitedStates.Thesehotsprings
D
harborlow-complexitycellularcommunitiesdominatedbyseveralspeciesofhyperthermophilicArchaea.Aviralmetagenomics o
approachwastakentoassemblesegmentsoftheseRNAvirusgenomesfromviralpopulationsisolateddirectlyfromhotspring w
n
samples.AnalysisoftheseRNAmetagenomesdemonstrateduniquegenecontentthatisnotgenerallyrelatedtoknownRNA lo
virusesofBacteriaandEukarya.However,genesforRNA-dependentRNApolymerase(RdRp),ahallmarkofpositive-strand a
d
RNAviruses,wereidentifiedintwocontigs.Oneofthesecontigsisapproximately5,600nucleotidesinlengthandencodesa e
d
polyproteinthatalsocontainsaregionhomologoustothecapsidproteinofnodaviruses,tetraviruses,andbirnaviruses.Phylo-

f
geneticanalysesoftheRdRpsencodedinthesecontigsindicatethattheputativearchaealvirusesformauniquegroupthatis ro
distinctfromtheRdRpsofRNAvirusesofEukaryaandBacteria.Collectively,ourfindingssuggesttheexistenceofnovelposi- m

tive-strandRNAvirusesthatprobablyreplicateinhyperthermophilicarchaealhostsandarehighlydivergentfromRNAviruses h
t
thatinfecteukaryotesandevenmoredistantfromknownbacterialRNAviruses.Thesepositive-strandRNAvirusesmightbe tp
:
directancestorsofRNAvirusesofeukaryotes. /
/
jv
i.
a
In contrast to viruses that infect Bacteria and Eukarya, little is overcomes many of the limitations of traditional culture-based sm
knownaboutthevirusesthatinfectArchaea.Fewerthan50ar- approachesforthedetectionofnewviruses.Ingeneral,viralmet- .
o
chaealviruseshavebeendiscovered,comparedtomorethan5,500 agenomicsstudieshaverevealedanenormousdiversityandabun- r
g
virusesknowntoinfecthostsfromtheothertwodomainsoflife dance of viruses. For example, more than 5,000 different viral /
o
(2). Of the few archaeal viruses that have been characterized, genotypeswereidentifiedin200litersofseawater(12).Although n

mainly those infecting members of the phylum Crenarchaeota, most viral metagenomics studies have focused primarily on A
manyhaverevealedbothunusualgenecontent(56)andunique dsDNAviruses(5,72),recentadvancesinmethodologyhaveen- p
r
virionmorphologies(42,54,55,65).Todate,allarchaealviruses abledtheimplementationofRNAviralmetagenomicsasanap- il
3
possessdouble-strandedDNA(dsDNA)genomes,withtheexcep- proachforinvestigatingRNAviralcommunities.Studiesinma- ,

2
tionofonesingle-strandedDNA(ssDNA)virusinfectingarchaea rine environments have revealed a diverse community of RNA
0
ofthegenusHalorubrum(52).NoRNAvirusesinfectingarchaea viruses broadly distributed throughout multiple viral taxa (16, 1
9
havebeendescribed.EukaryoticRNAvirusesareadominantviral 17).Inthesemetagenomicstudies,RNAvirusesinfectingbacteria
b
form:theyarenumerous,diverse,andwidespread,foundtoinfect orarchaeahavenotbeenidentified,supportingtheviewthatthe y

animals,plants,andmanyunicellularforms(17,28,38,40,70). majorhostsforRNAvirusesinthemarineenvironmentareuni- g
u
OnlytwonarrowgroupsofbacterialRNAvirusesareknown,and cellulareukaryotes(73).MetagenomicanalysesofRNAvirusesin e
s
thesedonotshowacloserelationshipwiththevirusesinfecting fresh water have shown that the majority of sequences did not t
eukaryotes (38, 40). Thus, the origin of the RNA viruses of eu- showsignificantsimilaritytoknownvirusesinpublicdatabases,
karyotesremainsanenigma.Inthiscontext,thesearchforRNA following the trend of marine environments. Those sequences
virusesinfectingarchaeaappearstobeofspecialinterestbecause thatdidmatchwerebroadlydistributedtonearly30familiesof
thediscoveryofsuchviruseswouldhavethepotentialtoshednew virusesinfectingavarietyofeukaryotesbutdidnotincludethefew
lightontheoriginofvirusesofeukaryotes. groups of identified RNA viruses infecting bacteria or putative
Onefactorlimitingthediscoveryofarchaealviruseshasbeen RNA viruses of archaea (19). The absence of evidence of RNA
therelianceonculture-dependentapproachesforvirusisolation.
ManyArchaeainhabitextremeenvironments,suchasthosewith
high-temperatureandhigh-salinityconditions,makingculture- Received6January2012 Accepted22February2012
dependentapproachesforvirusdiscoverydifficult.Overthepast Publishedaheadofprint29February2012
decade, direct sequencing of viral communities from environ- AddresscorrespondencetoMarkYoung,[email protected].
mentalsamples(viralmetagenomics)hasbeenusedtomorefully Supplementalmaterialforthisarticlemaybefoundathttp://jvi.asm.org/.
understandviraldiversityinnaturalenvironments,includingma- Copyright©2012,AmericanSocietyforMicrobiology.AllRightsReserved.
rine(13),sedimentary(10),humanandanimalfecal(11,14,33), doi:10.1128/JVI.07196-11
gut(23),andfreshwater(60)environments.Viralmetagenomics
5562 jvi.asm.org 0022-538X/12/$12.00 JournalofVirology p.5562–5573
EvidenceforHyperthermophilicRNAViruses
virusesinfectingarchaeainbothmarineandfreshwaterenviron- bion)treatment.AliquotsofRNA-enrichedextractedviralnucleicacids
ments(5,13,17,19)appearssurprisinginlightoftheindications weretreated(controls)ornottreatedwithRNaseOne(Promega)priorto
thatarchaeacompriseupto40%oftheplanet’smicrobialbiomass cDNAsynthesis.First-strandcDNAsynthesisreactionswerecarriedout
anduptoone-thirdoftheocean’sprokaryotes(34). using1mMrandomhexamersinthepresenceof10(cid:4)Ciof[(cid:6)-32P]dCTP
The recently described clustered regularly interspaced short (MPBioMedicals).Reactionmixtureswereincubatedat25°Cfor10min
andat50°Cfor90minandthenterminatedat85°Cfor5min,followedby
palindromicrepeats(CRISPR)adaptiveimmunitysystemcanbe
RNaseHtreatment.Trichloroaceticacid(TCA)-precipitatednucleicacids
usedtolinkviralgenomesequencestocellularhosts.TheCRISPR
were collected on glass fiber filters (Whatman G/F), and scintillation
systemappearstobeanactivedefensemechanismagainstinvad-
countsofprecipitatedandnonprecipitatedmaterialwerecollected.
ingnucleicacids,includingviruses,throughageneticinterference
Isolationofcellularfractionforcommunitysequenceanalysis.For
pathway (7, 29, 44, 45, 68). The CRISPR system is present in
communitysequenceanalysis(seeFig.S1inthesupplementalmaterial),
(cid:1)40%and(cid:1)90%ofsequencedbacterialandarchaealgenomes, cellswereisolatedfrom(cid:1)500mlofhotspringwaterbyfiltrationonto
respectively (41). A CRISPR locus is composed of a leader se- 0.45-(cid:4)m Pall filters (Millipore) and stored at (cid:7)20 C. Total DNA was
quence that directs the transcription of a noncoding RNA that isolatedfromcellsusingphenol-chloroform-isoamylalcohol25:24:1ex-
spans multiple copies of an (cid:1)25-nucleotide (nt) direct repeat tractionofthefiltersfollowedbyethanolprecipitation.DNAwasampli-
(DR)regionthatareseparatedby(cid:1)35-ntspacersequencesthat fiedusingGenomePlexwhole-genomeamplification(Sigma).Amplifica-
canformlongarrays.EachspacersequencewithinaCRISPRarray tion products were purified using a QIAquick PCR purification kit D
isunique.Immunityrequiresasequencematchbetweenthein- (Qiagen).Approximately10(cid:4)gofcellularDNAwasprovidedtotheUni- ow
versityofIllinoisChampaign-UrbanaSequencingCenter(Urbana,IL)
vadingnucleicacidandthespacersequencesthatliebetweenDRs. n
The sequence content of the DRs can often be used to assign a andsubjectedto454Titaniumpyrosequencing(Roche). lo
Isolationofenrichedviralnucleicacids.Approximately1.2litersof a
CRISPRtypeatthecellularfamilylevel(22).Ithasbeenshown hot spring water was filtered through two successive Pall 0.8-/0.2-(cid:4)m de
thattheCRISPRlociprovidearecordofacell’sinteractionwith filters(Millipore).ThefiltrateflowthroughwasconcentratedwithUltra d
viruses(45,64).Accordingly,theCRISPRDRandspacercontent centrifugal filters (Millipore) using a 100,000 molecular weight cutoff fr
presentinanenvironmentalsamplecanbeusedtoconnectthe (100K MWCO) to a volume of 500 (cid:4)l. Total viral nucleic acids were om
viruses present in that environment with bacterial or archaeal extractedfrom200(cid:4)lofeachsamplewiththePurelinkviralRNA/DNA
hosts. minikit(Invitrogen)andelutedtoafinalvolumeof60(cid:4)l. ht
t
TheacidichotspringsinYellowstoneNationalPark(YNP)in Isolation,preparationandsequencingofenrichedRNAviralfrac- p
:
theUnitedStatesofferanopportunitytosearchforarchaealRNA tion.Animprovedmethodtoisolatenucleicacidsfromtheenrichedviral //
vpirreuvsieosuisnraRnNeAnvgireonnemseeqnutewnhceereanAarlcyhsaise,atphreesedoemnviniraotne.mBeanstesdaorne frricahcteidonRNwAasvdireavleflroapcetidoanfst,e3r0in(cid:4)iltioafltRoTta-lPeCxtRrascctreedenniuncgle.iTcoacoibdtwaianstthreeaetend- jvi.a
s
inhabitedbymicrobialcommunitiesoflowcomplexitythatare with3URNase-freeDNaseI(Ambion)at37°Cfor20min,followedby m
dominatedbyfewerthan10archaealspecies(30).Intheselow-pH theadditionofethylalcohol(EtOH)to37%.Thismixturewasappliedto .o
(pH (cid:2) 4), high-temperature ((cid:3)80°C) springs, bacteria and eu- theRNA/DNAminikitcolumns(Purelink),eluted,andre-extractedfol- rg
lowingthemanufacturer’sprotocols.Theelutednucleicacidsweream- /
karyotes are scarce or, in many cases, absent (9, 30, 58). Using plifiedwiththeTransplexwhole-transcriptomeamplificationkit(Sigma). o
traditionalculture-dependentapproaches,manydsDNAarchaeal ThereactionmixtureswerepurifiedusingQIAquickPCRpurificationkits n
viruseshavebeenisolatedfromthesehigh-temperature,acidichot (Qiagen)followedbypassagethroughAmiconUltra-0.5filtercolumns A
p
springsinYNP(59)andotherthermalhotspringsworldwide(6, (100KMWCO).Approximately100ngofpurified,amplifiedviralcDNA r
48,53).Weusedaviralmetagenomicapproachtosearchdirectly productswereprovidedtotheBroadInstitute(Cambridge,MA)forse- il 3
for archaeal RNA viruses in several acidic hot springs found in quencingusingtheTitaniumchemistryonthe454FLXpyrosequencer ,
2
YNP.WereportherethedetectionofputativearchaealRNAvirus (Roche). 0
genomes. Isolation, preparation, and amplification of enriched DNA viral 1
fraction.ToobtaintheenrichedDNAviralfraction,10(cid:4)lofenriched 9
b
MATERIALSANDMETHODS viraltotalnucleicacidswasamplifiedwiththeGenomePlexwhole-ge- y
YNP hot spring sample sites. Twenty-eight YNP high-temperature, nomeamplificationkit(Sigma).Thereactionmixtureswerepurifiedas g
acidichotspringswereinitiallyscreenedforthepresenceofRNAinthe describedabovefortheenrichedRNAviralfractions.Approximately5(cid:4)g ue
enrichedviralfraction(seeTableS1inthesupplementalmaterial)be- of purified, amplified viral DNA was provided to the Broad Institute s
t
tweenOctoberandNovember2008.In-depthsamplingformetagenomic (Cambridge,MA)forsequencingaswiththeenrichedRNAviralfrac-
analysiswasperformedon3ofthesesites:NL10,NL17,andNL18.Atotal tions.
of7pairedDNAviralandRNAviralsampleswerecollected.SiteNL10in Sequenceassemblyandanalysis.DNAsequencesweretrimmedof
the Nymph Lake hot springs was sampled in October 2009, February bothprimerandtagsequences.Toaidinassembly,highlyrepresented
2010,andJune2010.NymphLakehotspringsitesNL17andNL18were duplicated sequences were identified and removed using CD-HIT-454
sampled in October 2009 and June 2010. Nymph Lake hot spring site (50)witha40-bpoverlapand98%sequenceidentity.All7viralRNAdata
NL10wasalsosampledinAugust2008and2009fortotalcellularDNA. setswerethencross-assembledusinggsAssemblerversion2.5(Roche)at
InitialscreeningofenrichedviralsamplesforRNAgenomesby32P- 98%sequenceidentitywitha50-bpoverlap.CellularandDNAviralmet-
labeling experiments (reverse transcriptase [RT]-dependent signal agenomicdatasetswereassembledusingthesameprocedures.Theas-
fromRNAviralfraction).Anenrichedviralfractionwascreatedfrom sembledRNAviralcontigs(greaterthan100bp)werecomparedagainst
eachsamplebyfiltrationof26mlofhotspringwaterthroughtwosucces- the Nymph Lake viral DNA metagenomes, as well as the GreenGenes
sive0.8-/0.2-(cid:4)mAcrodisc25-mmPFfilters(Millipore)toremovecells, rRNAdataset(18).SequencesmatchingeitherDNAviralreadsorribo-
collection of the virus particles in the flowthrough, centrifugation at somalsequenceswereexcludedfromfutureanalysis.ThesefilteredRNA
30,000(cid:5)gfor2h,andresuspensionoftheviruspelletinto200(cid:4)lsterile viral contigs were subsequently analyzed against the NCBI-nr and -nt
water.Totalviralnucleicacidswereextractedfromthevirus-enriched databasesusingBLASTN,BLASTX,TBLASTX,BLASTP,andPSI-BLAST
fractionusingthemirVanamiRNA(microRNA)isolationkit(Ambion). foriterativesearchofproteinsequencedatabases(3).TheRPSTBLASTN
Followingnucleicacidextraction,DNAwasremovedbyDNaseI(Am- programfromtheNCBIBLASTpackagewasusedtocomparecontigs
May2012 Volume86 Number10 jvi.asm.org 5563
Bolducetal.
againsttheconserveddomaindatabase.Theresultingmetagenomeswere faultparameters.ReadsidentifiedascontainingCRISPRsusingCRTwere
alsoanalyzedusingtheMG-RASTmetagenomicsanalysisserver(49)to thenanalyzedusingCRISPRFinder(25).AllreadsidentifiedasCRISPRs
assigntaxonomies.Contigsshowingsignificantmatches(E-valueof1(cid:5) withCRTwerealsoidentifiedasCRISPRswithCRISPRFinder.CRISPR-
10(cid:7)3)toRNA-dependentRNApolymerases(RdRps)andstructuralpro- containingreadswereparsedfordirectrepeats(DRs)andspacersgener-
teinsweresubjectedtoamorerigorousexaminationusingHHpred(67) atedfromCRT.TheresultingCRISPRspacerswerecomparedagainstthe
againstthepdb70database(November2010release).Sequenceassembly viral RNA and DNA metagenomes using BLASTN with an ungapped
andanalysisareoutlinedinFig.S2inthesupplementalmaterial. alignmentand100%nucleotideidentityacrosstheentirelengthofthe
Proteinstructureanalysis.SecondarystructurepredictionsforRdRp spacer.ReadswithspacersmatchingRNAcontigswerefurtheranalyzed
andCPsequenceswereperformedusingtheHHpredsoftware.Sequence- byextractionoftheirassociatedDRsandidentificationoftheirclosest
structurethreadingwasperformedusingthePhyreserver(35)andHH- referencemicrobialgenome.
pred, the three-dimensional (3-D) model was built with MODELLER ExaminingtheviabilityofaeukaryoticvirusinYNPhotsprings.To
(21),andvisualizationwasrenderedusingPyMOL(PyMOLMolecular testforpossibleexternalcontaminationofeukaryoticvirusesinthehot
GraphicsSystem,version1.3r1).Multiplealignmentof3-Dstructuresof springsexamined,10ng(4,800genomes)ofpurifiedcowpeachlorotic
capsidproteinswasextractedfromtheDALIdatabase(27). mottlevirus(CCMV),ahighlystablessRNAplantvirus,wasmixedwith
Phylogenetic analysis of RdRps. Seed alignments of viral RdRp 50mlofhotspringwaterinaFalcontubeandplacedinthehotsprings.
(cd01699) and group II intron reverse transcriptases (cd01651) were Aliquotsof50(cid:4)lweretakenfromthesampleatvarioustimeintervalsand
downloadedfromtheNCBICDD(ConservedDomainDatabase)(47). analyzedwithquantitativeRT-PCR(qRT-PCR)usingSSoFastEvaGreen D
Thesealignmentswereusedtogenerateposition-specificscoringmatrices supermix(LifeSciences)onaRotorGene-Qsystem(Qiagen). o
(PSSM)thatservedasqueriesforPSI-BLASTiterativesearch(4)against Comparing RNA metagenomes to other environments. To assess w
n
positive-strandssRNAvirussequencesintheNCBIRefSeqdatabase,pro- whethersimilarRNAviralgenomeswerepresentinYNPhigh-tempera- lo
ducing988matches.ThedetectedRdRpsequenceswereclusteredinto29 ture,near-neutralhotspringenvironmentsthataredominatedbybacte- a
ria,viralRNAmetagenomeswerecomparedagainsttranscriptomelibrar- d
groupsusingtheNCBIBLASTClustprogram(http://www.ncbi.nlm.nih e
.gov/Web/Newsltr/Spring04/blastlab.html), roughly corresponding to iesofMushroomandOctopusSpringsinYNP(36,43)usingBLASTN. d
varioustaxaofssRNAviruses.Additionally,aclusterof31nonredundant Nucleotidesequenceaccessionnumbers.Thenucleotidesequences fr
ofcontig0002andcontig0028havebeensubmittedtoGenBankunder o
sequencesofgroupIIintronreversetranscriptasesandaclusterwithtwo m
accessionnumbersJQ756122andJQ756123,respectively.
RdRpsequencesfromcontig00002andcontig00228wereaddedtothe
h
data set. Multiple sequence alignments within clusters were produced t
usingtheMUSCLEprogram(20).Clusteralignmentswereprogressively RESULTS tp
:
alignedusingtheHHalignprogram(66);ateachstep,thehighest-scoring Aninitialsurveyof28YNPhotspringswasperformedtodeter- //
pairofalignmentsreplacedthetwo“parent”alignments;alignmentposi- minewhichhotspringscontainedadetectableRNAsignalinthe jvi.
tionscontaininglessthan33%nongapcharacterswereremoved.Forthe isolatedRNAviralfraction(seeTableS1inthesupplementalma- a
s
purpose of phylogenetic analysis, alignment positions containing less terial).Thesurveyedsiteswereselectedbasedonbeinghigh-tem- m
than50%ofnongapcharacterswerefurthereliminatedfromthefinal perature((cid:3)80°C)andlow-pH((cid:2)4.0)springslikelytobedomi- .o
alignment.Thephylogenetictreeofthefulldataset(1,021sequences)was r
natedbyArchaea.Thisinitialscreenofviralfractionswasbasedon g
reconstructedusingtheFastTreeprogramwithdefaultparameters(57). /
Theoptimalsequenceevolutionmodelforthedatasetwasselectedusing an RNA-dependent reverse transcriptase (RT) assay where o
theProtTestprogram(1).Arepresentativedatasetof104sequenceswas [32P]dCTPincorporationintofirst-strandcDNAwasdetermined n
selectedusingthefulltreeforguidance;aphylogenetictreewasrecon- inRNase-treatedcontrolsanduntreatedenrichedviralsamples, A
p
structedusingtheRAxMLprogram(69)withthePROTGAMMALGF bothofwhichhadpreviouslybeenDNasetreated.While24sites r
evolutionarymodel,asindicatedbytheProtTestanalysis.Bootstrapanal- showedlittledifferencebetweentheamountsof[32P]dCTPincor- il 3
ysiswith100replicationsandtheShimodaira-Hasegawalog-likelihood porated by RNase-treated and untreated samples, 3 hot springs ,
2
testforalternativetreetopologiesderivedfromconstrainttreeswereper- showedreproducibleandstatisticallysignificantdifferences(Fig. 0
formedusingRAxML. 1). Resampling of these hot springs 12 months later resulted in 1
9
ValidationofselectedassembledcontigsfromenrichedRNAviral nearlyidenticaldetectionoftheRNAsignalintheRNAviralfrac- b
metagenomesbyRT-PCRandDNAsequencing.Upto2yearsafterthe tion, suggesting that RNA viral communities were being main- y
originalsampleswerecollectedfortheproductionoftheviralandcellular g
metagenomes,additionalNL10,NL17,andNL18cellularandviralfrac- tained in these hot springs. The three sites (NL10, NL17, and u
NL18) with the highest RT-dependent signals were selected for e
tionsampleswerecollectedinJune2011andSeptember2011asdescribed s
generatingmetagenomiclibraries. t
abovetodeterminewhetherselectedRNAviralgenomeswerestillpresent
inthesehotsprings.ViralnucleicacidswereextractedusingtheZRviral AstringentapproachwastakentoassembletheRNAviraland
RNAkit(ZymoResearch)incombinationwithon-columnDNasediges- DNAviralmetagenomes.Removalofhighlyduplicatedsequence
tion,asrecommendedbythemanufacturer.ForwardandreversePCR reads at 98% similarity with CD-HIT-454 resulted in a 41.6%
primersdesignedfromselectedmetagenomiccontigs(seeTableS2inthe reductioninreadsacrosstheviralmetagenomes(Table1).These
supplementalmaterial)wereusedwithSuperScriptIIIone-stepRT-PCR duplicatedsequencesareprobablyindicatorsofboththeamplifi-
withPlatinumTaq(Invitrogen).TotestforRTdependency,theRTwas cationbiasandthehighsequencingdepthoftherelativelylow-
excluded from the procedure. To test for strand specificity, only one complexity viral communities found in these hot springs. Re-
primerwasaddedduringthecDNAsynthesisstageandthesecondprimer
movalofthesehighlyrepresentedsequencingreadssignificantly
wasaddedafterthe94°Cheatkillofthereversetranscriptaseandactiva-
improved the overall assembly, resulting in much higher confi-
tionofPlatinumTaq.PCRproductswereclonedintothepCR2.1vector
dencecontigs.
usingtheTopoTAcloningkit(Invitrogen).Sequencingoftheproducts
The initial analysis of the viral RNA metagenomes indicated
wasperformedusingSangersequencingwithBigDyeTerminatorversion
thattheycontainedahighproportionofsequencesfoundinthe
3.1onanABI3100.
AnalysisofCRISPRincellularandviralmetagenomes.Readsfrom paired viral DNA metagenomes. This finding suggests that the
thecellularmetagenomeswereanalyzedforCRISPR-relatedsequences initialtreatmentoftheisolatedviralnucleicacidwithDNasewas
withtheCRISPRRecognitionTool(CRT)(8),usingtheprogram’sde- insufficienttoremove100%oftheviralDNAsequences,probably
5564 jvi.asm.org JournalofVirology
EvidenceforHyperthermophilicRNAViruses
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FIG1SelectedexamplesoftheresultsofscreeninghotspringsforthepresenceofRNAtemplatesintheRNAvirus-enrichedfractions.[32P]dCTPincorporation A
intoRT-dependentfirst-strandcDNAsynthesisisshown.Foreachhotspringsample,resultsareshownforpriorRNasetreatmentofthesample((cid:8)RNase), p
wpohsiicthivwecaosnpterrofolwrmasedakansoawconnptorosilt.iRvee-ssatmrapnldinsgsRoNfsAelveicrtuesd,hcoowtspperainchgslo1r2otmicomntohtstlelavteirrudse(mCoCnMstVra)t,eadntdhtehmenaiengtaetnivaenccoenotfroRlNwAassiwgantaelri(nHth2Oev).irEarlrforracbtaiorsns.hTohwe ril 3
standarddeviations. ,
2
0
1
9
duetoitsvastexcesscomparedtotheamountofRNAintheviral b
fraction.ToaddressthisbiastowardDNAviralsequences,RNA TABLE1Cellular,DNAviral,andRNAviralassemblystatistics y

contigswerecomparedagainsttheDNAviralmetagenomesusing RNAmetagenomesa gu
BLASTN.MatchesofRNAcontigs(E-valueof1(cid:5)10(cid:7)30)against e
DNA Pre-DNA Post-DNA Cellular s
reads from the DNA metagenomes were excluded from further Parameter metagenomesa filtration filtration metagenomesb t
analysis.Morethan72%ofthecontigsintheinitialRNAmetag-
Totalno.ofreads 1,075,407 3,568,86 90,227 632,479
enomicdatasetswereremovedasaresultofmatchingsequences
Totalno.ofbases 34.0 4.2 1.1 229
intheviralDNAdatasets(7,060oftheinitial9,696contigs).This (Mb)
screeningprocedure,whileremovingthemajorityofcontigs,pro- %ofreads 72.90 34.1 34.1 64.6
videdconfidencethattheremainingcontigsassembledfromthe assembled
RNAviralmetagenomeswerederivedfromRNAvirusespresent Largestcontig(bp) 21,541 5,846 5,846 23,333
inthathotspringandnotderivedfromviralDNAsequences. No.oflargecontigs 3,411 519 79 4,820
TheassemblyofsequencesfromtheNymphLakehotspring ((cid:3)1,000bp)
Totalno.ofcontigs 27,746 9,696 2,636 22,649
sitesresultedinalargenumberofcontigsunderstringentassem-
(cid:3)100bp
bly conditions (98% identity within at least 50 bp of overlap)
Averagecontig 573.9 434.3 409.5 716.8
(Table1).The14viralsamples(7enrichedRNAviralsamplesand
length(bp)
7enrichedDNAviralsamples)generated(cid:1)2.8millionreadsand
Avgcoverage(fold) 37.8 36.8 34.2 22.7
807Mbofsequence.Afterdeduplication,therewere877,000reads
aSampledfromhotspringsitesNL10,NL17,andNL18inOctober2009andFebruary
and590Mbofuniquesequence.TheassembledviralDNAmet-
andJune2010.
agenomesgenerated27,746totalcontigs,withanaveragelarge- bSampledfromhotspringsiteNL10inAugustof2008and2009.
May2012 Volume86 Number10 jvi.asm.org 5565
Bolducetal.
D
FIG2HierarchicalclassificationofcontigswithMG-RAST.Classificationofcellular(A),viralDNA(B),andviralRNA(C)sequencesbasedontheM5NR o
w
databaseinMG-RAST.Sequenceswithinsufficientsignificanceorthosethatdidnotmatchwereclassifiedas“NotAssigned/Unknown.”
n
lo
a
d
contig size ((cid:3)1,000 bp) of 1,898 bp. The assembled viral RNA ExaminationoftheputativeviralRNAcontigsshowedthatthe ed
metagenomesgenerated9,696totalcontigs,withanaveragelarge- majority of sequences (56%) did not align to known sequences f
r
contiglengthof1,361bp.Nearly73%ofthesequencereadsinthe (Fig.2C).Asmallfractionofthesequencesmatchedknownvi- o
m
DNAviralmetagenomeswereeitherfullyorpartiallyassembled ruses (3%). The remaining sequences matched sequences from

h
intocontigs,whereas36.8%ofsequencereadsintheRNAviral Archaea(20%),Bacteria(10%),andEukaryota(11%). t
t
metagenomesassembledintocontigs.TheassembledviralDNA Analysis of the RNA viral contigs using NCBI’s protein and p
:
metagenomic contigs had an average length of 574 bp with an nucleotidenonredundantdatabasesandtheCDD,aswellasUni- //
jv
averagereaddepthof38-fold.ThefinalassembledviralRNAmet- prot/Swissprot(31),detectedseveralsequenceswithsimilarityto i.
a
agenomeshadanaveragecontiglengthof410bpwithanaverage RNA-dependent RNA polymerases (RdRps), a positive-strand
s
readdepthof34-foldbasecoverage. single-strandedRNAvirus“hallmarkgene”(39).Thelargestcon- m
CellularDNAmetagenomes,comprising632,000sequencing tig in the RNA viral data set (5.6 kb) and smaller contigs with .o
r
readswhichrepresented229Mbofsequence,wereassembledun- similaritytoRdRpswereconfirmedtobepresentandpersistentin g
/
derthesamehigh-stringencyparametersastheviraldatasets(Ta- thehotspringsovertime.ResamplingoftheNL10andNL18RNA o
ble 1). The NL0808 and NL0908 cellular assemblies generated viralfractionsandextractionofthetotalRNAfromthecellular n

22,649 contigs with an average contig length of 716 bp and an fractions18monthsafterthelastsamplingforthemetagenomic A
p
averagereaddepthof36-foldbasecoverage.Sixty-fivepercentof analysisshowedthecontinuedpresenceoftheRNAgenomeseg- r
thereadsassembledintocontigs. ments.AnRT-PCRassaywithprimersdesignedfromtheseRNA il 3
Theanalysisoftheassembledcellularmetagenomesrevealed contigs was performed on samples taken from the same hot ,
2
that81.0%ofthecontigshadasignificantmatchagainsttheserv- springsoveran18-monthperiod(Fig.3).Theseassaysdemon- 0
1
er’s protein databases (Fig. 2A). The majority of the sequences strated that sequences were single stranded because amplifiable
9
(57%) matched to Archaea, primarily to the Crenarchaeota productwasgeneratedonlywhencDNAsynthesisusedaspecific, b
(86.9%) and to a lesser degree to the Euryarchaeota (11.6%). directional primer. The longest RT-dependent contig, con- y

g
Matches to Nanoarchaeota, Korarchaeota, and Thaumarchaeota tig00002,is5,662ntinlength,composedof258reads,andcon- u
were also detected. A smaller portion of the cellular contigs tainsasinglelargeopenreadingframe(ORF)thatencodesapu- e
s
matchedtoBacteria(20.6%),mostlytoDelta-andGammaproteo- tative viral polyprotein encompassing an RdRp and a putative t
bacteriaandFirmicutes(clostridiaandbacilli).However,overall capsidproteinasdetailedbelow.OmissionoftheRTortemplate
thesematchestotheBacteriawereweakcomparedtothematches orpretreatmentofsampleswithRNasepriortotheRT-PCRassay
totheArchaea.ThiscouldreflectsharedgenesbetweenArchaea eliminatedthesignal,indicatingthatthesourceofthesignalwas
and Bacteria and/or a statistical bias toward bacterial sequences anRNAtemplateintheviralfraction(Fig.3).Thisanalysissup-
duetotheiroverrepresentationcomparedtotherepresentationof portedtheoriginalcontigassembliesanddemonstratedthatthese
archaealsequencesinthepublicdatabases.Insupportofthispos- putativeRNAvirusesarestablemembersoftheviralcommunity
sibility, further analysis of the cellular metagenomes using the withinthehotspringsexplored.
ribosomal databases available on the MG-RAST server (Ribo- Further search for RdRps, as well as viral structural (capsid)
somal Database Project, SILVA rRNA Database Project, and proteins, was performed using a combination of BLAST, MG-
Greengenes)revealed4matchesagainsttheCrenarchaeota(Ther- RAST, and HHpred. Two contigs were identified as containing
moprotei), one match to the Nanoarchaea, and a single distal significantsimilaritytoknownRdRps.Asimilarsearchexamining
matchtoSulfurihydrogenibiumyellowstonense,abacteriumofthe theDNA-enrichedviralmetagenomedatasetrevealednosimilar-
phylumAquificaeoriginallyisolatedfromaYNPhotspring.Over- itytoRdRpsbutdidshowmanyhitstocapsidproteins,mainly
all,theseresultsconfirmthattheYNPhotspringsanalyzedhere thoseofarchaealDNAviruses,asexpected.
aredominatedbyArchaea. Contig00002containsasinglelongORFpotentiallycodingfor
5566 jvi.asm.org JournalofVirology
EvidenceforHyperthermophilicRNAViruses
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FIG3Detectionandsingle-strandednatureofRNAviralsequenceswithinhotspringsmonthsaftersamplingformetagenomicanalysis.Totalnucleicacidsof o
samplesobtained18monthsaftertheoriginalsamplingwereextractedfromeithertheviralfractionortotalcellularfractionofNL10(B)andNL18(A),DNase w
treated,andsubjectedtoRT-PCRformetagenomicanalysis.Thedetectionandstrand-specificnatureofcontig00009(A)andcontig00002(B)weredetermined n
usingcontig-specificprimersets(seeTableS2inthesupplementalmaterial).Eitherforward((cid:8)),reverse((cid:7)),orboth((cid:8)/(cid:7))primerswereaddedtotheinitial lo
first-strandcDNAsynthesismixandthemixtureincubatedasdescribedinMaterialsandMethods.Afterfirst-strandsynthesis,thecompleteprimerpairwas a
d
added(ifnecessary)forthePCRstage.Thecontrol,wherereversetranscriptasewasexcludedfromtheprocedure((cid:7)RT),andthemolecular-sizemarker(bp)are e
indicated. d

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a198-kDa(1,809aminoacids)polyproteinthatspansalmostthe portedthefindingofhighlysignificantsimilaritytoviralRdRps

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entirelengthofthecontig,suggestingthatacompleteornearly and,additionally,ledtothedetectionofsequencesthatweresim- t
t
completeRNAviralgenomewasassembled.Inthemiddlepartof ilartocapsidproteinsofpositive-strandeukaryoticRNAviruses p
:
this polyprotein (approximately between amino acids 800 and withinthenodavirusfamily.Thesequencewiththehighestsimi- //
jv
1020),aputativeRdRpdomainwasidentifiedusingseveralsearch laritytocapsidproteinsisapproximately90aminoacidsinlength i.
a
methods. A search of the Conserved Domain Database at the andislocatedCterminaltotheRdRp,spanningaminoacidresi-
s
NCBIusingtheRPS-BLASTprogramdetectedahighlysignificant dues1562to1652ofthepolyprotein.Thealignmentbetweenthis m
similarity(E-valueofapproximately6(cid:5)10(cid:7)11)totheRdRpdo- portionofthepolyproteinandthecapsidproteinsofeukaryotic .o
r
main profile (no significant similarity to any other domain was virusesdemonstratedalevelofsimilaritythatwasnotstatistically g
/
detected for this or other parts of the polyprotein). A BLASTP significant,althoughthealignmentsincludedapproximately30% o
searchofthenonredundantproteindatabaseattheNCBIyielded identicalaminoacidresidues.Nevertheless,amoredetailed,di- n
statisticallysignificantsimilarity(E-valueof2(cid:5)10(cid:7)4,23%iden- rectcomparisonofthecontig00002polyproteinsequencetothe A
p
tityinanalignmentof341aminoacidresidues)totheRdRpof nodaviruscapsidproteinsequences,aswellastherelatedcapsid r
Pariacatovirus,anodavirus,andlimited,not-significant(E-value proteinsequencesoftetravirusesandnodaviruses,allowedusto il 3
of approximately 0.1) similarity to the RdRps of several other extendthealignmentandidentifythemainstructuralelementsof ,
2
RNAviruses,includingtombusvirusesandpestiviruses.Thesec- thecapsidproteins(Fig.5).Thenodaviruscapsidproteinadopts 0
1
onditerationofthePSI-BLASTsearchresultedinhighlysignifi- an8-strandjellyrollfoldthatisdistantlyrelatedtothestructures
9
cantsimilaritytotheRdRpsofavarietyofpositive-strandRNA ofthecapsidproteinsofothersmallicosahedralpositive-strand b
virusesofeukaryotes.ThisputativeRdRpsequencecontainsall RNA viruses. In addition, unlike other well-characterized viral y

g
three highly conserved motifs, A (D-X[4,5]-D) and B ([S/T]G- capsid proteins, nodaviruses possess an autoproteolytic activity u
X[3]-T-X[4]-N[S/T)],whichareinvolvedinnucleotideselection thatisinvolvedintheprocessingofthecapsidproteinprecursor e
s
aswellasmetalbinding(24),andC,whichcontainsthehighly (the (cid:6) protein) into the mature large ((cid:9)) and small ((cid:10)) capsid t
conservedGDDmotifandisanessentialpartoftheMg2(cid:8)-bind- proteins(61,63).Thenodaviruscapsidproteinisthefounding
ingsite(Fig.4A)(32,37).ModelingoftheinferredproteinRdRp memberoftheA6peptidasesuperfamilythatemploysanunusual
onto the X-ray structure of the positive-strand single-stranded catalyticmechanisminvolvinganinteractionbetweentheactive
NorwalkvirusRdRpstructureshowedthattheputativearchaeal aspartateofthecapsidproteinprecursorandaconservedaspara-
virus RdRp contained the principal structural elements of the ginethatisproximaltothescissilepeptidebondintheC-terminal
Palm domain of the RdRps of eukaryotic positive-strand RNA partoftheprotein,attheboundarybetween(cid:9)and(cid:10)capsidpro-
viruses(Fig.4B).MatchestoRdRpswerealsodetectedforcon- teins (75). These residues, which are essential for autocatalytic
tig00228(comprising10reads),whichencompassedthreeover- cleavageofthenodaviruscapsidproteinprecursor,arealsocon-
lapping short ORFs, each of which showed approximately 70% servedinthesequencesofcapsidproteinsoftetraviruses.Coun-
amino acid sequence identity to the predicted RdRp of con- terpartstoboththecatalyticaspartateandthecleavagesiteaspar-
tig00002(Fig.4A).Thus,thiscontigappearstoencodeanRdRpof agineweredetectedinthecontig00002polyprotein,andregions
aputativearchaealvirusthatisrelatedtobutclearlydistinctfrom surroundingthesetwoconservedaminoacidswerefoundtohave
theputativevirusencodedbycontig00002. thehighestlevelsofsequencesimilarity(Fig.5).Exhaustiveanal-
Acomparisonofthecontig00002polyproteinsequencetoda- ysisofthepartsofthecontig00002polyproteinoutsidetheRdRp
tabasesofproteinfamilyprofilesusingtheHHPredprogramsup- andcapsidproteinregionsfailedtodetectsimilaritytoanyother
May2012 Volume86 Number10 jvi.asm.org 5567
Bolducetal.
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FIG4TheRdRpoftheputativearchaealviruses.(A)MultiplesequencealignmentoftheputativearchaealvirusRdRpswithhomologsfrompositive-strand
RNAvirusesofeukaryotesandbacteriaandreversetranscriptasesfrombacterialgroup2introns.The(nearly)universallyconservedaminoacidresiduesinthe
threesignaturemotifsoftheRdRpsarehighlightedincyan,andpartiallyconservedresiduesarehighlightedinyellow.Thetoptwosequencesarefromthe
putativearchaealvirusesidentifiedinthiswork.Therestofthesequencesincluderepresentativesofthe29clustersofviralRdRpsidentifiedasdescribedin
MaterialsandMethods.Thetwosequencesatthebottom(RTG2)arereversetranscriptases.EachsequenceisdenotedbytheGenBankidentifier(GInumber)
andthenameofthevirusgroup(typicallyfamily)andsubgroup(typicallygenus).Thenumbersdenotethelengths(numberofaminoacids)inless-well-
conservedregionsbetweentheconservedmotifsandthedistancesbetweentheendsoftherespectiveproteinsandthealignedsegments.(B)Structuralmodelof
thecentralcoreregionoftheputativearchaealvirusRdRpfromcontig00002(blue)usingthecalicivirusRdRpasatemplate(red;PDBID3bso)(74).
known proteins or domains. Collectively, the observation of a thattheyformedalineagedistinctfromknownRdRpsofeu-
large polyprotein (a common feature of eukaryotic positive- karyotic and bacterial RNA viruses (Fig. 6). The putative
strand RNA viruses) containing putative RdRp and capsid pro- RdRpsencodedbycontig00002andcontig00228didnotshow
teinsinadditiontoapossibleautoproteolyticactivitysuggestthat specificaffinitywithanyofthethreesuperfamiliesofeukaryote
thiscontigcorrespondstoanearfull-lengthgenomeofaprevi- positive-strand RNA viruses (picornavirus-like, alphavirus-like,
ouslyunknownpositive-strandRNAvirus. and flavivirus-like) or the only known bacterial lineage (levivi-
PhylogeneticanalysisoftheidentifiedRdRpsequencesshowed ruses), and statistical tests showed that association with any of
5568 jvi.asm.org JournalofVirology
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FIG5Thepredictedcapsidproteinoftheputativearchaealvirusanditspotentialautoproteolyticactivity.Multiplealignmentoftheputativearchaealviruscapsidproteinwith
thecapsidproteinsequencesofnodaviruses,tetraviruses,andbirnaviruses.SequencesareidentifiedeitherbyPDBIDorbyGenBankGInumbers.Secondarystructureisshown
forthethreeavailablecrystalstructuresdenotedbythecorrespondingPDBcodes(l,loop;H,helix;E,beta-strand).Alignmentcolumnswithhomogeneityof0.4orgreater(see
MaterialsandMethods)arehighlightedinyellow.ThecatalyticAspishighlightedincyan,andthecleavagesiteAsnishighlightedingreen.
May2012 Volume86 Number10 jvi.asm.org 5569
Bolducetal.
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FIG6 PhylogenetictreeoftheRdRps.TheunrootedphylogenetictreewasgeneratedasdescribedinMaterialsandMethods.Bootstrapsupportvaluesgreater o
n
than0.5areindicatedfordeepinternalbranches.Largegroupsofpositive-strandRNAvirusesfromeukaryotesandbacteria(Levigroup)andthebacterial
A
retrotranscribingelements(RT)areindicatedandshownbyuniquecolors.
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thesemajorlineagesoradditionaldistinctlineages,suchasnoda- tomicanalysisoftwohigh-temperatureYNPhotspringsatneu- ,
2
viruses,couldnotbeconvincinglyruledout(seethesupplemental tral or alkaline pHs (Mushroom and Octopus Springs) that are 0
1
material).Theseresultsarecompatiblewithacentralpositionof known to be dominated by thermophilic bacteria. These hot
9
thenewRdRpinthetreeand,hence,withitspossibleancestral springsharborathermophilicbacterialcommunityofrelatively b
status. highcomplexitythatconsistsofChloroflexi,Cyanobacteria,Acido- y

g
ToinvestigatethepossibilitythattheRNAvirusgenomesde- bacteria, and Chlorobi (36). A BLASTN analysis found that the u
tectedwerecontaminantsofthehotspringsintroducedfromex- greatmajorityofthecontigs(97%ofthetotalRNAviralcontigs) e
s
ternalsources,wetestedtheabilityofanRNAplantvirusknown didnothavestatisticallysignificantmatchestothemetatranscrip- t
foritshighstabilitytobemaintainedwithinthehotspringenvi- tomeoftheseneutraloralkalineYNPhotsprings.Threepercent
ronment.SamplesofCCMVweremixedwithhotspringwaterin oftheRNAviralcontigsdidshowasignificantmatch.However,
50-mlFalcontubesandplacedbackintothehotsprings.Aliquots the average length of the matching contigs was 342 bp, much
were taken at regular intervals for up to 30 min and quantified shorterthantheoverallaveragecontiglength.Thisanalysisindi-
usingaqRT-PCRassaywhichcandetectasfewas10viralgenome catesthatthereislittleoverlapbetweentheRNAviralcommunity
copies.Within1minofsampling,therewasnodetectableamount presentinthearchaea-dominatedacidichotspringsandthebac-
oftheCCMVRNA(datanotshown).Thisexperimentindicates teria-dominatedneutraloralkalinehotspringsandprovidesfur-
that it is unlikely that an externally introduced RNA virus can therevidencethattheputativeviralRNAgenomesdetectedare
survivelongenoughtobedetected,especiallyintheformoflong associatedwitharchaealhosts.
RNAsegments,underthehigh-temperatureacidicconditionsof Finally,weanalyzedtheCRISPRdirectrepeat(DR)andspacer
thehotspringsfromwhichtheputativearchaealvirussequences content present in our cellular metagenomic data sets in an at-
wereobtained. tempttolinktheputativeRNAviralgenomesdirectlytoaspecific
To further probe the possibility that the detected RNA viral hosttypeandtoinvestigatepotentialhostimmunitytoRNAvi-
genomesreplicateinarchaealhosts,thesesequenceswerecom- ruses.CRISPRDRsandspacerswereextractedfromthecellular
pared to the sequences produced by environmental transcrip- metagenomicdatasets.Thespacersequences(10,349)werecom-
5570 jvi.asm.org JournalofVirology
EvidenceforHyperthermophilicRNAViruses
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FIG7AnalysisofcellularCRISPRdirectrepeatunitsandnumbersofuniquespacersmatchingarchaealspeciesandRNAviralmetagenomecontigs.(A) r
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SchematicrepresentationoftheCRISPRlociindicatingthedirectrepeatstructureinterspacedwithspacerregions.(B)ThenumberofCRISPRspacersequences m
with100%matchtoviralRNAcontigsandthealignmentofthedirectrepeatstructures(topsequence)withtheclosestsequencedorganism(bottomsequence).
h
PercentagesofidentityandEvaluesareindicated.
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pared with the assembled viral RNA and DNA metagenomes. domains. Polyproteins of positive-strand RNA viruses of eu- i.
Forty-six spacers (0.44%), associated with 4 types of DRs, were karyotes,inparticularvirusesofthepicornavirus-likesuperfam- a
s
identicaltoRNAsequenceswithintheviralmetagenomicdataset ily,areprocessedbyviralproteasestoyieldindividualfunctional m
(Fig. 7). A similar comparison of the spacers to the DNA viral proteins.Adistinctproteasedomainwasnotdetectedinthevirus .o
metagenome revealed 628 (6.07%) identical spacers (data not polyproteinencodedincontig00002.However,thecatalyticsite rg
shown).ThemajorityofmatchingspacersequencesoftheRNA andthecleavagesiteofthenodaviruscapsidautoproteasewere /
o
metagenome(44/46)wererelatedtoDRsofthearchaealspecies conservedintheregionoftheputativearchaealviruspolyprotein n

SulfolobusislandicusandSulfolobusacidocaldarius.Thesewerethe thatissimilartotheviralcapsidproteins(Fig.5).Innodaviruses, A
p
onlysignificantmatchesfoundintheCRISPRfinderdirectrepeat thecapsidproteinprecursorisencodedinadistinctsegmentof r
database.ThesefindingssuggestthattheRNAviralgenomesrep- genomicRNAsuchthattheproteaseisinvolvedonlyintheauto- il
3
licateinanarchaealhostbelongingtotheSulfolobales,acelltype catalytic maturation of the capsid proteins (15, 62, 75). In the ,
2
commonlyfoundinNL10andacidichotspringsworldwide,and novelvirusdescribedhere,thisproteasemightperformmorever- 0
elicitaCRISPR-mediatedimmuneresponse.However,wecannot satilerolesbycatalyzingadditionalcleavageeventsandreleasing 1
9
categorically rule out the unlikely possibility that the CRISPR the RdRp, the capsid protein, and possibly, other mature viral b
spacermatchestotheRNAviralmetagenomeareinfacttoDNA proteinsthatremaintobeidentified. y

viral sequences that are still present in our viral RNA metag- Theresultsofthisstudydonotunequivocallydemonstratethe g
u
enomicdataset. existenceofarchaealRNAviruses.Itcannotberuledoutthatthe e
s
detectedRNAgenomesegmentsoriginatefromvirusesreplicating t
DISCUSSION inbacterialhostspresentinthesehotsprings.However,several
To our knowledge, this work is the first attempt at viral RNA linesofindependentexperimentalandcomparativegenomicevi-
communityanalysisinextremeenvironments.Theresultsdem- dencesuggestthattheRNAgenomesegmentsidentifiedprobably
onstratethepresenceofputativeRNAviralgenomesinhigh-tem- originatefromvirusesreplicatinginarchaealhosts.First,analysis
peratureacidichotspringsdominatedbyArchaea.Severallinesof ofboththeoverallcompositionofthecellularmetagenomesby
experimental and comparative genomic evidence support the MG-RASTandthe16SrRNAgenecontentshowsthatthemicro-
conclusionthattheassembledgenomesegmentsbelongtoRNA biota of the sampled hot springs consists primarily of Archaea.
viral genomes. In particular, two contigs encoded protein se- Second, the putative RNA viral sequences were recovered from
quenceswithaspecific,significantsimilaritytotheRdRp,ahall- multiplehotspringsatmultipletimepoints.Third,experimental
mark protein of positive-strand RNA viruses. One of these, the exposure of a plant RNA virus known for high stability to the
5.6-kbcontig00002,mightcomprisea(nearly)completeviralge- acidic hot springs environment shows that the virus is rapidly
nome with signature features of positive-strand RNA viruses of degradedundertheseconditions.Fourth,whenwecomparedthe
eukaryotes.Specifically,thiscontigencodesapolyproteinthatis RNAviralsequencesdetectedintheYNPacidichotspringswith
similarinsizetothepolyproteinsofdiverseeukaryotepositive- thetranscriptomesofYNPneutraloralkalinehotspringsthatare
strandRNAvirusesthatcontainboththeRdRpandcapsidprotein knowntobedominatedbybacteriaratherthanarchaea,therewas
May2012 Volume86 Number10 jvi.asm.org 5571