Characterization of a putative novel nepovirus from Aeonium sp.

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ContentslistsavailableatScienceDirect

Virus

Research

j ou rn a l h o m epa g e :w w w . e l s e v i e r . c o m / l o c a t e / v i r u s r e s

Short

communication

Characterization

of

a

putative

novel

nepovirus

from

Aeonium

sp.

Roberto

Sorrentino

a

_,

_Angelo

_De

_Stradis

b

_,

_Marcello

_Russo

b

_,

_Daniela

_Alioto

a,∗∗

,

Luisa

Rubino

b,∗

a_Università_di_Napoli_“Federico_II”,_via_Università_100,₈₀₀₅₅_Portici_(NA),_Italy b_Istituto_di_Virologia_Vegetale_del_CNR,_UOS_Bari,_Via_Amendola_165/A,₇₀₁₂₆_Bari,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29July2013

Receivedinrevisedform26August2013 Accepted28August2013

Available online 6 September 2013

Keywords: Aeonium Nepovirus Cytopathology Serology Sequencing

a

b

s

t

r

a

c

t

AviruswasisolatedfrompottedplantsofanunidentifiedspeciesofAeonium,asucculentornamentalvery commoninSouthernItaly,showingchloroticspotsandringsonbothleafsurfaces.Itwassuccessfully transmittedbysapinoculationtoalimitedrangeofhosts,includingNicotianabenthamianawhichwas usedforultrastructuralobservationsandviruspurification.Virusparticlesareisometric,ca.30nmin diameter,haveasingletypeofcoatprotein(CP)subunits54kDainsize,thatencapsidatesingle-stranded positive-senseRNAspeciesof7549(RNA1)and4010(RNA2)nucleotides.AthirdRNAmolecule3472nts insizeentirelyderivedfromRNA2wasalsofound.ThestructuralorganizationofbothgenomicRNAsand thecytopathologicalfeatureswerecomparabletothoseofnepoviruses.Inaddition,aminoacidsequence comparisonsofCPandthePro-Polregion(asequencecontainingpartsoftheproteinaseandpolymerase) withthoseofothernepovirusesshowedthattheAeoniumvirusbelongstothesubgroupAofthegenus

Nepovirusandisphylogeneticallycloseto,butserologicallydistinctfromtobaccoringspotvirus(TRSV). BasedonthespeciesdemarcationcriteriaforthefamilySecoviridae,thevirusunderstudyappearstobea novelmemberofthegenusNepovirusforwhichthenameofAeoniumringspotvirus(AeRSV)isproposed. © 2013 Elsevier B.V. All rights reserved.

Crassulaceae(ClassMagnoliopsida,OrderSaxifragales),afamily

ofwidelygrownsucculentornamentalplantscomprisesseveral

genera,amongwhichthegenusAeonium,anativeoftheCanary

Islandsthatincludes37species,mostofwhichareherbaceousand

perennial.

Recently,chloroticspotsandringsonbothleafsurfaceswere

observed on several potted plants of an unidentified Aeonium

speciesgrowinginprivategardensofthecityofScafati(Campania,

Italy)(SupplementaryFig.1).Anisometricviruswasrecoveredby

sapinoculationfromsymptomaticplants.Onthebasisofa

pre-liminarysequence analysisofca. 400 nucleotides(nt) ofthe3′

untranslated(UTR)regionoftheviralgenome(Sorrentinoetal.,

2012),itwasidentifiedasastrainoravariantoftobaccoringspot

virus(TRSV),thetypespeciesofthegenusNepovirus(family

Sec-oviridae,orderPicornavirales;Sanfac¸onetal.,2012).However,as

thestudiesprogressed,itbecameevidentthatthepropertiesof

thevirusunderstudy,hereafterreferredtowiththeprovisional

nameofAeoniumringspotvirus (AeRSV),differedenoughfrom

thoseofTRSVtowarrantamoreexhaustivecharacterization,which

constitutestheobjectofthispaper.

∗ Correspondingauthor.Tel.:+390805442936;fax:+390805442911. ∗∗ Correspondingauthor.Tel.:+390812539365;fax:+390812539367.

E-mailaddresses:alioto@unina.it(D.Alioto),l.rubino@ba.ivv.cnr.it(L.Rubino).

Sap from symptomatic Aeonium leaves was inoculated to a

herbaceoushostrangecomprising19speciesinsixfamilies.

Inoc-ulatedplantswerekeptinagreenhouseatca.25◦_C_and_observed

uptofourweeksforsymptomexpression.Mostoftheinoculated

plantswereinfectedshowingovertsymptoms(TableS1).In

par-ticular,Nicotianabenthamianareactedwithchlorotic/necroticlocal

lesionsoninoculatedleaves,frequentlyintheshapeofringspots,

followedseventotendaysafterinoculationbysystemicsymptoms

consistingofextensivemottlingandringspots.Arecoveryphase

tookplaceaboutoneweeklater,withproductionofsymptomless

leaves.Useful diagnostichostswereChenopodiumquinoaandC.

muralethatreactedwithlocallesionsfollowedbysystemic

infec-tion,tipnecrosisanddeathoftheplant.Theviruswasroutinely

propagatedinN.benthamiana,whichwasusedforvirus

purifica-tionaccordingtoPincketal.(1988).Purifiedvirusparticleswere

resuspendedinasmallvolumeof0.02Msodiumcitratebuffer,pH

6.0.Concurrently,aTRSVisolate(obtainedfromDr.M.Fuchs)was

initiallypropagatedinN.benthamiana.However,sinceatthe

green-housetemperature(25–26◦_C)_these_plants_showed_no_symptoms,

althoughsystemicallyinfected,C.quinoaplantswerepreferredfor

viruspurificationfollowingthesameprotocol(Pincketal.,1988).

AeRSVwasreadilypurified fromN.benthamianaplants with

average yieldsofca.0.1mg/g freshtissue.Purification wasalso

attemptedfrominfectedAeoniumplants,buttheyieldwasmuch

lower (ca. 0.01mg/g tissue). Preparations negatively stained in

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Fig.1.(A)NegativelystainedpurifiedunfractionatedAeRSVparticles,fully,partiallyornotpenetratedbythestain.(BandC)AeRSVparticlesexposedtothehomologousor toTRSVantiserum,respectively.(DandE)TRSVparticlesexposedtothehomologousortoAeRSVantiserum,respectively.Bars=50nm.

2% aqueous uranyl acetate and viewed at the electron

micro-scope containedisometricparticlesca.30nmindiameter,some

ofwhichpartiallyortotally(emptyshell)penetratedbythestain

(Fig.1A).Anantiserumwasproducedbyinjectingpurifiedvirus

preparations(0.5–2mg)mixedwithanequalvolumeofFreund’s

incomplete adjuvant in a New Zealand rabbit. Serological

test-ingwasdoneinagargeldouble diffusion(Noordam,1973)and

immunoelectron microscopy (Milne and Lesemann, 1984). The

antiserum toAeRSVhad atitreof 1:256asdeterminedin agar

geldouble-diffusiontests.Itgaveasingleclear-cutprecipitinline

with thehomologous antigen in agar plates and nodetectable

reaction with TRSV and healthy plantextracts (notshown). In

immunoelectronmicroscopythisantiserumdecoratedAeRSV

par-ticlesfromcrudesap(notshown)orpurifiedpreparations(Fig.1B),

butnotTRSVparticles(Fig.1E).Conversely,anantiserumagainst

TRSV decoratedpurifiedTRSV (Fig.1D),butnotAeRSV(Fig.1C)

particles.

The cytopathologicalfeatures ofAeRSV werestudiedin

sys-temicallyinfectedleavesofN.benthamianaprocessedaccordingto

standardprocedures(MartelliandRusso,1984).Comparedwith

the healthy control(not shown),the structuralorganization of

infectedN.benthamianacellswasfairlywellpreserved,exceptfor

thepresenceofconspicuousinclusionbodiesmadeupof

accumu-lationsofmembranousvesicleswithafibrillarcontent,anetwork

ofproliferatedendoplasmicreticulumstrands,lipiddropletsand

virus particles(Fig.2A).These cytopathologicalstructures were

usually appressed tothenuclei, theoutermembrane of which

wasdilatedinseveralpoints(Fig.2B).Virusparticleswereeither

scattered in the ground cytoplasmor arranged in rows within

tubular structures usually connected with plasmodesmata and

sometimes associated with cell wall outgrowths (Fig. 2C). The

observed cytopathologicalmodificationsare incomplete

agree-mentwiththosetypicallyinducedbynepovirusinfections(Martelli

andRusso,1984;Ritzenthaleretal.,2002;Gokalpetal.,2003).

Purifiedviruspreparationsweredissociatedinthepresenceof

Laemmli’sbuffer(1970).Thecoatprotein(CP)subunitsmigrated

asasinglebandwithanestimatedmol.wtof54kDa(Fig.3A)in

discontinuos12.5%acrylamidegels.

RNAwasextractedfromca.200mgpurifiedvirusparticlesby

adding1volofextractionbuffer(100mMglycine–NaOH,pH9.0,

containing100mMNaCl,10mMEDTA,2%sodiumdodecylsulfate

and1%sodiumlauroylsarcosine)and2volofwater-saturated

phe-nol.Theaqueousphasewasfurtherextractedwithequalvolumes

ofphenolandchloroform,thenchloroformalone,andprecipitated

with2.5volofethanolinthepresenceof0.3Msodiumacetate,pH

5.5.RNAwasresuspendedinsteriledeionizedwater.Denatured

RNAwaseithervisualizedbyethidiumbromidestainingor

trans-ferredtonylonmembranesand hybridizedwith a DIG-labelled

probecorrespondingtothelast200nucleotidesoftheviralRNAs.

AsshowninFig.3B,theencapsidatedviralgenomeseparatedinto

twobands(RNA1andRNA2).

Totalnucleicacids(TNA)wereextractedasdetailedinDalmay

etal.(1993).Briefly,about100mgleaftissueweremaceratedina

coldmortarinthepresenceof600mlextractionbufferand600ml

phenol,processedasdescribedaboveandresuspendedinsterile

deionizedwater.NorthernblotanalysisconfirmedthatRNA1and

RNA2weretheonlyvirus-relatedRNAspeciespresentbothinvirus

particles(Fig.3B)andTNAextractsfrominfectedN.benthamiana

tissues(Fig.3C,lane1).However,Northernblotanalysisofextracts

frominfectedAeoniumplantsrevealedthepresenceofanadditional

RNAspeciessmallerthanRNA2(Fig.3C,lane2).

Approximately2mgofRNAextractedfromvirusparticleswere

denaturedbyheatingat65◦_C_and_used_for_the_construction_of_a

cDNA librarywith a cDNASynthesis Module(Roche, USA).The

doublestrandedcDNAwasligatedtoaSmaI-linearized,

dephos-phorylatedpUC18,andclonedintoEscherichiacolistrainXL1-Blue

Fig.2. UltrastructuralaspectsofAeRSV-infectedN.benthamianamesophyllcells. (A)Vesiculate-vacuolatecytoplasmicinclusionbodies(IB)nexttothenucleus(N). (B)Portionofanucleus(N),showingenlargementoftheoutermembrane(arrow). (C)Virus-containingtubularstructuresconnectedwithplasmodesmata(arrow)and insidedevelopingcellwalloutgrowth(doublearrow).Bars=250nm.

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Fig.3.StructuralcomponentsofAeRSV.(A)SDS-polyacrylamidegelelectrophoresis ofdissociatedvirusparticles.AsingletypeofCPsubunitsca.54kDainsizeis visi-ble(lane2).Lane1,molecularweightmarkers.(B)GenomicRNA1andRNA2from purifiedunfractionatedparticlesvisualizedafterethidiumbromidestaining(left)or hybridizationwithaDIG-labelledprobe(right).(C)NorthernblotofRNAextracted frominfectedN.benthamiana(lane1)andAeonium(lane2)leaves;hybridization asinpanelB.TheasteriskmarkstheadditionalRNAspeciesfoundonlyinAeonium

extracts.

competentcells.The5′_terminus_of_the_viral_genome_was

deter-minedusinga5′_rapid_{amplification}_of_cDNA_ends_(RACE)_(Roche,

USA).The5′ _RACE_products_were_cloned_into_pGEM-T_Easy

vec-tor(Promega,USA).Recombinantplasmidswerepurifiedwiththe

NucleoSpinkit(Macherey-Nagel,Germany)andcustomsequenced

inbothdirections(EurofinsMWGOperon,Germany).Sequences

thusobtainedwerebridgedbyRT-PCRusingspecificprimersand

highfidelityTaqDNApolymerase(Roche,USA).Sequenceswere

assembledandanalyzedusingtheDNAStridersoftware(Marck,

1988)andfurtherexaminedbycomparisonwithknownnucleotide

andproteinsequencesusingBLAST(Altschuletal.,1990).Pairwise

comparisonsweremadeusingEMBOSSintheEBIpackage(Rice

etal.,2000).Sequencesoftheencodedproteinswerealignedand

tentativephylogenetictreeswereconstructedwithClustalW

pro-gramme(Thompsonetal.,1994)andvisualizedbytheTreeView

programme(Page,1996).

RNA1consistsof7549nts,excludingthepoly(A)tail(GenBank

accessionno.JX304792).ThefirstAUGstartcodonisatntposition

104inacontextfavourabletoenhancetranslationineukaryotic

cells,withanAinposition−3,aCinposition−2andaGinposition

+4(Kozak,2005).AssumingthattranslationbeginsatthisAUG,it

wouldterminateattheUAAstopcodonatposition7046–7048,thus

yieldingatranslationproduct(P1)of2314aminoacids(aa)with

acalculatedMr of257,168(257kDa)(Fig.4).Computer-assisted

analysisshowedthatP1containsthecharacteristicmotifsofthe

putativeviralproteasecofactor(PRO-co),theNTP-binding(NTP-B),

Fig.4.PutativeschematicgenomeorganizationofAeRSV.Greyzonesindicate conservedmotifsinnepoviruses.Putativecleavagesitesdeducedbysequence com-parisonareindicatedbytheverticallines.Dotsinthe2Aregionindicatetheposition oftwoproline-richsequences.BlackcirclesindicatetheputativeVPg.RNA2′

repre-sentsthesmallerRNA2species,havinga179aadeletionintheMP.

cysteineprotease(PRO)andtheRNA-dependentRNApolymerase

(POL)coredomains,indicatingthatthepredictedpolypeptideis

cleaved by a viral protease togive maturefunctional products

(Fig.4).Theputativeviralproteasecofactormotif(F-x21-W-x11

-L-x22-L-x-Tbetweenaaatpositions525–590)isslightlydifferent

from theconsensus sequence for this motif (Rottet al., 1995),

themajordifferencebeingthesubstitutionofthelastaa(EtoT).

Theputative NTP-bindingmotif(Gorbalenyaand Koonin,1989)

islocatedbetweenaa827and878,intwosites:siteAbetween

positions 827and 834(G-x4-GKS) and site B (DD)at positions

877–878.Theputativeviralcysteineproteasemotif(H-x35-E-x98

-CG-x8-G-x5-G) is locatedbetween aa positions1324 and 1475.

TheunderlinedaaH,EandCformtheputativecatalytictriadof

theenzyme(DessensandLomonossoff, 1991;Gorbalenyaetal.,

1989;MargisandPinck,1992).TheC-terminalregionofP1

con-tainsthesequencecharacterizingtheputativeRNA-dependentRNA

polymerase (RdRp) (Argos, 1988)(D-x4-D-x69-G-x3-T-x3-N-x33

-GDD-x33-DK)betweenaapostitions1782and1936.Theconsensus

sequencereportedbyMayoandFritsch(1994)forthe

genome-linkedprotein(VPg)wasnotfound.

Aminoacid alignmentsof AeRSV-encodedP1 with the

com-parable protein of selectednepoviruses allowed theprediction

ofputative cleavagesitesandpositionsofmatureproducts. For

instance,comparisonwiththeexperimentallyvalidatedstructure

oftheN-proximalregionofarabismosaicvirus(ArMV)P1(Wetzel

etal.,2008),indicatesthattheN-terminalproteasecofactorofthe

virusunderstudywouldbecleavedatthesiteC651_/G652_to_separate

fromtheNTBprotein,andanadditionalcleavagesite(C450_/S451₎

wouldproducetwoproteindomainsof450 and201aa,

respec-tively,denotedX1andX2.VPgislocatedbetweentheNTP-binding

and proteaseproteins,beginningataaposition1254,as

identi-fiedbycomparison withthechemicallydeterminedN-terminal

sequenceofTRSVVPg(Zallouaetal.,1996).However,therestof

thesequenceandthecleavagesitescouldnotbeestablishedwith

certaintyduetothelowdegreeofconservationofthissmallprotein.

AcleavagesiteC1499_/S1500_separating_the_protease_(ca.₂₁₅_aa)_and

RdRp(815aa)proteinsispredictedbycomparisonwiththe

corre-spondingregionofgrapevinefanleafvirus(GFLV)P1(Ritzenthaler

etal.,1991;Margisetal.,1994).

RNA2 is 4010 nts in length, excluding the 3′_-poly(A) _tail

(JQ670669).ItsfirstAUGstartcodonatposition124–126is

pre-cededbyaCinposition−2andfollowedbyaGinposition+4,i.e.

inalessfavourabletranslationcontextthanthatofRNA1.The

sin-gleORFofRNA2extendsfromnt124tont3508–3510,yieldinga

putativepolyproteinP2of1128aaandaMrof125,601(126kDa)

(Fig.4).Thepolypeptidecontainsthe“P”motifconservedinthe

movementproteins(MP)ofnepovirusesandotherplantviruses

(Kooninetal.,1991;Mushegian,1994)ataaposition442and,ataa

1120–1123themotifFWGR,thatisveryclosetothenepovirusCP

motifFYGR(LeGalletal.,1995).TheN-terminalpeptide

(approx-imately260aa)shareslowsequencehomologywithprotein2AHP

ofGFLVinvolvedinthereplicationofRNA2(Margisetal.,1993;

Gaireetal.,1999;Ritzenthaleretal.,2002),includingtheproline

motifsthatarealsopresentintomatoringspotvirus(ToRSV)P2

N-terminalprotein(Carrieretal.,2001),suggestingthisproteinto

havearoleinthelocalizationandreplicationofRNA2.Comparison

ofAeRSVCPsequencewiththechemicallydeterminedsequence

ofTRSVCP(Buckleyetal.,1993,1995)indicatedtheAresidueat

position616astheN-terminalaaandacleavagesiteM615_/A616

betweentheMPandCPproteins.SuchM/Acleavagesitebetween

MPandCPisunusualandwasreportedonlyforanothernepovirus

(Tomitakaetal.,2011).

ThesmallerRNAspeciespresentonlyininfectedAeoniumplants

(Fig.3C)wasamplifiedbyRT-PCRfrompurifiedviralRNAextracted

fromAeoniumusingprimerscorrespondingtothefirstandlast17

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RNAis3472ntsinlengthandhasasequenceidenticaltothatof

RNA2butlacks537ntsfromposition1286to1822.Thisadditional

RNA2(RNA2′₎_has_a_single_ORF_that_extends_from_the_AUG_at

pos-itions124–126andcodesforaputativeproteinof949aawithaMr

of105,726(106kDa)(Fig.4).Putativematurationproductsofthe

polyproteinencodedbyRNA2′_are_the_N-terminal_protein_(2A)_and

CPofthesamesizeasthoseencodedbyRNA2,andaMPwitha

dele-tionof179aa,fromposition389toposition567.Atleasttwomore

examplesofvariabilityofnepoviralRNAsareknown:onearising

fromRNA2ofArMV(Loudesetal.,1995;Wetzeletal.,2002)and onefromRNA1ofTBRV(Hasiów-Jaroszewskaetal.,2012).

How-ever,onlyinthelattercasesomeevidenceofapossibilebiological

significancewasobtained.ThebiologicalroleofAeRSVRNA2′_has

notyetbeeninvestigated.

Sequences ofthe5′ _UTR _regions _of _the_RNA1 ₍₁₀₃_nts) _and

RNA2(123nts)were53.2%identical.Inparticular,thefirst17nts

(UUGAAAAUUCUCUCACA) are100%identical andcontainthe5′

end consensussequencefor nepovirusRNAs (underlined;Fuchs

etal.,1989).Theconservedrepeatsofthemotifsequences

capa-bletoformstem-loopstructuresinthe5′_untranslated_regions_of

othernepoviruses(Wetzeletal.,2001)werenotfound.Comparison

betweenthe3′_UTR_of_RNA1₍₅₀₁_nts)_and_RNA2₍₅₀₀_nts)_showed

98.6%identity.

Particle morphology, cytopathological features and genome

organization support the classification of AeRSV in the genus

Nepovirus(familySecoviridae,orderPicornavirales;Sanfac¸onetal.,

2012), ataxoncomprisingthree subgroupsofspeciesidentified

bydifferencesinthecleavagesitesrecognizedbytheirproteinase

andsizeofRNA2:(i)subgroupA3700–4000nts;(ii)subgroupB

4440–4700 nts;(iii)subgroupC6400–7300nts.Basedonthese

parameters,AeRSVfitswellinsubgroupA,alikelihoodsupported

byitsallocationnexttoTRSVinacladeofthephylogenetictree

constructedwithCPsequences(Fig.S2).

SpeciesdemarcatingcriteriainthefamilySecoviridaesetbythe

International CommitteonTaxonomyofViruses(ICTV)include,

among others,lack ofserological relationhip andless than75%

Table1

Percentageofsequenceidentitybetweentheaminoacidsequencesofvarious regionsofAeRSVgenomeandthoseofothernepoviruses.

Virus P1 P2 Pro-Pol CP SubgroupA ArMV 31 23 44 27 GDefV 30 20 42 25 GFLV 30 20 42 25 MMMoV 31 29 44 29 RpRSV 28 23 31 25 TRSV 78 70 84 66 SubgroupB BRSV 26 17 38 20 CNSV 28 19 36 20 GCMV 27 20 38 21 TBRV 27 20 40 25 SubgroupC BRV 27 17 38 23 CLRV 25 15 37 21 GBLV 27 16 42 21 ToRSV 27 15 38 23

GenBank accession numbers are: ArMV (arabis mosaic virus; NC006057, NC006056);GDefV(grapevinedeformationvirus;NC017939,NC017938);GFLV (grapevine fanleafvirus;NC003615,NC003623);MMMoV (melonmild mot-tlevirus;AB518485,AB518486);RpRSV(raspberryringspotvirus;NC005266, NC005267);TRSV(tobaccoringspotvirus;NC005097,NC005096);BRSV(beet ringspot virus; NC003693, NC003694); CNSV (cycas necrotic stunt virus; NC003791,NC003792);GCMV (grapevine chromemosaic virus; NC003622, NC003621);TBRV(tomatoblackringvirus;NC004439,NC004440);BRV (black-currant reversion virus; NC003509, NC003502); CLRV(cherry leafroll virus; NC015414, NC015415); GBLV (grapevine Bulgarianlatent virus; NC015492, NC015493);ToRSV(tomatoringspotvirus;NC003840,NC003839).

identity in theaa sequence of CP and less than 80% in theaa

sequence of the Pro-Pol region, defined as “the sequence

con-tainedbetweentheproteaseCGandthepolymeraseGDDmotifs”

(Sanfac¸onet al., 2012). When these and the whole P1 and P2

sequenceswerecomparedwiththoseofnepovirusesofthethree

subgroups,thedivergencebetweenAeRSVandalltheotherviruses

appearedtobewellbelowthethresholdforCP,whereasthePro-Pol

domainofTRSVwasslightlyabove(84vs.80%)(Table1).However,

comparisonofeachsingledomainofTRSVandAeRSVpolyproteins

disclosedarelativelylowpercentageofidentitythroughout(Table

S2).

Themoleculardata,thedifferentreactionofinfectedN.

ben-thamianaplants,andthelackofserologicalrelationshipssupport

thenotionthatAeRSVisanovelnepovirusspeciesphylogenetically

closetobutclearlydistinctfromTRSV.

Acknowledgements

GratefulthanksareexpressedtoMrsA.Antonacciforthe

skil-fultechnicalhelp,Dr.D.Boscia(IVV,Bari,Italy)forthepreparation

oftheantiserum,Dr.M.Fuchs(CornellUniversity,Geneva,USA)

for providing the TRSV inoculum and antiserum and Prof. G.P.

Martelliforusefulsuggestionsandrevisionofthemanuscript.This

studywaspartiallysupportedbyMIPAAFprojectOIGA-IN.OR.QUA

(2009–2012).

AppendixA. Supplementarydata

Supplementary data associated with this article can be

found,intheonlineversion,athttp://dx.doi.org/10.1016/j.virusres.

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