2018 Coronavirus infectious bronchitis virus non-structural proteins 8 and 12 form stable complex independent of the non

2018·0.82 MB·English

Virology 513 (2018) 75–84
ContentslistsavailableatScienceDirect
Virology
journal homepage: www.elsevier.com/locate/virology
Coronavirus infectious bronchitis virus non-structural proteins 8 and 12
MARK
form stable complex independent of the non-translated regions of viral RNA
and other viral proteins
Yong Wah Tana,b,c,1, To Sing Funga,1, Hongyuan Shenc, Mei Huangb, Ding Xiang Liua,⁎
aSouthChinaAgriculturalUniversity,GuangdongProvinceKeyLaboratoryMicrobialSignals&DiseaseCo,andIntegrativeMicrobiologyResearchCentre,Guangzhou
510642,Guangdong,People'sRepublicofChina
bSchoolofBiologicalSciences,NanyangTechnologicalUniversity,60NanyangDrive,63755,Singapore
cInstituteofMolecularandCellBiology,61BiopolisDrive,Proteos138673,Singapore
A R T I C L E I N F O A B S T R A C T
Keywords: Thecleavageproductsfromcoronaviruspolyproteins,knownasthenon-structuralproteins(nsps),arebelieved
Coronavirus to makeup themajor components of theviral replication/transcription complex. In thisstudy, severalnsps
Infectiousbronchitisvirus encodedbyaviangammacoronavirusinfectiousbronchitisvirus(IBV)werescreenedforRNA-bindingactivity
IBV andinteractionwithitsRNA-dependentRNApolymerase,nsp12.Nsp2,nsp5,nsp8,nsp9andnsp10werefound
Non-structureprotein tobindtountranslatedregions(UTRs),whilensp8wasconfirmedtointeractwithnsp12.Nsp8hasbeenreported
RNA-dependentRNApolymerase
tointeractwithnsp7andfunctionsasaprimasesynthesizingRNAprimersfornsp12.Furthercharacterization
Proteininteraction
revealedthatnsp8-nsp12interactionisindependentoftheUTRsofviralRNA,andnsp8interactswithboththe
RNAsynthesis
Viralgenomereplication N-andC-terminalregionsofnsp12.Theseresultshavepromptedaproposalofhowthensp7-nsp8complexcould
possiblyfunctionintandemwithnsp12,formingahighlyefficientcomplexthatcouldsynthesizeboththeRNA
primerandviralRNAduringcoronavirusinfection.
1. Introduction (gRNA)andanestedsetofpositive-sensesgRNAs.Thekeyplayerinthis
part of the coronavirus life cycle is the virus-encoded replicase gene,
Coronaviruses are a family of enveloped viruses with single which makes up two thirds of the genome and is translated into two
stranded,positivesenseRNAgenomeof27–30kbinlength.Infectious polyproteins: pp1a and pp1ab. The two polyproteins are then auto-
bronchitis virus (IBV) is an avian gammacoronavirus that causes re- proteolyticallyprocessedinto16non-structuralproteins(nsps)bytwo
spiratorydiseaseinchickens,resultinginmajoreconomicburdentothe virus-encodedproteases,thepapain-likeproteaseencodedinnsp3and
poultry industry worldwide. In IBV-infected cells, a 3′-coterminal the 3C-like (main) protease encoded in nsp5 (Liu et al., 1994, 1995,
nested set of six mRNA species, including the genome-length mRNA 1997,1998;LiuandBrown,1995;NgandLiu,1998,2000,2002;Lim
(mRNA1)andfivesubgenomicmRNAspecies(mRNA2-6),isexpressed. andLiu,1998;Limetal.,2000;Xuetal.,2001;Fangetal.,2008;Chen
Four structural proteins, the type 1 glycoprotein spike (S), the small etal.,2009).Notably,duetothelackofacleavagesitethatusuallylies
membrane-associated envelope protein (E), the integral membrane between nsp1 and nsp2 in other coronaviruses, cleavage of the IBV
protein(M)andthephosphorylatednucleocapsid protein(N),areen- replicase only produces 15 nsps (nsp2-16) without the counterpart of
coded by subgenomic mRNA(sgRNA) 2,3,4 and6,respectively (Liu nsp1. Based on sequence comparison, biochemical and structural stu-
etal.,1991;LiuandInglis,1992a;Zhaoetal.,1993).Inaddition,four dies,theenzymaticactivitiesrequiredformostofthekeyprocessesin
accessory proteins, 3a, 3b, 5a and 5b are encoded by sgRNA 3 and 5 coronavirus RNA synthesis have been mapped to some of the nsps
(LiuandInglis,1992a,1992b). (Pasternak et al., 2006). Among them, the key enzyme critical to the
During coronavirus infection, full-length genome is replicated for viralabilitytoreplicateitsgenomeandpropagateinthehostcellisthe
packaging into progeny virions, while sgRNAs are transcribed for the RNA-dependentRNApolymerase(RdRP)ornsp12.Availableevidence
expression of structural proteins. These two processes are dependent suggests that the RNA polymerase activity of nsp12 may be primer-
uponviralabilitytosynthesizeboththepositive-sensegenomicmRNA dependent(teVelthuisetal.,2010)andrequiretheprimaseactivityof
⁎Correspondingauthor.
E-mailaddress:[email protected](D.X.Liu).
1Equalcontribution.
http://dx.doi.org/10.1016/j.virol.2017.10.004
Received28July2017;Receivedinrevisedform27September2017;Accepted2October2017
0042-6822/ © 2017 Published by Elsevier Inc.
Y.W.Tanetal. Virology 513 (2018) 75–84
nsp8(Imbertetal.,2006).Otherimportantenzymaticactivitiesinclude 2.2. Mammaliancellculture,virusinfectionandoverexpression
theRNAhelicaseofnsp13(Seybertetal.,2000;Ivanovetal.,2004a),
the methyltransferase activities of nsp14 and nsp16 (Y. Chen et al., H1299 cells were cultured in RPMI 1640 medium supplemented
2009;Maetal.,2015;Decrolyetal.,2008;Chenetal.,2011;Bouvet with5%FBSand1%PS.Cellsweregrownina37°Cincubatorsupplied
et al., 2010), the exoribonuclease activity of nsp14 (Ma et al., 2015; with 5% CO . For infection experiments, cells were collected by cen-
2
Minskaia et al., 2006) and the endoribonuclease activity of nsp15 trifugation at 600rpm for 5min, re-suspended in fresh serum-free
(Ivanovetal.,2004b;Bhardwajetal.,2004;Ricagnoetal.,2006). medium,andapproximately2.5×106cellswereseededintoa60mm
Informationonthespecificinteractionsamongnspsisalsoemerging dish.
based on biochemical, structural and functional studies of nsps from Amonolayerofcellsgrownto100%confluencywaswashedwith
othercoronaviruses(Imbertetal.,2006;Maetal.,2015;Imbertetal., serum-free medium twice and inoculated with virus at multiplicity of
2008;vonBrunnetal.,2007;Panetal.,2008;Brockwayetal.,2003; infection (MOI) of approximately 2, unless specified. Cells were in-
Subissi et al., 2014; Knoops et al., 2008). Distinct from the viral cubatedat37°Cwith5%CO beforeharvestedforanalysisatspecific
2
structuralproteins,thefunctionsservedbythenspsarefocusedonthe timepoints.Fortime-courseexperiments,0hsampleswereharvested
modulation ofhostenvironment tosupport viralreplication andviral afterthecellswereincubatedfor5min.
RNAsynthesis.Integralmembraneproteinsnsp3,nsp4andnsp6engage Vaccinia/T7 recombinant virus, used for overexpression of genes,
in homotypic and heterotypic interactions to drive membrane re- waspreparedbyinfectingVerocellsandharvestedbythreefreeze-thaw
arrangements to form the network of double membrane vesicles cycles,asdescribedpreviously(J.Wangetal.,2009).H1299cellswere
(DMVs) and convoluted membranes (CMs) (Angelini et al., 2013), or grown to 80–90% confluency and infected with the virus for 1h at
doublemembranespherulesfromzipperedERmembranes(Maieretal., 37°C,5%CO .PlasmidDNAwasdeliveredintotheinfectedcellswith
2
2013),thesitesofviralreplicationandalsoserveasthedockingsitefor Effectene® transfection reagent. The cells were incubated with the
viral replication complexes (Oostra et al., 2008). A recent study has transfectionmixfor20hin37°Cbeforebeinglysed.
identifiedcriticalresiduesonnsp7andnsp8ofsevereacuterespiratory
syndromecoronavirus(SARS-CoV),whichareessentialforefficientde 2.3. Polymerasechainreaction(PCR)andsite-directedmutagenesis
novoRNAsynthesisbynsp12,indicatingthatinteractionsamongthese
proteinscontributetotheprocessivityofRNAsynthesis(Subissietal., Amplification of DNA not exceeding 2000 base pairs (bp) was
2014). achievedusingKAPATaqDNApolymerase(KapaBiosystemsInc,USA)
On the other hand, interaction between the nsps and viral RNA orFermentasTaqDNApolymerase(ThermoScientific,USA).Ina50µl
genome has not been thoroughly studied. Viral protein-RNA interac- reaction, 150ng of template DNA was mixed with 1.5pmol of each
tionsareassumedtooccurbetweenthekeyreplicationenzymes,nsp7, primer,2nmolofeachdexoyribonucleotidetriphosphate,1mMMgSO
4
nsp8andnsp12,thusformingacomplexexhibitingreplicaseactivityin and1.5UofKODHotStartDNApolymerase.Eighteenthermalcycles
the presence of a RNA primer (Subissi et al., 2014). In addition, al- were used for amplification and each product was purified, digested
though some nsps have not been assigned with functions involved in with DpnI to remove template DNA and purified again before trans-
coronavirus RNA synthesis, they might still be participating in viral formationforselection.
RNAreplicationortranscription.Inthisstudy,atwo-prongedapproach
was adopted to study viral proteins and protein-RNA interactions in 2.4. InvitrotranscriptionandlabelingofRNAprobes
coronavirus RNA synthesis. To elucidate RNA-protein interactions, an
attemptwasmadetodetectthepresenceofRNA-bindingactivityinany All biotin-labeled probes used in biotin pull-down assay were
oftheIBVnsps,whichhadnotbeenpreviouslyreportedtopossesssuch transcribed with PCR products using appropriate primer pairs, as de-
an activity. This was done using available constructs over-expressing scribedpreviously(Tanetal.,2012).AstretchofpolyTisincludedin
individual nsps in a biotin pull-down assay with biotinylated probes. the5′endofreverseprimersforthetemplatesofpositivesenseRNAor
The second method used was to conduct a screen for protein-protein forwardprimersforthetemplatesofnegativesenseRNA,sothatthein
interactionsbetweenviralpolymerase,nsp12andothernsps. vitrotranscribedRNAwillcontainastretchofpolyAtailtofacilitate
RNA stability. Briefly, a template for EGFP (-) was amplified from
pEGFP-N1 using pT-EGFP_F (5′-TTTTTTTTTTTTTTTTTTTTTTGTATG-
2. Materialandmethod GTGAGCAAGGGCGAGG-3′) and T7-EGFP_510-528R (5′-TGTAATACG-
ACTCACTATAGGGCTGCCGTCCTCGATGTTG-3′). IBV 5′-UTR (+) and
2.1. Antibodies,enzymesandotherreagents IBV 5′-UTR (-) probes were generated from PCR fragments amplified
fromplasmidpKT-IBVAwiththeprimerpairsT7_i1-27(5′-TGTAATA-
Antibodies to β-tubulin, FLAG and HA tags were purchased from CGACTCACTATAGGACTTAAGATAGATATTAATATATATCT-3′)/
Sigma-Aldrich(St.Louis,MO,USA).AntiseraagainstIBVproteinswere pT_i507-528R (5′-TTTTTTTTTTTTTTTTTTTTTGTTGTCACTGTCTATT-
made by immunization of rabbits with bacterially expressed fusion GTATGT-3′)andpT_i1-29(5′-TTTTTTTTTTTTTTTTTTTTTACTTAAGA-
proteinsaspreviouslydescribed(Liuetal.,1991;Fangetal.,2005).All TAGATATTAATATATATGTAT-3′)/T7_i528-506R(5′-TGTAATACGACT-
horseradish peroxidase (HRP) conjugated secondary antibodies were CACTATAGGGTTGTCACTGTCTATTGTATGTC-3′),respectively.IBV3′-
purchasedfromDako(Glostrup,Denmark)andAlexaFluor®conjugated UTR(+)probewastranscribedfromthePCRproductamplifiedfrom
secondary antibodies were purchased from Invitrogen Molecular pGEM T easy-IBVE plasmid with primers T7_i27106-27125 (5′-TGTA-
Probes® (Eugene, OR, USA). T7 RNA polymerase and Biotin RNA La- ATACGACTCACTATAGGGTAACATAATGGACCTGTTG-3′) and LDX30
beling Mix were purchased from Roche Applied Science (Penzberg, (5′-TGATGCCGGCCACGATGCGTC-3′).
Upper Bavaria, Germany). Taq polymerase and KOD Hot Start DNA For in vitro transcription, template DNA fragments were purified
Polymerase were purchased from KAPA Biosystems (Cambridge, MA, andelutedinRNase-freewater.LinearizedplasmidDNA(1μg)orPCR
USA)andMerck(Darmstadt,Germany),respectively. fragment(200ng)wasincubatedwith10UT7RNApolymerase,10U
RPMI 1640 medium and Dulbecco’s Modified Eagle Medium Protector RNase Inhibitor and NTPs (1mM each of ATP, CTP, GTP,
(DMEM) were purchased from Invitrogen (Carlsbad, CA, USA). Fetal UTP)in1Xtranscriptionbufferprovidedwiththepolymerase,at37°C
Bovine Serum (FBS) was purchased from Thermo Scientific HyClone for 2h. The transcription mix was incubated with 5U of DNase I at
(Waltham,MA,USA).Penicillin-streptomycinsolution(PS)and0.25% 37°C for 15min to remove template DNA. For labeling of RNA pro-
Trypsin-EDTA solution were purchased from Gibco (Carlsbad, CA, ducts,NTPswerereplacedwithBiotinRNALabelingMix(1mMeachof
USA). ATP, CTP, GTP, 0.65mM UTP, 0.35mM biotin-16-UTP). Transcripts
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Y.W.Tanetal. Virology 513 (2018) 75–84
A B C Fig. 1.Construction and characterization of a re-
combinant IBV expressing HA-tagged RdRP (rIBV-
wt 0 4 8 H1A2 16 24 hpi 250- wt 8H A w t 2 4H hApi wtCLH A I P w: t - HHAA HHsio1An2-R9id9nRccPee)lll.lssawi.nefKreeicntieentdfiecwctaietndhawlryIisBtihVs-aoHpfApHr-RoAxd-iRRmPda.RteCPloyen2xflpuMreeOnstI-
250- 150- 215500-- ofwildtypeIBV(wt)andrIBV-HA-RdRP(HA),re-
spectively. Cellsinfected with rIBV-HA-RdRPwere
150- 100- 100- -nsp12 harvestedat0,4,8,12,16and24hpost-infection,
respectively. As acontrol, cells infected with wild
100- -HA-RdRP 75- 75- typeIBVwereharvestedat16hpost-infection.Total
celllysateswereprepared,andproteinsweresepa-
50-
50- ratedon8%SDS-PAGEandanalyzedbyWesternblot
75-
37- with anti-HA antibodies. Numbers on the left in-
N 37-
dicateproteinsizesinkilodalton.b.Replicationof
50- rIBV-HA-RdRPinH1299cells.ConfluentH1299cells
wereinfectedwithapproximately2MOIofwildtype
WB: -IBVN
WB: -HA WB: -nsp12 IBV(wt)andrIBV-HA-RdRP(HA),respectively.The
infectedcellswereharvestedat8and24hpost-in-
fection,respectively.Totalcelllysateswereprepared,proteinswereseparatedon15%SDS-PAGEandanalyzedbyWesternblotwithanti-Nantisera.Numbersontheleftindicateprotein
sizesinkilodalton.c.ImmunoprecipitationofHA-nsp12inrIBV-HA-RdRP-infectedH1299cellswithHA-beads.ConfluentH1299cellswereinfectedwithapproximately2MOIofwild
typeIBV(wt)andrIBV-HA-RdRP(HA),respectively.Theinfectedcellswereharvestedat16hpost-infectionandtotalcelllysateswereprepared.Proteinsinthetotalcelllysateswere
eitherseparatedon15%SDS-PAGEdirectlyorsubjectedtoimmunopecipitationwithanti-HAbeadsbeforebeingloadedontothegel.AnalysisofHA-nsp12expressionandim-
munoprecipitationefficiencywasdonebyWesternblotwithanti-nsp12antisera.Numbersontheleftindicateproteinsizesinkilodalton.
were purified with phenol:chloroform:isoamyl alcohol (25:24:1, v/v) 2.7. ConstructionofIBVnsp12truncationmutants
andstoredat−80°C.
Allnsp12truncationmutantswerecreatedbyamplificationofthede-
siredfragmentfrompXL-IBVC,whichcontainedtheIBVgenomefromnu-
2.5. Biotinpull-downassay cleotide 8689–15526. Fragments amplified with primer pairs i12339-
12360_BamHIF (5'-TTGGATCCGAATTATTTAAACGGGTACGGG-3') / i135
Allproteinsusedinthisassaywerewholecelllysatespreparedfrom 38_13516_XhoIR (5'-TTCTCGAGTTATAACGCACAAACACTAAAACAAG-3'),
Vaccinia/T7-infected H1299 cells over-expressing N-terminal FLAG- i13296-13316_BamHIF (5'-TTGGATCCATACCGCAGACTTCTTTCGGT-3') /
epitope tagged proteins. EGFP over-expressed with the same method i14498-14474_XhoIF (5'-TTCTCGAGTTAGCTCTTAATATTATCATAGACAA
wasusedasanegativecontrol.Ina200µlbindingreaction,0.1µMof TA-3') and i13926-13948_BamHIF (5'-TTGGATCCAAGAAGAATGTCC
biotin-labeled RNA was mixed with 150µl of total cell lysate in the TACCCACTAT-3') / i15128-15106_XhoIR (5'-TTCTCGAGTTATTGTAAAG
presenceof10mMDTT,20µgofyeasttRNA,200UofRNaseinhibitor TCGTAGGAGCTCTAT-3') were cloned into vector pXJ40-Myc generating
and nuclease-free water. The reaction was incubated at room tem- plasmids that could express N-terminally tagged proteins Myc-iNsp12n,
peraturewithgentlerotationfor30min.40µlofstreptavidinagarose Myc-iNsp12m,Myc-iNsp12c.
beads (Sigma-Aldrich, USA) was added directly into the tube and the
mixture was incubated at room temperature with gentle rotation for 3. Result
30min,andthencentrifugedat1000rpmfor5min.500µlofRNaseP
(RP) buffer (50mM KCl, 1mM MgCl2, 10mM HEPES, pH 7.5) were 3.1. ConstructionandcharacterizationofarecombinantIBVexpressingan
usedtowashthebeadstriceandthebufferwasremovedthoroughlyby HA-epitopetaggednsp12
gentlyaspirationafterthelastwash.25µlof2XSDSloadingdyewith
DTTwasmixedwiththebeadsandthemixtureincubatedat100°Cfor A recombinant IBV which expressed an N-terminally HA-epitope
10min,cooledatroomtemperaturebeforeloadedontoaPAGEgelof tagged nsp12 (rIBV-HA-RdRP) was constructed using reverse genetics
anappropriateconcentration. (Fangetal.,2008,2007,2010).TheHAtagwasinsertedatnucleotide
position12319whichistwoaminoacidsdownstreamofthecleavage
site between nsp10 and nsp11/12. The recovered recombinant virus
2.6. ProteinelectrophoresisandWesternblotanalysis grewwellinH1299cells.
Approximately 2 MOI of wild type and the recombinant IBV, re-
Polyacrylamidegelswerepreparedwith30%acrylamide/bissolu- spectively,wereusedtoinfectconfluentH1299cells.Theinfectedcells
tion (29:1) in 375mM Tris and 0.1% SDS. The electrophoresis was were harvested at 24h post-infection and total cell lysates were pre-
performed with 1X Tris-Glycine running buffer (25mM Tris, 250mM pared.TheexpressionkineticsoftheHA-taggednsp12incellsinfected
glycine, 0.1% SDS). Resolved proteins were visualized by 30min of with rIBV-HA-RdRP was studied in a time-course experiment. H1299
coomassiebluestaining(0.25%(w/v)CoomassieBrilliantBlueR-250, cellsinfectedwiththeviruswereharvestedat0,4,8,12,16and24h
20%methanol,10%glacialaceticacid)ortransferredontoamembrane post-infection,respectively,andanalyzedbyWesternblotwithanti-HA
forimmune-detection.Gelswereequilibratedin1Xwettransferbuffer monoclonalantibodies.CellsinfectedwithwildtypeIBVharvestedat
(48mM Tris, 39mM glycine, 20% methanol) before transferred onto 16h post-infection were also included as a control. The HA-tagged
either nitrocellulose or PVDF membranes. Transfer was performed in nsp12wasinitiallydetectedat12hpost-infectionandpeakedat16h
1Xwettransferbufferat4°C.Membranewasblockedinblockingso- post-infection(Fig.1a).Asexpected,thesamebandwasnotobservedin
lution(10%(w/v)non-fatmilkin1XPBS)for1hatroomtemperature cellsinfectedwithwildtypeIBV(Fig.1a).
and incubated with the primary antibody at an appropriate con- Cells infected with wild type and the recombinant rIBV-HA-RdRP
centration for 1h. The membrane was washed with PBS-T (1X PBS, harvestedat8and24hpost-infectionwerealsoanalyzedbyWestern
0.1%Tween®-20)threetimesandincubatedwithsecondaryantibodyat blotfortheexpressionlevelofIBVNproteinwithanti-Nantisera.As
a1:2000dilutionfor1h.ThemembranewaswashedwithPBS-Tthree shown inFig. 1b,Nprotein expression wasobserved incells infected
times and Western Lightning®-ECL from Perkin Elmer (Waltham, MA, withbothwildtypeIBVandrIBV-HA-RdRP.Infact,slightlyhigherlevel
USA)wasappliedontothemembranefordetection. of N protein expression was detected in cells infected with rIBV-HA-
RdRP (Fig. 1b), indicating that tagging of RdRP with an HA epitope
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Y.W.Tanetal. Virology 513 (2018) 75–84
doesnotaffectIBVreplicationincells. weightbandswerelikelypartiallyprocessedproductsofpp1aorpp1ab,
TheexpressionofwildtypeandHA-taggednsp12intheseinfected whichincludednsp8 andnsp10,respectively, andthus weredetected
cells as well as the immunoprecipitation efficiency and specificity of by the antibodies. Many antisera used during this screening were un-
HA-tagged nsp12 with α-HA coated beads (HA-beads) were then de- abletodetecttheircorrespondingproteinbandsinthecelllysates,due
termined by Western blot with a rabbit polyclonal antiserum against to the low expression level of these proteins during IBV infection in
nsp12(α-RdRP).ThiswasdonetoensurethatHA-taggednsp12could additiontothelowlevelsofantibodiesinthesera.
be efficiently pulled-down for detection of other co-precipitated viral
andcellularproteins.AscanbeseeninFig.1c,acomparablelevelof 3.3. Furtherconfirmationoftheinteractionbetweennsp8andnsp12
wildtypeandHA-taggednsp12wasdetectedincellsinfectedwiththe
respectiveviruses,butonlyHA-nsp12wasprecipitatedbyHA-beads. To further confirm that nsp8 interacts with nsp12, im-
munoprecipitationofnsp12wasrepeatedwithHA-beads.Inaddition,a
3.2. Screenfornon-structuralproteinsthatinteractwithnsp12 reciprocalprecipitationwasperformedusinganti-nsp8serum.Thely-
sateswerepreparedandthebead-boundproteinswereelutedwith2X
Wethendecidedtoidentifyviralstructural,non-structuralandac- SDSloadingdyeandresolvedby8%and15%SDS-PAGEgels.Asshown
cessoryproteinsthatwereco-precipitatedwiththeHA-taggedRdRPby inFig.3A,HA-nsp12wasdetectedincellsinfectedwithrIBV-HA-RdRP
Western blot with available antisera. Confluent H1299 cells grown in afterprecipitationwithanti-nsp8serum.Similarly,precipitationofcell
80cm2 flasks were infected with wild-type and rIBV-HA-RdRP, re- lysates with anti-HA antibodies led to the co-precipitation of nsp8 in
spectively,andharvestedat16hpost-infectionbylysiswith800µlof cells infected with the recombinant virus (Fig. 3B). It was also noted
LysisBufferforeachflask.HA-beads(20µlof50%slurry)wereadded thatadominantbandwithanapparentmolecularweightof64kDawas
to 500µl of each lysate to precipitate HA-nsp12 and any other inter- detectedinbothtotalcelllysatesandinprecipitatedsamplesfromrIBV-
actingproteinsfromthelysates.Theboundproteinswereelutedwith HA-RdRP-infected cells(Fig.3B).This mayrepresentthecleavage in-
55µlof 2X SDS loading dye andresolved by8% and 15% SDS-PAGE termediate covering nsp7/8/9/10. Although the interaction between
gel.Becausesimilarnumbersofcellswereusedforinfectionandvirus nsp8 and nsp12 has been reported before for SARS-CoV (von Brunn
replication was comparable, the protein levels of host housekeeping etal.,2007),itwasnotreportedinothercoronavirusesandtheinter-
genesweresimilarincellsinfectedwithwild-typerIBVandrIBV-HA- actionhasnotbeensubjectedtoanin-depthstudy.Inthesubsequent
RdRP, as determined by Western blot analysis of GAPDH and β-actin sections,theinteractionbetweenthesetwoproteinswasfurtherchar-
(data not shown). As shown in Fig. 1c, HA-tagged nsp12 could be acterized.
specifically detected in the cell lysate and co-precipitated samples in
cells infected with rIBV-HA-RdRP but not in cells infected with wild- 3.4. Interactionbetweennsp8andnsp12intheabsenceofotherviral
type rIBV. Detection of co-precipitated proteins was performed by structuralandnon-structuralproteins
WesternblotusingavailableantiseraforIBVnspsaswellasstructural
proteins S and N. We noted that some antisera (especially α-nsp15) Thephysicalinteractionbetween nsp8andnsp12wasfurtherstu-
pickeduponeormorenon-specificbands.Onlyifthetargetproteinof diedbyco-expressionofthetwoproteinsintheabsenceofotherviral
correctmolecularweightwasdetectedatsimilarlevelsinthewholecell proteinsandviral genomic RNA.Theproteinswere co-expressedasa
lysates, but could only be detected in the co-immunoprecipitated FLAG-tagged protein for nsp8 (FLAG-nsp8) and a Myc-tagged protein
samplefromrIBV-HA-RdRP-infectedcells,itwouldbeconsideredtobe for nsp12 (Myc-nsp12) inH1299 cells. As controls, dual vector trans-
apotentialinteractingpartnerofHA-taggednsp12. fected samples (FLAG + Myc) and single vector transfected samples
Theresultsoftheco-immunoprecipitationwerepresentedinFig.2. (FLAG-nsp8 + Myc and FLAG + Myc-nsp12) were included.
For the structural proteins, S protein was not detected in the im- Immunoprecipitationwasperformedwithanti-Myccoatedbeads(Myc-
munoprecipitated samples, while a similar level of N was detected in beads) and the bound proteins were resolved by SDS-PAGE. Western
cellsinfectedwithwildtypeIBVandrIBV-HA-RdRP(HA)(Fig.2,pa- blot was then performed using anti-FLAG-HRP and anti-Myc-HRP an-
nels anti-IBV SandN).Although itappeared that theamountco-pre- tibodiestodetecttheprecipitatedproteins.AsshowninFig.4,FLAG-
cipitated in rIBV-HA-RdRP-infected samples was slightly higher com- nsp8 wasdetected atalmostequal amounts whetheritwasexpressed
paredtowildtypeIBVinfectedsamples,theamountdetectedinthecell togetherwithMyconly(FLAG-nsp8+Myc)orwithMyc-nsp12(FLAG-
lysateswasalsoslightlyhigher.SoitwasunlikelyforNtohavebeen nsp8+Myc-nsp12),butwasdetectedintheMycpulled-downsamples
specifically co-precipitated by an interaction with nsp12. Nsp3, nsp5, onlywhenitwasco-expressedwithMyc-nsp12(Fig.4,leftpanel).The
nsp8 and nsp14, but not nsp10, nsp13 and nsp15/nsp16, were speci- ~50kDabanddetectedinallimmunoprecipitatedsampleswaslikely
fically detected in the precipitated samples of rIBV-HA-RdRP-infected tobetheheavychainelutedfromthebeadsduringsamplepreparation
cells(Fig.2).Aspreviouslyreported,adominantbandwithanapparent for SDS-PAGE. Also, Myc-nsp12 was detected in both the cell lysates
molecularweightof58kDacouldbedetectedbyWesternblotincells andimmunoprecipitatedsamples(Fig.4,rightpanel).AsFLAG-nsp8in
infected with wild type IBV with anti-nsp14 antiserum (Fang et al., the immunoprecipitated sample was detected only when Myc-nsp12
2007). This was proved to be a nonspecific band from our previous wasco-expressed,itwasconcludedthatFLAG-nsp8wascoprecipitated
work(Xuetal.,2010).Asnsp14bandisusuallyveryweakandmigrates withMyc-nsp12byinteractingwithit.Theseresultsconfirmthatnsp12
onSDS-PAGEgeljustbelowthisnon-specificband,muchmoresamples couldinteractwithnsp8intheabsenceofotherviralproteinsandviral
were added during precipitation and gel loading, leading to the de- genomic/subgenomicRNAs.
tectionofaspecificbandrepresentingnsp14inHA-beadsprecipitated
samples(Fig.2).However,thisbandcannotbeseparatedfromthenon- 3.5. EffectofRNaseAtreatmentontheinteractionbetweennsp8andnsp12
specificbandintotalcelllysates(Fig.2).Anti-nsp13antibodiesfailed
todetectaspecificbandrepresentingnsp13eitherintotalcelllysatesor WethentestediftreatmentofcelllysateswithRNaseAwouldaffect
in HA-beads precipitated samples (Fig. 2). This issue will be further the interaction between the two proteins. For this purpose, im-
studiedbyusingarecombinantviruswithHA-taggednsp13(Zhaietal., munoprecipitation was performed with cell lysates that were either
2005). treated(+RNaseA)ornottreated(-RNaseA)with50µg/mlofRNase
AdditionalhighermolecularweightbandswerealsodetectedinHA- A at 37°C for 1h before adding anti-Myc (Myc-beads) or anti-FLAG
beadsprecipitatedsampleswithanti-nsp8andanti-nsp10antisera,al- (FLAG-beads) coated beads. As shown in Fig. 5A, nsp8 was still pre-
thoughaspecificbandrepresentingthensp10wasnotdetected(Fig.2). cipitated whenRNAwasremoved,suggesting thattheinteraction be-
Based on their apparent molecular masses, these higher molecular tween the two proteins is not mediated through their simultaneous
78
Y.W.Tanetal. Virology 513 (2018) 75–84
CL IP: -HA CL IP: -HA CL IP: -HA CL IP: -HA CL IP: -HA
wt HA wt HA wt HA wt HA wt HA wt HA wt HA wt HA wt HA wt HA
250- 250- 250- 82.2- 82.2-
150- 64.2- -7/8/9/10 64.2-
-nsp3 48.8- 48.8-
150- 150- 100- 37.1- 37.1- -9/10/11
-7/8(8/9)
100- 100- 75- 25.9- -nsp8 25.9- -9/10
50-
75- 19.4- 19.4-
75- -nsp2
37- -nsp10
-nsp5 14.8- 14.8-
50-
50- 25-
37- 6.0- 6.0-
WB: -nsp2 WB: -nsp3 WB: -nsp5 WB: -nsp8 WB: -nsp10
CL IP: -HA CL IP: -HA CL IP: -HA CL IP: -HA CL IP: -HA
wt HA wt HA wt HA wt HA wt HA wt HA wt HA wt HA wt HA wt HA
250-
250- 150- 250- 250- 250-
150- 150-
150- 100- 100- 100- 150-
S
75- 75- 75- 100-
100- --nnss*p14
75-
75- 50- 50- 50-
-(nsp13) 37- N
50-
50- -nsp15 37-
37-
25- -nsp16 37-
WB: -nsp13 WB: -nsp14 WB: -nsp15 WB: -IBV N WB: -IBV S
Fig.2.Co-precipitationIBVnon-structuralproteinswithHA-nsp12inrIBV-HA-RdRP-infectedH1299cells.ConfluentH1299cellswereinfectedwithapproximately2MOIofwildtype
IBV(wt)andrIBV-HA-RdRP(HA),respectively.Theinfectedcellswereharvestedat16hpost-infectionandtotalcelllysateswereprepared.Proteinsinthetotalcelllysateswereeither
separatedon8%and15%SDS-PAGEdirectlyorsubjectedtoimmunopecipitationwithanti-HAbeadsbeforebeingloadedontothegel.IBVproteinsexpressedintheinfectedcellsandco-
precipitatedwithHA-nsp12wereanalyzedbyWesternblotwithantiseraspecifiedundereachpanel.Antiserawhichwereusedbutnotpresentedwereforthefollowingproteins:nsp7,
ORF3a,ORF3b,ORF5aandE.Arepresentativeresultoftwoindependentexperimentswasshown.Numbersontheleftindicateproteinsizesinkilodalton,andtheidentitiesoftheIBV
proteinsareindicatedontheright.
A B Fig.3.CoprecipitationofIBVnsp8withHA-nsp12
Vero H1299 Vero H1299 and vice versa in both Vero and H1299 cells. a.
ConfluentH1299andVerocellswereinfectedwith
CL IP: -nsp8 CL CL IP: -HA CL approximately2MOIofwildtypeIBV(wt)andrIBV-
wt HA wt HA wt HA wt HA wt HA wt HA wt HA wt HA HA-RdRP(HA),respectively.Theinfectedcellswere
115.5- harvestedat16hpost-infectionandtotalcelllysates
181.8- 64.2- -7/8/9/10 wereprepared.Proteinsinthetotalcelllysateswere
48.8- eitherseparatedon8%SDS-PAGEdirectlyorsub-
115.5- -HA-nsp12 jected to immunopecipitation with anti-nsp8 anti-
25.9- -nsp8
82.2- serumbeforebeingloadedontothegel.Analysisof
the expression of HA-nsp12 and the im-
64.2- 19.4- munoprecipitation efficiencywasdonebyWestern
-HC
48.8- blot with anti-HA antibodies. Numbers on the left
indicate protein sizes in kilodalton. b. Confluent
37.1- H1299andVero cellswereinfectedwithapproxi-
14.8-
mately2MOIofwildtypeIBV(wt)andrIBV-HA-
-LC RdRP (HA), respectively. The infected cells were
harvestedat16hpost-infectionandtotalcelllysates
WB: -HA WB: -nsp8 wereprepared.Proteinsinthetotalcelllysateswere
eitherseparatedon15%SDS-PAGEdirectlyorsub-
jectedtoimmunopecipitationwithanti-HAbeadsbeforebeingloadedontothegel.Analysisoftheexpressionofnsp8andtheimmunoprecipitationefficiencywasdonebyWesternblot
withanti-nsp8antibodies.Numbersontheleftindicateproteinsizesinkilodalton.
interactions with RNA. Similar result was also obtained in the re- subgenomicviralRNA.
ciprocalco-immunoprecipitationusinganti-FLAGantibody(Fig.5B).A
lowlevelofMyc-nsp12wasdetectedinthewholecelllysate,duetothe
3.6. Nsp8interactswiththeN-andC-terminalregionsofnsp12
loadingofthesmallamountofcelllysateasinput,ascomparedwith
the large amount of cell lysate used for co-immunoprecipitation. The
two reciprocal co-immunoprecipitation experiments thus confirmed Wethensetuptomapthedomain(s)onnsp12responsibleforthe
interaction between the twoproteins. ThreeMyc-tagged deletion mu-
thatnsp8interactsdirectlywithnsp12andfurthersupportedthatthis
tantsofnsp12werecreated,asshowninFig.6a.Becausethetruncated
interaction is not dependent on the presence of the genomic and
mutantsencodepeptidefragmentswithupto400aminoacidresidues,
79
Y.W.Tanetal. Virology 513 (2018) 75–84
A B Fig. 4.Formation of FLAG-nsp8 and Myc-nsp12
IP:α-Myc IP:α-Myc
complex in cells over-expressing the two epitope-
12 12 12 12 12 12 12 12 tagged proteins. H1299 cells were grown to 80%
Myc Myc M-nsp M-nsp Myc Myc M-nsp M-nsp Myc Myc M-nsp M-nsp Myc Myc M-nsp M-nsp ccoonmflbuinenancytviarnudsfoirnf1echteadt3w7i°tCh,5V%acCciOn2ia./PTl7asmried-
181.8 FLAG+ F-nsp8+ FLAG+ F-nsp8+ FLAG+ F-nsp8+ FLAG+ -8+Fnsp 181.8 FLAG+ F-nsp8+ FLAG+ F-nsp8+ FLAG+ F-nsp8+ FLAG+ F-nsp8+ DEc3u7ffNbe°AaCctteewidnnaecws®ubiTtdahretaolnitrvhsefeberecetftdroiaornneisnfRtteohceetaiytgohenwentem.irneiTxfehlcyeftoseecrdde2ll0wcseihwltlhseirnLewytiishtnihes-
115.5 115.5 -M-nsp12 buffer. Total cell lysates were either separated on
82.2 82.2 15% SDS-PAGE directly or subjected to im-
64.2 64.2
48.8 48.8 munopecipitationwithanti-Mycbeadsbeforebeing
37.1 37.1 loadedontothegel.Analysisoftheexpressionofthe
over-expressed proteins and their co-im-
25.9 -F-nsp8 25.9 munoprecipitation was done by Western blot with
19.4 19.4 anti-FLAG(A)andanti-Myc(B)antibodies,respec-
tively.Numbersontheleftindicateproteinsizesin
14.8 14.8 kilodalton.
WB:α-FLAG WB:α-Myc
we hypothesized that critical domains and/or protein folding motifs itsmiddleregion.TheabsenceofMyc-taggedproteinco-precipitation
wouldremainintact,andthusthestructuralfeaturesandstabilityofthe insampleswhichwereco-expressedwithFLAG-epitope,indicatedthat
mutants would not be drastically affected. The three mutants encode theco-precipitationofMyc-nsp12nandMyc-nsp12cweredependenton
IBV nsp12 fragments with considerable overlaps, to ensure that po- thepresenceandtheirinteractionwithFLAG-nsp8,aswasthecasefor
tential nsp8 interacting domain(s) disrupted by the margin of a frag- Myc-nsp12.
mentwouldstillremainintactinanothermutant.
The three mutants and full-length nsp12 (Myc-nsp12) were either
co-expressedwithFLAG-nsp8.Thecelllysateswereprobedtoassessthe 3.7. Biotinpull-downscreenforRNA-bindingactivityofIBVnon-structural
expression levels of the different nsp12 mutants. As shown in Fig. 6b proteins
(left),full-lengthMyc-nsp12wasdetectedathighlevelsintransfected
cells.ProteinlevelsofMyc-nsp12nandMyc-nsp12cwereslightlylower, PlasmidsbasedonthevectorpXJ40-FLAGinsertedwithnucleotide
whilethatofMyc-nsp12mwasconsiderablylowercomparedwiththe sequencesofnspseitherintandem(nsp7/8fusionandnsp8/9fusion)
full-length protein, suggesting that the deletion mutants were not as orindividually were usedto over-express theproteins inVaccinia/T7
stableasthefull-lengthprotein.Thecelllysateswerealsoprecipitated virus-infectedH1299cells.Duetoeithertheverylowexpressioneffi-
withFLAG-beadsandtheboundproteinswereresolvedbySDS-PAGE ciencyorthelack ofdistinctive band,thescreendatafornsp3,nsp4,
anddetectionwasmadewithanti-Myc-HRPantibody.Asexpected,the nsp6andnsp13werenotincluded.Totalcelllysateswereusedinbiotin
full-lengthMyc-nsp12interactedstronglywithFLAG-nsp8andwasef- pull-down assays with biotinylated 5′-UTR (+), 5′-UTR (-) or 3′-UTR
ficientlyco-precipitated.Ontheotherhand,thelevelofco-precipitated (+)RNAprobes.AsthefocusofthestudywasonnegativestrandRNA
protein was lower for Myc-nsp12n and considerably lower for Myc- synthesis, only the positive sense UTRs and the 5′-UTR (-), which
nsp12c,comparedwiththatoffull-lengthnsp12.Nodetectablelevelof contains the anti-leader TRS important for strand-transfer during dis-
Myc-nsp12m could be co-precipitated (Fig. 6b, right). Therefore, the continuous transcription of negative strands, were included in the
nsp8 interacting domain(s) in nsp12 was attributed mainly by the N- screen.Theproteinsboundtothestreptavidinbeadswereresolvedby
terminalregion,andtoalesserdegreebytheC-terminalregion,butnot SDS-PAGE and detected by Western blot using anti-FLAG-HRP anti-
body.EGFPwasalsoincludedasanon-bindingproteinnegativecontrol
A B Fig. 5.Co-precipitation of FLAG-nsp8 and Myc-
IP:α-Myc IP:α-FLAG
nsp12intotalcelllysateswithorwithoutRNaseA
Myc Myc M-nsp12 M-nsp12 Myc Myc M-nsp12 M-nsp12 Myc Myc M-nsp12 M-nsp12Myc Myc M-nsp12 M-nsp12 tflvriuereauntsmcfyeonratn.1dHhi1na2fte9c39te7dc°eCwl,lsit5h%wVerCaecOc2gin.rioPawl/aTns7mtiordec8Do0mN%bAincwoaannst-
+ + + + + + + + + + + ++ + + + delivered into the infected cells with Effectene®
FLAG F-nsp8 FLAG F-nsp8 FLAG F-nsp8 FLAG F-nsp8 FLAG F-nsp8 FLAG F-nsp8FLAG F-nsp8 FLAG F-nsp8 TthreantsrfaencstfieocntiRoneamgeinxt.foTrh2e0cehllisnwtheree3i7nc°uCbiantceudbwatiothr
181.8 181.8 beforetheywerelysedwithLysisbuffer.Aportionof
115.5
82.2 115.5 -M-nsp12 thelysateswastreatedwith50µg/mlofRNaseAat
64.2 82.2 37°Cfor1h.Bothtreatedanduntreatedcelllysates
48.8
+RNaseA 37.1 were either separated on 8% and 15% SDS-PAGE
25.9 -F-nsp8 +RNaseA64.2 directly or subjected to immunopecipitation with
19.4 48.8 -HC anti-Myc(A)andanti-FLAG(B)beads,respectively
before being loaded onto the gel. Analysis of the
14.8 37.1 expressionoftheover-expressedproteinsandtheir
181.8 co-immunoprecipitation was done by Westernblot
11852..52 115.5 -M-nsp12 withanti-FLAG(A)andanti-Myc(B)antibodies,re-
4648..28 82.2 spectively.Numbersontheleftindicateproteinsizes
37.1 64.2 inkilodalton.
-RNaseA 25.9 -F-nsp8 -RNaseA -HC
19.4 48.8
14.8 37.1 -LC
WB:α-FLAG WB:α-Myc
80
Y.W.Tanetal. Virology 513 (2018) 75–84
A Myc Fig.6.Mappingtheregionsonnsp12responsibleforinteraction
Myc-nsp12 withnsp8.a.SchematicdiagramofMyc-taggednsp12truncation
1 940 mutantproteins.Numeralsindicateaminoacidpositions.b.The
Myc-nsp12n N- and C-terminal portions of IBV nsp12 co-precipitated with
1 400 FLAG-nsp8 using FLAG-beads. Full-length and truncated nsp12
proteinsorMyc(vectorcontrol) wereco-expressedwithFLAG
Myc-nsp12m
328 729 (vectorcontrol)orFLAG-nsp8.Thecelllysateswereprecipitated
withFLAG-beadsandboundproteinswereanalyzedwithwestern
Myc-nsp12c blotting using α-Myc. Full-length nsp12 and mutants nsp12n
538 938
(residues 9 – 400), nsp12c (residues 538 – 938) were co-pre-
cipitated by the beads. A representative result of three in-
dependentexperimentswasshown.
B lysate IP:α-FLAG
Myc-nsp12M M-nsp12n M-nsp12m M-nsp12cMyc M-nsp12 M-nsp12n M-nsp12m M-nsp12c Myc-Mnsp12 M-nsp12n M-nsp12m Mnsp-12cMyc M-nsp12 M-nsp12n M-nsp12m M-nsp12c
++ + + ++ + + + + ++ + + ++ + + + +
FLAGFLAG FLAG FLAG FLAG-Fnsp8 F-nsp8 F-nsp8 F-nsp8 F-nsp8 FLAGFLAG FLAG FLAG FLAGF-nsp8 F-nsp8 F-nsp8 F8-nsp F-nsp8
181.8
115.5 -M-nsp12
82.2
64.2
48.8 -M-nsp12n
M-nsp12c
37.1
25.9
19.4
WB: -Myc
ineachscreen.ForthescreenconductedusingbiotinylatedIBV5′-UTR hamperedbythedifficultyofexpressing/detectingcertainnsps,suchas
(+), individual nsps, two fusion proteins, nsp7/8 and nsp8/9, and nsp4andnsp13,aswellastheinsolubilityofnsp6,aproteincontaining
EGFP were over-expressed in H1299 cells infected with Vaccinia/T7 multipletransmembranedomains(Oostraetal.,2008).
recombinant virus on 60mm dishes. Cells were lysed with 250µl of One previously undocumented interaction between nsp2 and viral
Lysis Buffer and used immediately. Biotin pull-down assay was per- RNA was founded by the screen. This finding adhered to a previous
formedwith0.1µM(finalconcentration)biotinylatedIBV5′-UTR(+) reportofnsp2possiblybeingaweakantagonistofPKR(X.Wangetal.,
probewith150µlcelllysate,andstreptavidinbeadswereusedtopurify 2009),whichrequiresdouble-strandedRNAforactivation.Thescreen
the biotinylated RNA from the mixture. Proteins bound to the beads alsoshowed that nsp9 andnsp10 only bindto some butnot allthree
through interactions with the bound RNA were eluted with 2X SDS probes tested. This is unexpected as both proteins were previously
loadingdye.ForWesternblotanalysis,10µlofcelllysateandallofthe documentedtohaveRNA-bindingactivitieswithanon-specificnature
elutedproteinsweredenaturedandresolvedbySDS-PAGE.Detectionof (B. Chen et al., 2009; Egloff et al., 2004; Joseph et al., 2006). This
theFLAG-taggednspsandEGFPwereperformedwithanti-FLAG-HRP could, however, be explained by the generally weak interactions of
and anti-EGFP antibodies, respectively. As shown in Fig. 7a, proteins thesetwoproteinsandtheRNAprobesthatcouldescapedetectionin
that were able to co-purify with the biotinylated probe in the assay oneormoreofthescreens.Theinteractionbetweennsp8andIBV3′-
werensp2,nsp10andtoalesserextent,nsp5. UTR (+) may have provided another piece of evidence that nsp8 is
Similarbiotinpull-downassayswithbiotinylated5′-UTR(-)and3′- heavilyinvolvedinviralRNAsynthesis,asaRNA-primersynthesizing
UTR (+) probes were performed to screen for interacting proteins. enzymefornsp12.Itshouldalsobenotedthattheobserveddifferential
Proteinswhichco-purifiedwiththebiotinylated5′-UTR(-)RNAprobe interactions of these nsps with UTRs in cells overexpressing these
werensp2,nsp5andnsp10(Fig.7b).Nsp2,nsp5,nsp8andnsp9were proteinsmaybeduetothefactthattheamountsoftheover-expressed
shown to be interacting with the biotinylated 3′-UTR (+) probe nsps may be likely rather different from those expressed in virus-in-
(Fig.7c).Amongthem,nsp5showedstronginteraction,nsp2,nsp8and fectedcells.
nsp9showedamoderatelevelofinteractionwithIBV3′-UTR(Fig.7c). Attempts to depict a more complete map of viral protein-protein
interactionswerealsohamperedbythelimitedavailabilityofsensitive
antiseratosomeviralproteins,especiallythoseofthelessimmunogenic
4. Discussion nspsandaccessoryproteins.Compoundedbythefactthatmanynon-
structural and accessory proteins were expressed in much lower
Inthisstudy,wehaveshownthatIBVnsp12isabletocomplexwith amountscomparedtostructuralproteins,theirdetectionincelllysates
anumberofothernon-structuralproteins,includingnsp3,nsp5,nsp8 provedtobemuchmoredifficult.Outofthesixnspswhichcouldbe
and nsp14. Further characterization of the nsp8-nsp12 complex de- detected by the available antisera, nsp3, nsp5, nsp8 and nsp14 were
monstratedthattheformationofthiscomplexisindependentofthe5′- identified as binding partners to nsp12. Nsp8-nsp12 interaction was
and3′-UTRsofviralRNAandothernsps.Nsp8interactswithnsp12at reportedinanORFeomescreenforSARS-CoV(vonBrunnetal.,2007).
twopoints,boththeN-andC-terminialregionsoftheprotein.Analysis Sinceithasnotbeenproventoexistinothercoronaviruses,reporting
of viral RNA-protein interactions using thebiotin pull-down assay re- thesameinteractioninIBVsuggeststhatitishighlyprobablethatthe
vealedthatnsp2,nsp8,nsp9andnsp10exhibitRNA-bindingactivityto interactionholdstrueforallothercoronaviruses.
eitheroftheUTRs.Theeffortstodrawamorecompletepicturewere
81
Y.W.Tanetal. Virology 513 (2018) 75–84
A
EGFP nsp2 nsp5 nsp7 nsp7/8 nsp8/9 nsp10 nsp12 nsp14 nsp15 nsp16 etal.,2012).Thensp7-nsp8hexadecamericcomplexwhichwasshown
250 C E C E C E C E C E C E C E C E C E C E C E tobethesecondRdRPofcoronavirusrequiringnoprimertofunction
115000 andsynthesizingshortlengthRNAnon-specificallyfulfilledtheroleas
75
50 theprimasefornsp12(Imbertetal.,2006).
37 +)
R( Alikely,althoughpremature,modelthatwouldfitthemappedin-
221505 5-UT’ tteerramcitnioanlsrewgioounlsdoifndniscpa1t2e awidthiretchteinnstper7a-cntsipo8ncboemtwpeleexn.tIhnethNis- manoddeCl-,
thensp7-nsp8complexwouldfirstsynthesizeaRNA-primerofasuffi-
B EGFP nsp2 nsp5 nsp7 nsp8 nsp9 nsp10 nsp12 nsp14 nsp15 nsp16 cient length, which would be exposed to and binds to the N-terminal
C E C E C E C E C E C E C E C E C E C E C E regionofnsp12,broughtincloseproximitythroughitsinteractionwith
125500 nsp8.TheextensionoftheRNAchaincouldthenbecatalyzed bythe
10705 polymerase domain in the C-terminal region of nsp12, which would
5307 R-)( also be oriented in close proximity to the end of the RNA-primer
25 UT through its interactions at its N-terminal region with nsp8. This ar-
20 5’- rangement of the two RdRPs would ensure the high efficiency of the
15
replicase complex as the RNA primers synthesized by nsp8 could be
utilizedbynsp12immediatelyfortranscriptionelongation.Thisputa-
C
EGFP nsp2 nsp5 nsp7 nsp8 nsp9 nsp10 nsp12 nsp14 nsp15 nsp16 tivemodelissupportedbystructuralstudiesshowingthatnsp8isavery
C E C E C E C E C E C E C E C E C E C E C E elongatedmolecule(Xiaoetal.,2012),itcouldbeeasilyspanningthe
64.2 N-andtheC-terminalregionsofnsp12.Furthercharacterizationofthe
48.8
3275..19 R(+) ipnrtoeproascetidonmobdeetwliesevnalinds.pI8naadndditinosnp,1i2deinstirfiecqautiiroendantodcahscaerartcatienriziaftitohne
T
19.4 U oftheinteractionsbetweennsp12andothernspsaswellastheinter-
14.8 3’- actionsamongothernspswouldberequiredtodepictamorecomplete
6.0
pictureoftheviralreplication/transcription complex.Attemptstoco-
Fig.7.ScreenforIBVnon-structuralproteinsthatinteractwiththe5′-UTR(+),5′-UTR expressallnspsinthesamecellsweremade,butnopresentabledata
(-)and3′-UTR(+)ofIBV.a.IBVnsp2,nsp5andnsp10showedbindingactivitytoits5′- weregenerated,possiblyduetothelowexpressionefficiencyofseveral
UTR(+).Biotinpull-downassayofnon-structuralproteinswithIBV5′-UTR(+).Cell nspsandtheevenlowerexpressionefficiencywhenco-expressingthem.
lysatesofH1299cellsover-expressingdifferentnon-structuralproteinswereincubated Structural information, especially the resolution of the nsp12
withbiotinylated5′-UTR(+)probesandpurifiedusingstreptavidinbeads.Boundpro- structurewouldbeofparticularimportanceinconfirmingiftheinter-
teinswereresolvedbySDS-PAGEandwesternblotwasperformed.Proteinswhichdidnot
actions detected between its N- and C-terminal regions and nsp8 are
expresswerenotpresented.C:celllysate,E:elution.Arepresentativeresultoftwoin-
spatiallypossible.However,asthestructuresofcoronavirusnsp12are
dependentexperimentswasshown.b.IBVnsp5andnsp10showedbindingactivitytoits
5′-UTR(-).Biotinpull-downassayofnon-structuralproteinswithIBV5′-UTR(-).Cell notsolvedtilldate,onlypredictionsofitsstructureareavailable.This
lysatesofH1299cellsover-expressingdifferentnon-structuralproteinswereincubated wouldbeoneofthemostimportantstudiesrequiredforelucidationof
withbiotinylated5′-UTR(-)probesandpurifiedusingstreptavidinbeads.Boundproteins thecooperationbetweennsp8andnsp12.Likewise,theinteractionsite
wereresolvedbySDS-PAGEandwesternblotwasperformed.Theexpressionofnsp2was onnsp8shouldalsobemappedasitsinteractionwithnsp12wouldonly
notdetectableforthisassayandwasexcluded.C:celllysate,E:elution.Arepresentative be meaningful if it is confined to the exterior of the hexadecameric
resultoftwoindependentexperimentswasshown.c.IBVnsp5,nsp8andnsp9showed complex.Subissietal.haveidentifiedP183andR190ontheSARS-CoV
bindingactivitytoIBV3′-UTR(+).Biotinpull-downassayofnon-structuralproteinswith
IBV 3′-UTR (+). Cell lysates of H1299 cells over-expressing different non-structural nsp8 to be essential for its interaction with SARS-CoV nsp12 (Subissi
proteinswereincubatedwithbiotinylated3′-UTR(+)probesandpurifiedusingstrep- et al., 2014). It would be interesting to examine whether the corre-
tavidinbeads.BoundproteinswereresolvedbySDS-PAGEandwesternblotwasper- sponding residues in IBV nsp8 is also required for nsp12-binding. In
formed.C:celllysate,E:elution.Arepresentativeresultoftwoindependentexperiments addition,functionalstudiesbyintroducingmutationsattheinteraction
wasshown. sites could be performed to ascertain if the interaction between nsp8
andnsp12iscrucialfortheprocessivityofthereplicasecomplex.
Thensp8bindingactivityinnsp12appearedtobepresentinboth Coronavirus nsp9 was shown to be a weak, non-specific single-
Myc-nsp12n and Myc-nsp12c, its N- and C-terminally truncated mu- stranded nucleic acid binding protein (ssDNA and ssRNA) (B. Chen
tants. Coincidentally, it was reported for SARS-CoV that both a p12 etal.,2009;Egloffetal.,2004).Itwasinterestingthatnsp9wasfound
fragmentinitsN-terminalandap64fragmentinitsC-terminalregions tointeractweaklyonlywithIBV3′-UTR(+)andnotatallwiththe5′-
wererequiredfortheRdRPactivityofnsp12(Chengetal.,2005).The UTRinbothpolarity. There isstillapossibility ofnsp9 beingable to
p12andp64fragmentswereshowntoformastablecomplexandthe bind particular regions of the coronavirus genome in a sequence spe-
p12fragmentmayplayaroleinprimer-bindingwhilethep64fragment cificmanner,and3′-UTR(+)couldhavebeenitsspecificinteraction
contains the polymerase activity. Therefore, the ability of nsp8 to in- partner.Conversely,thelowaffinitybindingcouldimplythattheweak
teract with Myc-nsp12n (residues 9–400) seemed to add a piece of affinitybindingtothebiotinylatedprobewasofanon-specificnature.
supportingevidencefortheroleofthep12fragmentinprimer-binding. Further studies would be required to verify the specificity of the in-
This offers an interesting perspective on the coordination between teraction.
coronavirusnsp8andnsp12inviralRNAsynthesis. Theactualstructureofnsp10iscontroversial.Inonestudy,nsp10is
Information pertaining to how the initiation of coronavirus nega- describedasadodecamer,butthesamestructurewasnotobservedin
tive-strand synthesis is limited and only in the recent years has more another study (Su et al., 2006). In fact, no dodecamer formation has
information been available with regards to the source of the primer beendescribedforthisproteininsolution(Josephetal.,2006).Nsp10
requiredforRdRP(nsp12)tobefunctioning(teVelthuisetal.,2010). isacofactorforboththe2′O-methyltransferaseactivityofnsp16,and
ThestructureofSARS-CoVnsp7-nsp8complexhasbeensolvedbytwo theN7-guanine-methyltransferase/exoribonucleaseactivitiesofnsp14
groups(Zhaietal.,2005;Xiaoetal.,2012),revealingahexadecameric (Decroly et al., 2008; Bouvet et al., 2012). As a stimulatory factor,
8:8 complex between the two proteins, and a 2:1 heterotrimer of the nsp10mayenhancethe2′-O-methyltransferaseactivityofnsp16which
twoproteinsinthecrystalaswellasinsolution,respectively.denovo catalyzestheconversionofthecap-0structureonm7GpppA-RNAtoa
RNAsynthesishasalsobeendemonstratedfornsp7andnsp8fromboth cap-1structure(Decrolyetal.,2008).Nsp10wasalsoshowntoexhibit
SARS-CoV andfelinecoronavirus(FeCoV)(Xiaoetal.,2012;Velthuis low-affinity binding to ssRNA, dsRNA and dsDNA in the absence of
82
Y.W.Tanetal. Virology 513 (2018) 75–84
other viral proteins (Joseph et al., 2006). Hence, in a case similar to Fang,S.G.,Shen,H.,Wang,J.,Tay,F.P.L.,Liu,D.X.,2008.Proteolyticprocessingof
nsp9,thebindingofnsp10toIBV5′-UTR(-)couldalsobeoflow-spe- polyproteins1aand1abbetweennon-structuralproteins10and11/12of
Coronavirusinfectiousbronchitisvirusisdispensableforviralreplicationincultured
cificity. cells.Virology379(2),175–180.
Nsp2wasfoundtointeractwithIBV5′-UTR(+)anditcouldnotbe Imbert,I.,Guillemot,J.,Bourhis,J.,Bussetta,C.,Coutard,B.,Egloff,M.,etal.,2006.A
concludedifitboundtoIBV5′-UTR(-)and3′-UTR(+).Althoughnsp2 second,non‐canonicalRNA‐dependentRNApolymeraseinSARSCoronavirus.EMBO
J.25(20),4933–4942.
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