2016 p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PL_ _sup

2016·1.79 MB·English

p53 down-regulates SARS coronavirus replication and
pro
is targeted by the SARS-unique domain and PL via
E3 ubiquitin ligase RCHY1
YueMa-Lauera,b,JavierCarbajo-Lozoyab,MarcoY.Heinc,1,MarcelA.Müllerd,WenDenge,JianLeia,BenjaminMeyerd,
YuriKusova,BrigittevonBrunnb,DevRajBairadb,SabineHüntenf,ChristianDrostend,HeikoHermekingf,
HeinrichLeonhardte,MatthiasMannc,RolfHilgenfelda,andAlbrechtvonBrunnb,2
aInstituteofBiochemistry,CenterforStructuralandCellBiologyinMedicine,UniversityofLübeckandGermanCenterforInfectionResearch,partnersite
Luebeck,23538Luebeck,Germany;bMax-von-PettenkoferInstitute,Ludwig-Maximilians-UniversityMunichandGermanCenterforInfectionResearch
(DZIF),partnersiteMunich,80336Munich,Germany;cDepartmentofProteomicsandSignalTransduction,Max-PlanckInstituteofBiochemistry,82152
Martinsried,Germany;dInstituteofVirology,UniversityofBonnMedicalCentreandGermanCenterforInfectionResearch,partnersiteBonn,53127Bonn,
Germany;eDepartmentofBiologyandCenterforIntegratedProteinScience,Ludwig-Maximilians-UniversityMunich,82152Planegg-Martinsried,
Germany;andfExperimentalandMolecularPathology,InstituteofPathology,Ludwig-Maximilians-UniversityMunich,80337Munich,Germany
EditedbyKennethI.Berns,UniversityofFloridaCollegeofMedicine,Gainesville,FL,andapprovedJune27,2016(receivedforreviewMarch15,2016)
Highly pathogenic severe acute respiratory syndrome coronavirus (Y2H) methodologies to screen for important virus–host protein–
(SARS-CoV) has developed strategies to inhibit host immune proteininteractions(PPIs),weidentifiedring-fingerandCHY
recognition.WeidentifycellularE3ubiquitinligasering-fingerandCHY zinc-fingerdomain-containing1(RCHY1)andcalcium/calmodulin-
zinc-fingerdomain-containing1(RCHY1)asaninteractingpartnerof dependentproteinkinaseIIdelta(CAMK2D)astwointeracting
theviralSARS-uniquedomain(SUD)andpapain-likeprotease(PLpro), partners of the SARS-unique domain (SUD), which is part of
and,asaconsequence,theinvolvementofcellularp53asantagonistof SARS-CoVnonstructuralprotein3(Nsp3).Containingvarious
coronaviralreplication.Residues95–144ofRCHY1and389–652ofSUD subdomains [ubiquitin-like (Ubl) globular fold, acidic domain,
(SUD-NM)subdomainsarecrucialforinteraction.AssociationwithSUD catalyticallyactiveADPribose-1′′-phosphatase(X-domain),SUD,
increasesthestabilityofRCHY1andaugmentsRCHY1-mediatedubiq- Ubl, catalytically active papain-like protease (PLpro) domain,
uitinationaswellasdegradationofp53.Thecalcium/calmodulin- nucleicacid-bindingdomain,G2Mmarkerdomain,twopredicted
dependent protein kinase II delta (CAMK2D), which normally double-pass transmembrane domains, a putative metal binding
influencesRCHY1stabilitybyphosphorylation,alsobindstoSUD.In region,Ydomainofunknownfunction],Nsp3proteinrepresents
vivophosphorylationshowsthatSUDdoesnotregulatephosphory- thelargestNspofSARS-CoV(5)andplaysanessentialrolefor
lationofRCHY1viaCAMK2D.SimilarlytoSUD,thePLprosfromSARS- theformationofviralreplicationcomplexes.Twomacrodomains
CoV,MERS-CoV,andHCoV-NL63physicallyinteractwithandstabilize ofSUD(SUD-Nand,inparticular,SUD-M)havebeenshownto
RCHY1,andthustriggerdegradationofendogenousp53.TheSARS- bind oligo(G) nucleotides (both deoxynucleotides and ribonu-
CoVpapain-likeproteaseisencodednexttoSUDwithinnonstructural cleotides)thatareabletoformG-quadruplexes(6).Interestingly,
protein3.ASUD–PLprofusioninteractswithRCHY1moreintensively
andcausesstrongerp53degradationthanSARS-CoVPLproalone.We Significance
showthatp53inhibitsreplicationofinfectiousSARS-CoVaswellasof
repliconsandhumancoronavirusNL63.Hence,humancoronaviruses
Severeacuterespiratorysyndromecoronavirus(SARS-CoV)isone
antagonizetheviralinhibitorp53viastabilizingRCHY1andpromot-
ofthemostpathogenichumancoronaviruses.Virulenceisreflec-
ingRCHY1-mediatedp53degradation.SUDfunctionsasanenhancer
tedinthemolecularinterplaybetweenvirusandhostcells.Here
tostrengtheninteractionbetweenRCHY1andnonstructuralprotein3,
we show a strategy of how SARS-CoV antagonizes the host
leadingtoafurtherincreaseininp53degradation.Thesignificanceof
antiviralfactorp53,whichimpairsviralreplication.Thepapain-like
these findings is that down-regulation of p53 as a major player in
protease of the nonstructural protein 3 of SARS-CoV and other
antiviralinnateimmunityprovidesalong-soughtexplanationfor
coronaviruses physically interact with and stabilize E3 ubiquitin
delayedactivitiesofrespectivegenes. ligase ring-finger and CHY zinc-finger domain-containing 1
(RCHY1), thereby augmenting RCHY1-mediated degradation of
| | |
p53antiviralactivity SARS-CoVSUD papain-likeprotease p53.TheSARS-uniquedomain(SUD)enhancestheseeffects.
|
E3ubiquitinligaseRCHY1 coronavirusreplication Knockoutofp53promotesreplicationofSARS-CoVrepliconsand
of infectious virus. Taken together we identify cellular p53 as
The global outbreak of severe acute respiratory syndrome antiviralmeasureofcoronavirus-infectedcells,whichiscounter-
coronavirus (SARS-CoV) in 2003 caused 774 deaths among actedviathestabilizationofRCHY1byviralSUDandpapain-like
8,098 infected individuals, although human coronaviruses (HCoVs) protease(PLpro)proteinsandviaubiquitinationofp53.
had always been regarded relatively harmless until then (1, 2). To
date, six coronaviruses (CoVs) are known to infect humans; Authorcontributions:Y.M.-L.,J.C.-L.,M.Y.H.,M.A.M.,andA.v.B.designedresearch;Y.M.-L.,
theseareHCoV-229EandHCoV-NL63belongingtothegenus J.C.-L.,M.Y.H.,M.A.M.,W.D.,J.L.,B.M.,B.v.B.,andD.R.B.performedresearch;J.L.,B.M.,Y.K.,
AlphacoronavirusaswellasSARS-CoV,MiddleEastrespiratory S.H.,C.D.,H.H.,H.L.,M.M.,andR.H.contributednewreagents/analytictools;Y.M.-L.,J.C.-L.,
M.Y.H.,M.A.M.,W.D.,J.L.,B.v.B.,D.R.B.,H.L.,andA.v.B.analyzeddata;andY.M.-L.andA.v.B.
syndromeCoV(MERS-CoV),HCoV-HKU1,andHCoV-OC43of wrotethepaper.
theBetacoronavirusgenus.MERS-CoVisanotherhighlypathogenic Theauthorsdeclarenoconflictofinterest.
coronavirus connected with an even higher case/fatality rate.
ThisarticleisaPNASDirectSubmission.
Despiteadecadeofresearchefforts,thereareneitherapproved
FreelyavailableonlinethroughthePNASopenaccessoption.
antiviraltreatmentseitherspecificforSARS-CoVorwithabroad-
1Presentaddress:CellularandMolecularPharmacology,UniversityofCalifornia,SanFrancisco,
spectrumprofileforallhumancoronaviruses,noranyvaccineavail- CA94158.
able (2–4). Therefore, it is necessary to further study coronavirus– 2Towhomcorrespondenceshouldbeaddressed.Email:[email protected].
host relations to discover new targets and signaling pathways for
Thisarticlecontainssupportinginformationonlineatwww.pnas.org/lookup/suppl/doi:10.
antiviral intervention. Applying high-throughput yeast-2-hybrid 1073/pnas.1603435113/-/DCSupplemental.
E5192–E5201 | PNAS | PublishedonlineAugust12,2016 www.pnas.org/cgi/doi/10.1073/pnas.1603435113
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amino acid residues of SUD-M that have been shown to be in- SUD–RCHY1interactions,identifyp53asanegativeregulatorof P
volvedinG-quadruplexbinding(6)arealsoessentialforthefunc- CoV replication, and characterize the RCHY1-dependent mecha-
tionofthedomaininSARS-CoVreplicationandtranscription(7). nismforSARS-CoVtoabolishp53antiviralactivity.
PLpro (corresponding to Nsp3 residues720–1039)is the C-terminal
neighbortotheSUD.PLproand3C-likeproteinase(3CLpro)process Results
the viral replicase polyproteins into 16 replicase proteins. Many SUDInteractswithRCHY1andCAMK2DinVivo.Studyingvirus–host
CoVs encode two Papain-like proteases (PLPs) [PLP1 (cleaving interactions, we have recently performed Y2H screens of a com-
Nsp1/Nsp2 and Nsp2/Nsp3), PLP2 (cleaving Nsp3/Nsp4)] within plete SARSCoV ORF collection (ORFeome) with human cDNA
Nsp3. SARS-CoV PLpro and most of other CoV PLpros display libraries(34).SeveralE3ubiquitinproteinligasesincludingRCHY1
deubiquitinating and deISGylating activities, thus acting as IFN were identified as interacting partners of SUD. Independently,
plasmidsexpressinganectopicGFP–SUDfusionweretransfected
antagonistsandcontributingtoevasionofinnateimmuneresponse.
intoHEK293cells.Togetherwithitshost-bindingpartners,the
RCHY1isanE3ubiquitinligasemediatingproteasomaldegra-
dationof itstarget proteins;itstargetsincludethetumorproteins GFP–SUD fusion protein was subsequently enriched with a
p53,p63,andp73(8–10).RCHY1regulatescell-cycleprogression GFP-trapconsistingofbeadscoatedwithanti-GFPantibodies.
andisinduciblebyp53(8,11);italsoformsahomodimerandhas Afterquantitativemass-spectrometryanalysisoftheproteinsbound
self-ubiquitination activity (12). RCHY1 is a short-lived protein. to the beads (35, 36), RCHY1 showed up again as an interacting
Inhibition of RCHY1 ubiquitination via interaction with measles partnerofSUD(Fig.1A–C).Inaddition,CAMK2Dwasalso
virusphosphoproteincanenhancethestabilityofRCHY1(13).In identified to bind to SUD in the same assay. RCHY1–SUD in-
addition,phosphorylationbycyclin-dependentkinase9(CDK9)or teractionwasfurtherconfirmedbyfluorescence-3-hybridassay(Fig.
CAMKIIcanalsoregulatethestabilityofRCHY1.Phosphorylation S1).Mostinterestingly,RCHY1isasubstrateofCAMKII(14).
byCAMKIIatThr154/Ser155increasesinstabilityofRCHY1and ToconfirmthatSUDalsobindstothosenovelpartnersinvivo,
impairstheE3ubiquitinligaseactivityofRCHY1towardp53(14). split-YFP protein–protein interaction assays were carried out in
CAMK2D belongs to the calcium/calmodulin-dependent serine/ humanlivingcells(HEK-293).SUDanditsinteractingcandidates
threonineproteinkinaseII(CAMKII)familyinvolvedinmanysig- were fused to the C-terminal (YFPC) and N-terminal (YFPN)
nalingpathways.CAMKIIisaholoenzymecomposedofCAMK2A/ YFPfragments,respectively.IfSUDbindstoatestedcandidatein
2B/2G/2Disoforms(15).Thesehaveverysimilarstructures,including thisassay,theN-terminalandC-terminalYFPfragmentswillas-
an N-terminal kinase domain, a central regulatory domain with a semble, leading to fluorescence emission (37). As a result, SUD
calmodulin-bindingregion,andaC-terminalassociationdomain(15– wasdemonstratedtobindtobothRCHY1andCAMK2Dinthe
17). Important substrates of CAMKII include RCHY1 and signal cytosolofHEK293livingcells(Fig.1DandE).
transducer and activator of transcription 1 (STAT1). CAMKII im- CAMKII isoforms can phosphorylate RCHY1 at T154/S155
pairstheE3ligaseactivityofRCHY1viaphosphorylation(14). in vitro (14), but a physical interaction between RCHY1 and
p53 regulates a plethora of target genes that mediate tumor CAMK2D has never been examined in vivo. For this reason, a
suppressionbyinducingmultipleprocessessuchascell-cyclearrest, split-YFP assay was performed. However, no clear YFP signal
DNArepair,apoptosis,andsenescence(18,19).Theproteasomal representingassociationofRCHY1andCAMK2Dwasdetected.
degradationofp53isregulatedbyseveralE3ubiquitinligasessuch TheinvivoexpressionlevelofRCHY1wassubsequentlyfoundto
as RCHY1 and MDM2 (8, 20, 21). RCHY1 binds to the central beextremelylowinthepresenceofCAMK2D,comparedwiththe
region of p53, ubiquitinates p53, and promotes p53 degradation expressionlevelinthepresenceofSUD.Alternatively,theRCHY1–
independentlyofMDM2(8).LikeRCHY1,MDM2isalsoazinc CAMK2D interaction was tested by a highly sensitive coimmuno-
fingerandRINGdomain-containingE3ubiquitinligase(22).High precipitation (coIP) assay. As shown in Fig. 1 F and G, the
levelsofMDM2inducepolyubiquitinationanddegradationofp53, SUD–RCHY1andSUD–CAMK2Dinteractionswerefurther
whereaslowlevelsofMDM2leadtomonoubiquitinationandnu- confirmed(Fig.1F,comparelanes2and4withlane1;Fig.1G, Y
clearexportofp53(23).CDK9phosphorylatesRCHY1atSer211/ compare lanes 6 and 5). Furthermore, the RCHY1–CAMK2D OG
Thr217andMDM2atSer395,causingdegradationofRCHY1and interactionwasdetected(Fig.1F,comparelane3and4withlane1). OL
attenuatedactivityofMDM2towardp53,respectively(24,25).p53 Negativecontrols(lane1inFig.1Fandlane5inFig.1G)showed OBI
canalsobedirectlyphosphorylatedbyCDK9atSer392,leadingto nounspecificbindingofCAMK2D–HAorSUD–RFPtotheGFP- MICR
p53accumulation(26).Inadditiontoitstumorsuppressiveactivity, trap.Inconclusion,thereisdirectphysicalinteractionbetweeneach
p53alsoservesashostdefensemoleculeduringviralinfection(27). pairofproteinsamongCAMK2D,RCHY1,andSUD.
Notably, p53 was identified by its interaction with SV40 large T
antigen(28).Overexpressionofp53reducesHIV-1replicationvia RCHY1 Residues 95–144 Bind to SUD. The RCHY1 protein as an
preventing phosphorylation of RNA polymerase II and conse- interacting partner of SUD consists of 261 aa residues. The N-ter-
quentlyestablishingadelayedRNApolymeraseIIcomplexatthe minalfragmentcomprisingresidues1–94containsaCHYzinc-finger
LTRofHIV-1(29).Arecentstudyreportedthatectopicexpression domain(residues20–94)thatisresponsibleforbindingtocalmodulin
of HCoV-NL63 PLP2 promotes p53 degradation by interaction (14).TheC-terminalfragmentcomprisingresidues145–261contains
with the cellular ubiquitin ligase MDM2 and that p53 exhibits aRING-fingerdomain(residues145–186)withintrinsicE3ubiquitin
antiviral activity onthereplication ofSendai virus (30).Further- ligaseactivityandazincribbon(residues190–250).Themiddlere-
more,p53functionsasanantiviralfactoragainstinfluenzaAvirus gionofRCHY1comprisingresidues120–137isrequiredforbinding
(31).Toantagonizethenegativeeffectofp53,someviruseshave to tumor protein p53 and to CDK9 (8, 24). Phosphorylation by
evolved strategies to induce p53 degradation. Epstein–Barr virus CAMKII at T154/S155 or by CDK9 at S211/T217 enhances self-
(EBV) applies a double strategy to counteract p53. First, EBV ubiquitinationofRCHY1andshortensitshalf-life(14,24).A
nuclearantigen3CdeubiquitinatesandstabilizesMDM2,thereby schematicdomainpresentationofRCHY1isshowninFig.2A.
enhancing ubiquitination and degradation of p53 (32). Second, TodeterminewhichregioniscrucialforinteractionwithSUD,we
during the lytic infection of EBV, p53 is phosphorylated and in- clonedtwotruncatedRCHY1fragmentsintosplit-YFPdestination
capable of interacting with MDM2. However, the viral BZLF1 vectorsand tested theirinteractionswith SUD.One fragment con-
proteincaninteractwithp53andsubsequentlyrecruitp53,espe- tainedresidues1–144andwasthuslackingtheRING-fingerdomain
ciallyphosphorylatedp53,toECSE3ubiquitinligasecomplexfor and the zinc ribbon; the other contained residues 95–261 and was
ubiquitin-mediateddegradation(33). devoidofthecalmodulin-bindingregion(Fig.2A).Interestingly,both
WehaverecentlyestablishedaSARS-CoV–hostinteractomeby truncated RCHY1 fragments interacted with SUD (Fig. 2B). The
screening interactions of individual viral proteins against human region in common between these two fragments, residues 95–144,
cDNA libraries, leading to the identification of important cellular wasconsequentlysupposed toberequiredforbindingtoSUD.In-
pathwaysandtargetsforthepreventionofCoVreplication(34).In deed,it wassubsequentlydemonstrated that theRCHY1 fragment
the present study, we investigate the SUD–CAMK2D as well as 95–144interactedwithSUDinthecytosolofHEK293cells(Fig.2B).
Ma-Laueretal. PNAS | PublishedonlineAugust12,2016 | E5193
Fig.1. InteractionsamongSUD,RCHY1,andCAMK2D.
Affinity-enrichment mass spectrometry screen (A–C):
SUDwithaC-orN-terminalGFPtagwasrecombinantly
expressedinHEK293cellsandenrichedfromcelllysates
using GFP-trap agarose beads (ChromoTek). Volcano
plotsshowenrichmentfactorsofallquantifiedproteins
in C-terminally (A) or N-terminally (B) tagged SUD-
expressing samples over vector-only controls, plotted
againstPvaluesdeterminedfromatwo-samplettest
betweenthreereplicateseach.Significancecurveswere
determinedasdescribed(36)usingc=0.5andx0=0.5
cutoff parameters. (C) Label-free quantification in-
tensitiesofselectedproteinsacrossallsamples.Tocal-
culateenrichmentfactors,missingquantificationswere
imputedwithnoisesimulatingthedetectionlimit(35).
(D) Split YFP: YFPN (YFP N-terminal fragment, amino
acids1–155)wasfusedtotheNterminusofRCHY1,and
YFPC(YFPC-terminalfragment,aminoacids156–239)
was fused to the N terminus of SUD. At 24 h after
cotransfection of both plasmids into HEK-293 cells,
strongYFPsignalwasdetectedinthecytosol.Negative
controls are YFPN–RCHY1 in combination with YFPC
vectorandYFPC–SUDincombinationwithYFPNvector.
(E) Split YFP: YFPN was fused to the N terminus of
CAMK2D, and YFPC was fused to the C terminus of
SUD.At24haftercotransfection,clearYFPsignalwas
detectedinthecytosolofHEK293cells.(F)Co-IP:plas-
midsexpressingtheindicatedgenesweretransfected
intoHEK293cellsgrownin10-cmdishes.At24hafter
transfection,cellswerelysedandthelysateswerepu-
rifiedwithGFP-trapconsistingofagarosebeadscoated
withanti-GFPantibodies.IPsampleswereanalyzedby
12%(wt/vol)SDS/PAGEandWesternblotting(WB)with
anti–SARS-CoVNsp3,anti-HA,andanti-GFPantibodies.
(G)Co-IP:plasmidsexpressingCAMK2D–HAfusionto-
getherwithplasmidsexpressingGFPorGFP–SUDwere
cotransfectedintoHEK293cellsgrownin10-cmdishes.
At24haftertransfection,cellswerelysed.Thelysates
werepurifiedwithGFP-trapandsubsequentlyanalyzed
byWBwithanti-HAandanti-GFPantibodies.
Ofnote,theRCHY1bindingsitesforp53andCDK9arewithinthis causep53issubjecttoubiquitinationbyRCHY1,bothinvivoandin
region(8,24). vitro (8), we examined whether binding to SUD influences the E3
ubiquitin ligase activity of RCHY1. As mentioned previously, the
SUD-NMInteractswithRCHY1.SUDislocatedbetweentheX-domain regionofRCHY1involvedinbindingtop53iswithintheRCHY1
andthePLprodomainofSARS-CoVNsp3(Fig.S2A);itconsistsof fragment (residues 95–144) that binds to SUD. Therefore, we first
three subdomains: SUD-N, SUD-M, and SUD-C. SUD-N and, in assumed SUD to compete with the RCHY1–p53 interaction and
particular, SUD-M have been demonstrated to bind oligo(G) consequentlyimpairRCHY1-mediatedubiquitinationofp53.How-
nucleotidescapableofformingG-quadruplexes(6).Inaddition, ever,theinvivoubiquitinationassayrevealedthatSUDdramatically
SUD-CexhibitsRNA-bindingactivity(38).Theinteractionbetween promotedtheubiquitinationofp53(Fig.3A).Thenegativecontrol
RCHY1anddiverseSUDsubdomainfragmentswastestedbysplit- (Fig.3A,lane1)showsthatGFPwasnotubiquitinatedbyRCHY1.
YFP microscopy (Fig. S2B). SUD-NM did bind to RCHY1, Thus,themultiplebandsappearingintheanti-HAblotofFig.3A,
whereas neither SUD-N nor SUD-M alone did. SUD-MC did lane2,onlyrepresentp53ubiquitination;comparedwithlane2,lane
notbindtoRCHY1either.TheresultsindicatethatbothSUD-N 3showsthatSUDstronglyaugmentedubiquitinationofp53.
andSUD-MareindispensablefortheinteractionwithRCHY1. As shown in Fig. S2A, the PLpro domain occurs next to the C
MERS-CoVisanotherhighlypathogeniccoronavirus.TheMERS terminusoftheSUDwithinSARS–CoVNsp3.ThePLproreleases
Nsp3containsahomologtoSUD-MC,butnottoSUD-N(Fig.S3). the nonstructural proteins Nsp1, Nsp2, and Nsp3 from the viral
The interaction between RCHY1 and MERS-MC (homologous to polyproteins. In addition to being a protease, PLpro possesses deu-
SUD-MC)wasalsoanalyzedbysplit-YFPfluorescencemicroscopy. biquitinating(DUB)anddeISGylatingactivities(39,40).Initially,we
Consistent with the lacking interaction between SUD-MC and thoughtthatSUDpromotingp53ubiquitinationwasinconflictwith
RCHY1,MERS-MCdidnotbindtoRCHY1(Fig.S2C). the DUB activity of PLpro. The SARS–CoV PLpro and the SUD–
PLprofusionwerethereforeclonedtotestwhetherp53isasubstrate
SUD Augments Ubiquitination of p53. As shown previously, the E3 ofthePLpro.Theinvivoubiquitinationassaydemonstratedthatthe
ubiquitin protein ligaseRCHY1 physically interactswith SUD.Be- PLprowasnotabletodeubiquitinatep53(Fig.3B).Thetwoanti-HA
E5194 | www.pnas.org/cgi/doi/10.1073/pnas.1603435113 Ma-Laueretal.
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by hiding the phosphorylation sites of RCHY1), an in vivo phos- P
phorylationassaywascarriedout(Fig.4A);thisshowedthatSUD
doesnotregulateCAMK2D-mediatedphosphorylationofRCHY1
(comparelane3withlane2).Theanti–phospho-Ser/Thrblotofthe
immunoprecipitates did not reveal phosphorylation of GFP by
CAMK2D (Fig. 4A, lane1); similarphosphorylation intensities of
RCHY1 were detected in the absence (lane 2) and presence of
SUD (lane 3). Phorbol-12-myristate-13-acetate (PMA) and ion-
omycinwereaddedtothecellstoopenthecalciumchannelsonthe
plasma membrane, in view of the calcium and calmodulin de-
pendence of CAMK2D. In conclusion, the interaction between
SUDandCAMK2Ddoesnotinhibittheproteinkinaseactivityof
CAMK2DtowardRCHY1.
SUD Stabilizes RCHY1. Phosphorylation of RCHY1 by CAMKII
abrogatestheE3ubiquitinligaseactivityofRCHY1towardp53;it
also leads to enhanced self-ubiquitination and instability of
RCHY1(14).However,SUDdoesnotblockthephosphorylation
ofRCHY1despitethefactthatSUDaugmentsubiquitinationof
p53.TofindoutthemechanismofhowSUDpromotesRCHY1-
mediatedubiquitinationofp53,theproteinlevelofRCHY1was
examined in the absence and presence of SUD. RCHY1 has a
shorthalf-lifeof∼3.5h(14).Therefore,theexpressionlevelofthe
RCHY1–RFP fusion was very weak, despite the presence of the
strong CMV promoter (Fig. 4B, Upper). However, SUD clearly
enhanced the protein level of RCHY1–RFP fusion (Fig. 4B,
Lower). Western blot has also demonstrated that SUD leads to
accumulationofMyc-YFPN-RCHY1(Fig.4C).
SUD–PLproFormsaComplexwithRCHY1andp53.p53couldbepulled
downbyRCHY1inacoIPassay(8).RCHY1islocatedinboththe
nucleusandthecytosol,whereasp53canalsoshuttlebetweenthese
twocompartments(14,41).IthasnotbeenreportedwhereRCHY1
andp53interactwitheachother.Therefore,weperformedasplit-
YFP assay to answer this question. However, the YFP signal was
not detectable due to the low expression level of RCHY1. The
RCHY1fragment1–144containingboththeSUDandp53binding
sitesdisplayedamuchhigherexpressionlevel.Thesplit-YFPassay
revealedthatp53interactswithRCHY1fragment1–144mainlyin
thenucleusandonlytoasmallextentinthecytosol(Fig.4D).Thus,
in the cytosol, RCHY1 can interact with both SUD and p53. To
Y
investigatewhetherp53andSUDcompeteforbindingtoRCHY1, G
O
anexcessiveamountofplasmidsectopicallyexpressingthep53–RFP OL
fusionwascotransfectedwithYFPN-RCHY1andYFPC-SUD OBI
plasmids into HEK293 cells(Fig. 4E). Asa result, the split-YFP CR
MI
signalrepresentingtheinteractionbetweenRCHY1andSUDwas
notdiminished,indicatingthattherewasnocompetitionbetween
p53andSUDforbindingtoRCHY1.
Subsequently,wetestedwhetherp53canformacomplexsimul-
Fig.2. RCHY1aminoacids95–144andSUD-NMarecrucialforinteraction. taneouslywithRCHY1andSUD.Tobettermimictherealsituation
(A)SchemeofRCHY1.(B)SplitYFP:YFPNwasfusedtotheNterminusof oftheviralproteininteractingwithRCHY1inSARS-CoV–infected
truncatedRCHY1fragments,andYFPCwasfusedtotheCterminusofSUD. cells, the SUD–PLpro fusion protein instead of SUD was applied.
At24haftertransfectionoftheexpressionplasmidsintoHEK293cells,YFP p53-GFPwasabletopulldownSUD–PLprointhecoIPassayonlyif
signalwasexaminedbyfluorescencemicroscopy. RCHY1wasalsoexpressed(Fig.4F,comparelane3withlane2);
this indicates that p53 forms a complex with RCHY1 and SUD–
PLprobutthereisnodirectinteractionbetweenp53andSUD–PLpro.
blots of input and immunoprecipitation samples showed that the Thenegativecontrol(Fig.4F,lane1)showsnounspecificbindingof
PLpro deubiquitinated some endogenous proteins but not p53 RCHY1orSUD–PLprototheGFP-trap.
(compare lane 4 with lanes 1 and 2 in Fig. 3B). Like SUD, the CDK9isanotherimportantproteinkinaseinvolvedinRCHY1–
SUD–PLprofusionproteinwasalsoabletoaugmentubiquitination p53signaling.SimilartoCAMK2D,CDK9phosphorylatesRCHY1
of p53 (compare lane 3 with lane 2 in Fig. 3B). Interestingly, as and enhances the self-ubiquitination of this protein. However, dif-
shownintheanti-Mycblot,theproteinlevelofMyc-YFPN–RCHY1 ferently from CAMK2D, CDK9 can directly phosphorylate p53 at
wasup-regulatedbybothPLproandSUD–PLpro. Ser392(24).WethereforetestedapossiblebindingofSUD–PLproto
CDK9butcouldnotdetectanyinteraction(Fig.4F,lane4).
SUD Does Not Interfere with Phosphorylation of RCHY1. CAMKII
phosphorylatesRCHY1at T154/S155and therebyimpairstheE3 PLproBindstoRCHY1andPromotesItsAccumulation.Toconfirmthat
ubiquitinligaseactivityofRCHY1towardp53(14).CAMK2D,asa SUD–PLpro binds to RCHY1 in vivo, plasmids expressing YFPN-
memberoftheCAMKIIfamily,bindstoRCHY1(Fig.1F).SUD SUD–PLpro and YFPC–RCHY1 were transfected into HEK293
interacts with both RCHY1 and CAMK2D (Fig. 1). To find out cells for a split-YFP assay. A vector expressing YFPN–PLpro was
whetherSUDinterfereswithCAMK2D-mediatedphosphorylation appliedasoneofthecontrols.Bychance,aninteractionbetween
ofRCHY1(e.g.,byblockingtheenzymaticdomainofCAMK2Dor RCHY1 and SARS–CoV PLpro was discovered although the
Ma-Laueretal. PNAS | PublishedonlineAugust12,2016 | E5195
interaction was weaker than the interaction between the SUD–
PLprofusionandRCHY1(Fig.5A).
AcoIPassaywassubsequentlyperformedtofurtherconfirmthe
binding of PLpro to RCHY1. HA-tagged PLpro was pulled down
together with GFP–RCHY1 by the GFP-trap containing beads
coated with anti-GFP antibodies (Fig. 5B), implying a physical
interactionbetweenSARS–CoVPLproandRCHY1.Similarlyto
SUD, PLpro also influences the stability of RCHY1 (Fig. 5C).
Interestingly,bothSUDandPLproledtoaccumulationofRCHY1
at the protein level but not the mRNA level (Fig. 5C, compare
lanes2and3withlane1).ComparedwiththeSUD–PLprofusion
and PLpro, SUD promoted a slightly higher accumulation of
RCHY1whenexpressedseparately(Fig.5C,comparelane4with
lanes 2 and 3). Because it is known that viral SARS-CoV PLpro
acts as a deubiquitinase on cellular proteins and inhibits innate
immuneresponses,wetestedtheeffectofPLproonRCHY1inan
in vitro deubiquitination assay (Fig. S4). As RCHY1 was not
deubiquitinatedbyPLpro,theproteinmustbestabilizedbyadif-
ferent,as-yet-unknownmechanism.
SUD–PLproLeadstoDegradationofp53.Ubiquitination ofp53can
regulatetheproteininmanydifferentaspects,suchasprotease-
mediateddegradation,degradation-independentnuclearexport,
and functional alteration (41). To find out the consequence of
enhancedp53ubiquitinationpromotedbySUDandSUD–PLpro,
the endogenous p53 protein level was tested (Fig. 5D). Ectopic
HA-taggedRCHY1wastransfectedintocellsasapositivecontrol.
Overexpression of RCHY1 resulted in p53 degradation (Fig. 5D,
comparelane2withlane1).Likethephenotypeobtainedfromthe
positive control, SUD–PLpro also led to dramatic enhancement of
the degradation of p53 (Fig. 5D, compare lane 3 with lane 1).
Subsequently,PLproalonewasalsofoundtocausep53degradation
(Fig.5D,comparelane5withlane4).Moreover,theSUD–PLpro
fusionresultedinfurtherdegradationofp53comparedwithPLpro
alone (Fig. 5D, compare lane 6 with lane 5), which indicates that
SUDaugmentsp53degradation.
P53InhibitsReplicationofaSARS-CoV.Thetumorsuppressorprotein
p53hasbeendemonstratedtopossessaninhibitoryeffectonsome
virusessuchasHIV-1andhumanpapillomavirus(42,43).Ectopic
expressionof HCoV-NL63 PLP2wasshownto promotep53deg-
radation by interaction with the cellular ubiquitin ligase MDM2,
leadingtoreducedreplicationofSendaivirus(30).However,toour
knowledge,adirecteffectofp53oncoronavirusreplicationhasnot
been shown before. To investigate how SARS-CoV benefits from
p53degradationpromotedbySUD–PLpro,theinfluenceofp53on
viral growth of SARS-CoV was examined. Human ACE2-trans-
genicp53-expressingHCT116cells(HCT116/ACE2p53+/+)aswell
as p53 knockout cells (HCT116/ACE2 p53−/−) were infected with
SARS-CoV,andviralreplicationwasmonitoredbyreal-timePCR.
AsshowninFig.6,SARS-CoVgrewtoan∼1,000-foldhighertiter
in p53−/− cells compared with p53+/+ (Fig. 6A). To exclude that
differential metabolism or cell growth was responsible for differ-
encesinvirusreplication,cellswereinfectedwithasecondvirusin
parallel.Vesicularstomatitisvirus(VSV)grewequallywellinboth
p53−/−and p53+/+ HCT116/ACE2 cells, suggesting that facilitated
growthwasSARS-CoVspecific(Fig.6B).
Loss-of-function assays were also performed to examine the
effect of p53 on replication of the SARS-CoV replicon. The
SARS-CoV replicon DNA pBAC-SARS-Rep-R(enilla)Luc (7)
and the SARS-CoV replicon RNA in vitro transcribed from
pBAC-SARS-Rep-M(etridia)Luc (34) carrying a secreted Met-
ridia luciferase reporter were tested in p53-expressing HCT116
lysateswerepurifiedwithGFP-trapconsistingofbeadscoatedwithanti-GFP
antibodies.ThesamplesofIPandinputwereanalyzedby12%(wt/vol)SDS/
Fig.3. SUDaugmentsp53ubiquitinationandstabilizesRCHY1.(A)Plasmids PAGEandWBwithanti-HA,anti-GFP,anti–SARS-CoVNsp3,anti-Myc,andanti-
expressingHA-taggedubiquitin,c-Myc-taggedYFPN-RCHY1,GFPorp53–GFP laminAantibodies.(B)Plasmidsexpressingindicatedproteinswerecotrans-
fusion, and RFP or SUD–RFP fusion were cotransfected into HEK293 cells fectedintoHEK293cellsgrowingin10-cmdishes.Theinvivoubiquitination
growingin10-cmdishes.At24haftertransfection,cellswerelysedandthe assaywascarriedoutasdescribedinA.
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Fig.4. SUDdoesnotinterferewithphosphorylationofRCHY1andstabilizesit.(A)TheindicatedplasmidswerecotransfectedintoHEK293cellsgrowingin Y
10-cmdishes.At20hposttransfection,PMAtoafinalconcentrationof20ng/mLandionomycintoafinalconcentrationof1μMwereaddedtothecells.Two OG
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hoursafterthetreatment,cellswerelysedandthelysateswerepurifiedwithGFP-trap.ThesamplesofIPandinputwereanalyzedby12%(wt/vol)SDS/PAGE/ O
WBandprobedwithanti–phospho-Ser/Thr,anti-GFP,anti-HA,anti-Myc,andanti–SARS-CoVNsp3antibodies.(B)PlasmidsexpressingRCHY1–RFPandplas- OBI
R
m2%ids(wetc/tvoopl)icpaallryafeoxrpmrealsdsienhgydSeUD(PFwAe)raencdotsrtaainnsefdecwteitdhinDtAoPHI.ETKh2e9s3amceplllsesgwroewrenfoinnaclloyveexrasmlipinseindaw2it4h-waeflluloprlaestcee.nAttm2i4crhosacfotpeert(rLaenicsafeDcMtio4n0,0c0eBll;s1w0×eroebfjiexcetdivew)i.tAh MIC
representativeareaofpicturestakenrandomlyisshown.(C)Split-YFPplasmidsexpressingMyc-YFPN–RCHY1andexpressingYFPCorSUD–YFPCweretransfectedto
HEK293cellsgrowinginasix-wellplate.At24haftertransfection,cellswerelysed.ThelysateswereWesternblottedwithanti-Mycandanti-laminAantibodies.
(D)YFPNwasfusedtotheNterminusofRCHY1aminoacids1–144,andYFPCwasfusedtotheNterminusofp53.Split-YFPfluorescencemicroscopywasperformed
asdescribedbefore.(E)The300-ngplasmidsectopicallyexpressingRFPorp53-RFPtogetherwith100-ngplasmidsexpressingYFPN–RCHY1andYFPC–SUDwere
cotransfectedintoHEK293cellsgrowingoncoverslipsina24-wellplate.Thesplit-YFPexperimentwascarriedoutasdescribedbefore.(F)Indicatedplasmidswere
transfectedintoHEK293cellsgrowingin10-cmculturedishes.TheGFP-trap-basedcoIPwasperformedasdescribedbefore.
p53+/+andHCT116p53–knockoutp53−/−cells(44)aswellasin replicationoftheSARS-CoVrepliconby65%,whereasithadno
adoxycycline(DOX)-dependentp53-induciblecelllineSW480(19) effect on the pcDNA3.0 Renilla luciferase (pcDNA3.0 RLuc)
(Fig. 6 C and D). p53 expression was monitored by Western blot control(Fig.S6).Theresultsdemonstratethatp53isanegative
usinganti-p53antibodiesintherespectivecelllines(Fig.S5).Both regulatorofSARS-CoVreplication.
of the replicons replicated better in HCT116 p53−/− than in
HCT116p53+/+,andbetterinSW480withoutDOXinductionthan MERS-CoV PLpro and HCoV-NL63 PLP1/2 Interact with and Stabilize
inSW480withDOXinduction.Theobservedfolddifferencesbe- RCHY1 and Cause Endogenous p53 Degradation. As its name im-
tweentheuseofrepliconsandvirusinfectionareattributedtothe plies, the SUD is unique for the SARS-CoV, whereas PLpro do-
commonly lower replication efficiencies of coronavirus replicons mainsarerelativelywell-conservedincoronaviruses(39).Among
(>27 kb), because transfection/electroporation efficiencies do not thehumancoronaviruses,onlySARS-CoVandMERS-CoVhave
reach 100% of cells. On the contrary, viable virus is much more one PLpro domain, whereas most other HCoVs have two PLpro
efficient due to multicycle infection and exponential growth when domains, PLP1 and PLP2 (39, 45, 46). To investigate whether
usinglowmultiplicitiesofinfections(MOIs,0.001). other HCoV PLpros also target RCHY1, similarly to what the
ToexploretheefficiencyofgenomereplicationofSARS-CoV SARS-CoVPLprodoes,weclonedtheMERS-CoVPLproandthe
replicon under the pressure of p53 the plasmid pBAC-SARS- HCoV-NL63PLP1/2toperformacoIPRCHY1-bindingassay.As
Rep-RLucwastransfectedintoHEK293cellsatincreasingcon- a result, MERS-CoV PLpro and NL63 PLP1 showed clear in-
centrationsofap53-expressingplasmid.p53stronglyreduced teractionwithRCHY1(Fig.7A,comparelanes2and3withlane1).
Ma-Laueretal. PNAS | PublishedonlineAugust12,2016 | E5197
Fig.5. SARSPLprointeractswithRCHY1andleadsto
accumulationofRCHY1aswellasp53degradation.
(A)YFPNandYFPCwerefusedtotheNterminusof
SARS PLpro and to the N terminus of RCHY1, re-
spectively. At 24 h after transfection, HEK293 cells
werefixedwithPFAandstainedwithDAPI.Samples
wereinspectedwithafluorescentmicroscope(Leica
DM4000 B, 40× objective; Upper). Pictures for sta-
tistical analysis were taken from at least five ran-
domlychosenareas.Intensitiesofsplit-YFPandDAPI
signalsweremeasuredwithImageJsoftware(***P<
0.001;Lower).(B)PlasmidsexpressingHA-taggedSARS
PLproandGFPorGFP–RCHY1fusionweretransfected
intoHEK293cellsgrownin10-cmculturedishes.The
coIPassaywasperformedasdescribedbefore.(C)WB
(Upper)PlasmidsectopicallyexpressingSUD,PLpro,or
SUD–PLpro fusion were transfectedintoHEK293 cells
growninasix-wellplate.Celllysateswereprepared24h
posttransfectionandsubjectedtoWesternblotting
with anti-Myc and anti-lamin A antibodies. qPCR:
(Lower)PlasmidsectopicallyexpressingSUD,PLpro,or
SUD–PLprofusionweretransfectedintoHEK293cells
grownina24-wellplate.At24hposttransfection,cells
were harvested for total RNA extraction, first cDNA
synthesis,andsubsequentSYBRGreenqPCR.Theex-
periment was carried out in quadruplicates. (D) In-
dicatedplasmids were transfectedinto HEK293 cells
growninasix-wellplate.At24hposttransfection,cells
were lysed for Western blotting with anti-p53 and
anti-laminAantibodies.
MostoftheGFP-NL63PLP2wasdegradedwithonlyalittleintact coronavirusessharethisstrategytoantagonizetheantiviralhost
proteinremaining(Fig.7A,lane4).Nevertheless,asmallamount factorp53.SUDalonestronglybindstoandstabilizesRCHY1,
ofHA-RCHY1couldbecoimmunoprecipitatedbyGFP-NL63 leading to stimulation of p53 ubiquitination and degradation.
PLP2(Fig.7A,comparelane4withlane1),implyinginteraction The SUD–PLpro fusion interacts with RCHY1 more intensively
betweentheseproteins.Tofurtherconfirmtheinteraction,asplit- and causes stronger endogenous p53 degradation than SARS-
YFPassaywasperformed(Fig.S7).BothMERS-CoVPLproand CoV PLpro alone. Hence, SUD functions as an enhancer for
NL63PLP2showedclearinteractionwithRCHY1inthenucleus SARS-CoVNsp3tocounteractp53,andthismightcontributeto
andthe cytosol, whereas NL63PLP1 interacted with RCHY1 at thehighvirulence ofSARS-CoV.
theenodplasmicreticulum(ER). The mechanism of how SUD and PLpro/PLPs stimulate the ac-
Subsequently, we examined whether MERS-CoV PLpro and cumulation of RCHY1 remains unclear. It is known that phos-
NL63PLP1/2alsostabilizeRCHY1.AsshowninFig.7B,allofthe phorylation by CAMKII or CDK9 leads to polyubiquitination and
testedPLprosstimulatedRCHY1accumulation(comparelanes2,3, subsequent degradation of RCHY1 (14, 24), but SUD does not
and4withlane1).Indeed,thestabilizationofRCHY1bytheNL63 impair the phosphorylation activity of CAMK2D toward RCHY1.
PLP2wasdramatic(Fig.7B,comparelane4withlane1).Consis- Becausepapain-likeproteasesofSARS-CoV,MERS-CoV,and
tent with this result, all of the tested PLpros led to degradation of NL63possessdeubiquitinatingactivity(39,46,47)andcanstabilize
endogenous p53 as well (Fig. 7C). Thus, similarly to SARS-CoV RCHY1,weoriginallyhypothesizedthatthelattermaybeapossible
PLpro,MERS-CoVPLproandNL63PLP1/2alsotargetedRCHY1 substrate for these enzymes. However, an in vitro deubiquitination
forp53degradation. assay has shown that SARS-CoV PLpro does not deubiquitinate
Tofindoutwhetherp53inhibitsviralreplicationofotherHCoVs RCHY1. Thus, SUD and PLpro should stabilize RCHY1 through
inadditiontoSARS-CoV,theinfluenceofp53onNL63replication othermechanisms,e.g.,bycounteractingthebindingofRCHY1to
wastested.Duetoanextremelylowtransfectionefficiencyofp53 CDK9orinterferingwiththehomodimerizationofRCHY1forself-
intotheNL63-infectedhost Caco2 cells,HEK293 cellstransiently ubiquitination.BecausebothSUDandPLprophysicallyinteractwith
expressingtheNL63receptorACE2wereusedasasubstitutefor RCHY1, their mechanisms of RCHY1 stabilization might also be
Caco2.Real-timequantitativePCR(q-PCR)analysisrevealedthat differentfromoneanother.
NL63replicationwasreducedby∼30%incellsoverexpressingp53, The protein level of RCHY1 is dramatically increased in the
compared with control (Fig. 7D); this implies that p53 is an in- presenceof SUD. Asa result,RCHY1-mediatedubiquitinationof
hibitoryfactornotonlyforSARS-CoVbutalsoforotherHCoVs. p53 is enhanced and p53 degradation is stimulated. However, en-
hanced p53 degradation should not be the only consequence of
Discussion RCHY1accumulation.Besidesp53,thesubstratetargetsoftheE3
PLP1/2 of the alphacoronavirus NL63 as well as the PLpros of ubiquitinligaseRCHY1includetranscriptionfactorsp63,p73,and
SARS-CoV and MERS-CoV from the genus betacoronavirus c-Myc; checkpoint kinase Chk2; DNA polymerase polH; histone
physically interact with and stabilize RCHY1, and lead to p53 deacetylaseHDAC1;andCDKinhibitorp27Kip1(9,10,48–52).It
degradation. Because PLpros and PLPs are relatively well con- canbehypothesizedthatduringHCoVinfection,theproteinlevels
served among coronaviruses, it is highly likely that all of the ofvariousgenesmightalsobedown-regulated.SUDalsophysically
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Fig.6. p53inhibitsreplicationofaSARS-CoV.(AandB)IncreasedSARS-CoVgrowthinp53−/−ACE2-transgenicHCT116cells.HCT116/ACE2withp53(+/+)and
withoutp53(−/−)wereseededat2×105cellspermL.After24h,cellswereinfectedinquadruplicatewithSARS-CoV(Frankfurt)andVSV(Indiana)atanMOI
of0.001.After1hat37°C,cellswerewashedwithPBSandMcCoyculturemediumwasreplenished.Samplesweretakenat0and24hpostinfection.Viral
RNAwasextractedbyaMacherey-NagelviralRNAkit.Real-timePCRwasperformedusingvirus-specificprobesandprimerswiththeOne-StepSuperScriptIII
Kit(LifeTechnologies)accordingtopreviousprotocols.(CandD)HCT116wild-type(p53+/+)andknockoutcells(p53−/−)weretransfectedwithHCoVSARS
DNAandRNAreplicons.p53expressioninSW480cellswasinducedbyadding100ng/mLDOX(DOX+)24hbeforetransfection.SW480cellswithoutDOX
induction(DOX−)wereappliedascontrol.pBAC-SARS-Rep-R(enilla)LucDNAwastransfectedintocellsusingLipofectamine3000,andRenillaluciferaseac-
tivitywasmeasured from celllysates after 24 h.ThepBAC-SARS-Rep-M(etridia)Luc RNA was cotransfected with N-RNA by electroporationof cells, and
Metridianluciferaseactivitywasdeterminedfrommediumsupernatantafter20h(*P<0.05,**P<0.01,***P<0.001).
interacts with CAMK2D, although it does not disturb phosphory- MaterialsandMethods
lation of RCHY1 by CAMK2D. However, RCHY1 is neither the PlasmidConstructs.Thefragmentsc-Myc-YFPN(aminoacids1–155)andHA-
only substrate nor the only interacting partner of CAMK2D. The YFPC(aminoacids156–239)wereamplifiedwithdesignedprimers(TableS1)
association of SUD and CAMK2D might alter the affinity of from the template plasmids pSPYNE-35Sand pSPYCE-35S(37), respectively.
CAMK2Dforotherproteins,e.g.,foritscandidatebindingpartner TheobtainedPCRfragmentsweresubsequentlyclonedintothepTREX-dest30-
IFN-γ receptor2(IFNGR2),whichwasdiscoveredbyY2Hscreen PrAGateway-compatiblevectorviaAgeI/XhoIcleavagesitesorintothepTREX-
(53). Alternatively, the interaction between SUD and CAMK2D dest30-ct-PrAGateway-compatiblevector(60)viaSpeI/ApaIsitestoreplacethe
might interfere with phosphorylation of STAT1 by CAMK2D, proteinAtag,yieldingpDEST-c-Myc-YFPN(c-Myc-YFPNfusedtoNterminusof
which is required for maximum IFN-γ–stimulated gene (ISG) atestgene)andpDEST-HA-YFPc(HA-YFPCfusedtoNterminusofatestgene)
expression (54). In addition to stimulation of p53 degradation, orpDEST-ct-c-Myc-YFPN(c-Myc-YFPNfusedtoCterminusofatestgene)and
pDEST-ct-HA-YFPc(HA-YFPCfusedtoCterminusofatestgene)split-YFPvectors.
SUD probably features even more complex interferences with
SUD,RCHY1,MERS-CoVMC,SARS-CoVPLpro,MERS-CoVPLpro,NL63PLP1/2,and
host cellular signaling pathways.
thetruncatedfragmentsofSUDandRCHY1amplifiedwithcorrespondingprimers
To date, p53 was found to serve as an antiviral factor against
(TableS1)werefirstBPGateway-clonedintothepDONR207vectorandconse-
diversepositive-sensesingle-strandedRNA(ssRNA)virusessuch quentlyLRGateway-clonedintothepDEST-c-Myc-YFPN,pDEST-HA-YFPc,pDEST-ct-
ashepatitisCvirus(HCV)andpoliovirus;negative-sensessRNA c-Myc-YFPN, pDEST-ct-HA-YFPc vectors, yielding constructs for split-YFP assays.
virusessuchasinfluenzaAvirus;andtheretrovirusHIV-1(29,31, CAMK2Dandp53wereselectedfromahumancDNAlibraryof5,000different,
55,56).p53antiviralactivityonSendaivirusreplicationwasreported individuallyclonedgenesinpDONR223vectors,asdescribedbefore(34).CAMK2D
in the context of deubiquitination of MDM2 by coronaviral PLpro andp53werethenclonedfrompDONR223tosplit-YFPvectorsviatheLRreaction.
andubiquitinationofp53byMDM2(30).Inthismainlybiochemical To generate RFP- or GFP-tagged constructs, the RFP and GFP fragments Y
G
study,theinhibitoryeffectofp53wasnottestedforcoronaviruses. withoutNLS(nuclearlocalizationsignal)wereamplifiedwithdesignedprimers O
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TheauthorshadhypothesizedregulatoryinfluenceofHCoV-NL63 (Table S1) from the template plasmids pNLS-TagRFP and pNLS-TagGFP, re- BIO
PLP2onthestabilityofMDM2,due(i)tostructuralsimilaritiesof spectively(61).ThePCRproductsweresubsequentlyclonedintopTREX-dest30- RO
PLP2,PLpro,andthecellulardeubiquitinaseHAUSP,and(ii)tothe PrAviaAgeI/XhoIsitesorintopTREX-dest30-ct-PrAviaSpeI/ApaIsitestoreplace MIC
preferential deubiquitination of MDM2 by HAUSP. Indeed, the protein A tag, yielding pDEST-RFP and pDEST-GFP or pDEST-ct-RFP and
interactionofPLP2andMDM2,deubiquitinationofMDM2by pDEST-ct-GFPGateway-compatibledestinationvectors.RCHY1,SUD,SARS-CoV
PLpro, SUD–PLpro fusion, MERS-CoV PLpro, NL63 PLP1/2, and p53 were sub-
PLP2, and ubiquitination of p53 leading to proteasomal deg-
sequentlyclonedintotheobtainedRFP-andGFP-taggeddestinationvectorsvia
radation was demonstrated.
Applyingunbiasedprotein–proteininteractionY2Hscreens(34), the LRreaction.CDK9 was selected from the human cDNA librarydescribed
aboveand,finally,LRGateway-clonedintothepDEST-ct-GFPvector,yieldingthe
we identified interaction of SUD with the cellular ubiquitin ligase CDK9–GFPfusionconstruct.ThepDEST-C-tag-HAandpDEST-N-tag-HAGateway-
RCHY1, which exerts similar ubiquitination activities on p53 as compatible destination vectors were used to generate HA-tagged fusions.
MDM2. We directly demonstrate, to our knowledge for the first Ubiquitinwasamplifiedwithdesignedprimers(TableS1)fromthesynthesized
time,thatp53inhibitsreplicationofcoronavirusesandprovideevi- firstcDNAofHEK293cellsandclonedintothepDONR207vectorviatheBP
dencethatSUDandPLpro/PLPsantagonizethehostdefensefactor reaction.CalmodulinwasselectedfromthehumancDNAlibraryasdescribed
p53viastabilizingtheE3ubiquitinligaseRCHY1.Sofar,wehave above.Ubiquitin,calmodulin,CAMK2D,RCHY1,SUD–PLpro,andSARS-CoVPLpro
nohintsonaconceivableinvolvementofMDM2,thoughitmaywell werefinallyclonedintopDEST-HAorpDEST-ct-HAvectorsviatheLRreaction.
bethatCoVshavedevelopeddifferentwaystodown-regulatep53.
In addition, the deISGylating activity, exhibited by PLpro do- CellCulturesandTransfection.HEK293andHCT116p53+/+/p53−/−cellswere
mains, might further interfere with the ISGylation status of host culturedatstandardconditions(SIMaterialsandMethods).Forintroductionof
proteins (57, 58). Decreased ISGylation of host proteins seems to theACE2receptorintoHCT116p53+/+andp53−/−,cells2×105cells/mLwere
positively mediate virus infection. It has been demonstrated that transducedwithhumanACE2-carryinglentivirusesatanMOIof0.005.Lenti-
viruseswereprovidedinMcCoy’s5Asupplementedwithpolybrene(1μg/mL).
cellularISGylationcontributestoinhibitionofreplicationofmurine
After48h,thesupernatantwasremoved,cellswerewashedwithPBS,and
hepatitis virus strain 3 (59). Thus, CoV Nsp3 proteins containing
McCoy’s5Acontaining1μg/mLpuromycinwasaddedforselection.Selective
SUDPLpro/PLPscounteractthehostdefenseintwodistinctman-
mediumwaschangedevery3d.After10d,survivingcellswereexpandedand
ners—oneisp53independent:thePLpro/PLPswithinNsp3exerta
tested for hACE2 protein expression by immunofluorescence assay and
directdeISGylationactivitytointerferewithcellularISGylation;the Westernblotanalysisasdescribedpreviously(62).
otherisp53dependent:Nsp3proteinsleadtop53degradationby
stabilizing RCHY1. These results may open new possibilities to Split-YFP. Fluorescence microscopy was performed according to standard
therapeuticallytargetSARSinfectionsinthefuture. proceduresandisdescribedindetailinSIMaterialsandMethods.
Ma-Laueretal. PNAS | PublishedonlineAugust12,2016 | E5199
protocol under native conditions (SI Materials and Methods). Detailed
Westernblotprotocolsweredescribedelsewhere(63).
qPCR.QuantificationNL63viralRNA(63)andofcellularmRNAsisdescribed
indetailinSIMaterialsandMethods.
InVivoUbiquitinationAssay.PlasmidsexpressingHA-taggedubiquitin,c-Myc-
YFPN-tagged RCHY1 (c-Myc-YFPN-RCHY1), substrate protein (GFP control or
p53-GFP),andeffectorprotein(RFPcontrol,SUD–RFPfusion,RFP-SUD–PLpro
fusion,orRFP–PLprofusion)werecotransfectedintoHEK293cellsusingthe
polyethylenimine(PEI)method.At24hposttransfection,cellswerelysedwith
lysisbuffercontaining10mMTris·HCl(pH7.5),150mMNaCl,0.5mMEDTA,
0.5%NonidetP-40,10mMN-ethylmaleimide,andproteaseinhibitor(Com-
pleteULTRATablets;Roche).Subsequently,GFP-Trap_Abeads(ChromoTek)
wereusedtopurifyGFPandp53-GFPfromthecelllysates.ThepurifiedGFP
and p53-GFPwereexaminedwith anti-HAand anti-GFP(ChromoTek) anti-
bodiesbyWesternblotforthetestofinvivoubiquitinationofp53.
InVitroDeubiquitinationAssay. HEK293cellsgrowingon10-cmdisheswere
cotransfectedwithGFP-RCHY1,HA-ubiquitin,calmodulin1-RFP,andCAMK2D-c-
Myc-YFPN. At 23 h posttransfection, cells were treated with PMA to a final
concentrationof20ng/mLandionomycintoafinalconcentrationof1μMto
activateCAMK2D.At90minafterthetreatment,cellswereharvestedandGFP-
RCHY1proteinwaspurifiedwithGFP-trap_Abeads(ChromoTek).Thepurified
bead-boundGFP-RCHY1wassubsequentlyincubatedwith10μMSARS-CoVPLpro
proteinat25 °Cfor30 mininabuffercontaining10 mMTris·HCl(pH7.5),
150mMNaCl,0.5mMEDTA,and2μMDTT.Afterincubation,thebeadswere
washedtwicewithwashingbuffercontaining10mMTris·HCl(pH7.5),150mM
NaCl,and0.5mMEDTA.Thebead-boundGFP-RCHY1wasfinallyexaminedwith
anti-HAandanti-GFP(ChromoTek)antibodiesbyWesternblot.
Purification of Recombinant SARS-CoV PLpro. The PLpro of SARS-CoV (strain
TOR2;GenBankaccessionno.AY274119)ispartofNsp3andcomprises319aa
residues,correspondingtoGlu1541–Tyr1859ofpp1a.Agenecodingforthe
PLpro was amplified by the PCR using the forward primer 5′-CTAGCTAGC-
GAGGTTAAGACTATAAAAGTGTTC-3′andthereverseprimer5′-CCGCTCGAGT
TAATACGACACAGGCTTGATGGTTGTAG-3′. The resulting PCR product was
digested with restriction enzymes NheI and XhoI for ligation into pET-28a
(Novagen).CloningwasdesignedtoincludeanN-terminalhexahistidine(His6)
tagandathrombincleavagesite.Therecombinantplasmidwasverifiedby
DNAsequencing.ExpressionofthePLprogeneandpurificationoftheprotein
werecarriedoutbyamethoddescribedpreviously(64).
InVivoKinaseActivityAssay.Plasmidsexpressingcalmodulin-HA,CAMK2D-c-
Myc-YFPN,substrateprotein(GFPcontrolorGFP–RCHY1),andeffectorprotein
(RFPcontrolorSUD–RFPfusion)werecotransfectedintoHEK293cellsusingthe
PEImethod.Mediumwaschanged20hposttransfection.Thenthecellswere
treatedwithPMAtoafinalconcentrationof20ng/mLandionomycintoafinal
concentrationof1μM.Twohoursafterthetreatment,cellswereharvestedand
GFP-trap_Abeads(ChromoTek)wereusedtopurifyGFPandtheGFP–RCHY1
fusionfromthecelllysates.PurifiedGFPandGFP–RCHY1fusionproteinswere
subsequently examined with anti–phospho-Ser/Thr (QIAGEN) and anti-GFP
(ChromoTek)antibodiesbyWesternblotforinvivophosphorylation.
LuciferaseActivityAssay.TransfectedcellswereharvestedwithRenillaLu-
ciferaseAssaySystemKit(Promega).Theluciferaseactivitywasthenmea-
suredwithaFLUOstarOPTIMAfluorescenceplatereader(BMGLABTECH).
Fig.7. MERSPLproandNL63PLP1/2leadtoaccumulationofRCHY1andp53
degradation.(A)TheindicatedplasmidsweretransfectedtoHEK293cellsgrowing Statistical Analysis. qPCR and luciferase measurement data are shown as
in10-cmculturedishes.CoIPwasperformedasdescribedbefore.(B)Theindicated mean±SDoftri-orquadruplicatedvaluesandanalyzedbyone-wayortwo-
plasmidsweretransfectedtoHEK293cellsgrowinginasix-wellplate.At24h
wayANOVAwithBonferroni´sposttest(orbyttest)usingPrism5(GraphPad
posttransfection,cellswerelysed.ThelysateswereWesternblottedwithanti-HA
SoftwareInc.).Pvaluesof0.05orlesswereconsideredsignificant.
andanti-laminAantibodies.(C)At24 haftertransfectionwiththe indicated
plasmidsintoHEK293,cellswereharvestedandWesternblottedwithanti-p53and
VirusInfectionExperiments.Cellcultureswereinfectedinquadruplicatewith
anti-laminAantibodies.(D)HEK293cellswerecotransfectedwithplasmidsover-
eitherSARS-CoV(strainFrankfurt)undVSV(strainIndiana)atamultiplicityof
expressingRFP-ACE2andplasmidsoverexpressingGFPcontrolorp53-GFP.At24h
infectionof0.001.Aftervirusadsorption,cellswerewashedwithPBSandcell
aftertransfection,cellswereinoculatedwithNL63virusesofMOI0.4at37°Cfor
culturemediumwasreplenished.Samplesweretakenat0and24hpostinfection.
1h.At24hafterinfectionwithNL63virus,totalRNAwasextractedfromcelllysates.
ViralRNAwasextractedbyaMacherey-NagelviralRNAkit.Real-timePCRwas
ViralRNAwasanalyzedbyqPCRwithNL63-specificprimersandstandardizedwith
RFP-ACE2intensity.Experimentswereperformedinsextuplicates.***P<0.001. performedusingvirus-specificprobesandprimerswiththeOne-StepSuperScript
IIIkit(LifeTechnologies)accordingtopreviousprotocols(34).
ACKNOWLEDGMENTS.HCT116celllinesandderivativeswerekindlyprovided
CoIPandWesternBlot.GFPfusionsandcomplexedproteinswereimmuno- byBertVogelstein(JohnsHopkinsMedicalSchool).Thisworkwassupportedby
precipitatedbyGFP-Trap_Abeads(ChromoTek)accordingtomanufacturer’s theBundesministeriumfürBildungundForschungoftheGermanGovernment,
E5200 | www.pnas.org/cgi/doi/10.1073/pnas.1603435113 Ma-Laueretal.
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ZoonosisNetwork,ConsortiumonEcologyandPathogenesisofSARSProject sitesMunich)ProjectCode8025801803(toA.v.B.);andLübeckProjectCode P
Code01KI1005A,F(toA.v.B.);GermanCenterforInfectionResearch(partner 8000204-3(toR.H.).
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Ma-Laueretal. PNAS | PublishedonlineAugust12,2016 | E5201