2014 Coronavirus virulence genes with main focus on SARS-CoV envelope gene

2014·3.53 MB·English

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VirusResearchxxx(2014)xxx–xxx

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Virus Research

journal homepage: www.elsevier.com/locate/virusres

Coronavirus virulence genes with main focus on SARS-CoV envelope
1

gene
2
3 Q1 MartaL.DeDiegoa,1,JoseL.Nieto-Torresa,JoseM.Jimenez-Guarden˜oa,

JoseA.Regla-Navaa,CarlosCastan˜o-Rodrigueza,RaulFernandez-Delgadoa,
4
Fern an doUserab,Lu isEnju anesa,∗
5

6 aDepartmentsofMolecularandCellBiology,NationalCenterofBiotechnology(CNB-CSIC),CampusUniversidadAutonomadeMadrid,Madrid,Spain
7 bDepartments of Biosafety, Nati onal Centero fBiotech nology (C NB-CSIC),Cam pusUniversi dadAuto nomadeMa drid,Madri d, Spain

8
a r t i c l e i n f o a b s t r a c t
927

10
11 Articlehistory: Coronavirus(CoV)infectionisusuallydetectedbycellularsensors,whichtriggertheactivationofthe
12 Availab leonlinexxx innateimmu nesy stem.Nev er theless, CoVshav ee volvedv iralprot einsth attarg etd ifferentsig na ling

13 pathwaystocounteractinnateimmuneresponses.SomeCoVproteinsactasantagonistsofinterferon

14 Keywords: (IFN)byinhibitingIFNproductionorsignaling,aspectsthatarebrieflyaddressedinthisreview.After
15 Coronavirus CoVi nfe ction,pote ntc ytokinesre lev antincon trolling virus inf ections andprimin g adap tiveimm une
16 Virulence resp onsesare alsogen erated.H owever,a n uncontrolled indu ctionofth esep roinflam matoryc ytokines
1178Q3 SMAERRSS--CCooVV can lead t o pa thog enesis and disease se ve rity as describ ed for SA RS -CoV a nd MERS-CoV. Th e cellular
pathwaysmediatedbyinterferonregulatoryfactor(IRF)-3and-7,activatingtranscriptionfactor(ATF)-
1290 IInnnflaatme mimamtiounnity 2/jun, acti vator prot ein (AP)-1, nu clear facto r kapp a-light- chai n-e nhancer of activated B c ells (N F-(cid:2)B),
2212 IInnfltearmfemroantory cytokines aafntde rnvuicrlaelainr ffaeccttoiorn osf, awctitivhaNteFd-(cid:2) TB cpelaltsh (wNFa-yAtTh)e, amreo tshtefr meqauinen dtrliyvaecrsti ovfa ttheed .inKfleyamComVaptororyte riensspionnvsoelv teridgginerthede

23 Envelopeprotein regulationofthesepathwaysandtheproinflammatoryimmuneresponsearerevisitedinthismanuscript.
24 Ionchann el Ithasbe en show nthatthe enve lop e(E)proteinplays avariab leroleinC oV morphog en esis ,depending
2256 APDRDZ Sbinding motif oCnoV t shesu C cohVa sg eMnEuRs,S b-C eoinVg) ,abbus tonluotteilny oets hseenr sti(agle innu ss o(cid:4)mCeo cVa ssessu c(hgeans uMs H(cid:3) V C oorVSs A sRuSc-hC aosV T).GAEcVo, man pdre gheennussiv (cid:4)e

accumulationofdatahasshownthattherelativelysmallEproteinelicitsastronginfluenceonthe

interactionofSARS-CoVwiththehost.Infact,afterinfectionwithvirusesinwhichthisproteinhasbeen

deleted,increasedcellularstressandunfoldedproteinresponses,apoptosis,andaugmentedhostimmune

responseswereobserved.Incontrast,thepresenceofEproteinactivatedapathogenicinflammatory

responsethatmaycausedeathinanimalmodelsandinhumans.

ThemodificationordeletionofdifferentmotifswithinEprotein,includingthetransmembranedomain

thatharborsanionchannelactivity,smallsequenceswithinthemiddleregionofthecarboxy-terminus

ofEprotein,anditsmostcarboxy-terminalend,whichcontainsaPDZdomain-bindingmotif(PBM),is

sufficienttoattenuatethevirus.Interestingly,acomprehensivecollectionofSARS-CoVsinwhichthese

motifshavebeenmodifiedelicitedfullandlong-termprotectioneveninoldmice,makingthosedele-

tionmutantspromisingvaccinecandidates.Thesedataindicatethatdespiteitssmallsize,Eprotein

drasticallyinfluencesthereplicationofCoVsandtheirpathogenicity.AlthoughEproteinisnotessential

forCoVgenomereplicationorsubgenomicmRNAsynthesis,itaffectsvirusmorphogenesis,budding,

assembly,intracellulartrafficking,andvirulence.Infact,Eproteinisresponsibleinasignificantpropor-

tionoftheinflammasomeactivationandtheassociatedinflammationelicitedbySARS-CoVinthelung

parenchyma.Thisexacerbatedinflammationcausesedemaaccumulationleadingtoacuterespiratory

distresssyndrome(ARDS)and,frequently,tothedeathofinfectedanimalmodelsorhumanpatients.

©2014PublishedbyElsevierB.V.

∗
Correspondingauthorat:DepartmentofMolecularandCellBiology,CentroNacionaldeBiotecnología(CNB-CSIC),Darwin3,CampusUniversidadAutónomadeMadrid,

Q2 28049Madrid,Spain.Tel.:[phone];fax:[phone].

E-mailaddresses:[email protected],[email protected](L.Enjuanes).
1 Presen taddress: CenterforVaccineBiolo gyandImmunology,Unive rsit yofRochesterMedicalCenter,601ElmwoodAvenue,14642Rochester,NY,USA.

http://dx.doi.org/10.1016/j.virusres.2014.07.024
0168-1702/©2014PublishedbyElsevierB.V.

Pleasecitethisarticleinpressas:DeDiego,M.L.,etal.,CoronavirusvirulencegeneswithmainfocusonSARS-CoVenvelopegene.Virus

Res.(2014),http://dx.doi.org/10.1016/j.virusres.2014.07.024
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2 M.L.DeDiegoetal./VirusResearchxxx(2014)xxx–xxx

28 1. Introduction pathogen-associatedmolecularpatterns(PAMPs)inthecellcyto- 91

plasm.Ontheotherhand,tolllikereceptorsdetectPAMPsinthe 92

29Q4 Anoverviewofthesensorsdetectingvirusinfectionispresented cellsurfaceandinendosomalcompartments. 93

30 first,followedbyadescriptionofthemechanismselicitedbyCoV TheRNAhelicases-dependentcytoplasmicIFNinductionpath- 94

31 proteinstocounteractinnateimmuneresponses.SomeCoVpro- waysusetheadaptormoleculemitochondrialantiviralsignaling 95

32 teins act as antagonists of interferon (IFN) production, whereas protein(MAVS)(Fig.1).MAVSpromotestheactivationofacomplex 96

33 othersinhibitIFNsignaling.Asaconsequence,acollectionofpotent comprisingtheproteinsTNFreceptor-associatedfactor3(TRAF-3), 97
34 cytokin esrele van tincontro llin g virusinfectio ns andprim ing adap- TRAF family m ember-as soci ated NF-(cid:2)B activato r (TAN K ), TANK- 98
35 tiveimmu nerespo ns esaregene rated (LeBonan dT ough,20 02). bindi ngkina se1(TBK-1)andIkap paBkin ase(cid:5)(IKK (cid:5)).Activ eTBK1 99
36 V iruspath ogenesisis fre quentlyass oci ated with anexa cerbated andIKK(cid:5) direct ly phosph oryla tethetr anscrip ti onfacto rsIRF- 3and 100

37 inductionofproinflammatorycytokinesthatismainlydrivenbythe IRF-7, promoting homodimerization (Sharma et al., 2003). Then, 101

38 activationofatleastoneofthefollowingfivepathways:IRF-3and- theIRF-3andIRF-7dimersareimportedintothenucleus,leading 102
39 7,ATF-2/ju n, ju n/fos (AP- 1) ,NF -(cid:2)BandNF -AT .Amongthe m,th eNF - toIR F-3a ndIR F-7-d epende nttr anscriptio n.In add ition,MA VStrig- 103
40 (cid:2)B pathway isthem ostfre quentl yac tivated (Hatad aetal .,20 00; ge rsthe NF- (cid:2)Bpathwaythrou ghIKK(cid:3)andI KK (cid:4)activat ion(K awai 104
41 Mo gensenan d Palu dan,2 001).NF-(cid:2) Bisahete rogeneo us coll ection and Akir a,2007 ). 105

42 ofdimers,composedofvariouscombinationsofmembersoftheRel The TLRs-dependent IFN induction pathways use the adaptor 106
43 fam ily,wh ichineuk ar yotesin cludep50(NF -(cid:2) B1),p52( NF -(cid:2)B 2), molecu les TIR-domain-c onta ining ada pter-induc ing IFN- (cid:4) (TRIF) 107

44 Rel(c-Rel),p65(RelA)andRelB.Anexacerbatedimmuneresponse andmyeloid-differentiationprimaryresponse88(MyD88)(Fig.1) 108

45 andaweakIFNresponsehavebeenassociatedwithvirulentCoVs (Kawai and Akira, 2007). TRIF-dependent pathway leads to the 109
46 such a sSAR S-Co VandME RS-Co V(Ba asetal.,20 08;La uetal.,2 013; activati ono fIRF-3 and-7, andNF-(cid:2)B.The activation ofIRF -3 and 110

47 Smitsetal.,2010). IRF-7ismediatedbythephosphorylationofthesefactorsbyTBK- 111
48 Th em ain focusofthisreviewistheanalysisoftheroleofthe 1 and IK K(cid:5), whic h p rom ote their activati on , as d escribed a bove. 112
49 CoVen velope (E)p rot eini nvirus pa thog enesis.E p rote inco nta ins TR IF a lso m ediates NF-(cid:2)B a ctiva tion throug h the activa tion of 113
50 seve ral active mo tifs des pit e its small size, bet w een 76 and 109 IKK(cid:3) and IKK(cid:4).MyD 88-med iatedpath wayactiv ates thetranscr ip- 114
51 aminoa cidsde pendin gonthe Co V.The mod ificationo rde letio nof tionf acto rsNF- (cid:2)B,AP-1andATF- 2/jun,th roughthe act ivationof 115

52 EproteinindifferentCoVshasledtoviruseswithdifferentphe- mitogen-activated protein kinases (MAPKs) (Herlaar and Brown, 116
53 n otypesa nd uniquea lterat ion ofv iru s–hosti ntera ctions,su chas 1999;Whitmarsha ndDavi s,1996) .NF-(cid:2)Bis alsoacti vated inthis 117

54 theinductionofstressandunfoldedproteinresponses,orchanges pathwaythroughIKKs(KawaiandAkira,2007). 118

55 incellularionconcentrationsduetotheionchannelactivityofE IRF-3andIRF-7,withthehelpofothertranscriptionfactorslike 119
56 pr otein.Al lthe seactivitiesha vehi gh imp act onCoVp athogen es is NF-(cid:2)B,a ndA P-1,in itiat eth etra ns criptio nofIFN-(cid:4)an dselec ted 120
57 (DeDieg oet al.,20 11;Nieto -Torr eset al.,201 4). IFN-(cid:3)g enes .IFN- (cid:3)andIF N-(cid:4) proteinsaret he nsecre ted fromthe 121

58 E protein PDZ-binding motif (PBM), which during SARS-CoV cell and can act in either an autocrine or a paracrine fashion to 122

59 infection could potentially target more than 400 cellular PDZ amplifytheIFNresponse(Fig.1). 123

60 motifspresentwithincellularproteins,conferstoEproteinvirus CoVs have devised a number of cell type-specific strategies 124

61 pathogenicity modulating properties. Interestingly, deletion or to inhibit type I IFN production (Table 1; Fig. 1). These viruses 125
62 modification o f E protein PBM and in ternal regions within the en codea2 (cid:3)-O-m e thy lase(non-str uctura lpr otei nns p16)t hatcre- 126
63 carboxy-term inu s of E pro tein most frequen tly resul ts in at ten- atesa5 (cid:3)-c apstructureana logoustothece llularmR NAson the viral 127

64 uatedCoVsthataregoodvaccinecandidates(Jimenez-Guarden˜o mRNAs, thereby escaping detection by MDA5 (Zust et al., 2011). 128

65 etal.,2014;Regla-Navaetal.,2014).Inaddition,theidentification MERS-CoV accessory protein 4a is a dsRNA binding protein that 129
66 of sig naling pathways,s uc has NF-(cid:2)B -m ediateds ign aling,respon- blocks IFN induction by supp res sin g PACT-i nduced activatio n of 130

67 sible for CoV pathogenicity has led to the selection of antivirals RIG-IandMDA5(Niemeyeretal.,2013;Siuetal.,2014).TheORF4b 131

68 thatconsiderablyincreasethesurvivalofinfectedanimalmodels encodedaccessoryproteinsofMERS-CoVandtworelatedbatCoVs 132

69 (DeDiegoetal.,2014). localizetothecellnucleusandinhibittypeIIFNproductionand 133
NF-(cid:2)Bs ign alin gpa thway(M att hewset al.,2 0 14) .Interesting ly,a 134

70 1.1. CoronavirusproteinsinhibitingtypeIinterferonproduction MERS-CoV lacking 4a and 4b proteins grew about 10-fold lower 135

thantheparentalvirusinIFNcompetentinfected-cells(Almazan 136

71 IFNsarepotentcytokinesrelevantinthecontrolofvirusinfec- et al., 2013). However, the specific effect of 4a and 4b proteins 137

72 tionsandintheprimingofadaptiveimmuneresponses(LeBonand IFNantagonisticactivityinvirusgrowthandvirulencestillneeds 138

73 Tough,2002).TreatmentwithtypeIIFNinhibitsCoVgrowthintis- to be determined. SARS-CoV membrane (M) protein impairs the 139
74 suecul turean dinanima lmo dels s uch ascyno molg usmac aq ues for ma tion of TRAF 3/TANK/TB K1/IKK(cid:5) co mpl ex, inhib iting IF N-(cid:4) 140

75 andmice(Barnardetal.,2006;Dahletal.,2004;Fuchizakietal., production (Siu et al., 2009). SARS-CoV structural nucleocapsid 141
76 200 3;Haa gmanset al .,20 04;Ku maki et al., 2011; Mahlakoiv et al., (N) protein bloc ks IFN- (cid:4) prod uction afte r induction with Sendai 142

77 2012;Sainzetal.,2004;Stroheretal.,2004;Zhengetal.,2004). virusandpolyI:C,butnotupstreamofcomponentssuchasRIG- 143
78 To cir cumve nt the inhib ition of vir us replica tion, m an y v iruses, I,MD A5,M AVS,IK K(cid:5), TBK 1orTRIF,i nd icatingthatN prot ein acts 144

79 includingCoVs,encodeviralproteinsinhibitingIFNproductionor afterthesesignalingmediators(Kopecky-Brombergetal.,2007;Lu 145

80 signaling (Table 1). However, most of the studies describing the etal.,2011).SARS-CoVpapain-likeprotease(PLP)domainofnsp3 146
81 IFNantag onistac tiv ityofcoron aviru s-e nco dedprot einshaveb een in hibi ts RIG- I and TLR3 -dependen t IFN-(cid:4) p roduc tion, be ing this 147

82 conductedincellstransientlyexpressingtheviralproteins.There- activityindependentofthedeubiquitinatingandproteaseactivi- 148

83 fore,additionalanalysesinthecontextofthevirusinfectionare ties (Clementz et al., 2010), and most probably mediated by the 149

84 required. interactionofPLPdomainwiththeproteinstimulatorofIFNgenes 150

85 Type I IFN production is controlled by two major path- (STING),whichisaproteinthatstimulatesphosphorylationofIRF3 151

86 ways dependent on RNA helicases or toll-like receptors (TLRs) bythekinaseTBK1(Sunetal.,2012).TheinhibitionofIFNproduc- 152

87 (Arpaia and Barton, 2011; Rathinam and Fitzgerald, 2011; tionhasalsobeendescribedfornsp3PLP2ofHCoV-NL63(Clementz 153

88 Sen, 2001) (Fig. 1). RNA helicases containing the cytoplas- etal.,2010;Sunetal.,2012),MHV(Wangetal.,2011;Zhengetal., 154

89 mic CARD domain, retinoic acid-inducible gene 1 (RIG-I) and 2008),andforthePLPdomainofMERS-CoV,whichblocksIFNpro- 155

90 melanoma differentiation-associated protein 5 (MDA5), sense ductionbyinhibitingIRF3phosphorylationandtranslocationinto 156

Pleasecitethisarticleinpressas:DeDiego,M.L.,etal.,CoronavirusvirulencegeneswithmainfocusonSARS-CoVenvelopegene.Virus

Res.(2014),http://dx.doi.org/10.1016/j.virusres.2014.07.024
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Table1
Q7 Coron avirusproteinsaffectinginnateimmuneresponses.

Protein CoV Immunefunction References

Nsp1 SARS-CoV AntagonizestypeIIFNproductionandsignalingbyinducinghostmRNAs Watheletetal.(2007),Kamitanietal.

shutoff,promotingthedegradationofhostmRNAsandpreventing (2009),Huangetal.(2011),Tanaka

phosphorylationofSTAT1 etal.(2012)

UpregulatesCCL5,CXCL10,andCCL3inhumanlungepithelialcellsviathe Lawetal.(2007)
activationof NF-(cid:2)B

Nsp3 SARS-CoV PreventsIFNproductionbyblockingIRF3phosphorylation,mostprobably Devarajetal.(2007),Friemanetal.

byinteractingwithSTING (2009),Sunetal.(2012),Clementz

etal.(2010)

MHV AntagonizestypeIIFN Zhengetal.(2008),Wangetal.(2011)

MERS-CoV AntagonizestypeIIFN Yangetal.(2014)

Nsp7 SARS-CoV AntagonizestypeIIFN Friemanetal.(2009)

Nsp15 SARS-CoV AntagonizestypeIIFN Friemanetal.(2009)
S SARS-CoV Inducesthee xpre s sion ofIL6,IL8,CXCL10andTNFthroughNF-(cid:2)B Wanget al. (2 007),Doschetal.(2009)

activationinmacrophages
M SARS-CoV BlocksIFN -(cid:4) productionbyimpairingtheformationof Siuetal.(2009)
TRAF3– TANK –TBK1/IKK(cid:5) co mplex

N SARS-CoV AntagonizestypeIIFNproductionbyblockingIRF-3phosphorylation Luetal.(2011),Kopecky-Bromberg

etal.(2007)
ActivatesNF-(cid:2)BandupregulatestheexpressionofIL-6 Lia oe tal.(2005),Zhangetal.(2007)

ActivatesAP-1 Heetal.(2003)

InducestheexpressionofIL8viaAP-1activation Changetal.(2004)

3a SARS-CoV DownregulatestheexpressionofthetypeIIFNreceptor(IFNAR),leading Minakshietal.(2009)

toablockadeontypeIIFNsignaling
Inc r easesNF- (cid:2)B andJ N Kac tivityandupregulatesTNF,IL8andCCL5 Obitsuetal.(2009),Kanzawaetal.

production (2006)
3b SARS-CoV AntagonizestypeIIFNproductionbyblockingIRF-3phosphorylation. Kopecky-Brombergetal.(2007),

InhibitsIFNsignaling Freundtetal.(2009)

InducestranscriptionalactivityofAP-1,throughactivationofJNKandERK VarshneyandLal(2011),Varshney

pathways,leadingtoCCL2upregulation etal.(2012)

6 SARS-CoV AntagonizestypeIIFNproductionbyblockingIRF-3phosphorylation Kopecky-Brombergetal.(2007),

Friemanetal.(2009)

InhibitsIFNsignalingbyblockingthenucleartranslocationofthe Friemanetal.(2007)

transcriptionfactorSTAT1
7a SARS-CoV ActivatesNF- (cid:2)Band upregulatestheexpressionoftheproinflammatory Kanzawaetal.(2006)

mediatorsIL8andCCL5

Nsp3 NL63 AntagonizestypeIIFN Clementzetal.(2010)

Nsp1 MHV AntagonizestypeIIFN Zustetal.(2007)

N MHV ActsasaninterferonantagonistandpreventsRNAdegradationby Yeetal.(2007)

inhibitingRNaseLactivity

2 MHV AntagonizestypeIIFNsignalingandpreventsactivationofthecellular Zhaoetal.(2011,2012)

endoribonucleaseRNaseL

5a MHV AntagonizestypeIIFN Koetzneretal.(2010)

4a MERS-CoV BlockinterferoninductionatthelevelofMDA5activationpresumablyby Niemeyeretal.(2013)

directinteractionwithdouble-strandedRNA

4b MERS-CoV AntagonizestypeIIFN Matthewsetal.(2014)

7 TGEV Reducestheexpressionofgenesinvolvedintheimmuneresponse,the Cruzetal.(2013)

interferonresponse,andinflammation

7a FIPV AntagonizestypeIIFN Dedeurwaerderetal.(2013)

157 thenucleus(Yangetal.,2014).SARS-CoVnsp7andnsp15blockIFN- IRF-9,toformtheIFNstimulatedgenefactor3(ISGF3)complex. 176
158 (cid:4)p roductio nthro ug ha nunid entifiedm echa nism (Friem ane tal., The IS GF 3 het erot rime r transloca tes to the n uc leus and triggers 177

159 2009).SARS-CoVproteinsN,3band6preventIFNproductionby thetranscriptionofIFN-stimulatedgenes(ISGs)thatwilldrivethe 178

160 blockingIRF-3phosphorylation(Devarajetal.,2007;Freundtetal., antiviralresponse. 179

161 2009;Friemanetal.,2009;Kopecky-Brombergetal.,2007). CoronaviruseshavedevelopedstrategiestointerferewithIFN 180

162 TGEV protein 7 inhibits IFN production as it has been shown signaling at different levels. SARS-CoV affects the initial stages 181

163 that a TGEV lacking protein 7 grew with similar titers than the of the cascade by down regulating the expression of IFNAR, and 182

164 wtvirus,butinducedexpressionofgenesinvolvedintheimmune inhibiting the translocation of STAT1 to the nucleus, through 183

165 responseandinterferonresponsetoahigherextentthanthewt proteins 3a and 6, respectively (Frieman et al., 2007; Kopecky- 184

166 virus(Cruzetal.,2013).Inaddition,protein7preventshosttrans- Brombergetal.,2007;Minakshietal.,2009).Inaddition,SARS-CoV 185

167 lationalshutoffandRNAdegradationthroughtheinteractionwith nsp1 affects STAT1 phosphorylation and induces a host trans- 186

168 proteinphosphatase1(PP1)(Fig.1)(Cruzetal.,2011). lational shut off promoting the degradation of cellular mRNAs, 187

furtheraffectingIFNantiviralsignaling(Huangetal.,2011;Jauregui 188

etal.,2013;Kamitanietal.,2009;Tanakaetal.,2012;Watheletetal., 189
169 1.2. CoV proteins inhibiting type I IFN signaling 20 07; Zust etal.,200 7) .SA RS-Co Vprot ein 3b inhib itsIFNsi gn al- 190

ingwithoutinhibitingSTAT1phosphorylation(Kopecky-Bromberg 191
170 Type I IFN signaling starts with its binding to IFNAR receptors eta l.,2007). SARS-CoV nsp1a ntagonizestype IIFNproductionand 192
171 at the cell surface, which leads to the activation of the JAK–STAT sig na lingby inducingh ostm RNAsshut off,p r omo tingthede gra- 193
172 pathway (Samuel, 2001) (Fig. 2). The members of the Janus Kinase dationof cell ularmRN Asan dpreve nting pho sphorylatio nof STAT1 194
173 (JAK) family JAK-1 and protein tyrosine kinase 2 (TYK-2) phospho- (Huang e t al., 20 11; Kam ita ni et al., 20 09; Tanaka et a l., 2012; 195
174 rylate the signal transducer and activators of transcription (STATs) Wathele te tal .,2007 ;Zusteta l.,2 007 ).Inhib itionof do wns tream 196
175 whichbecomeactivated.PhosphorylatedSTAT1andSTAT2recruit

Pleasecitethisarticleinpressas:DeDiego,M.L.,etal.,CoronavirusvirulencegeneswithmainfocusonSARS-CoVenvelopegene.Virus

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Fig.1. Effectofcoronavirusproteinsoncellularsignalingpathwaysassociatedwiththeinnateimmuneresponse.PAMPssuchasssRNA,dsRNA,orviralproteins,trigger
the act ivation o ftranscriptio nfactors lea dingto proinflam matorycy tokineand type IIF Nindu ction.PA MPsactiva tetheP KR,l ead ingto eIF2(cid:3)p hos phor ylationan dhost
tran slationinh ib ition,and2(cid:3)–5 (cid:3)OAS,l eadingto R NaseLtriggeringa ndRNAd egra datio n .Th eactivation ofRIG -IandMD A-5 trigg ersthe act ivation ofIRF-3,IRF-7a ndN F-(cid:2)B
throughMA VS.Inadd ition ,TLRsacti vatethe M yD88a n dTRIF-dep end entp athways,act ivat ingthetra nsc riptio nfa ctorsIR F-3,IRF- 7,N F-(cid:2)B,and AP -1.Th estep sinh ibited

orpromotedbyCoVproteinsareindicatedinredboxes.Besidetheseproteins,otherproteinsthatinhibitorpromotetheIFNsignalingandproductionandinflammatory

cytokineexpression,throughanidentifiedmechanism,areindicatedinTable1.

197 effectorsofIFNsignalingpathway(ISGs)hasalsobeendescribed 1.3. Coronavirusproteinsaffectingtheinductionof 211

198 duringcoronavirusinfection.MHVNandns2aswellasSARS-CoV proinflammatorysignals 212

199 N proteins prevent the activation of RNase L, blocking viral RNA

200 degradation(Fig.1)(Yeetal.,2007;Zhaoetal.,2012). Proinflammatory cytokines and chemokines are a part of the 213

201 Other CoV proteins confer IFN-resistance, however, whether necessary initial immune response to pathogens. However, an 214

202 theyinhibitIFNproductionorsignalingisunknown.MHVnsp1is exacerbatedimmuneresponsehasbeenassociatedwiththehigh 215

203 anefficientinterferonantagonistinmice,asreplicationandspread virulenceofSARS-CoV(Baasetal.,2008;Smitsetal.,2010).Expres- 216

204 ofannsp1mutantviruswererestoredalmosttowild-typelevels sionlevelsofproinflammatorycytokines,suchasIL-1,IL-2,IL-6, 217

205 in type I IFN receptor-deficient animals (Zust et al., 2007). MHV andIL-8,andchemokinessuchasCXCL10andCCL2areelevatedin 218
206 pr otein 5 aor itshomologuesfro mrelate dgenu s (cid:4)co ronavi ruses, peri phera lbl oodandlung sof SA RSpatie nts, anda sso ciatedwi th 219

207 conferIFN-resistancetothevirus(Koetzneretal.,2010).FIPV7a diseaseseverity(Cameronetal.,2007;Chienetal.,2006;Jiangetal., 220

208 proteinprotectsthevirusfromtheantiviralstateinducedbyIFN, 2005;Tangetal.,2005;Wongetal.,2004). 221

209 but it needs the presence of ORF3 encoded proteins to exert its Themostimportantsignaltransductionpathwaysactivatedby 222

210 antagonisticfunction(Dedeurwaerderetal.,2013). virusesleadingtotheexpressionofproinflammatorycytokinesare 223

Pleasecitethisarticleinpressas:DeDiego,M.L.,etal.,CoronavirusvirulencegeneswithmainfocusonSARS-CoVenvelopegene.Virus

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via AP-1 in lung epithelial cells (Chang et al., 2004). The acces- 252

sory protein 3a upregulates mRNA and fibrinogen levels in lung 253

epithelialcells(Tanetal.,2005).Inaddition,3aproteinincreases 254
NF-(cid:2)B an d JNK acti vit ies and up reg ulates th e T NF, IL8 and CCL5 255

productioninmurinemacrophagesandlungcelllines(Kanzawa 256

et al., 2006; Obitsu et al., 2009). Similarly, SARS-CoV 7a protein 257
als oa ctivate sNF-(cid:2)B an du pregula testheex pressionof th eproin- 258

flammatorymediatorsIL8andCCL5inalungcellline(Kanzawa 259

etal.,2006).Theaccessoryprotein3binducestranscriptionalactiv- 260

ityofAP-1,throughactivationofJNKandERKpathways,leadingto 261

CCL2upregulationinahumanhepatomacellline(Varshneyetal., 262

2012;VarshneyandLal,2011).Thepresenceofprotein7inTGEV 263

reducedtheexpressionofproinflammatorygenes,comparedtoa 264

viruslackingthisprotein,indicatingthatTGEVprotein7inhibits 265

proinflammatorycytokineexpression(Cruzetal.,2013). 266

In summary, several structural and non-structural SARS-CoV 267

proteinsaffecttheexpressionofproinflammatorysignals,mostfre- 268
quentlyb ymo dula tingtheNF -(cid:2) Bpathwayand,t oalowe rext ent, 269

byaffectingAP-1signaling. 270

1.4. Virulenceofrecombinantcoronaviruseslackingspecificviral 271

proteins 272
The generation of viral mutants lacking specific proteins or 273

domains, or containing point mutations is an invaluable tool to 274

studythecontributionofaparticularproteintothevirulenceofthe 275

virus.Thesesystemsofferadvantagesincomparisontooverexpres- 276

sionsystemsbecauseinthiscasethestudiesareperformedinthe 277

contextofinfection,inthepresenceoftheotherviralproteins,in 278

ascenarioinwhichtheonlydifferenceisthepresenceofasingle 279

mutatedordeletedviralprotein.Incontrast,theover-expressionof 280

specificproteinsfrequentlyyieldsoverwhelmingamountsofpro- 281
(cid:4)Figp.r o2t. eEinffsecatr eofs eccorreotneadvfirroums ptrhoetecienlsl aonnd tyapmep Il iIfFyNt hseigInFaNlinregs. pTohnes eIFaNc-t(cid:3)iv aatnindg IFtNhe- tein tha t may res ult toxic to t he viru s–host cell inte raction re qu ired 282
IS GF3comp lex formedb ySTA T1, STA T2an dIRF-9, lead ing totheexp ressionof the for a balanced virus replication. 283
interfe ron-stim ulatedg en es(ISG) .Thest eps inhibit edorpr om ote dbyCoVpr ote ins TostudytheroleofSARS-CoVgroupspecificprotein6during 284
areindicatedinredbo xes. viral i nfectio n t wo a pp roaches ha ve bee n used. In one of them, 285

SARS-CoVprotein6hasbeenexpressedinthecontextofanatten- 286

uatedmousehepatitisvirus(MHV).Thisrecombinantvirusgrew 287
224 mediatedbyfactorsIRF-3and-7,ATF-2/jun,AP-1,NF-(cid:2)BandNF- more rapidly andtohig hert itersinc ellc ultureandin them urine 288

225 AT(MogensenandPaludan,2001).Theactivationofthesefactors centralnervoussystemthanthecontrolvirus,leadingtoincreased 289

226 hasbeenbrieflydescribedabove.NF-ATisconstitutivelypresent mortalityinmice(Hussainetal.,2008;Netlandetal.,2008;Pewe 290

227 inthecytoplasminalatentphosphorylatedform.Increasinglevels etal.,2005).Intheotherapproach,aSARS-CoVlackingprotein6 291

228 ofcytoplasmiccalciumactivatethecalmodulin-dependentphos- wasengineered.TheSARS-CoVdeletionmutantgrewwithlower 292

229 phatase calcineurin that activates NF-AT by dephosphorylation titersbutessentiallymaintaineditsvirulenceintransgenicmice 293

230 (Crabtree,1999). expressingthehumanreceptorforSARS-CoVhACE-2,asitkilled 294
231 Activat ionofNF-(cid:2)Bisahallmarkofmostinfectionsincluding 100%ofthe mic ewitha delayof1 da y(Zhaoeta l.,2009). Th es edata 295

232 viral infections, leading to pathological outcomes. In fact, SARS- indicatedthatSARS-CoVprotein6,inthecontextoftheinfectionby 296

233 CoV-infected-aged macaques develop a more severe pathology, thevirusofwhichitisastructuralcomponent(Huangetal.,2007), 297

234 withanincreaseindifferentialexpressionofgenesassociatedwith doesnotseemtohaveahighinfluenceonSARS-CoVvirulence. 298
235 inflam m ation,w ith NF-(cid:2)Basa centralpl ay er,and areductio nin In fect ion of im mun e supp ressed h am sters with recombinant 299
236 theexpression ofty peIIFN -(cid:4) (S mitset al.,201 0). SARS-CoVvi rus esbearin gdisruption sinthege ne7c odingregion 300

237 SeveralCoV-encodedproteinsinterferewiththeproductionof showednosignificantchangesinreplication,tissuetropism,mor- 301

238 inflammatorymediators.SARS-CoVnsp1playsanimportantrole bidity,ormortalitysuggestingthatthe7aand7bproteinsarenot 302

239 inCCL5,CXCL10,andCCL3upregulationinhumanlungepithelial essentialforviruspathogenesis(Schaecheretal.,2008).Deletion 303
240 ce llsvia theactiv atio nofN F-(cid:2)B(Lawet al .,2007) .The nsp3PLP ofeachof gen es3a ,6,7a,and7bf romSARS-C oV did notaf fectvirus 304
241 dom ain disru ptsNF-(cid:2)B sig naling ,mos tp roba blyby inhi biting the gr owth in mice to ah igh ext en t(You ntetal.,2 005 ).A SARS -CoV 305
242 degrada tionofph ospho rylatedI(cid:2)B -(cid:3),w hichdimi nis hestheind uc- lacking the grou ps pe cific genes6 ,7a,7b ,8a ,8b ,and9 bg rewsimi- 306

243 tion of proinflammatory cytokines, leading to virus attenuation larlytotheparentalvirusandinducedaslightlydiminishedweight 307

244 (Friemanetal.,2009).ThestructuralSARS-CoVNproteinactivates lossandadelayinthetimeofdeathintransgenicmiceexpress- 308
245 NF-(cid:2)B-dr ive nt ranscri ptio nandupre gulatesthe e xpressio nofIL-6 ing hACE -2 ,whi ch are highl ys uscept ibl etothedis ease( DeDiego 309
246 byfacilitating thetransloca tion ofNF-(cid:2)Bfro mc ytosolton uc leus eta l.,2008) .Althou gh further analysisus ing oth eranim almodels 310

247 (Liaoetal.,2005;Zhangetal.,2007).Inaddition,theexpressionof shouldbeperformed,thesedatasuggestedthatthecontribution 311

248 Nprotein,butnottheMprotein,activatestheAP-1pathway(He ofproteins6,7a,7b,8a,8band9btothevirulenceofSARS-CoVis 312

249 etal.,2003).Similarly,SARS-CoVSproteininducestheexpressionof limited. 313
250 TN F,I L6,IL8 ,andCXCL 10through N F-(cid:2)Ba ctivation in macrophag es A recombinant MHV with a deletion in nsp1 (a homolog of 314

251 (Doschetal.,2009;Wangetal.,2007),andtheexpressionofIL-8 SARS-CoVnsp1)grewnormallyintissueculture,butwasseverely 315

Pleasecitethisarticleinpressas:DeDiego,M.L.,etal.,CoronavirusvirulencegeneswithmainfocusonSARS-CoVenvelopegene.Virus

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316 attenuatedinvivo.Interestingly,replicationandspreadofthensp1 pergramoftissue,inVeroE6cellsorinlungsofinfectedBALB/c 379

317 deletionmutantviruswasrestoredalmosttowild-typelevelsin mice,respectively,inareproduciblefashion(DeDiegoetal.,2007, 380

318 typeIIFNreceptor-deficientmice,indicatingthatnsp1interferes 2008; Fett et al., 2013). Nevertheless, the presence of E protein 381

319 efficiently with the type I IFN system in vivo (Zust et al., 2007). optimizesSARS-CoVyields.ThecontributionofEproteintoCoV 382

320 Similarly,amutantviruslackingaconserveddomainofMHVnsp1, morphogenesis could be mediated through its interaction with 383

321 showednogrowthdefectsincellculture,butwashighlyattenuated other virus structural proteins within the virus envelope (M, 3a, 384

322 invivo(Leietal.,2013). 3b,6,7b,and9b)(Arndtetal.,2010;Boscarinoetal.,2008;Chen 385

323 Deletion of group specific proteins ns2, HE, 4ab, and 5a from etal.,2009;Neumanetal.,2008;Panetal.,2008;vonBrunnetal., 386

324 MHV led to attenuated viruses in the natural host, the mice (de 2007).Eproteinhasthreepotentialpalmitoylationresiduesinits 387

325 Haanetal.,2002).AMHVmutantmissingproteinns2wasunable carboxy-terminus.Thepalmitoylationofthesesitesisessentialfor 388

326 toreplicateintheliverortoinducehepatitisinwild-typemice,but theformationofvesiclesincludingEproteinthatcontributetoCoV 389

327 was highly pathogenic in RNase L deficient mice, indicating that morphogenesis(Boscarinoetal.,2008;Lopezetal.,2008).Also,the 390

328 proteinns2increasesthepathogenicityofthevirusbyanRNaseL extent of E protein palmitoylation affects its interaction with M 391

329 dependentmechanism(Zhaoetal.,2011,2012). protein(Boscarinoetal.,2008). 392
330 Genus(cid:3) CoVssucha sTGEV m issi nggen e7,orFIPVlackingatthe Thep resenceof Ep rot eininCoVsparticlesisverylowingeneral 393

331 sametimegenes3abcand7abshowedmodificationoftheinflam- (around20moleculespervirion)(Godetetal.,1992),althoughthis 394

332 matory response and virulence. TGEV 7 protein deletion mutant couldvarydependingonthespecies(LiuandInglis,1991).Inter- 395

333 increased proinflammatory responses and acute tissue damage estingly,Eproteinishighlyabundantinthecytoplasmofinfected 396

334 afterinfection,leadingtoamorepathogenicvirus(Cruzetal.,2011, cells,whatmaybeduetoitsroleinvirustransportandmorpho- 397

335 2013).Incontrast,FIPVdeletionmutantwasattenuatedincatsand genesis(Ortegoetal.,2007).Aroleinintracellulartraffickinghas 398

336 induced protection against feline infectious peritonitis (Haijema beendescribedforCoVEprotein.ThehydrophobicdomainofIBV 399

337 etal.,2004).InthiscasetheeffectofFIPVproteins3abcor7bdele- Eproteinseemsimportantfortheforwardtraffickingofcargoto 400

338 tiononitsvirulenceprevailedoverthedeletionofFIPVprotein7a, theplasmamembrane.Infact,Eproteinaltersthehostsecretory 401

339 whichistheproteinequivalenttoTGEVprotein7. pathwaytotheapparentadvantageofthevirus,increasingtheeffi- 402

cacyofinfectiousvirusrelease(RuchandMachamer,2011,2012). 403

ThereforeEproteinseemstoplayaroleinvirusegress. 404

340 2. RequirementofcoronavirusEproteinincoronavirus E protein oligomerizes and forms ion channels that influence 405

341 replicationandmorphogenesis theelectrochemicalbalanceinsomesubcellularcompartmentsof 406

hostcells,asdescribedbelow. 407

342 CoVEproteinismultifunctional,affectingseveralstepsofthe

343 viral cycle. SARS-CoV can infect mouse brain, whereas in the

344 absence of E protein this tissue tropism has not been observed 3. EffectofSARS-CoVEgenedeletiononviralpathogenesis 408

345 (DeDiegoetal.,2008).However,inthiscase,theinvolvementofE

346 proteininentryisnotnecessarilyrequiredtoexplaintheobserved To study the effect of SARS-CoV E protein on viral pathogen- 409
347 differen ce ,asth e rest rictioncoul doperat ea talater ste p.Epro- esis, a SARS -CoV lacki ng the full-le ng th E gen e ( rSAR S-CoV-(cid:2)E) 410

348 teinexpressionisnotinvolvedinCoVgenomereplication,asboth wasengineered.ThedeletedviruswasattenuatedingoldenSyrian 411

349 SARS-CoVwithandwithoutEproteinsynthesizethesameamounts hamsters,andintransgenicmiceexpressingtheSARS-CoVreceptor 412

350 ofgenomicandsubgenomicmRNAs(DeDiegoetal.,2011). hACE-2(DeDiegoetal.,2007,2008).Inaddition,amouseadapted 413
351 Therequ irem entofEpro teinin CoVmorp ho gen esishasbeen SARS-Co Vlacking E gen e(rSA RS-Co V-M A15-(cid:2)E ) wasatt enuated 414

352 underdebate.Infact,Eproteinseemsnecessaryforviruslikepar- inconventionalyoungandagedBALB/cmice(DeDiegoetal.,2014; 415

353 ticleformationusingsomeexperimentalsystems(Hoetal.,2004; Fettetal.,2013),indicatingthattheexpressionofEgeneincreases 416
354 Mort olaandRo y,2004 )but notothers(Hu angetal. ,200 4). Dif ferent viru sp ath ogenici ty.rSARS-C oV- (cid:2)Et itersdecrea se d inviv o,incom- 417

355 CoVshaveshownvariablerequirementsforEproteinduringmor- parisontoparentalvirustiters.However,intrinsicpropertiesofE 418

356 phogenesis,resultinginthreedifferentphenotypes.Oneofthem protein,andnotjustadecreaseinvirustiters,mayincreasetheviral 419
357 isshownby genus(cid:3) cor onavi ruses,like TGEV,anda lsob y genus pathoge nesi s.In fac t, viralmu tan tsla ckingE pro teinion cha nnel 420
358 (cid:4) MERS-C oV , whic h in the absence of E prote in ar e re plic ation- activity and P BM , gro w si milarly to the w t v irus, an d n everthe- 421

359 competent propagation-defective viruses (Almazan et al., 2013; lessareattenuated(seeSections5and6)(Jimenez-Guarden˜oetal., 422

360 Curtis et al., 2002; Ortego et al., 2002, 2007). Both TGEV and 2014;Nieto-Torresetal.,2014). 423
361 MERS- Co Vm issing Eprotei nw ere propa gatedin pack aging cells To identifymech an ism sleadingtorSARS-CoV-(cid:2)Eattenuation, 424

362 byprovidingEproteinintrans,leadingtohighvirustiters.Inthis geneexpressionwascomparedincellsinfectedwiththeattenu- 425
363 cas e,theleve lo frecove re dvirus eswasp ro porti onalt othea mo unt ated (cid:2)Evirusan dinw tvirus-in fec tedc ells.Stres sresp ons egenes 426

364 ofEproteinprovidedbythepackagingcellline(Ortegoetal.,2002). were preferentially upregulated during infection in the absence 427

365 AsecondphenotypeofCoVsmissingEprotein,isrepresentedby ofEgene.Interestingly,expressionofEproteinintransreduced 428
366 ge nus(cid:4)M HV,with ar educt ionofvi ru stitersh ig herthan100 0- th e stress responseinc ellsinfected w it hrSARS -Co V-(cid:2)E orwith 429

367 fold(KuoandMasters,2003).Thethirdphenotypewasobserved respiratorysyncytialvirus,orincellstreatedwithdrugs,suchas 430
368 forg enus (cid:4)SA RS-CoV,i nwhich del etion mutantsmis sing Eprotein tunicamyci n and tha psigar gin , t hat e licit cel l stre ss by d iffere nt 431

369 onlyreducedtheirreplicationbetween20and200-fold,leadingto mechanisms(DeDiegoetal.,2011).Inaddition,SARS-CoVEprotein 432

370 virusesthatreplicatebothincellcultureandinvivo,anddisplayan down-regulatedthesignalingpathwayinositol-requiringenzyme 433

371 attenuatedphenotype(DeDiegoetal.,2007,2008,2014;Enjuanes 1(IRE-1)oftheunfoldedproteinresponse,andlimitedcellapo- 434

372 et al., 2008). The assembled viral particles could be the base for ptosis. The expression of proinflammatory cytokines was lower 435
373 sa fev accine cand idates,once add itionalsa fetygu ard sha veb een inrSAR S-Co V-(cid:2)E-infec ted cellscomparedt orSARS-Co V-in fected 436

374 incorporatedatadistalpositionintheCoVgenome. ones,suggestingthattheincreaseinstressresponsesandthereduc- 437

375 WhereasthedeletionofEproteinindifferentCoVsmayaffect tionofinflammationintheabsenceoftheEgenecontributedto 438
376 virusproduc tion todiffer en t extents, it isclearth atfor CoVs such the att enuation of rS AR S-C oV-(cid:2)E ( De Dieg o et al ., 2011). The se 439

377 as SARS-CoV, E protein is not essential, since SARS-CoV missing results were confirmed in mice. A reduced expression of proin- 440
378 E p rotein can p roduce v ir us ti ters close to 1× 106pfu p er ml or flamma tory c ytokines, d ec reased n umber o f neutrophi ls i n lung 441

Pleasecitethisarticleinpressas:DeDiego,M.L.,etal.,CoronavirusvirulencegeneswithmainfocusonSARS-CoVenvelopegene.Virus

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442 infiltrates,anddiminishedlungpathologywereobservedinSARS- 5. SARS-CoVEproteinPDZbindingdomainandSARS-CoV 503
443 CoV-MA15 -(cid:2)E -infectedm ice,c omparedt othe wtvirus- in fected vi rulence 504

444 ones (DeDiego et al., 2014), indicating that lung inflammation

445 contributes to SARS-CoV virulence. Furthermore, infection with A functional PDZ-binding motif (PBM) has been identified 505
446 rSARS-CoV- (cid:2)E resultedin adecrease dactivationo fthetrans crip- at th e carboxy- terminus end of E protein usi ng in vitro and 506
447 tionfactorNF- (cid:2)B. Impo rta n tly,treatm entwithN F- (cid:2)B inhibitors, in vivo approaches(Fig.4 )(Jim en ez -Guarden ˜oeta l.,2 014; Teoh 507

448 ledtoareductionininflammationinbothSARS-CoV-infectedcul- et al., 2010). PDZ motifs are abundant modules involved in pro- 508

449 turedcellsandmice,andsignificantlydiminishedlungpathology. tein–proteininteraction,whichconsistof80–90aminoacidsthat 509

450 These changes increased mice survival after SARS-CoV infection recognizeaspecificpeptidesequence(PBM)foundintheextreme 510
451 (DeDie goetal. ,2014).Th eseda taindica tedth atNF-(cid:2)Ba ctivation C-termini o ftargetp roteins (Hungand Sheng ,2002 ).I nth ehuman 511

452 isamajorcontributortotheinflammationinducedafterSARS-CoV genome,morethan900PDZdomainsarefoundinover400pro- 512
453 in fe ction, andthatdru gs inh ibitingNF-(cid:2)Ba ctivation are promising teins(Sp aller,2 006) .Itha sbe endescrib edt hatpr ote insco ntai ning 513

454 antivirals to treat SARS-CoVinduced disease, and most probably PDZ domains can be involved in cellular processes of relevance 514

455 theinflammationcausedbyotherpathogeniccoronaviruses,such for viruses, such as cell–cell junctions, cellular polarity and sig- 515

456 asMERS-CoV. nal transduction pathways (Javier and Rice, 2011). According to 516

457 Interestingly,hamstersimmunizedwiththeattenuatedrSARS- thisdata,severalviruses,suchasinfluenzaAvirus(Jacksonetal., 517
458 CoV-(cid:2)E develop ed high s erum-neutr alizin g an tibody tite rs, and 200 8),tic k-borne encepha litisv iru s(TBEV)( M elike tal.,2012 ),a nd 518

459 wereprotectedafterthechallengewithhomologous(Urbani)and humanpapillomavirus(HPV)(Kiyonoetal.,1997)encodeproteins 519

460 heterologous(GD03)SARS-CoVstrains(Lamirandeetal.,2008).In withPBMsthattargetcellularPDZmotifscarryingproteinsduring 520

461 addition, SARS-CoV missing E protein partially protected trans- infection.Throughtheseinteractions,cellularpathwaysinfluence 521

462 genic mice against challenge with virulent SARS-CoVs (Netland viralreplication,disseminationinthehost,andpathogenesis(Javier 522
463 et al. , 2010 ). More over, rSAR S-CoV -MA15-(cid:2) E totally protected andR ice,2011). 523

464 young and old (up to 2 years) BALB/c mice against the viru- ToidentifySARS-CoVEproteincellulartargetscontainingPDZ 524

465 lent mouse adapted virus (Fett et al., 2013), by inducing high domains, yeast two-hybrid based studies were undertaken. The 525

466 humoralandcellularimmuneresponses.Thesedataindicatedthat protein associated with Lin Seven 1 (PALS1), a tight junction- 526

467 theviruseslackingEgenearepromisingliveattenuatedvaccine associatedprotein,wasthefirstPDZproteinidentifiedasatarget 527

468 candidates. of E protein PBM, and this interaction was confirmed using co- 528

immunoprecipitation studies in mammalian cells (Teoh et al., 529

2010).PALS1isakeycomponentofthecomplexthatcontrolspolar- 530

ityestablishmentandtightjunctionformationinepithelia.Studies 531

using Vero E6 cells infected with SARS-CoV showed that E pro- 532
446790 4m. oSdAifiRcSa-tCiooVn Ea npdr ovtieriuns aamtteinnou aatnido ncarboxy-terminus ttheien e rc etloopciacl iezxepd r PesAsLi So1n toof tEh pe r EoRteGinI C i na nMdD GCoKlg ei prietghieolnia. lI nce allds d l ietdio tno, 553334
delayedtightjunctionandpolarityestablishment.Theresultssug- 535
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Pleasecitethisarticleinpressas:DeDiego,M.L.,etal.,CoronavirusvirulencegeneswithmainfocusonSARS-CoVenvelopegene.Virus

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Fig.3. EngineeredrSARS-CoVs-MA15withpointmutationsanddeletionsinEgene.TheorganizationofEproteinisshown.Eproteinsequenceisdividedintothreedomains:

theaminoterminal(N-terminal),thetransmembraneandthecarboxy-terminal(C-terminal).ThefigureillustratesthedeletionsandpointmutationsengineeredwithinE

protein.Theasterisk(*)indicatesmutationsintheresiduesS3A,V5L,T9A,T11A.Grayboxatthebottomindicatesmutantvirulence:(+)indicatesavirulentphenotypeand
(−)indic ates anatten ua tedpheno type.

568 bydeletingEproteinPBMcouldbethebasisforthedevelopment studies(Parthasarathyetal.,2008;Pervushinetal.,2009;Torres 593

569 ofrecombinantvaccines,asthosedescribedbydeletingthewhole etal.,2006). 594

570 SARS-CoVEproteinorinternaldomainsofthisprotein(Fettetal., The first functional evidence of SARS-CoV E protein acting as 595

571 2013;Lamirandeetal.,2008;Netlandetal.,2010;Regla-Navaetal., aviroporinwasprovidedafteritsexpressioninbacteria,whereE 596

572 2014). proteinoligomerizedandmodifiedmembranepermeability(Liao 597

et al., 2004, 2006). Direct measurement of E protein IC activity 598

573 6. IonchannelactivityofSARS-CoVE,3aand8aproteins was first reported using synthetic peptides representing full-length 599
574 an dvir ulence SARS-CoV E protein or its N-terminal 40 amino acids, including 600
thetransmembranedomain,inartificiallipidmembranes(Wilson 601

575 A wide range of animal viruses encode hydrophobic proteins et al., 2004). This IC activity was confirmed, and mutations that 602
576 thato ligom erize inh ostcell membr aneslea dingtostructu reswith suppressed this function were identified (Torres et al., 2007; 603
577 ion c hannel (IC) ac tivity . Th ese protein s, name d v iroporins, may Verdia-Baguena et al., 2012). In addition, compounds that inhibit 604
578 infl uence vi ral r eplicatio n and assembly , as wel l as virus p arti- the SARS-CoV E protein ion conductivity were described, although 605
579 cle entry and r elease from infe cted cells. Vir opori ns have a high their efficacy in the context of a viral infection was not reported 606
580 imp acton rel evantho stcel lphysiol ogical processes (Nieva e tal., (Pervushin et al., 2009). Initially, it was considered that SARS-CoV 607
558812 2vi0r1a2l)in. fTehcet iroenfosr.e, th ese prot eins are usefu l targets t o coun ter act Eca ptrioontesi no vfoerrm meodn aonv aICle wntit ahn aino nesn,h aanndc efodr s Nelae+ctoivveitry K fo+ri monosn (oWvaillseonnt 660089
583 M ostoftheRNAvirusesencodingtheseproteinsonlyhaveone et al., 2004). However, recent studies showed that the selectivity 610
584 viroporin i nth eirge nome( Castan˜o-R odrig uezetal .,201 4).H ow- of SARS-CoV E protein IC was dependent on the charge of the lipid 611
585 ever,SAR S-C oVen codesth reeproteinswithIC ac tiv ity:E,3 aand membranes in which the pore was reconstituted, which strongly 612
586 8a,w hichindica testhat SARS- CoVisthe RNA vi rusexpre ss ing the suggested that the lipid head-groups are an integral component 613
587 hig hestnu mberofv irop orinsknow n up toda te. of the channel pore (Fig. 5) (Verdia-Baguena et al., 2012, 2013). 614
588 TheI Cactivit yo fEprotein isthem ost ex tensivelycharacterized This novel finding highlights the relevance of the lipid membrane 615
589 among th ethree SA R S-CoVv iro por insus ingstructu ral,functional composition in the SARS-CoV ion channel structure and activity. 616
590 and ph ysio logica l assays. E protein ha s a si ngle transm embrane The influence of SARS-CoV E protein IC activity in cell ion 617
591 dom aintopology andits m onomers olig om erize inapentameric homeostasis is highly dependent on its subcellular localization. 618
592 ioncond uctivepo re,a sd eterminedb ylineardich ro is mandNMR After SARS-CoV infection, E protein mainly accumulates in the 619

Fig.4. RecombinantSARS-CoVswithEproteinPBMtruncatedormutatedbyreversegenetics.SARS-CoVEproteinsequenceanditscorrespondingdomainsareshownatthe
top. Be low,sequence scorrespon ding to theen dofE proteinar es hownin bo xesfort hediffere ntvirusm u tants.SA RS-CoV-E -wt ,w ildtypesequen ce.InSAR S-C oV-E-(cid:2) P BM

andSARS-CoV-E-mutPBMvirusmutants,EproteinPBMwaseliminatedbytheintroductionofdeletionsorpointmutations,reducingorkeepingthefullproteinlength,

respectively.InSARS-CoV-E-potPBM,fouraminoacidsofEproteinwerereplacedbyalanine,togenerateanewpotentialPBM.RedboxeshighlightPBMswithinEprotein.
Grayboxont he rightindicatesthevir ulen ceofth emu tan ts :(+)ind icate savirulen tp henotyp ea nd(−)in d icates anatten uated phen otype .

Pleasecitethisarticleinpressas:DeDiego,M.L.,etal.,CoronavirusvirulencegeneswithmainfocusonSARS-CoVenvelopegene.Virus

Res.(2014),http://dx.doi.org/10.1016/j.virusres.2014.07.024
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M.L.DeDiegoetal./VirusResearchxxx(2014)xxx–xxx 9

vectorialtransportofNa+ ionsdrivenbyepithelialsodiumchan- 665
nels(ENa C)andNa +/ K+ ATPase isesta bli shed(Holl enhorst etal., 666

2011).VirusesdisplayingEproteinionchannelactivitycausedan 667

increased damage within pulmonary epithelia, which correlated 668

with edema accumulation (Fig. 6) (Nieto-Torres et al., 2014). In 669

addition,SARS-CoVEproteindecreasedthelevelsandactivityof 670

ENaCinlungepithelialcells,viatheactivationofdistinctPKCiso- 671

forms,decreasingbothENaCexocytosisandendocytosisrates(Ji 672

etal.,2009).ThesedataindicatedthattheactivationofPKCbySARS- 673

CoVEprotein,whichmayleadtodecreasedlevelsandactivityof 674

ENaCattheapicalsurfaceoflungepithelialcells,andtheICactivity 675

ofEproteincontributetothelungedemaobservedafterSARS-CoV 676

infection. 677
Pulmonary epithelial damage is associated with a deleterious 678

exacerbated inflammatory response triggered in the lungs after 679

SARS-CoV infection. Evaluation of key inflammatory cytokines 680

involvedinepithelialdamageandedemaaccumulationrevealed 681
rFeigp.r 5es. eSnttreudc tiunr bel oufe S, AanRdS -EC poVro Ete pinro mteoinn opmroetreso alirpei dsihco iwonn c ahsa rnende cl.y Plihnodseprhs.o Nlipotide st haraet wthaayt sILo-f1t (cid:4)h,e TmNiFc eanindf eIcL t-e6d awmiothu nthtse w vierures eisn cdriespaslaeydi ning Ethper olutenign aioirn- 668823
lipidheadgroups(blueellipses)alsofacetheionchannellumen.
conductivitycomparedtotheinfectionwiththemutantslackingIC 684
activity(Niet o-Torrese ta l.,20 14).IL-1 (cid:4)iso neo fthemo stimpo r- 685

620 endoplasmicreticulum–Golgiintermediatecompartment(ERGIC) tantproinflammatorycytokinesinvolvedinARDSdisease(Meduri 686
621 regionofthe infectedcells,w herevirusmo rphogenesisa ndbud- etal .,1995;Puginet al.,1996). IL-1(cid:4)act iva tiono ccursw henthe 687

622 dingtakeplace(Nieto-Torresetal.,2011).Inartificialmembranes, inflammasome complex is stimulated by viral proteins with ion 688

623 mimickingtheERGICmembranecomposition,whereEproteinis channel activity (Ichinohe et al., 2010; Ito et al., 2012; McAuley 689

624 mainlyinserted,Eproteinshowedaslightselectivityforcations etal.,2013;Triantafilouetal.,2013).Theinflammatoryresponse 690
625 over an ions, wit h no pre ference fo r a sp ecific catio n ( Verdia- eli cite dbyIL -1(cid:4)isaccom pa nied byani ncre aseinTNF,and bothsig- 691

626 Baguenaetal.,2012).IthasbeensuggestedthatEproteincould nalsareamplifiedbytheaccumulationofIL-6,whicharekeyevents 692

627 alsobelocatedatthecellplasmamembrane,whichcouldinflu- duringARDSprogressionafterSARS-CoVinfection(Tisonciketal., 693

628 encecelldepolarization(Liaoetal.,2006;Pervushinetal.,2009). 2012;Wangetal.,2005).Webelievethatthisexacerbateddele- 694

629 EffortsdonebyourgrouptoidentifythepresenceofEproteinin teriousresponseisacausalagentoftheobserveddamageinthe 695

630 thecellsurface,ortodetectICactivityinthecellsurfacebyusing lungparenchymaofanimalsinfectedwiththevirusesdisplaying 696

631 patch-clamptechnologyshowedtheabsenceofthisactivityinthe ionchannelactivity. 697

632 plasmamembrane(Nieto-Torresetal.,2011).Accordingly,anaddi- Insummary,inhibitionofSARS-CoVEproteinICactivity,with- 698

633 tionalstudyindicatedthatEproteindoesnotformionchannelsat out significantly affecting virus growth, led to a virus inducing 699

634 thecellsurface(Jietal.,2009).Therefore,wehaveconcludedthatE an attenuated pathogenesis. Attenuation correlated with a mod- 700

635 proteinionchannelactivityisonlyshownintheintracellularstruc- erateinflammatoryresponseleadingtolessepithelialdamageand 701

636 tures,whereEproteinhasbeenlocated(Nieto-Torresetal.,2011; edemaaccumulation.Thesefindingsmayhaveimplicationsforthe 702

637 RuchandMachamer,2012). otherviroporinsencodedbySARS-CoVand,mostimportantly,for 703

638 Ionicimbalanceswithincellscaninterferewithinnateimmu- theidentificationoftherapiestoprotectagainsthighlypathogenic 704

639 nityandaffectviruspathogenesis.Interestingly,disruptionofion CoVssuchasSARS-CoVandMERS-CoV,orothervirusesencoding 705

640 gradientswithintheendoplasmicreticulumandGolgiapparatus proteins with IC activity. For example, hexamethylene amiloride 706

641 byviralproteinswithICactivitydelayedproteintransportprevent- (HMA),aninhibitoroftheHIV-1Vpuproteinionchannelactivity, 707

642 ingMHCmoleculesfromreachingtheplasmamembrane(Cornell alsoinhibitedSARS-CoV,HCoV-229EandMHVEproteinionchan- 708

643 etal.,2007;deJongetal.,2006).Recently,ithasbeendescribedthat nelconductance(Pervushinetal.,2009;Wilsonetal.,2006)and, 709

644 ionicimbalancescontrolledbyviroporinsaresensedbytheinflam- asaconsequence,suppressedthereplicationofthewtHCoV-229E 710

645 masome, which triggers the activation of key pro-inflammatory andMHV(Wilsonetal.,2006).Therefore,thisionchannelinhibitor 711
646 cytokines sucha sIL-1(cid:4), am ajordeterm in anto fdiseaseprogres- may bean efficien t ant iviralc ompound toco ntr olthere plication 712

647 sion(Ichinoheetal.,2010;Itoetal.,2012;McAuleyetal.,2013; severalmembersoftheCoronaviridaefamily. 713

648 Triantafilouetal.,2013). 8aand3aproteinsareSARS-CoVviroporinsaswell,buttheir 714

649 TheintroductionofpointmutationsthatinhibitedSARS-CoVE ionchannelactivitiesaremuchlessstudiedthanthatofEprotein.A 715

650 proteinICactivityledtoattenuatedviruses,withoutsignificantly 29ntdeletionoccurredinORF8whenthevirusfirstinfectedhuman 716

651 affectingvirusproduction(Nieto-Torresetal.,2014).Furthermore, beings,splittingORF8intoORF8aandORF8b.ORF8aencodesa39- 717

652 viruses in which E protein IC activity was suppressed quickly amino-acid-longpolypeptidewhosefirst35residuesareidentical 718

653 evolvedbyincorporatingmutationsthatrestoredionconductivity totheN-terminalpartoftheORF8primaryproduct(Oostraetal., 719

654 andavirulentphenotype(Nieto-Torresetal.,2014).Afterinfec- 2007).ORF8ashowsICactivitywhenreconstitutedintoartificial 720

655 tionwithvirusesdisplayingEproteinIC,increaseddamagewithin lipidbilayers(Chenetal.,2011),butthisactivityhasnotbeeniden- 721

656 pulmonary epithelia and edema accumulation within lung air- tifiedincells.Arolefor8aproteininvirusreplicationandinvitro 722

657 ways (Fig. 6), the ultimate determinant of ARDS, was observed, apoptosisthroughamitochondrial-dependentpathwayhasbeen 723

658 comparedtomiceinfectedwithviruseslackingEproteinIC(Nieto- suggested(Chenetal.,2007)buttheexperimentswereperformed 724

659 Torres et al., 2014). Enhanced liquid levels within lung airways withaHAtaggedvariantof8aproteinandsomeoftheresultsare 725

660 avoidproperoxygenexchangeleadingtoseverehypoxemiaand atvariancewiththosepreviouslyreported(Oostraetal.,2007).A 726

661 eventually to death (Matthay and Zemans, 2011). Ionic balances variantofSARS-CoVwithadeletionof415ntresultingintheloss 727

662 playacentralroleincontrollingliquidamountspresentwithinair ofORF8,wasisolatedtowardtheendoftheSARSepidemicand,in 728

663 spaces.Lungepitheliacreateanosmoticgradientbetweentheinte- spiteofthisdeletion,someoftheinfectedpatientsdied,suggesting 729

664 rioroftheairwaysandtheinterstitialspaces.Toresolveedema,a thatORF8isnotessentialforviruspathogenicity(Chiuetal.,2005). 730

Pleasecitethisarticleinpressas:DeDiego,M.L.,etal.,CoronavirusvirulencegeneswithmainfocusonSARS-CoVenvelopegene.Virus

Res.(2014),http://dx.doi.org/10.1016/j.virusres.2014.07.024
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10 M.L.DeDiegoetal./VirusResearchxxx(2014)xxx–xxx

Fig.6. EffectofSARS-CoVEproteinionchannelactivityinlungpathology.Thelunghistopathologyinmiceinfectedwithavirusdisplaying(EIC+)orlacking(EIC−)E

proteinionchannelactivityat4dayspostinfection(dpi)isshownatthetop.Lungsectionswereanalyzedbyhematoxylinandeosinstainingatanoriginalmagnificationof
20×.Air spa ceswhe reedem aw a sacc umu latedare indica te dwith as teri sks. Immu nofluore scenc estaining of lungsections ,an ddeta ilofbron ch iol arepith eliaat4dpi,at a
magn ificationo f40×a nd190 ×,re spectivelyis sho wnatthe botto m.Lunge pitheliawaslabeledu singana nt iNa+ /K+ATPas ean tibody (g reen)andSA RS-CoVi nfe c tion wa s
trackedwitha na nti- Npr oteina ntibody(red ).N ucleia re sho wninbl ue.De squamat edep ithelial cellsa nd cell debrisareobse rvedinlu ngairw ays afterEIC+v irusinfec tion

(whitearrows).

731 3a protein is a 274 aa SARS-specific structural component bytheself-oligomerizationofsyntheticpeptidescorrespondingto 736

732 of the virus with three transmembrane domains (TMDs) in its eachofthethreeTMDsintoartificiallipidbilayers.OnlyTMD2and 737

733 N-terminus. 3a protein forms a potassium ion channel after TMD3peptidesrestoredICactivity(Chienetal.,2013).However, 738

734 tetramerizationviainter-monomerdisulfidebridges(Cys133)(Lu additionalstudiesarerequiredtocharacterizetheICselectivityof 739

735 etal.,2006).TheICactivityof3aproteinhasbeencharacterized thereconstitutedviroporin.SARS-CoV3aproteininfluencesvirus 740

Pleasecitethisarticleinpressas:DeDiego,M.L.,etal.,CoronavirusvirulencegeneswithmainfocusonSARS-CoVenvelopegene.Virus

Res.(2014),http://dx.doi.org/10.1016/j.virusres.2014.07.024