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1、Monitoring of S Protein Maturation in the Endoplasmic Reticulum by Calnexin Is Important for the Infectivity of Severe Acute Respiratory Syndrome Coronavirus Masaya Fukushi,a,b,c,dYoshiyuki Yoshinaka,eYusuke Matsuoka,aSeisuke Hatakeyama,aYukihito Ishizaka,fTeruo Kirikae,a Takehiko Sasazuki,gand Tohr
2、u Miyoshi-Akiyamaa Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo, Japana; Disease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo, Japanb; Deputy Director-Generals Laboratory, Research Institute, Nati
3、onal Center for Global Health and Medicine, Tokyo, Japanc; Department of Virology, Institute of Biomedical Department of Molecular Virology, Bio-Response, Graduate School, Tokyo Medical and Dental University, Tokyo, Japane; Department of Intractable Diseases, Research Institute, National Center for
4、Global Health and Medicine, Tokyo, Japanf; and National Center for Global Health and Medicine, Tokyo, Japang Severeacuterespiratorysyndromecoronavirus(SARS-CoV)istheetiologicalagentofSARS,afatalpulmonarydisorderwithno effectivetreatment.WefoundthatSARS-CoVspikeglycoprotein(Sprotein),akeymoleculeforv
5、iralentry,bindstocalnexin,a molecularchaperoneintheendoplasmicreticulum(ER),butnottocalreticulin,ahomologofcalnexin.Calnexinboundtomost truncatedmutantsofSprotein,andSproteinboundtoallmutantsofcalnexin.PseudotypedviruscarryingSprotein(S-pseudo- virus)producedbyhumancellsthatweretreatedwithsmallinter
6、feringRNA(siRNA)forcalnexinexpression(calnexinsiRNA- treatedcells)showedsignifi cantlylowerinfectivitythanS-pseudovirusesproducedbyuntreatedandcontrolsiRNA-treatedcells. S-pseudovirusproducedbycalnexinsiRNA-treatedcellscontainedSproteinmodifi edwithN-glycansidechainsdifferently fromothertwoSproteins
7、andconsistedoftwokindsofviralparticles:thoseofnormaldensitywithlittleSproteinandthoseof highdensitywithabundantSprotein.Treatmentwithpeptide-N-glycosidaseF(PNGaseF),whichremovesalltypesofN-glycan sidechainsfromglycoproteins,eliminatedtheinfectivityofS-pseudovirus.S-pseudovirusandSARS-CoVproducedinth
8、epres- enceof?-glucosidaseinhibitors,whichdisrupttheinteractionbetweencalnexinanditssubstrates,showedsignifi cantlylower infectivitythaneachvirusproducedintheabsenceofthosecompounds.InS-pseudovirus,theincorporationofSproteininto viralparticleswasobviouslyinhibited.InSARS-CoV,viralproductionwasobviou
9、slyinhibited.Thesefi ndingsdemonstrated thatcalnexinstrictlymonitorsthematurationofSproteinbyitsdirectbinding,resultinginconferringinfectivityonSARS-CoV. S evere acute respiratory syndrome (SARS), a pulmonary infec- tiousdiseasewithsignifi cantmorbidityandmortality,became epidemic worldwide, especia
10、lly in China, Southeast Asia, and Canada, in 2002 to 2003 (5, 8). Its etiological agent was shown to be SARS coronavirus (SARS-CoV) (11, 23, 37), a new type of enveloped virus belonging to the Coronaviridae family, group 2, andcontaininga29.7-kb,single-stranded,positive-senseRNAge- nome (30, 40). Th
11、e SARS-CoV spike, located on the viral surface, is composed of a trimer of S glycoprotein (S protein) and is im- portant for viral entry into target cells (28, 43, 47). S protein is synthesized as a 1,255-amino-acid precursor polypeptide, with glycosylation at 23 amino acids occurring in the endopla
12、smic re- ticulum(ER)andGolgiapparatus(13,30,40,46).Sproteincanbe proteolytically cleaved into noncovalently associated regions, i.e., the N-terminal half (S1 domain), which contains the receptor- binding domain (RBD) (residues 318 to 510), and the C-terminal half (S2 domain), which is responsible fo
13、r fusion activity (3, 14, 26). The RBD binds to host angiotensin-converting enzyme 2 (ACE2),ahomologofACEandareceptorforSARS-CoV(24,27, 38, 45, 46). The host lectin L-SIGN also acts as a receptor for SARS-CoV infection by binding to another site distinct from the RBD (17, 22). Calnexin is a transmem
14、brane protein that functions as a mo- lecularchaperoneintheER(2).Calnexin,alongwithitshomologs calreticulin and ERp57, transiently binds to newly synthesized polypeptides containing monoglucosylated N-glycan side chains. Calnexin prevents the aggregation and premature degradation of substrateprotein
15、s,ensuringtheircorrectfoldingbeforethesepro- teins continue along their intracellular traffi cking pathways (16, 20, 34). The ?-glycosidase inhibitors N-nonyl-deoxynojirimycin (NN-DNJ), N-butyl-deoxynojirimycin (NB-DNJ), and castano- spermine, which target ?-glycosidases I and II in the ER, disrupt
16、theinteractionofcalnexinwithitssubstrateproteinsbyinterrupt- ingglycanchainprocessingofimmatureglycoproteins(12,31,35, 41, 49). We previously suggested that the N-terminal part of the S2 domain (S2-N) of S protein was important for SARS-CoV infec- tion (42). We therefore attempted to identify the cellular proteins that bind to S2-N and examined the effect of this interaction on SARS-CoV infection. MATERIALS AND METHODS Cell lines and viruses. Wild-type and calnexin gene-defi cient primary mouse
