Significant Redox Insensitivity of the Functions of the SARS-CoV Spike Glycoprotein

Abstract

The capacity of the surface glycoproteins of enveloped viruses to mediate virus/cell binding and membrane fusion requires a proper thiol/disulfide balance. Chemical manipulation of their redox state using reducing agents or free sulfhydryl reagents affects virus/cell interaction. Conversely, natural thiol/disulfide rearrangements often occur during the cell interaction to trigger fusogenicity, hence the virus entry. We examined the relationship between the redox state of the 20 cysteine residues of the SARS-CoV (severe acute respiratory syndrome coronavirus) Spike glycoprotein S1 subdomain and its functional properties. Mature S1 exhibited ∼4 unpaired cysteines, and chemically reduced S1 displaying up to ∼6 additional unpaired cysteines still bound ACE2 and enabled fusion. In addition, virus/cell membrane fusion occurred in the presence of sulfhydryl-blocking reagents and oxidoreductase inhibitors. Thus, in contrast to various viruses including HIV (human immunodeficiency virus) examined in parallel, the functions of the SARS-CoV Spike glycoprotein exhibit a significant and surprising independence of redox state, which may contribute to the wide host range of the virus. These data suggest clues for molecularly engineering vaccine immunogens. The capacity of the surface glycoproteins of enveloped viruses to mediate virus/cell binding and membrane fusion requires a proper thiol/disulfide balance. Chemical manipulation of their redox state using reducing agents or free sulfhydryl reagents affects virus/cell interaction. Conversely, natural thiol/disulfide rearrangements often occur during the cell interaction to trigger fusogenicity, hence the virus entry. We examined the relationship between the redox state of the 20 cysteine residues of the SARS-CoV (severe acute respiratory syndrome coronavirus) Spike glycoprotein S1 subdomain and its functional properties. Mature S1 exhibited ∼4 unpaired cysteines, and chemically reduced S1 displaying up to ∼6 additional unpaired cysteines still bound ACE2 and enabled fusion. In addition, virus/cell membrane fusion occurred in the presence of sulfhydryl-blocking reagents and oxidoreductase inhibitors. Thus, in contrast to various viruses including HIV (human immunodeficiency virus) examined in parallel, the functions of the SARS-CoV Spike glycoprotein exhibit a significant and surprising independence of redox state, which may contribute to the wide host range of the virus. These data suggest clues for molecularly engineering vaccine immunogens. SARS 3The abbreviations used are: SARS, severe acute respiratory syndrome; CoV, coronavirus; ACE2, angiotensin-converting enzyme 2; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; Env, envelope; GFP, green fluorescent protein; HIV, human immunodeficiency virus; MLV, murine leukemia virus; MPB, 3-(N-maleimidylpropionyl)biocytin; PBS, phosphate-buffered saline; PDI, protein-disulfide isomerase; SH, free sulfhydryl; SU, surface subunit; Tg, thyroglobulin. -CoV is a new human coronavirus that causes a severe acute respiratory syndrome, sometimes with a fatal outcome. Its Spike glycoprotein is the major surface antigen and induces potent neutralizing antibodies (1Temperton N.J. Chan P.K. Simmons G. Zambon M.C. Tedder R.S. Takeuchi Y. Weiss R.A. Emerg. Infect. Dis. 2005; 11: 411-416Crossref PubMed Scopus (121) Google Scholar, 2Xiao X. Dimitrov D.S. CMLS Cell. Mol. Life Sci. 2004; 61: 2428-2430Crossref PubMed Scopus (26) Google Scholar, 3Petit C.M. Melancon J.M. 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Chouljenko V.N. Colgrove R. Farzan M. Knipe D.M. Kousoulas K.G. Virology. 2005; 341: 215-230Crossref PubMed Scopus (49) Google Scholar). The S1 region interacts with angiotensin-converting enzyme 2 (ACE2), the primary cellular receptor (9Li F. Li W. Farzan M. Harrison S.C. Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1382) Google Scholar, 10Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzuriaga K. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Crossref PubMed Scopus (4100) Google Scholar, 11Fukushi S. Mizutani T. Saijo M. Matsuyama S. Miyajima N. Taguchi F. Itamura S. Kurane I. Morikawa S. J. Gen. Virol. 2005; 86: 2269-2274Crossref PubMed Scopus (91) Google Scholar, 12Ng M.L. Tan S.H. See E.E. Ooi E.E. Ling A.E. J. Gen. Virol. 2003; 84: 3291-3303Crossref PubMed Scopus (96) Google Scholar), L-SIGN (13Jeffers S.A. Tusel S.M. Gillim-Ross L. Hemmila E.M. Achenbach J.E. Babcock G.J. Thomas W.D. Thackray L.B. Young M.D. Mason R.J. Ambrosino D.M. Wentworth D.E. Demartin J.C. Holmes K.V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15748-15753Crossref PubMed Scopus (470) Google Scholar), and L-SECtin (14Gramberg T. Hofmann H. Moller P. Lalor P.F. Marzi A. Geier M. Krumbiegel M. Winkler T. Kirchhoff F. Adams D.H. Becker S. Munch J. Pohlmann S. Virology. 2005; 340: 224-236Crossref PubMed Scopus (163) Google Scholar), whereas S2 mediates membrane fusion (2Xiao X. Dimitrov D.S. CMLS Cell. Mol. Life Sci. 2004; 61: 2428-2430Crossref PubMed Scopus (26) Google Scholar, 3Petit C.M. Melancon J.M. Chouljenko V.N. Colgrove R. Farzan M. Knipe D.M. Kousoulas K.G. Virology. 2005; 341: 215-230Crossref PubMed Scopus (49) Google Scholar). Here, we investigated the functional role of the 20 conserved cysteine residues of mature S1. A variety of evidence has shown that the capacity of the viral envelope glycoproteins to mediate virus/cell membrane fusion depends on a precise thiol/disulfide balance within the viral surface complex (15Ryser H.J. Levy E.M. Mandel R. DiSciullo G.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4559-4563Crossref PubMed Scopus (236) Google Scholar, 16Gallagher T.M. J. Virol. 1996; 70: 4683-4689Crossref PubMed Google Scholar, 17Pinter A. Kopelman R. Li Z. Kayman S.C. Sanders D.A. J. Virol. 1997; 71: 8073-8077Crossref PubMed Google Scholar, 18Sanders D.A. Subcell. Biochem. 2000; 34: 483-514Crossref PubMed Google Scholar, 19Fenouillet E. Barbouche R. Courageot J.C. Miquelis R. J. Infect. Dis. 2001; 183: 744-752Crossref PubMed Scopus (95) Google Scholar, 20Wallin M. Ekstrom M. Garoff H. EMBO J. 2004; 23: 54-65Crossref PubMed Scopus (110) Google Scholar, 21Krey T. Thiel H.J. Rümenapf T. J. Virol. 2005; 79: 4191-4200Crossref PubMed Scopus (64) Google Scholar, 22Sturman L.S. Ricard C.S. 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Virology. 1997; 230: 179-186Crossref PubMed Scopus (9) Google Scholar). On one hand, manipulation of the native disulfide network of mature envelopes at the virus surface using reducing or alkylating reagents has important consequences for their subsequent capacity to carry out the virus/cell interaction process (15Ryser H.J. Levy E.M. Mandel R. DiSciullo G.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4559-4563Crossref PubMed Scopus (236) Google Scholar, 16Gallagher T.M. J. Virol. 1996; 70: 4683-4689Crossref PubMed Google Scholar, 19Fenouillet E. Barbouche R. Courageot J.C. Miquelis R. J. Infect. Dis. 2001; 183: 744-752Crossref PubMed Scopus (95) Google Scholar, 20Wallin M. Ekstrom M. Garoff H. EMBO J. 2004; 23: 54-65Crossref PubMed Scopus (110) Google Scholar, 23Abell B.A. Brown D.T. J. Virol. 1993; 67: 5496-5501Crossref PubMed Google Scholar, 24Anthony R.P. Paredes A.M. Brown D.T. Virology. 1992; 190: 330-336Crossref PubMed Scopus (43) Google Scholar, 26Barbouche R. Miquelis R. 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Biol. Chem. 2003; 278: 3131-3136Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 27Markovic I. Stantchev T.S. Fields K.H. Tiffany L.J. Tomic M. Weiss C.D. Broder C.C. Strebel K. Clouse K.A. Blood. 2004; 103: 1586-1594Crossref PubMed Scopus (126) Google Scholar). For HIV, gentle chemical reduction of Env efficiently prevents viral infectivity by inhibiting the capacity of the surface subunit (SU) to bind its ligands on the target cell surface (15Ryser H.J. Levy E.M. Mandel R. DiSciullo G.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4559-4563Crossref PubMed Scopus (236) Google Scholar, 19Fenouillet E. Barbouche R. Courageot J.C. Miquelis R. J. Infect. Dis. 2001; 183: 744-752Crossref PubMed Scopus (95) Google Scholar). In contrast, after receptor binding, fusion mediated by the transmembrane subunit occurs solely following reduction of SU by a cell-surface protein-disulfide isomerase (PDI) activity (19Fenouillet E. Barbouche R. Courageot J.C. Miquelis R. J. Infect. Dis. 2001; 183: 744-752Crossref PubMed Scopus (95) Google Scholar, 25Gallina A. Hanley T.M. Mandel R. Trahey M. Broder C.C. Viglianti G.A. Ryser H.J. J. Biol. Chem. 2002; 277: 50579-50588Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 26Barbouche R. Miquelis R. Jones I.M. Fenouillet E. J. Biol. Chem. 2003; 278: 3131-3136Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 27Markovic I. Stantchev T.S. Fields K.H. Tiffany L.J. Tomic M. Weiss C.D. Broder C.C. Strebel K. Clouse K.A. Blood. 2004; 103: 1586-1594Crossref PubMed Scopus (126) Google Scholar). For these reasons, reducing or free sulfhydryl reagents and nonpermeant inhibitors of PDI (e.g. the thiol reagent DTNB and bacitracin, an oxidoreductive inhibitor) block entry (15Ryser H.J. Levy E.M. Mandel R. DiSciullo G.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4559-4563Crossref PubMed Scopus (236) Google Scholar, 19Fenouillet E. Barbouche R. Courageot J.C. Miquelis R. J. Infect. Dis. 2001; 183: 744-752Crossref PubMed Scopus (95) Google Scholar). A proper disulfide network within MLV- and human T-leukemia virus-Env is also a determinant of virus entry. Incubation in the presence of dithiothreitol (DTT) of cell-bound virus inhibits Env fusogenicity. Sulfhydryl-blocking reagents also prevent virus/cell fusion, but entry can be rescued following disruption of a SU-transmembrane protein disulfide by chemical reduction in A. Kopelman R. Li Z. Kayman S.C. Sanders D.A. J. Virol. 1997; 71: 8073-8077Crossref PubMed Google Scholar, 20Wallin M. Ekstrom M. Garoff H. EMBO J. 2004; 23: 54-65Crossref PubMed Scopus (110) Google Scholar, 28Wallin M. Ekstrom M. Garoff H. J. Virol. 2005; 79: 1678-1685Crossref PubMed Scopus (34) Google Scholar). has to the that fusion requires for the protein disulfide between transmembrane protein and a on SU to be within the by the oxidoreductase activity by the In to these the protein of the C. Linder M. Wengler G. Virology. 1997; 230: 179-186Crossref PubMed Scopus (9) Google and the envelope of viral a exhibit The during the to at an following disulfide reduction an T. Thiel H.J. Rümenapf T. J. Virol. 2005; 79: 4191-4200Crossref PubMed Scopus (64) Google Scholar). For the virus envelope disulfide important for the and of the The of DTNB fusion, whereas using reducing reagents that the of a role during the entry B.A. Brown D.T. J. Virol. 1993; 67: 5496-5501Crossref PubMed Google Scholar, 24Anthony R.P. Paredes A.M. Brown D.T. Virology. 1992; 190: 330-336Crossref PubMed Scopus (43) Google Scholar). It is also important to for the that redox changes conformational rearrangements of coronavirus during cell interaction L.S. Ricard C.S. Holmes K.V. J. Virol. 1990; 64: 3042-3050Crossref PubMed Google Scholar). DTNB virus entry by with a free sulfhydryl in its S2 whereas chemical reduction with of the fusion process T.M. J. Virol. 1996; 70: 4683-4689Crossref PubMed Google Scholar, 22Sturman L.S. Ricard C.S. Holmes K.V. J. Virol. 1990; 64: 3042-3050Crossref PubMed Google Scholar). these that manipulation of the redox of viral envelope glycoproteins by reducing or free sulfhydryl reagents cell entry by a variety of enveloped bacitracin, DTT, and other reagents The thiol reagent S1 is to an antigen that by Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzuriaga K. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Crossref PubMed Scopus (4100) Google Scholar). using a and by protein A the using a and by R. H. Jones I.M. Fenouillet E. Mol. 2005; 67: PubMed Scopus Google Scholar). A of an fluorescent protein fusion protein also using to the of is a sulfhydryl reagent to its interaction with protein can be using a S1 with or using and The by a and with phosphate-buffered the using and R. Miquelis R. Jones I.M. Fenouillet E. J. Biol. Chem. 2003; 278: 3131-3136Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, R. H. Jones I.M. Fenouillet E. Mol. 2005; 67: PubMed Scopus Google Scholar). of the using to the presence of in the Tg, or in of a with PBS, 20 and with the with to the with for and after using by using a and its The relationship between the and the thiol of the of using a of Tg, to reduction following and used glycoprotein a to to S1 and of the a between and into its and cysteine cysteine we a that enabled the of a specific thiol to the of the within the range of to R. Miquelis R. Jones I.M. Fenouillet E. J. Biol. Chem. 2003; 278: 3131-3136Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, R. H. Jones I.M. Fenouillet E. Mol. 2005; 67: PubMed Scopus Google Scholar). The Chemical with of at and to its thiol For binding the with at to with at which with receptor The for receptor binding and R. Miquelis R. Jones I.M. Fenouillet E. J. Biol. Chem. 2003; 278: 3131-3136Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, R. H. Jones I.M. Fenouillet E. Mol. 2005; 67: PubMed Scopus Google Scholar). with ACE2 binding, with ACE2 of for at The with S1 in and with ACE2 in the for to in for and the with For binding in in 26Barbouche R. Miquelis R. Jones I.M. Fenouillet E. J. Biol. Chem. 2003; 278: 3131-3136Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), HIV SU for 2 in with for 2 binding the by various used in and to a B. J. J. 2003; PubMed Scopus Google Scholar). using a with the various viral envelope and a the that and used for envelope following used for and envelope with envelope in a and with of virus in a of used for and used for The or reagents a during the the with the virus or for to of the and the in for at by cell The infectivity of with or neutralizing antibodies with the infectivity using a and to on Mature investigated the in disulfide of the 20 cysteine residues on a of S1. We and a glycoprotein to that in 10Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzuriaga K. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Crossref PubMed Scopus (4100) Google for the S1 antigen be a virus of protein used for the of ACE2 the virus receptor W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzuriaga K. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Crossref PubMed Scopus (4100) Google and in a of subsequent binding and the role of the of S1 S.K. Li W. Moore M.J. Choe H. Farzan M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, W. C. Sui J. Moore M.J. S. Wong S.K. Xu K. Vasilieva N. A. Y. Y. Choe H. Farzan M. EMBO J. 2005; PubMed Scopus Google Scholar). The of the and the presence of free examined using and A the S1 antigen to with thiol reagent is to and its with the protein using and We the of free on S1 following and S1 and using by and the free sulfhydryl to the using a and R. Miquelis R. Jones I.M. Fenouillet E. J. Biol. Chem. 2003; 278: 3131-3136Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, R. H. Jones I.M. Fenouillet E. Mol. 2005; 67: PubMed Scopus Google Scholar). We that an of S1 with the that of 20 cysteine residues of mature S1 unpaired cysteines in contrast to HIV Env, examined in parallel, cysteine residues The data for HIV SU, which a of cysteine residues in its in with R. Miquelis R. Jones I.M. Fenouillet E. J. Biol. Chem. 2003; 278: 3131-3136Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). of S1 to Chemical examined the of S1 to of and the thiol following the chemical reduction HIV Env examined in S1 exhibited a to the reducing HIV Env these a relationship between the of and the thiol of the protein by the presence of the reducing and we a of reduced with the to the thiol of using and thiol and in R. H. Jones I.M. Fenouillet E. Mol. 2005; 67: PubMed Scopus Google The of the with and the reducing The antigen for receptor S1 displaying of its cysteine residues unpaired residues a of to bind ACE2 in contrast to reduced HIV Env, which receptor binding one disulfide SU the data R. Miquelis R. Jones I.M. Fenouillet E. J. Biol. Chem. 2003; 278: 3131-3136Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). of unpaired cysteine residues on S1 the ACE2 binding a specific interaction still S1 exhibited of its cysteine residues unpaired reduction binding capacity with with human antibodies The using a and a native virus of the Spike the of ACE2 binding reduction in In addition, we examined the of chemical reduction on the fusion capacity of the Spike We an to the entry into of viruses with Spike and the In by target is following the virus entry and by is to the activity of inhibitors the specific For chemical we used reagent is used for in cell that we examined the of on virus entry. of the of the or of the reducing during the of and the subsequent of Thus, an S1 antigen of its cysteine residues unpaired residues thiol of the antigen following the the capacity of the Spike complex to mediate fusion of that the reduction of the S1 in these or virus entry that these the fusion of Spike in contrast to other viral envelopes for which fusion capacity is by gentle chemical reduction we the of DTT, which to an S1 antigen of its cysteine residues unpaired of the virus or of the reducing during the of virus and virus entry. of with by to hence virus that a with a cell-surface These data with S1 of its cysteine residues unpaired residues ACE2 of or has on the binding of S1 to ACE2 S.K. Li W. Moore M.J. Choe H. Farzan M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google leading to the that the binding and fusion capacity of the Spike complex reduction These that the unpaired cysteine residues of S1 and ACE2 binding and role in the activity of mature S1 S.K. Li W. Moore M.J. Choe H. Farzan M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). with the that unpaired cysteines and binding of redox which may occur within the Spike complex or between the complex and cell-surface to fusogenicity, hence to entry. We examined using DTNB, a that with free and bacitracin, a PDI the that unpaired cysteines and redox within Spike cell role during entry. We that enabled the of DTNB and by with HIV Env, in with the role of by with cell-surface PDI by in HIV entry the SU reduction by PDI (15Ryser H.J. Levy E.M. Mandel R. DiSciullo G.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4559-4563Crossref PubMed Scopus (236) Google Scholar, 19Fenouillet E. Barbouche R. Courageot J.C. Miquelis R. J. Infect. Dis. 2001; 183: 744-752Crossref PubMed Scopus (95) Google DTNB, bacitracin, inhibits fusion, which is with an disulfide by within Env that entry (20Wallin M. Ekstrom M. Garoff H. EMBO J. 2004; 23: 54-65Crossref PubMed Scopus (110) Google Scholar). The Spike glycoprotein of SARS-CoV is a major target for neutralizing and the of a vaccine molecularly of SARS-CoV Spike (1Temperton N.J. Chan P.K. Simmons G. Zambon M.C. Tedder R.S. Takeuchi Y. Weiss R.A. Emerg. Infect. Dis. 2005; 11: 411-416Crossref PubMed Scopus (121) Google Scholar, 2Xiao X. Dimitrov D.S. CMLS Cell. Mol. Life Sci. 2004; 61: 2428-2430Crossref PubMed Scopus (26) Google Scholar, 3Petit C.M. Melancon J.M. Chouljenko V.N. Colgrove R. Farzan M. Knipe D.M. Kousoulas K.G. Virology. 2005; 341: 215-230Crossref PubMed Scopus (49) Google Scholar, 4Bisht H. Roberts A. Vogel L. Subbarao K. Moss B. Virology. 2005; 334: 160-165Crossref PubMed Scopus (92) Google Scholar, 5Faber M. Lamirande E.W. Roberts A. Rice A.B. Koprowski H. Dietzschold B. Schnell M.J. J. Gen. Virol. 2005; 86: 1435-1440Crossref PubMed Scopus (85) Google Scholar, 6He Y. Zhu Q. Liu S. Zhou Y. Yang B. Li J. Jiang S. Virology. 2005; 334: 74-82Crossref PubMed Scopus (90) Google Scholar, 7Yao Y.X. Ren J. Heinen P. Zambon M. Jones I.M. J. Infect. Dis. 2004; 190: 91-98Crossref PubMed Scopus (24) Google Scholar, 8Pang H. Liu Y. Han X. Xu Y. Jiang F. Wu D. Kong X. Bartlam M. Rao Z. J. Gen. Virol. 2004; 85: 3109-3113Crossref PubMed Scopus (57) Google Scholar). In the of is important to on the of the Spike that its The SARS-CoV envelope is into the S1 region that the and the S2 transmembrane which mediates membrane fusion (2Xiao X. Dimitrov D.S. CMLS Cell. Mol. Life Sci. 2004; 61: 2428-2430Crossref PubMed Scopus (26) Google Scholar, 3Petit C.M. Melancon J.M. Chouljenko V.N. Colgrove R. Farzan M. Knipe D.M. Kousoulas K.G. Virology. 2005; 341: 215-230Crossref PubMed Scopus (49) Google Scholar). viral including of and exhibit a functional these and in contrast to the entry of several viruses that the of the of cysteine residues of mature Spike in and redox changes within SARS-CoV Spike and cell-surface for virus entry. the that of mature S1 for ACE2 binding out the that the a role during the and a role may The independence of ACE2 binding disulfide and the that of several residues affects receptor binding S.K. Li W. Moore M.J. Choe H. Farzan M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, S. P. X. Dimitrov D.S. Virol. J. 2005; PubMed Scopus Google with the residues in the between the S1 and the and cysteine residues and within the receptor binding which the with ACE2 (9Li F. Li W. Farzan M. Harrison S.C. Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1382) Google Scholar). These the that the S1 protein is a (2Xiao X. Dimitrov D.S. CMLS Cell. Mol. Life Sci. 2004; 61: 2428-2430Crossref PubMed Scopus (26) Google and the of its disulfide may be that the envelope complex to be and on redox by cell-surface to entry. independence the redox changes may be of the for the wide host range of the virus and to the that to human Chan K.H. Wong T.M. S.K. Y. J. Virol. 2005; 79: PubMed Scopus Google Scholar, T. R.A. G. W. Nature. 2003; PubMed Scopus Google Scholar). The of of the free of the SARS-CoV envelope protein using DTNB the of the functions of the unpaired cysteine the cysteine residues in the Spike complex conserved between and human (9Li F. Li W. Farzan M. Harrison S.C. Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1382) Google Scholar), the of an by DTNB that to a functional role of a virus/cell interaction at a or during redox In to to a of SARS virus entry by that of other the of vaccine on the Spike In with data (9Li F. Li W. Farzan M. Harrison S.C. Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1382) Google Scholar), suggest that the engineering of cysteine residues to or may the of the The of the unpaired cysteine residues in is but is in a their is by the protein unpaired cysteine residues for the of additional at of envelope and may to the capacity of coronavirus to Chan K.H. Wong T.M. S.K. Y. J. Virol. 2005; 79: PubMed Scopus Google Scholar, T. R.A. G. W. Nature. 2003; PubMed Scopus Google and to human cell-surface ligands (9Li F. Li W. Farzan M. Harrison S.C. Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1382) Google Scholar, W. C. Sui J. Moore M.J. S. Wong S.K. Xu K. Vasilieva N. A. Y. Y. Choe H. Farzan M. EMBO J. 2005; PubMed Scopus Google Scholar). We to for and to and for SARS-CoV