Functional analysis of an epitope in the S2 subunit of the murine coronavirus spike protein: involvement in fusion activity

Abstract

The monoclonal antibody (MAb) 5B19.2, which has virus-neutralizing and fusion inhibition activities, binds to an epitope (S2A) consisting of nine hydrophobic amino acids in the S2 subunit of the mouse hepatitis virus (MHV) spike (S) protein. This suggests that the S2A epitope may be involved in binding the virus to the MHV receptor and/or in virus-cell fusion. Co-immunoprecipitation analyses demonstrated that while the binding of virus to the receptor was blocked by anti-S1 MAbs, it was not blocked by the S2A antiserum, indicating that S2A was not involved in receptor-binding. The S proteins prepared in this study with mutations in the S2A epitope were either fusogenic or nonfusogenic and their fusogenicity did not correlate with the hydrophobic feature of the S2A epitope. All of these wt and mutated S proteins were similarly transported onto the cell membrane independent of their fusogenicity capability. These results suggest that S2A may mediate the fusion activity of the MHV S protein during virus entry into cells.The spike (S) protein of mouse hepatitis virus (MHV), a member of the family Coronaviridae, forms a spike projecting from the virion surface. The spike comprises two molecules of the S protein, each of which is a heterodimer consisting of two non-covalently bound subunits, S1 and S2 (Sturman et al., 1985). The S1 subunit makes up the globular part of the spike and the S2 subunit is the stalk portion (De Groot et al., 1987).An important biological function of the S protein is binding to the virus receptor, which is a member of the carcinoembryonic antigen gene family (Dveksler et al., 1991). The receptor-binding domain is located in the N-terminal 330 amino acids of the S1 subunit (S1N330) (Kubo et al., 1994 ;Suzuki & Taguchi, 1996), rather than on subunit S2 Author for correspondence : Fumihiro Taguchi.Fax j81 423 46 1754. e-mail taguchi!ncnp.go.jp (Taguchi, 1995), as is expected from the topologies of these subunits. Although the receptor-binding site has been mapped to S1N330, it is possible that additional regions in S1 and\or S2 may play an important role in virus-receptor interactions. Another important function is cell-cell and virus-cell fusion (Collins et al., 1982 ;Taguchi et al., 1992). Various regions in S2 are involved in fusion activity (Bos et al., 1995 ; Gallagher, 1996 ; Gallagher et al., 1991 ;Luo & Weiss, 1998).Monoclonal antibody (MAb) 5B19.2, which is specific for S2, has been reported to have virus-neutralizing (VN) and fusion-inhibition (FI) activities (Collins et al., 1982). Its binding site, epitope A (S2A), is composed of nine hydrophobic amino acids, Leu " (Fig. 1). The S2A epitope is located upstream of heptad repeat 1 (Luytjes et al., 1989) and forms a major antigenic determinant in the S2 subunit (Daniel et al., 1993). Antibodies specific to this epitope play an important role in protection in mice (Koolen et al., 1990). Although MAb 5B19.2 was isolated from mice immunized with MHV-4 (JHM strain), it is cross-reactive with other MHV strains (Talbot & Buchmeier, 1985). ...

Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis

“…Notably, MHV S contains what appears to be an insertion of a stretch of residues upstream of the cleavage site (table 3), which are clearly linked to fusion activity (Taguchi and Shimazaki, 2000). In this regard, there are also parallels with influenza virus, where peptide insertions upstream of the cleavage site/fusion peptide modulate the fusion activity of H7 influenza viruses (Hamilton et al, 2012; Perdue, 2008).…”

Inhibition of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infectivity by peptides analogous to the viral spike protein

“…Like other enveloped viruses, CoVs enter target cells by inducing fusion between the viral and cellular membranes, a process mediated by the viral spike (S) glycoprotein (Gallagher and Buchmeier, 2001). The S1 subunit of the S protein mediates receptor binding (Cavanagh and Davis, 1986;Taguchi, 1995), while the S2 subunit is responsible for driving viral and target cell membrane fusion (Taguchi and Shimazaki, 2000). In the case of SARS-CoV, the S1 subunit binds to the mammalian receptor angiotensin-converting enzyme 2 (ACE2) (Huang et al, 2006;Li et al, 2003Li et al, , 2005Wang et al, 2004;Wong et al, 2004) and/or CD209L (Jeffers et al, 2004), initiating the entry process via a cathepsin L-dependent pathway (Huang et al, 2006).…”

Identification of Porcine haemagglutinating encephalomyelitis virus receptor in PK cell membranes

“…S1 mediates binding to host cell receptors and S2 mediates viral to host cell fusion. There are many epitope sites in the S1 region, which are closely correlated with immune reactions (Taguchi and Shimazaki, 2000;Callagher and Buchmeier, 2001;Krueger et al, 2001;Matsnyama and Taguchi, 2002). Therefore, we expressed HEV-S1 protein in P. pastoris, which could be specially recognized by polyclonic antibody against HEV in Western-blot analysis.…”