National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan1
National Veterinary Assay Laboratory, 1-15-1 Tokura, Kokubunji, Tokyo 185-8511, Japan2
Author for correspondence: Fumihiro Taguchi. Fax +81 423 46 1754. e-mail taguchi{at}ncnp.go.jp
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Abstract |
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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 (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 virusreceptor interactions. Another important function is cellcell and viruscell 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, Leu1-Leu2-Gly3-Cys4-Ile5-Gly6-Ser7-Thr8-Cys9 (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
). These findings suggest that S2A plays a critical role in the life-cycle of MHV, presumably in either virusreceptor interactions and/or virus entry into cells. In the present study, we analyse the receptor-binding and fusion activities of the S2A epitope.
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Since S2A antiserum neutralizes JHMV infectivity, albeit weakly, we examined whether this neutralization was due to the inhibition of MHV binding to its receptor CEACAM1a (MHVR1) (Dveksler et al., 1991 ), although S2 has been shown not to bind to a receptor (Taguchi, 1995
). MHVR1 tagged with the Influenza virus HA epitope (Ohtsuka et al., 1996
) was bound to protein ASepharose beads via anti-HA antibodies. JHMV (5x105 p.f.u. in 100 µl) was incubated with 20 VN units of either anti-S1 MAbs or S2A antiserum at room temperature for 2 h. In this experiment, 20 VN units of each antibody neutralized more than 95% of JHMV. The virus stock treated with anti-S1 MAbs was also incubated with anti-mouse IgG Fc antibodies in order to prevent direct binding of anti-JHMV-S1 MAb complexes to the protein ASepharose beads via the Fc portion of the anti-S1 MAbs. The treated virus stocks were then allowed to interact with the MHVR1-bound protein ASepharose beads. The beads were collected by centrifugation, washed, subjected to SDSPAGE and blotted onto a transfer membrane. As shown in Fig. 2
, S protein was still bound by MHVR1 when viruses were treated with S2A antiserum, MAb 13 or control serum. This shows that these antibodies failed to block the binding of viruses to the receptor. In contrast, only trace amounts of the S protein were detected following the treatment of the JHMV stock with MAbs 3, 6 or 93, indicating that these three MAbs blocked the binding of virus to the receptor. These results indicate that the S2A antiserum fails to block the binding of virus to the receptor and argue strongly against the involvement of the S2A epitope in receptor-binding activity. Neutralization of S1-specific MAb 13 was shown to be independent of the interference of virus binding to the receptor. Its VN activity is, however, presumably not due to the inhibition of viruscell fusion, since its FI activity was slightly lower relative to that of other MAbs. MAb 13 could inhibit virus internalization, as reported for several MAbs against human immunodeficiency virus and influenza virus; these MAbs neutralize these viruses without interfering with the receptor binding of the viruses (Armstrong et al., 1996
; Outlaw & Dimmock, 1993
; Skinner et al., 1988
).
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It was recently reported that a region, PEP1, in the heptad repeat 1 of MHV strain A59 could work as a fusion peptide (Luo & Weiss, 1998 ). The fusion peptide motif has several common features: it is relatively hydrophobic, it adopts an alpha-helical conformation and it is rich in Ala and Gly (White, 1990
). Further, substitution of hydrophobic amino acids in the fusion peptide with hydrophilic residues usually destroys fusion activity (Bosch et al., 1989
; Gething et al., 1986
; White, 1990
). The PEP1 fusion peptide has these features (Luo & Weiss, 1998
). However, the hydrophobic feature of S2A is not critical for fusion activity. Moreover, the S2A epitope is unable to form an alpha-helix according to computer analysis. These findings suggest that S2A plays an important role in fusion activity, yet the mechanism by which the S2A epitope operates in fusion events appears to be different from that of the ordinary fusion peptide (White, 1990
). Completion of MHV fusion events may require several distinct steps of interaction between the viral envelope protein and the cell membrane, with some functions performed by the PEP1 fusion peptide and others by S2A.
MAb 5B19.2 cross-reacted with other MHV strains suggesting that the S2A epitope exists in these MHV strains (Talbot & Buchmeier, 1985 ). JHMV and MHV-A59 S proteins have an identical 15 amino acid cluster encompassing the S2A epitope (Luytjes et al., 1987
; Parker et al., 1989
; Schmidt et al., 1987
). Four other MHV strains have a stretch almost identical to this cluster with one to four amino acid substitutions (Kunita et al., 1995
; Yamada et al., 1997
; Yamada & Yabe, 2000
). Moreover, coronaviruses classified in group 2, bovine coronavirus and human coronavirus OC43, do have a similar stretch with four or five substitutions out of 15 amino acids (Boireau et al., 1990
; Mounir & Talbot, 1993
; Parker et al., 1990
). However, coronaviruses classified in groups 1 and 3 lack such a sequence. This suggests that the hydrophobic region including the S2A epitope may be an important site for fusion activity for the coronaviruses classified in group 2.
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References |
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Received 18 April 2000;
accepted 1 September 2000.