Acquired Fusion Activity of a Murine Coronavirus MHV-2 Variant with Mutations in the Proteolytic Cleavage Site and the Signal Sequence of the S Protein

Abstract

The spike (S) protein of a nonfusogenic murine coronavirus, MHV-2, was compared to the S protein of a variant with fusion activity, MHV-2f. Two amino acids differed between the S proteins of these viruses; one was located in the signal sequence and the other was in the putative cleavage site. The amino acid at position 12 in the signal sequence was S in MHV-2 and C in MHV-2f. The amino acid sequence of the cleavage site of MHV-2 was HRARS, while that of MHV-2f was HRARR, showing one amino acid replacement at position 757. In DBT cells infected with MHV-2, the S protein was not cleaved, while the S protein of MHV-2f was cleaved. The S protein of MHV-2f expressed in a transient vaccinia virus expression system was cleaved and was fusogenic in contrast to the nonfusogenic activity of uncleaved MHV-2 S protein. Because the signal sequence is assumed to be removed from the mature S protein soon after synthesis, and because the S protein of MHV-2 was expressed on the cell surface in the same way as the S protein of MHV-2f, the difference in the signal sequence seemed to have had little effect on the transportation and the fusion activity of the S protein. These results showed that MHV-2 does not fuse cells due to the lack of cleavage of its S protein. This conclusion differs from studies on the activity of syncytium formation by the S proteins of fusogenic MHV-JHM and -A59 strains. Possible reasons for these differences in fusion activity are discussed.

Communication between S1N330 and a Region in S2 of Murine Coronavirus Spike Protein Is Important for Virus Entry into Cells Expressing CEACAM1b Receptor

“…However, the amino acid changes in S1N330-III alone did not enhance fusion activity, as seen for srr7Aa or srr7Ba, but a simultaneous mutation at position 1114 was inevitable. These findings strongly suggest that the combination of S1N330-III and the amino acid at 1114, presumably 1114 and its neighbors common to various MHV strains (Kunita et al, 1995;Luytjes et al, 1987;Parker et al, 1989;Taguchi et al, 1992;Yamada et al, 1997;Yamada and Yabe, 2000), determines the fusogenic feature of the S protein. It is not evident at present how S1 and S2…”

Unique N-linked glycosylation of murine coronavirus MHV-2 membrane protein at the conserved O-linked glycosylation site

“…MHV-2 does not induce cell-to-cell fusion on cultured DBT cells forming syncytium, as opposed to most MHV strains. However, it seems unlikely that MHV-2 M protein is responsible for non-syncytium formation, since the S protein has been demonstrated to be the only element that determines the phenotype of MHV strain with regard to syncytium formation (Yamada et al, 1997). The M protein of MHV-2 does not seem to have sufficient role in changing pathogenicity or tissue tropism, since an A59based recombinant virus containing the entire M gene of MHV-2 produced encephalitis and hepatitis similar to parental A59 following intracerebral inoculation (Lavi et al, 1998).…”

Structural Basis for Coronavirus-mediated Membrane Fusion

“…Most of the enveloped proteins with fusion activity contain two noncovalently associated subunits: S1 ϩ S2 in coronaviruses, HA1 ϩ HA2 in influenza viruses, gp120 ϩ gp41 in HIV/SIV, GP1 ϩ GP2 in Ebola virus, and F1 ϩ F2 in paramyxoviruses, all of which are generated by proteolytic cleavage. Nevertheless, some enveloped proteins have no cleavage in their precursors and yet still maintain fusion activity, such as the S proteins of some coronaviruses (9) and GP of Ebola virus (10).…”