Abstract
The spike (S) glycoprotein of mouse hepatitis virus (MHV) plays a major role in the viral pathogenesis. It is often processed into the N-terminal S1 and the C-terminal S2 subunits that were evidently important for binding to cell receptor and inducing cell-cell fusion, respectively. As a consequence of cell-cell fusion, most of the naturally occurring infections of MHV are associated with syncytia formation. So far, only MHV-2 was identified to be fusion-negative. In this study, the S gene of MHV-2 was molecularly cloned, and the nucleotide sequence was determined. The MHV-2 S protein lacks a 12-amino acid stretch in the S1 hypervariable region from amino acid residue 446 to 457 when compared with the fusion-positive strain MHV-JHM. In addition, there are three amino acid substitutions in the S2 subunit, Tyr-1144 to Asp, Glu-1165 to Asp, and Arg-1209 to Lys. The cloned MHV-2 S protein exhibited the fusion-negative property in DBT cells as the intrinsic viral protein. Furthermore, similar to the fusion-positive MHV-JHM strain, proteolytic cleavage activity was detected both in DBT cells infected with the fusion-negative MHV-2 and in the transfected cells that expressed the cloned MHV-2 S protein. Domain swapping experiments demonstrated that the 12-amino acid stretch missing in the MHV-2 S1 subunit, but not the proteolytic cleavage site, was critical for the cell-fusion activity of MHV.
Highlights
Mouse hepatitis virus (MHV)1 is a member of the Coronaviridae family that causes inapparent enteric and respiratory infection, hepatitis, and acute and chronic demyelinating diseases of the central nervous system [1]
By replacing the BspEI–ClaI fragment of the MHV-2 S gene that lacks the 12-amino acid stretch with the cognate fragment of MHV-JHM, the hybrid S-m2/j protein was found to cause syncytia of cultured DBT cells 48 h posttransfection (Fig. 3, C and D), whereas by replacing the BspEI–ClaI fragment of MHV-JHM S gene with the cognate fragment of MHV-2, no polykaryon formation was evident, the hybrid S-j/m2 protein was expressed well to the S-m2/j in the transfected cells (Fig. 3, A and B). These results indicated that the 12 amino acid residues in the hypervariable region of the S1 subunit between 446 and 457 are important, but the three variable amino acid residues in the C-terminal domain may have little contribution for the cell fusion activity of MHV S protein
The MHV-2 S protein possesses the predicted proteolytic cleavage sequence RRARR and was capable of undergoing cleavage both in MHV-2-infected cells and cells transfected with the S-encoding plasmid
Summary
Fusion-negative Property of MHV-2 cells, a recombinant vaccinia virus that encodes the T7 RNA polymerase [38] and a recombinant plasmid containing the cDNA of MHV S protein driven by the promoter of T7 RNA polymerase were used in this study. We found that the 12amino acid stretch from residue 446 to 457 of the MHV-JHM was important for the cell fusion activity. The S protein of MHV-2 was capable of undergoing proteolytic cleavage, but it lost the ability to induce cell-cell fusion
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