Identification and stability of a 30-kDa nonstructural protein encoded by mRNA 2 of mouse hepatitis virus in infected cells.

A bacterial expression vector encoding a fusion protein containing almost the entire first open reading frame (ORF1) of mRNA 2 of MHV-A59 has been constructed. The purified fusion protein was used to raise antibodies to the protein encoded by mRNA 2 ORF1. Specificity of the antibodies was verified by immunoprecipitation of the in vitro translation product of ORF1, which was reconstructed downstream of a T7 promoter. In vivo the antiserum reacted specifically with a 30-kDa protein synthesized in MHV-A59- and MHV-JHM-infected cells. This 30-kDa protein could not be identified in purified virions and is therefore a nonstructural viral protein. The expression pattern of this 30-kDa nonstructural viral protein in infected cells was shown to be identical to that of the viral structural proteins. However, in comparison to the nucleocapsid protein pulse-chase studies revealed a relative short half life for this 30-kDa protein in vivo.