Expression of reovirus p14 in bacteria and identification in the cytoplasm of infected mouse L cells

Reovirus genome segment S1 is transcribed by the virion-associated polymerase to form a single mRNA species that codes for two polypeptides: the 49-kDa cell-attachment protein, σ1, starting from the first A-U-G in the S1 transcript, and a 14-kDa nonstructural, basic protein initiated from the second A-U-G in a different reading frame ( Ernst and Shatkin, 1985; Jacobs et al., 1985 ; Shatkin, 1985). To confirm that p14 is made in reovirus-infected cells, determine its intracellular location, and generate sufficient amounts of the polypeptide to begin an analysis of its presumptive role in the virus life cycle, the p14 coding sequence of an S1 cDNA clone was subcloned into the EcoRI site downstream of the λP L promoter in the bacterial expression vector, pEV-vrfl. The vector was modified to align the ribosome binding site with the p14 initiator codon, and transcription was placed under control of λ cIts in a compatible plasmid. Transformed Escherichia coli RRI incubated at 42° produced a new polypeptide of ∼14 kDa as determined by SDS-PAGE. This polypeptide reacted specifically with rabbit antisera made against synthetic peptides corresponding to exposed regions of authentic p14 as predicted from the S1 cDNA sequence. Antipeptide sera also precipitated a ∼14-kDa polypeptide in lysates of reovirus-infected mouse L cells, demonstrating the synthesis of p14 in vivo. Immunofluorescence experiments indicate that p14 accumulates in the cytoplasm of infected L cells.