THE INTERNAL OPEN READING FRAME WITHIN THE NUCLEOCAPSID GENE OF MOUSEHEPATITIS-VIRUS ENCODES A STRUCTURAL PROTEIN THAT IS NOT ESSENTIAL FOR VIRAL REPLICATION

The coronavirus mouse hepatitis virus (MHV) contains a large open read ing frame embedded entirely within the 5' half of its nucleocapsid (N) gene. This internal gene (designated I) is in the +1 reading frame wi th respect to the N gene, and it encodes a mostly hydrophobic 23-kDa p olypeptide. We have found that this protein is expressed in MHV-infect ed cells and that it is a previously unrecognized structural protein o f the virion. To analyze the potential biological importance of the I gene, we disrupted its expression by site directed mutagenesis using t argeted RNA recombination. The start codon for I was replaced by a thr eonine codon, and a stop codon was introduced at a short interval down stream. Both alterations created silent changes in the N reading frame . In vitro translation studies showed that these mutations completely abolished synthesis of I protein, and immunological analysis of infect ed cell lysates confirmed this conclusion. The MHV I mutant was viable and grew to high titer. However, the I mutant had a reduced plaque si ze in comparison with its isogenic wild-type counterpart, suggesting t hat expression of I confers some minor growth advantage to the virus. The engineered mutations were stable during the course of experimental infection in mice, and the I mutant showed no significant differences from wild type in its ability to replicate in the brains or livers of infected animals. These results demonstrate that I protein is not ess ential for the replication of MHV either in tissue culture or in its n atural host.