The internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication.
The coronavirus mouse hepatitis virus (MHV) contains a large open reading frame embedded entirely within the 5' half of its nucleocapsid (N) gene. This internal gene (designated I) is in the +1 reading frame with respect to the N gene, and it encodes a mostly hydrophobic 23-kDa polypeptide. We have found that this protein is expressed in MHV-infected cells and that it is a previously unrecognized structural protein of the virion. To analyze the potential biological importance of the I gene, we disrupted its expression by site-directed mutagenesis using targeted RNA recombination. The start codon for I was replaced by a threonine codon, and a stop codon was introduced at a short interval downstream. 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 infected cell lysates confirmed this conclusion. The MHV I mutant was viable and grew to high titer. However, the I mutant had a reduced plaque size in comparison with its isogenic wild-type counterpart, suggesting that 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 essential for the replication of MHV either in tissue culture or in its natural host.
Fischer
F
F
Department of Biomedical Sciences, State University of New York at Albany, New York 12237, USA.
Peng
D
D
Hingley
S T
ST
Weiss
S R
SR
Masters
P S
PS
D000818:Animals; D015967:Gene Expression Regulation, Viral; D051379:Mice; D006517:Murine hepatitis virus / Q000502:physiology*; D019251:Nucleocapsid / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics*
D015678:Viral Structural Proteins