Detection of the ORF3 polypeptide of feline calicivirus in infected cells and evidence for its expression from a single, functionally bicistronic, subgenomic mRNA

Feline calicivirus (FCV) is a small positive-stranded RNA virus within the family Caliciviridae. Its genome is 7690 nucleotides in length and encodes three open reading frames (ORFs). The smallest, ORF3, is located at the extreme 3' end of the genome and can potentially encode a polypeptide of approximately 12 kDa. In this paper, we report the identification of an ORF3-encoded polypeptide in FCV-infected cells using an antiserum raised against a bacterially-expressed bacteriophage T7 gene 10-ORF3 fusion protein. Although a small mRNA of 0.5 kb, which could potentially encode ORF3, has been described, reports on the number and size of FCV subgenomic RNAs have varied considerably. To clarify the situation, RNAs from FCV-infected cells were labelled in vivo using [P-32]orthophosphate, an approach which provided definitive data. Only two RNA species were detected, the genomic RNA and a subgenomic mRNA of 24 kb. The 5' end of the subgenomic mRNA was mapped to position 5227 on the genomic RNA using RNA sequencing and primer extension methods. RNA isolated from FCV-infected cells in which no subgenomic RNA smaller than 24 kb was detectable directed the synthesis in rabbit reticulocyte lysate of the ORF3-encoded polypeptide. Furthermore, a synthetic RNA copy of the 24 kb subgenomic mRNA of FCV, containing both ORF2 and 3, directed the synthesis of both ORF2 and ORF3 polypeptides in the in vitro translation system. These data strongly suggest that ORF3 is expressed from the 24 kb subgenomic RNA and that this RNA is functionally bicistronic. The possible mechanisms by which ORF3 is expressed are discussed.