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 [32P]orthophosphate, an approach which provided definitive data. Only two RNA species were detected, the genomic RNA and a subgenomic mRNA of 2·4 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 2·4 kb was detectable directed the synthesis in rabbit reticulocyte lysate of the ORF3-encoded polypeptide. Furthermore, a synthetic RNA copy of the 2·4 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 2·4 kb subgenomic RNA and that this RNA is functionally bicistronic. The possible mechanisms by which ORF3 is expressed are discussed

Identification of a protein linked to the genomic and subgenomic mRNAs of feline calicivirus and its role in translation

125I protein labelling of oligo(dT)-selected RNA from feline calicivirus (FCV)-infected cells revealed that the genomic and 2.4 kb subgenomic RNAs of FCV are linked to a 15 kDa protein (VPg). Proteinase K treatment of FCV RNA, to remove VPg, led to a decrease in the translatability of the RNA, but there was no obvious change in the site of RNA initiation. Addition of the cap analogue 7-methylGTP to in vitro translations had no effect on the translation of FCV RNA, suggesting that FCV RNA is translated by a cap-independent mechanism. Further evidence that FCV RNA is translated by an unusual mechanism was obtained by translating FCV RNA in vitro at a range of K+ concentrations. FCV RNA was able to direct translation at K+ concentrations at which cellular RNA translation was inhibited

Comparison of acetate inhibition on growth of host and recombinant E. coli K12 strains

10.1007/BF01027012Biotechnology Letters14121115-1118BILE