Identification and Genomic Mapping of the ORF3 and VPg Proteins in Feline Calicivirus Virions

Abstract

Two minor proteins with molecular masses of 8.5 and 15.5 kDa were identified in feline calicivirus (FCV) virions. Direct sequence analysis showed that the N-terminal sequence of the 8.5-kDa protein was identical to that of the predicted protein encoded by open reading frame 3 (ORF3) of the FCV genome. The N-terminal sequence of the 15.5-kDa protein corresponded to amino acids 961–980 of the FCV ORF1 polyprotein and mapped to the genomic location of the calicivirus VPg. Antisera raised against recombinant ORF3 protein or the N-terminal 20 amino acids of the putative VPg reacted with the corresponding proteins present in both a Western blot analysis of purified FCV virions and an immunofluorescence assay of FCV-infected cells. A comparative analysis of radioactivity incorporated into virion proteins during in vivo labeling experiments indicated that the ORF3 protein is likely present in one or two copies per virion. The mobility of the ORF3 protein present in virions was similar to that of the ORF3 protein found in FCV-infected cells or expressed in bacteria. Direct N- and C-terminal sequence analysis of the purified ORF3 protein obtained by expression in bacteria demonstrated the presence of intact, uncleaved termini, suggesting that the observed difference between the calculated and the apparent masses in SDS–PAGE was not due to proteolytic processing of the protein.