Characterization of the Nonstructural and Spike Proteins of the Human Respiratory Coronavirus OC43: Comparison with Bovine Enteric Coronavirus

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Abstract
The nucleotide sequence of the region between the spike (S) and the membrane (M) protein genes, and sequences of the S and ns2 genes of the OC43 strain of human coronavirus (HCV-OC43) were determined. The ns2 gene comprises an open reading frame (ORF) encoding a putative nonstructural (ns) protein of 279 amino acids with a predicted molecular mass of 32-kDa. The S gene comprises an ORF encoding a protein of 1353 amino acid residues, with a predicted molecular weight of 149,918. Sequence comparison between HCV-OC43 and the antigenically related bovine coronavirus (BCV) revealed more sequence divergence in the putative bulbous part of the S protein (S1) than in the stem region (S2). The cysteine residues near the transmembrane domain and the internal predicted protease cleavage site are conserved in the HCV-OC43 S protein. Nucleotide sequence analysis of the region between the S and M gene loci revealed the presence of an unexpected intragenomic partial leader sequence and two ORFs encoding potential proteins of 12.9 and 9.5-kDa. These two proteins were identified as nonstructural by comparison with the homologous BCV genes. In vitro translation analyses demonstrated that the HCV-OC43 9.5-kDa protein, like its BCV counterpart, is poorly translated when situated downstream of the 12.9-kDa ORF, but is expressed in infected cells, as shown by immunofluorescence. Interestingly, two ORFs, potentially encoding 4.9 and 4.8-kDa ns proteins in BCV are absent in HCV-OC43, indicating that they are not essential for viral replication in HRT-18 cells.