Human Coronavirus OC43 RNA 4 Lacks Two Open Reading Frames Located Downstream of the S Gene of Bovine Coronavirus

Nucleotide sequences between the spike (S) and membrane (M) protein genes of the OC43 strain of human coronavirus were obtained from POP-amplified vital mRNAs. Sequence analysis of this region revealed the presence of two ORFs encoding proteins of 12.9 and 9.5 kDa. These two proteins were identified as putatively nonstructural (ns) due to their homology to the corresponding BCV ns gene products. Northern blot analysis indicated that each of these two genes was present on a separate mRNA (5 and 5-1, respectively). 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, although immunofluorescence studies did confirm its presence in infected cells. Sequence analysis showed that a large portion of the 3′-end of the leader sequence is present within the viral genome, upstream of the 12.9-kDa ORF. In addition, two ORFs encoding potential 4.9- and 4.8-kDa ns proteins in BCV are absent in HCV-OC43, although a corresponding mRNA 4 was found at a very low level. These results demonstrate that these two putative ns proteins are not essential for virus replication, at least in HRT-18 cells.