The 5'-most gene, gene 1, of the genome of murine coronavirus, mouse hepatitis virus (MHV), is presumed to encode the viral RNA-dependent RNA polymerase. We have determined the complete sequence of this gene of the JHM strain by cDNA cloning and sequencing. The total length of this gene is 21,798 nucleotides long, which includes two overlapping, large open reading frames. The first open reading frame, ORF 1a, is 4488 amino acids long. The second open reading frame, ORF 1b, overlaps ORF 1a for 75 nucleotides, and is 2731 amino acids long. The overlapping region may fold into a pseudoknot RNA structure, similar to the corresponding region of the RNA of avian coronavirus, infectious bronchitis virus (IBV). The in vitro transcription and translation studies of this region indicated that these two ORFs were most likely translated into one polyprotein by a ribosomal frameshifting mechanism. Thus, the predicted molecular weight of the gene 1 product is more than 800,000 Da. The sequence of ORF 1b is very similar to the corresponding ORF of IBV. In contrast, the ORF 1a of these two viruses differ in size and have a high degree of divergence. The amino acid sequence analysis suggested that ORF 1a contains several functional domains, including two hydrophobic, membrane-anchoring domains, and three cysteine-rich domains. It also contains a picornaviral 3C-like protease domain and two papain-like protease domains. The presence of these protease domains suggests that the polyprotein is most likely processed into multiple protein products. In contrast, the ORF 1b contains polymerase, helicase, and zinc-finger motifs. These sequence studies suggested that the MHV gene 1 product is involved in RNA synthesis, and that this product is processed autoproteolytically after translation. This study completes the sequence of the MHV genome, which is 31 kb long, and constitutes the largest viral RNA known.

The 5'-most gene of the murine coronavirus genome, gene A, is presumed to encode viral RNA-dependent RNA polymerase. It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5'-most 5.3 kilobases of murine coronavirus mouse hepatitis virus strain JHM were sequenced and subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. The sequence was found to encode a single long open reading frame continuing from near the 5' terminus of the genome. Although p28 is encoded from the first 1 kilobase at the 5' end of the genome, translation of in vitro-transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3-kilobase region was synthesized. Translation of RNAs of 3.9 kilobases or smaller yielded proteins which contained the p28 sequence, but p28 was not cleaved. This suggests that the sequence in the region between 3.9 and 5.3 kilobases from the 5' end of the genomic RNA is essential for proteolytic cleavage and contains autoproteolytic activity. The p28 protein could not be cleaved from the smaller primary translation products of gene A, even in the presence of the larger autocleaving protein. Cleavage of the p28 protein was inhibited by addition of the protease inhibitor ZnCl2. This study thus identified a protein domain essential for autoproteolytic cleavage of the gene A polyprotein.

We have previously shown that some strains of the murine coronavirus mouse hepatitis virus (MHV) synthesize an additional mRNA species (mRNA 2b, previously called mRNA 2a) with a size intermediate between that of mRNAs 2 and 3, suggesting the presence of an optional transcriptional initiation site. This transcriptional start is dependent on the leader sequence of the virus strains. To study the mechanism of coronavirus transcriptional regulation, we have cloned and sequenced the region of the viral genome corresponding to the 5' unique coding region of mRNA 2 of the JHM strain of MHV. In addition to the open reading frame (ORF) predicted to encode the viral nonstructural protein p30, a second complete ORF, with the potential to encode a 439-amino-acid polypeptide, was discovered. The transcriptional initiation sites of both mRNA 2a (formerly called mRNA 2) and mRNA 2b were determined by primer extension studies and RNA sequencing. The data indicated that transcription of mRNA 2a initiated at a site, UCUAUAC, that resembled the consensus intergenic sequence. In contrast, the start signal of the optional mRNA 2b, UAAUAAAC, deviated from the consensus sequence. mRNA 2b is a functional mRNA, as shown by in vitro translation studies of mRNA and ORF 2b and by the detection of an additional viral structural protein, gp65, in the JHM strain that synthesized this mRNA. Although the A59 strain of MHV was found to retain ORF 2b, it lacked the correct transcriptional and translational start signals for this gene. This study has therefore identified an optional gene product for murine coronaviruses and provided insights into the mechanism of regulation of MHV RNA transcription.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.