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The sequence of the MHV-A 59 non-structural gene 4 (ns 4) reveals two open reading frames. The upstream ORF potentially encodes a 19 amino acid (2.2 kDa) polypeptide and the downstream ORF potentially encodes a 106 amino acid (11.7 kDa) polypeptide. This is in contrast to MHV-JHM gene 4 which expresses a 15 kDa protein. Cell free translation of a synthetic mRNA containing both ORFs of MHV-A 59 ns 4 results in the synthesis of a 2.2 kDa polypeptide; the predicted 11.7 kDa product of the MHV-A 59 downstream ORF is not detected during cell free translation nor in infected cells. These results add to the recent data suggesting that expression of some of the ns proteins of MHV is not necessary for efficient growth in tissue culture.

Several polypeptide products of MHV-A59 ORF 1a were characterized in MHV-A59 infected DBT cells, using antisera directed against fusion proteins encoded in the first 6.5 kb of ORF1a. These included the previously identified N-terminal ORF 1a product, p28, as well as 290-, 240-, and 50-kDa polypeptides. P28 was always detected as a discrete band without larger precursors, suggesting rapid cleavage of p28 immediately after its synthesis. Once p28 was cleaved there was little degradation of the protein over a 2-hr period. The intracellular cleavage of p28 was not inhibited by the protease inhibitor leupeptin, in contrast to results obtained during in vitro translation of genome RNA (Denison and Perlman, 1986). These data suggest that different protease activities may be responsible for the cleavage of p28 in vitro and in vivo. The 290-kDa protein was an intermediate cleavage product derived from a precursor of greater than 400 kDa. The 290-kDa product was subsequently cleaved into secondary products of 50 and 240 kDa. The intracellular cleavage of the 290-kDa polypeptide was inhibited by leupeptin at concentrations which did not inhibit the early cleavage of p28 or the cleavage of the 290-kDa product from its larger polyprotein precursor. In the presence of zinc chloride, a product of greater than 320 kDa was detected, which appears to incorporate p28 at its amino terminus. This suggests that at least two protease activities may be necessary for processing of ORF1a proteins, one of which cleaves p28 and is sensitive to zinc chloride but resistant to leupeptin, and the other which cleaves the 290-kDa precursor and is sensitive to both inhibitors. Both the 290- and 240-kDa proteins should contain sequences predicted to encode two papain-like protease activities.

A 266-bp fragment of cDNA from within gene B, ORF 2a, of MHV-A59 was used to construct a vector encoding a bacterial/viral fusion protein. Antiserum raised against this fusion protein specifically immunoprecipitates a 30K protein from infected 17Cl-1 mouse fibroblasts. The protein is localized primarily in the cytosol and not in the membranes. This is consistent with its predicted sequence and potential role as an RNA binding protein.

Mouse hepatitis virus, strain A59 cDNAs were inserted into the procaryotic fusion vector pGE374. RecA/viral/LacZ tripartite fusion proteins were synthesized from these plasmids and purified from E. coli. Antisera were raised in rabbits against these fusion proteins. Viral nonstructural proteins were detected in infected murine fibroblasts and glial cells. The anti-gene B, ORF1 sera detect a 30K cytoplasmic protein while the anti-gene E, ORF2 sera detect a 9.6K protein. Sera raised against proteins encoded in cDNAs from 5' portions of gene A immunoprecipitate the 200-250K polypeptides synthesized in vitro from genome RNA. Antisera raised against proteins encoded in both 5' and 3' portions of gene A immunoprecipitate membrane associated polypeptides of 150K and greater than 600K from MHV infected cells.

Complementary DNA (cDNA) libraries were constructed representing the genome RNA of the coronavirus mouse hepatitis virus, strain A59 (MHV-A59). From these libraries clones were selected to form a linear map across the entire gene A, the putative viral polymerase gene. This gene is approximately 23 kb in length, considerably larger than earlier estimates. Sequence analysis of the 5' terminal region of the genome indicates the presence of the 66-nucleotide leader that is found on all mRNAs. Secondary structure analysis of the 5' terminal region suggests that transcription of leader terminates in the region of nucleotide 66. The sequence of the first 2000 nucleotides is very similar to that reported for the closely related JHM strain of MHV and potentially encodes p28, a basic protein thought to be a component of the viral polymerase (L. Soe, C. K. Shieh, S. Baker, M. F. Chang, and M. M. C. Lai, 1987, J. Virol., 61, 3968-3976). Gene A contains two of the consensus sequences found in intergenic regions. One is adjacent to the 5' leader sequence and the other is upstream from the initiation codon for translation of gene B.

A prokaryotic vector, pGE374, containing the recA and lacZ genes, out-of-frame, was used for the expression of cDNA derived from the putative polymerase-encoding gene of the coronavirus mouse hepatitis virus strain A59 (MHV-A59). The pGE374/viral recombinant vector generates a tripartite bacterial/viral protein composed of a segment of the RecA protein at the N terminus, the coronaviral sequences in the middle, and an enzymatically active beta-galactosidase at the C terminus. Rabbits immunized with such recombinant proteins generated antibodies to the MHV-A59 portion of the tripartite protein. Because the MHV-A59 polymerase proteins have been difficult to identify during infection, we used a novel method to demonstrate the viral specificity of the antiserum. The viral cDNA was excised from the expression vector, and transferred to a pGem vector, downstream from and in-frame with a portion of the cat gene. This construct contained a bacteriophage RNA polymerase promoter that enabled the cell-free synthesis of a fusion protein that was used to verify that antibodies were generated to the expressed viral DNA. This strategy was shown to successfully result in the specific generation of antibodies to the encoded information of the viral cDNA. Furthermore, this method has general applicability in the generation and characterization of antibodies directed against proteins encoded in cDNAs.