The composition and structure of the mouse hepatitis virus (MHV)-specific RNA in actinomycin D-treated, infected L-2 cells were studied. SEven virus-specific RNA species with molecular weights of 0.6 X 10(6), 0.9 X 10(6), 1.2 X 10(6), 1.5 X 10(6), 3.0 X 10(6), 4.0 X 10(6), and 5.4 X 10(6) (equivalent to the viral genome) were detected. T1 oligonucleotide fingerprinting studies suggested that the sequences of each RNA species were totally included within the next large RNa species. The oligonucleotides of each RNA species were mapped on the 60S RNA genome of the virus. Each RNA species contained the oligonucleotides starting from the 3' end of the genome and extending continuously for various lengths in the 3' leads to 5' direction. All of the viral RNA species contained a polyadenylate stretch of 100 to 130 nucleotides and probably identical sequences immediately next to the polyadenylate. These data suggested that the virus-specific RNAs are mRNA's and have a stairlike structure similar to that of infectious bronchitis virus, an avian coronavirus. A proposal is presented, based on the mRNA structure, for the designation of the genes on the MHV genome. Using this proposal, the sequence differences between A59, a weakly pathogenic strain, and MHV-3, a strongly hepatotropic strain, were localized primarily in mRNA's 1 and 3, corresponding t genes A and C.

The mouse hepatitis virus strain A59 codes for seven RNA species in the infected cells. These virus-specific RNAs were found to be polysome associated and therefore likely to represent mRNA's. All of them have common 3'-end sequences (Lai et al., J. Virol. 39:823-834, 1981). Their structure was further studied with respect to their 5'-end sequences. It was found that all of these mRNA's contained cap structures at their 5' ends. Furthermore, the capcontaining oligonucleotides which represent the sequences immediately adjacent to the 5' ends were found to be the same for most, if not all, of the seven virusspecific mRNA's. These sequences are also identical to the 5'-end sequences of the virion RNA genome. The 5'-end sequences were tentatively determined to be 5'-cap-N-UAAG. The presence of the common nucleotides in all of the virusspecific RNAs in mouse hepatitis virus strain A59 suggests several possible mechanisms of synthesis for these RNAs. The significance of these findings is discussed. Mouse hepatitis virus (MHV) is a member of Coronaviridae and contains a positive singlestranded 60S RNA _enome with a molecular weight of 5.4 x 10 (5). This RNA contains polyadenylic acid sequences at the 3' end (5, 16, 18) and a "cap" structure at the 5' end (6). It codes for at least three structural proteins, gp 90/ 180, pp 60, and gp 23 (12, 14), and probably three nonstructural proteins (1, 2, 10). The mode of synthesis of the viral RNA is still not clear. Recently, several laboratories have shown that seven virus-specific RNAs could be detected in the cells infected with MHV (4, 6a, 11; H. Wege, S. Siddell, M. Sturm, and V. ter Meulen, in V. ter Meulen, S. Siddell, and H. Wege, ed., Biochemistry and Biology of Coronaviruses, in press). These RNAs include the genomic RNA and six subgenomic RNA species. All of them contain polyadenylic acid sequences, suggesting that they represent mRNA's. By oligonucleotide mapping of these RNAs, we have further shown that the sequences of each virus-specific RNA are included within the next-larger RNA spe

To determine the structure and the mechanism of synthesis of mouse hepatitis virus mRNA, the map positions of the large RNase T1-resistant oligonucleotides of the seven mouse hepatitis virus strain A59 intracellular mRNA species were studied. We found that all but one of the oligonucleotides were mapped at the positions within each mRNA consistent with the nested-set, stairlike structure of mouse hepatitis virus mRNA (Lai et al., J. Virol. 39:823-834). However, one oligonucleotide, 10, was mapped near the 5' ends of every mRNA and virion genomic RNA. In other words, oligonucleotide 10 and, therefore, the sequences around the 5' ends of the mRNAs are not colinear with the genomic sequences. Because this oligonucleotide is present only once in the genomic RNA, this result indicates that oligonucleotide 10 is not transcribed from multiple sites on the genomic template, but rather represents a leader RNA sequence which is joined to the body sequences of the different mRNAs during mRNA transcription. This provides the most direct evidence thus far for the presence of leader sequences in the mRNAs of mouse hepatitis virus, which is a cytoplasmic virus. Several possible mechanisms of RNA synthesis are discussed.

There are seven virus-specific mRNA species in mouse hepatitis virus-infected cells (Lai et al., J. Virol. 39:823-834, 1981). In this study, we examined virusspecific negative-stranded RNA to determine whether there are corresponding multiple negative-stranded RNAs. Intracellular RNA from mouse hepatitis virusinfected cells was separated by agarose gel electrophoresis, transferred to nitrocellulose membranes, and hybridized to positive-stranded genomic 60S [32P]RNA. Only a single RNA species of genomic size was detected under these conditions. This RNA was negative stranded. No negative-stranded subgenomic RNA was detected. We also studied double-stranded replicative-form RNA in the infected cells. Only one replicative-form of genomic size was detected. When the double-stranded RNA isolated without RNase treatment was analyzed, again only one RNA species of genomic size was detectable. Furthermore, most of the virusspecific mRNAs could be released from this RNA species upon heating. These results suggest that all of the mouse hepatitis virus-specific RNAs are transcribed from a single species of negative-stranded RNA template of genomic size.