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Both genomic and subgenomic replicative intermediates (RIs) and replicative-form (RF) structures were found in 17CL1 mouse cells that had been infected with the A59 strain of mouse hepatitis virus (MHV), a prototypic coronavirus. Seven species of RNase-resistant RF RNAs, whose sizes were consistent with the fact that each was derived from an RI that was engaged in the synthesis of one of the seven MHV positive-strand RNAs, were produced by treatment with RNase A. Because the radiolabeling of the seven RF RNAs was proportional to that of the corresponding seven positive-strand RNAs, the relative rate of synthesis of each of the MHV positive-strand RNAs may be controlled by the relative number of each of the size classes of RIs that are produced. In contrast to alphavirus, which produced its subgenome-length RF RNAs from genome-length RIs, MHV RF RNAs were derived from genome-and subgenome-length RIs. Only the three largest MHV RF RNAs (RF,, RF1I, and RFIII) were derived from the RIs that migrated slowest on agarose gels. The four smallest RF RNAs (RFIV, RFv, RFvl, and RFVIl) were derived from RIs that migrated in a broad region of the gel that extended from the position of 28S rRNA to the position of the viral single-stranded MHV mRNA-3. Because all seven RIs were labeled during very short pulses with [3H]uridine, we concluded that the subgenome-length RIs are transcriptionally active. These findings, with the recent report of the presence of subgenome-length negative-strand RNAs in cells infected with porcine transmissible gastroenteritis virus (P. B.

We have developed a new model for corona virus transcription, which we call discontinuous extension, to explain how subgenome-Iength negatives stands are derived directly from the genome. The current model called leader-primed transcription, which states that sub genomic mRNA is transcribed directly from genome-length negative-strands, cannot explain many of the recent experimental findings. For instance, sub genomic mRNAs are transcribed directly via transcription intermediates that contain subgenome-length negativestrand templates; however subgenomic mRNA does not appear to be copied directly into negatives strands. In our model the subgenome-Iength negative strands would be derived using the genome as a template. After the polymerase had copied the 3'-end of the genome, it would detach at anyone of the several intergenic sequences and reattach to the sequence immediately downstream of the leader sequence at the 5'-end of genome RNA. Base pairing between the 3'-end of the nascent subgenome-length negative strands, which would be complementary to the intergenic sequence at the end of the leader sequence at the 5'-end of genome, would serve to align the nascent negative strand to the genome and permit the completion of synthesis, i.e., discontinuous extension of the 3'-end of the negative strand. Thus, sub genome-length negative-strands would arise by discontinuous synthesis, but of negative strands, not of positive strands as proposed originally by the leader-primed transcription model. Coronaviruses contain an unusually long (27-32,000 ribonucleotides) positive-sense RNA genome (1, 2) that is polyadenylated at the 3' end and capped at the 5' end. During the coronavirus replication cycle, genome and sub genomic mRNAs are produced that together comprise a 3' co-terminal nested set. At their 5' ends they also all possess an identical sequence, called the leader RNA, of 60-80 nucleotides. The original proposal (3) that transcription of subgenomic mRNA was discontinuous was based on the published report (4) that only genome-length negative-strand templates existed in infected cells. Splicing of

Coronaviruses contain an unusually long (27-32,000 ribonucleotide) positive sense RNA genome that is polyadenylated at the 3' end and capped at the 5' end. In addition to the genome, infected cells contain subgenomic mRNAs that form a 3' co-terminal nested set with the genome. In addition to their common 3' ends, the genome and the subgenomic mRNAs contain an identical 5' leader sequence. The transcription mechanism that coronaviruses use to produce subgenomic mRNA is not known and has been the subject of speculation since sequencing of the subgenomic mRNAs showed they must arise by discontinuous transcription. The current model called leader-primed transcription has subgenomic mRNAs transcribed directly from genome-length negative strands. It was based on the failure to find in coronavirus infected cells subgenome-Iength negative strands or replication intermediates containing subgenome-length negative strands. Clearly, these structures exist in infected cells and are transcriptionally active. We proposed a new model for coronavirus transcription which we called 3' discontinuous extension of negative strands. This model predicts that subgenome-length negative strands would be derived directly by transcription using the genome RNA as a template. The subgenome-length templates would contain the common 5' leader sequence and serve as templates for the production of subgenomic mRNAs. Our findings include showing that: I. Replication intermediates (RIs) containing subgenome-length RNA exist in infected cells and are separable from RIs with genome-length templates. The RFs with subgenome-length templates are not derived by RNase treatment of RIs with genome-length templates. 2. The subgenome-length negative strands are formed early in infection when RIs are accumulating and the rate of viral RNA synthesis is increasing exponentially. 3. Subgenome-Iength negative strands contain at their 3' ends a complementary copy of the 72 nucleotide leader RNA that is found in the genome only at their 5' end. 4. RIs with subgenomic templates Coronaviruses and Arteriviruses, edited by Enjuanes et af.

The coronavirus replicase-transcriptase complex is an assembly of viral and cellular proteins that mediate the synthesis of genome and subgenome-sized mRNAs in the virus-infected cell. Here, we report a genetic and functional analysis of 19 temperature-sensitive (ts) mutants of Murine hepatitis virus MHV-A59 that are unable to synthesize viral RNA when the infection is initiated and maintained at the non-permissive temperature. Both classical and biochemical complementation analysis leads us to predict that the majority of MHV-A59 ORF1a replicase gene products (non-structural proteins nsp1–nsp11) form a single complementation group (cistron1) while the replicase gene products encoded in ORF1b (non-structural proteins nsp12–nsp16) are able to function in trans and comprise at least three, and possibly five, further complementation groups (cistrons II–VI). Also, we have identified mutations in the non-structural proteins nsp 4, nsp5, nsp10, nsp12, nsp14, and nsp16 that are responsible for the ts phenotype of eight MHV-A59 mutants, which allows us to conclude that these proteins are essential for the assembly of a functional replicase-transcriptase complex. Finally, our analysis of viral RNA synthesis in ts mutant virus-infected cells allows us to discriminate three phenotypes with regard to the inability of specific mutants to synthesize viral RNA at the non-permissive temperature. Mutant LA ts6 appeared to be defective in continuing negative-strand synthesis, mutant Alb ts16 appeared to form negative strands but these were not utilized for positive-strand RNA synthesis, and mutant Alb ts22 was defective in the elongation of both positive- and negative-strand RNA. On the basis of these results, we propose a model that describes a pathway for viral RNA synthesis in MHV-A59-infected cells. Further biochemical analysis of these mutants should allow us to identify intermediates in this pathway and elucidate the precise function(s) of the viral replicase proteins involved.