In our studies of murine coronavirus transcription, we continue to use defective interfering (DI) RNAs of mouse hepatitis virus (MHV) in which we insert a transcription consensus sequence in order to mimic subgenomic RNA synthesis from the nondefective genome. Using our subgenomic DI system, we have studied the effects of sequences flanking the MHV transcription consensus sequence on subgenomic RNA transcription. We obtained the following results. (i) Insertion of a 12-nucleotide-long sequence including the UCUAAAC transcription consensus sequence at different locations of the DI RNA resulted in different efficiencies of subgenomic DI RNA synthesis. (ii) Differences in the amount of subgenomic DI RNA were defined by the sequences that flanked the 12-nucleotide-long sequence and were not affected by the location of the 12-nucleotide-long sequence on the DI RNA. (iii) Naturally occurring flanking sequences of intergenic sequences at gene 6-7, but not at genes 1-2 and 2-3, contained a transcription suppressive element(s). (iv) Each of three naturally occurring flanking sequences of an MHV genomic cryptic transcription consensus sequence from MHV gene 1 also contained a transcription suppressive element(s). These data showed that sequences flanking the transcription consensus sequence affected MHV transcription.

Insertion of a region, including the 18-nucleotide-long intergenic sequence between genes 6 and 7 of mouse hepatitis virus (MHV) genomic RNA, into an MHV defective interfering (DI) RNA leads to transcription of subgenomic DI RNA in helper virus-infected cells (S. Makino, M. Joo, and J. K. Makino, J. Virol. 66:6031-6041, 1991). In this study, the subgenomic DI RNA system was used to determine how sequences flanking the intergenic region affect MHV RNA transcription and to identify the minimum intergenic sequence required for MHV transcription. DI cDNAs containing the intergenic region between genes 6 and 7, but with different lengths of upstream or downstream flanking sequences, were constructed. All DI cDNAs had an 18-nucleotide-long intergenic region that was identical to the 3\' region of the genomic leader sequence, which contains two UCUAA repeat sequences. These constructs included 0 to 1,440 nucleotides of upstream flanking sequence and 0 to 1,671 nucleotides of downstream flanking sequence. An analysis of intracellular genomic DI RNA and subgenomic DI RNA species revealed that there were no significant differences in the ratios of subgenomic to genomic DI RNA for any of the DI RNA constructs. DI cDNAs which lacked the intergenic region flanking sequences and contained a series of deletions within the 18-nucleotide-long intergenic sequence were constructed to determine the minimum sequence necessary for subgenomic DI RNA transcription. Small amounts of subgenomic DI RNA were synthesized from genomic DI RNAs with the intergenic consensus sequences UCUAAAC and GCUAAAC, whereas no subgenomic DI RNA transcription was observed from DI RNAs containing UCUAAAG and GCTAAAG sequences. These analyses demonstrated that the sequences flanking the intergenic sequence between genes 6 and 7 did not play a role in subgenomic DI RNA transcription regulation and that the UCUAAAC consensus sequence was sufficient for subgenomic DI RNA transcription.

A plaque-cloned mouse hepatitis virus mutant, MHV-S No. 8, was isolated from Ki-BALB cells persistently infected with MHV-S. The mRNAs 1 to 6 were larger in the mutant, whereas there was no difference between the two viruses in the size of the smallest mRNA, mRNA 7. Sequence analyses of the genomic RNA, mRNA 6, and mRNA 7 of the two viruses revealed that an additional 111 nt were inserted just upstream of the intergenic consensus sequence preceding the N gene in MHV-S No. 8. The inserted region consisted of two different parts; the 3\'-most 30 nt corresponded to nucleotides 28 to 57 of the leader sequence and the 5\'-most 81 nt corresponded to nucleotides 58 to 138 of mRNA 7. This structure of No. 8 was most likely generated by RNA-RNA recombination between genomic RNA and subgenomic RNA species. The nucleotide insertion in the intergenic sequence between genes M and N resulted in two consensus sequences separated by 111 nt. Primer extension analysis revealed that the amount of a slightly larger, subgenomic mRNA resulting from initiation of synthesis at the upstream consensus sequence was only 5% of the usual sized mRNA 7 initiated from the downstream consensus sequence.

Previously, a system in which an intergenic region from mouse hepatitis virus (MHV) inserted into an MHV defective interfering (DI) RNA led to transcription of a subgenomic DI RNA in helper virus-infected cells was established. In the present study, a DI cDNA containing one UCUAAAC consensus sequence in the middle of the 0.3-kb-long intergenic region located between genes 6 and 7 was constructed. From this DI cDNA clone, 21 mutant DI RNAs were constructed so that each of the seven consensus sequence nucleotides was changed individually to the three alternative bases. These mutants were used to define how changes in the integrity of MHV transcription consensus sequence UCUAAAC affected mRNA transcription. Except for two mutants with the sequences UGUAAAC and UCGAAAC, all of the mutants supported efficient subgenomic DI RNA transcription. This indicated that MHV transcription regulation was sufficiently flexible to recognize altered consensus sequences. Next, these and other mutants were used to examine the leader-body fusion site on the subgenomic DI RNAs. Sequence analysis demonstrated that all subgenomic DI RNAs analyzed contained two pentanucleotide sequences; the first sequence seemed to be contributed by the leader, and the leader-body fusion most likely took place at either the first or the second nucleotide of the second sequence. This observation was not consistent with the proposed coronavirus transcription model (S. C. Baker and M. M. C. Lai, EMBO J. 9:4173-4179, 1990) which states that nucleotide mismatch can be corrected by RNA polymerase proofreading activity.