A system that exploits defective interfering (DI) RNAs of mouse hepatitis virus (MHV) for deciphering the mechanisms of coronavirus mRNA transcription was developed. A complete cDNA clone of MHV DI RNA containing an inserted intergenic region, derived from the area of genomic RNA between genes 6 and 7, was constructed. After transfection of the in vitro-synthesized DI RNA into MHV-infected cells, replication of genomic DI RNA as well as transcription of the subgenomic DI RNA was observed. Si

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... lease protection experiments, sequence analysis, and Northern (RNA) blotting analysis revealed that the subgenomic DI RNA contained the leader sequence at its 5' end and that the body of the subgenomic DI RNA started from the inserted intergenic sequence. Two subgenomic DI RNAs were synthesized after inserting two intergenic sites into the MHV DI RNA. Metabolic labeling of virus-specific protein in DI RNA replicating cells demonstrated that a protein was translated from the subgenomic DI RNA, which can therefore be considered a functional mRNA. Transfection study of gel-purified genomic DI RNA and subgenomic DI RNA revealed that the introduction of genomic DI RNA, but not subgenomic DI RNA, into MHV-infected cells was required for synthesis of the subgenomic DI RNA. A series of deletion mutations in the intergenic site demonstrated that the sequence flanking the consensus sequence of UCUAAAC affected the efficiency of subgenomic DI RNA transcription and that the consensus sequence was necessary but not sufficient for the synthesis of the subgenomic DI RNA. Mouse hepatitis virus (MHV), a coronavirus, is an enveloped virus containing a single-stranded positive-sense RNA genome of approximately 31 kb (13, 14) . The virion is composed of four structural proteins, three of which are integral envelope proteins, including the peplomer-forming S (spike) protein, M (membrane) protein and HE (hemagglutinin-esterase) protein. The fourth structural protein, N, is an internal component of the virus. It is a phosphoprotein of 50 kDa (37, 38) and binds to virion RNA (38), forming the helical nucleocapsid of the virion. In MHV-infected cells seven to eight species of virusspecific subgenomic mRNAs composing a 3'-coterminal nested-set structure (10, 15) are synthesized, and from each of these a viral protein is translated. These subgenomic mRNAs are named mRNAs 1 to 7, according to decreasing order of size (10, 15). mRNA 1 is structurally identical to the genomic RNA which is detected in MHV particles, whereas the other subgenomic mRNAs are not packaged into virions (13, 20). The 5' end of the MHV genomic RNA contains a 72to 77-nucleotide-long leader sequence (9, 11, 36). The 3' region of the leader sequence contains a pentanucleotide sequence, UCUAA, that is repeated two to four times in different MHV strains (19). Downstream of the leader sequence are the MHV-specific genes, each of which is separated by a special short stretch of sequence, the intergenic sequence. All MHV intergenic sequences analyzed so far include the unique consensus sequence of UCUAAAC or a very similar sequence (33). A sequence identical to the leader sequence, of 72 to 77 nucleotides, is also found at the 5' end of each and every MHV mRNA species, and these leader sequences are fused with the mRNA body sequence * Corresponding author. which starts from the intergenic site consensus sequence (9, 11, 33, 36) . The leader fusion site on a given species of MHV subgenomic mRNA is heterogeneous (22), and that heterogeneity is due to a variation in the number of pentanucleotide (UCUAA) repeats at the leader fusion site (22). It has been proposed that coronavirus utilizes a unique leader RNA-primed transcription in which a leader RNA is transcribed from the 3' end of the genome-sized negativestrand template RNA, dissociates from the template, and then rejoins the template RNA at downstream intergenic regions to serve as the primer for mRNA transcription (8). This transcription model is based on the observation that the negative-stranded RNA of MHV is of genomic size (12), and a body of evidence indeed supports this transcription model (1, 2, 8, 23) . However, Sethna et al. demonstrated the presence of subgenomic-size negative-stranded RNAs containing the antileader sequence in transmissible gastroenteritis virus and bovine coronavirus-infected cells (30, 31). Also, subgenomic replicative intermediate RNAs corresponding to each MHV subgenomic mRNA species have been demonstrated in MHV-infected cells (29). These recent discoveries cannot be explained by the original leader RNA-primed transcription mechanism. The mechanism of coronavirus transcription remains to be elucidated. There is a rough correlation between the degree of intergenic site nucleotide homology with the leader sequence and the amount of mRNA transcribed (33). The effect on transcription of both the sequences at the 3' end of the leader and the intergenic site has been demonstrated by using several recombinant MHVs and mutant MHVs with differing pentanucleotide repetitions (19, 32) . These data strongly indicate that the complementary sequences present at the leader RNA and the intergenic site play an essential role(s) in MHV transcription. 6031 on March 18, 2020 by guest