A 5'-proximal RNA sequence of murine coronavirus as a potential initiation site for genomic-length mRNA transcription.
Coronavirus transcription is a discontinuous process, involving interactions between a trans-acting leader and the intergenic transcription initiation sequences. A 9-nucleotide (nt) sequence (UUUAUAAAC), which is located immediately downstream of the leader at the 5' terminus of the mouse hepatitis virus (MHV) genomic RNA, contains a sequence resembling the consensus intergenic sequence (UCUAAAC). It has been shown previously that the presence of the 9-nt sequence facilitates leader RNA switching and may enhance subgenomic mRNA transcription. It is unclear how the 9-nt sequence exerts these functions. In this study, we inserted the 9-nt sequence into a defective interfering (DI) RNA reporter system and demonstrated that mRNA transcription could be initiated from the 9-nt sequence almost as efficiently as from the intergenic sequence between genes 6 and 7. Sequence analysis of the mRNAs showed that the 9-nt sequence served as a site of fusion between the leaders and mRNA. The transcription initiation function of the 9-nt sequence could not be substituted by other 5'-terminal sequences. When the entire 5'-terminal sequence, including four copies of the UCUAA sequence plus the 9-nt sequence, was present, transcription could be initiated from any of the UCUAA copies or the 9-nt sequence, resulting in different copy numbers of the UCUAA sequence and the deletion of the 9-nt sequence in some mRNAs. All of these heterogeneous RNA species were also detected from the 5'-terminal region of the viral genomic-length RNA in MHV-infected cells. These results thus suggest tha the heterogeneity of the copy number of UCUAA sequences at the 5' end, the deletion of the 9-nt sequence in viral and DI RNAs, and the leader RNA switching are the results of transcriptional initiation from the 9-nt site. They also show that an mRNA species (mRNA 1) that lacks the 9-nt sequence can be synthesized during MHV infection. Therefore, MHV genomic RNA replication and mRNA 1 transcription may be distinguishable.
Zhang
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Department of Neurology, University of Southern California School of Medicine, Los Angeles 90033-1054, USA.
Lai
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MM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral; D017384:Sequence Deletion; D015533:Transcriptional Activation
D012333:RNA, Messenger; D012367:RNA, Viral