Importance of coronavirus negative-strand genomic RNA synthesis prior to subgenomic RNA transcription

Abstract

The (-)-strand viral RNAs that result from after infection of cells with coronaviruses, which possess RNA genomes of message polarity, are genomic-sized and subgenomic-sized. Each of the (-)-strand subgenomic RNAs corresponds in size to each of the subgenomic mRNA species that are made in infected cells. We tested whether (-)-strand subgenomic RNAs might initially be synthesized from the input single-stranded (+)-strand genomic RNA prior to the production of subgenomic mRNAs. We used a mouse hepatitis virus (MHV) defective interfering (DI) RNA. from which subgenomic RNA was produced in DI RNA-replicating cells, because this DI RNA had a functional MHV intergenic region inserted in its interior. MHV samples containing the DI particles were irradiated with UV-light and then superinfected into cells that had been infected with MHV 4 h prior to superinfection. Northern blot analysis of intracellular RNAs that were extracted 3 h after superinfection showed that genomic DI RNA and subgenomic DI RNA had similar UV-target sizes, indicating that (-)-strand genomic DI RNA synthesis from input genomic DI RNA probably occurred prior to the subgenomic-size DI RNA synthesis. We discuss why, in the course of coronavirus transcription, (-)-strand genomic-length coronavirus RNA synthesis might occur before subgenomic-sized RNAs of either polarity are made.

Subgenomic Negative-Strand RNA Function during Mouse Hepatitis Virus Infection

“…Using a model system that may be reflective of conditions present in heavily immunosuppressed xenograph recipients, we previously isolated an MHV variant (MHV-H2) that replicated efficiency (10 7 to 10 8 PFU/ml) in mouse, hamster, and primate cell lines (5). It has been suggested that the subgenomic negative strands represent dead-end products of transcription (16,24,40). Accordingly, efficient virus replication in alternative host species may alter the molar ratio or, perhaps, not require the synthesis of subgenomic-length negative-strand RNAs.…”

“…Clearly, transcription attenuation is an attractive hypothesis which deserves serious attention, as it is consistent with the available data presented in this and other reports and represents a more direct mechanism to regulate subgenomic RNA synthesis (9,14,18,25,38). Recent UV transcription-mapping studies with mRNA transcribed from DI RNAs, however, have suggested that the subgenomic mRNAs directly originate from genome-length templates (16,24). Unfortunately, these studies did not directly determine whether full-length or subgenomic-length templates were functioning during infection.…”

“…Nascent labeling experiments and temperature shift experiments with a temperature-sensitive (ts) mutant defective in negative-strand synthesis strongly suggest that the subgenomic-length negative-strand RNAs are transcriptionally active after 6 h postinfection (30, 33). Data from other groups have suggested that the subgenomic negative strands may represent dead-end transcriptional products involved in the synthesis of a single mRNA (16,24,40).It has also been proposed that the subgenomic-length negative strands may be synthesized directly from the incoming genomic RNA, either by trans splicing of full-length negative strands or by transcription attenuation within the IG elements (3,22,23,30). While trans splicing is less attractive since full-length replicative intermediates (RIs) cannot be degraded into subgenomic-length RF RNA (3,30), the transcription attenuation model readily accounts for most of the observations central to coronavirus discontinuous transcription (30).…”

“…Nascent labeling experiments and temperature shift experiments with a temperature-sensitive (ts) mutant defective in negative-strand synthesis strongly suggest that the subgenomic-length negative-strand RNAs are transcriptionally active after 6 h postinfection (30, 33). Data from other groups have suggested that the subgenomic negative strands may represent dead-end transcriptional products involved in the synthesis of a single mRNA (16,24,40).…”

Mitochondrial Aconitase Binds to the 3′ Untranslated Region of the Mouse Hepatitis Virus Genome

“…Following uncoating, coronaviruses express the largest known replicase polyproteins, which in turn are proteolytically processed to yield a large number of mature proteins, including RNA-dependent RNA polymerase. The RNA-dependent RNA polymerase, perhaps in association with host proteins, directs the synthesis of negative-sense full-length and subgenomic RNA from the 3Ј end of the viral genome (40). Several alternative models have been described to explain the mechanism of MHV RNA synthesis (25,54,61).…”