Abstract
The subgenomic (sg) mRNAs of arteriviruses (order Nidovirales) form a 5'- and 3'-coterminal nested set with the viral genome. Their 5' common leader sequence is derived from the genomic 5'-proximal region. Fusion of sg RNA leader and "body" segments involves a discontinuous transcription step. Presumably during minus-strand synthesis, the nascent RNA strand is transferred from one site in the genomic template to another, a process guided by conserved transcription-regulating sequences (TRSs) at these template sites. Subgenomic RNA species are produced in different but constant molar ratios, with the smallest RNAs usually being most abundant. Factors thought to influence sg RNA synthesis are size differences between sg RNA species, differences in sequence context between body TRSs, and the mutual influence (or competition) between strand transfer reactions occurring at different body TRSs. Using an Equine arteritis virus infectious cDNA clone, we investigated how body TRS activity affected sg RNA synthesis from neighboring body TRSs. Flanking sequences were standardized by head-to-tail insertion of several copies of an RNA7 body TRS cassette. A perfect gradient of sg RNA abundance, progressively favoring smaller RNA species, was observed. Disruption of body TRS function by mutagenesis did not have a significant effect on the activity of other TRSs. However, deletion of body TRS-containing regions enhanced synthesis of sg RNAs from upstream TRSs but not of those produced from downstream TRSs. The results of this study provide considerable support for the proposed discontinuous extension of minus-strand RNA synthesis as a crucial step in sg RNA synthesis.
Highlights
The synthesis of subgenomic mRNAs is a common mechanism that positive-strand RNA viruses have evolved to express structural and auxiliary proteins that do not have to be translated directly from the genomic mRNA
For the arterivirus Equine arteritis virus (EAV), we recently established that the stability of the leader transcription-regulating sequences (TRSs)-body TRS duplex is an important determinant of sg RNA abundance, it is clear that other factors are involved [34, 35, 56]
One to four copies of this cassette were cloned head to tail directly downstream of the EAV replicase gene, the 3Ј-proximal cassette being the “natural” ORF7 and the others replacing the part of the genome that contains all envelope protein genes and body TRSs (Fig. 2A)
Summary
The synthesis of subgenomic (sg) mRNAs is a common mechanism that positive-strand RNA viruses have evolved to express structural and auxiliary proteins that do not have to be translated directly from the genomic mRNA (reviewed in reference 29). According to the leader-primed transcription model, sg RNA synthesis would be primed by free leader molecules that attach, by means of TRS-TRS base pairing, to the body TRS complements in the genomic minus strand, after which the leader primer would be elongated to produce an sg mRNA [3, 23, 49]. This model was partly based on the fact that sg minus-strand RNAs were initially not detected in cells infected with the coronavirus Mouse hepatitis virus (MHV) [25]. Strand transfer reactions at 3Ј-proximal TRSs would suppress the transcriptional activity of moreupstream (in the plus sense), 3Ј-distal TRSs, but not vice versa
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