Abstract
The gene expression of plus-strand RNA viruses with a polycistronic genome depends on translation and replication of the genomic mRNA, as well as synthesis of subgenomic (sg) mRNAs. Arteriviruses and coronaviruses, distantly related members of the nidovirus order, employ a unique mechanism of discontinuous minus-strand RNA synthesis to generate subgenome-length templates for the synthesis of a nested set of sg mRNAs. Non-structural protein 1 (nsp1) of the arterivirus equine arteritis virus (EAV), a multifunctional regulator of viral RNA synthesis and virion biogenesis, was previously implicated in controlling the balance between genome replication and sg mRNA synthesis. Here, we employed reverse and forward genetics to gain insight into the multiple regulatory roles of nsp1. Our analysis revealed that the relative abundance of viral mRNAs is tightly controlled by an intricate network of interactions involving all nsp1 subdomains. Distinct nsp1 mutations affected the quantitative balance among viral mRNA species, and our data implicate nsp1 in controlling the accumulation of full-length and subgenome-length minus-strand templates for viral mRNA synthesis. The moderate differential changes in viral mRNA abundance of nsp1 mutants resulted in similarly altered viral protein levels, but progeny virus yields were greatly reduced. Pseudorevertant analysis provided compelling genetic evidence that balanced EAV mRNA accumulation is critical for efficient virus production. This first report on protein-mediated, mRNA-specific control of nidovirus RNA synthesis reveals the existence of an integral control mechanism to fine-tune replication, sg mRNA synthesis, and virus production, and establishes a major role for nsp1 in coordinating the arterivirus replicative cycle.
Highlights
Plus-strand RNA (+RNA) viruses are ubiquitous pathogens of plants, animals, and humans
The genome is translated into a single polyprotein precursor comprising all viral proteins, while the expression of genomes containing multiple open reading frames commonly depends on the production of additional, subgenomic mRNAs
Arteriviruses and coronaviruses secure the expression of their structural protein genes by generating an extensive nested set of subgenomic mRNAs, which are copied from a set of complementary minus-strand templates
Summary
Plus-strand RNA (+RNA) viruses are ubiquitous pathogens of plants, animals, and humans. Nidoviruses share important traits in their genome organization and gene expression mechanisms, and their key replicative enzymes are presumed to be evolutionarily related (for a review, see [3]) Their polycistronic genomes are 59capped, 39-polyadenylated, and the two 59-most open reading frames (ORFs) – ORF1a and ORF1b, encode the viral replicase subunits segregated in two large replicase polyproteins, pp1a and pp1ab, the expression of the latter controlled by a 21 ribosomal frameshift (Fig. 1A). Autoproteolytic processing of these precursors generates between 13 and 16 non-structural proteins (nsps) that direct viral RNA synthesis. Arteri- and coronavirus RdRp-containing complexes mediate the synthesis of a distinctive nested set of sg mRNAs that are both 59- and 39coterminal with the viral genome and consist of sequences that are noncontiguous in the genomic RNA (Fig. 1B)
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