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Abstract
In previous studies, differences in the amount of genomic and subgenomic RNA produced by coronaviruses with mutations in the programmed ribosomal frameshift signal of ORF1a/b were observed. It was not clear if these differences were due to changes in genomic sequence, the protein sequence or the frequency of frameshifting. Here, viruses with synonymous codon changes are shown to produce different ratios of genomic and subgenomic RNA. These findings demonstrate that the protein sequence is not the primary cause of altered genomic and subgenomic RNA production. The synonymous codon changes affect both the structure of the frameshift signal and frameshifting efficiency. Small differences in frameshifting efficiency result in dramatic differences in genomic RNA production and TCID50 suggesting that the frameshifting frequency must stay above a certain threshold for optimal virus production. The data suggest that either the RNA sequence or the ratio of viral proteins resulting from different levels of frameshifting affects viral replication.
Highlights
Coronaviridae, along with Arteriviridae and Roniviridae, belong to the order Nidovirales
We have shown that alterations to the SARS coronavirus frameshift signal affect frameshifting efficiency [9,11]
In an effort to further our understanding of how the SARS frameshift signal functions we performed deletion and mutagenesis studies an analyzed the effects on frameshifting efficiency, RNA structure and viral RNA production
Summary
Coronaviridae, along with Arteriviridae and Roniviridae, belong to the order Nidovirales. The proteins encoded in ORF1a and ORF1a/b function as the replicase, making subgenomic RNAs and new copies of the genomic RNA [5]. Production of the pp1ab polyprotein requires the translating ribosome to change reading frame at the frameshift signal that bridges ORF1a and ORF1a/b. We have previously suggested that the ratio of the pp1a and pp1ab proteins might affect the regulation and production of genomic and subgenomic. Unlike other frameshift-stimulating pseudoknots the SARS pseudoknot contains an additional internal stem loop [8,9,10] The function of this structure, called stem 3, is unknown. Analyses using synonymous protein coding mutations demonstrate that the region of the genome that harbors the frameshift signal affects the regulation of genomic and subgenomic RNA production without altering protein sequence.
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