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Abstract
A wide range of RNA viruses use programmed −1 ribosomal frameshifting for the production of viral fusion proteins. Inspection of the overlap regions between ORF1a and ORF1b of the SARS-CoV genome revealed that, similar to all coronaviruses, a programmed −1 ribosomal frameshift could be used by the virus to produce a fusion protein. Computational analyses of the frameshift signal predicted the presence of an mRNA pseudoknot containing three double-stranded RNA stem structures rather than two. Phylogenetic analyses showed the conservation of potential three-stemmed pseudoknots in the frameshift signals of all other coronaviruses in the GenBank database. Though the presence of the three-stemmed structure is supported by nuclease mapping and two-dimensional nuclear magnetic resonance studies, our findings suggest that interactions between the stem structures may result in local distortions in the A-form RNA. These distortions are particularly evident in the vicinity of predicted A-bulges in stems 2 and 3. In vitro and in vivo frameshifting assays showed that the SARS-CoV frameshift signal is functionally similar to other viral frameshift signals: it promotes efficient frameshifting in all of the standard assay systems, and it is sensitive to a drug and a genetic mutation that are known to affect frameshifting efficiency of a yeast virus. Mutagenesis studies reveal that both the specific sequences and structures of stems 2 and 3 are important for efficient frameshifting. We have identified a new RNA structural motif that is capable of promoting efficient programmed ribosomal frameshifting. The high degree of conservation of three-stemmed mRNA pseudoknot structures among the coronaviruses suggests that this presents a novel target for antiviral therapeutics.
Highlights
Severe acute respiratory syndrome (SARS) first appeared in Guangdong Province, China, late in 2002
The À1 programmed À1 ribosomal frameshift (PRF) signal presented in that study contained a typical mRNA pseudoknot composed of two double-helical, Watson– Crick basepaired stems connected by two single-stranded loops (Figure 1A)
The presence of a long, 29-nt loop 2 seemed to be unusual, prompting us to subject the sequence from positions 13392– 13472 to additional computational analyses in an effort to further define the structure of this mRNA pseudoknot
Summary
Severe acute respiratory syndrome (SARS) first appeared in Guangdong Province, China, late in 2002. The rapid response of the World Health Organization is credited with containing this contagion by late June 2003, and only a few cases were reported during the winter cold season of 2003– 2004. The severity of this crisis mobilized the scientific community as well: by March 24, 2003, scientists at the Centers for Disease Control and Prevention and in Hong Kong had announced that a new coronavirus had been isolated from patients with SARS (reviewed in [1]). We present computational, comparative genomic, molecular, biophysical, and genetic evidence demonstrating that the SARS-CoV frameshift signal includes a new type of highly ordered three-stemmed mRNA pseudo-
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