Characterization of the SARS-CoV frameshift signal

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DESCRIPTION (provided by applicant): Viruses utilize programmed ribosomal frameshifting (PRF) to post-transcriptionally regulate the expression of multiple genes that are encoded on monocistronic viral mRNAs. Studies in Retro- and Totiviruses have shown that maintaining correct PRF efficiencies is critical for virus propagation, thus identifying this mechanism as a potential target for antiviral therapeutics. PRF has previously been shown to be utilized by the Coronaviruses (CoV), and primary sequence analysis of the rapidly emerging SARS-CoV, the etiological agent of Severe Acute Respiratory Syndrome (SARS), reveals the presence of a putative PRF signal that is predicted to shift elongating ribosomes by one base in the -1 or 5' direction (-1 PRF). Initial comparative, structural and functional analysis of this sequence in our laboratory suggests that the -1 PRF signal of SARS-CoV (and possibly of the entire Coronavirus family) utilizes a complex, heretofore unknown mRNA pseudoknot structure that contains three stem loops as opposed to the usual two-stem loop variety. Further, our findings suggest that the function of the third stem-loop may be to regulate -1 PRF efficiency, and hence the expression of viral proteins. Thus, small molecules that interact with this structure may potentially have antiviral properties. The broad aim of the proposed research is to characterize important features of the SARS-CoV -1 PRF signal using a combination of phylogenetic, molecular, structural, and viral assays. Specifically, we will 1) characterize how changes in the mRNA pseudoknots of the SARS and Mouse Hepatitis CoV's affect frameshifting efficiency using an in vivo human epithelial cell based assay system, 2) determine the effects of changes in frameshift efficiency on propagation of the SARS-CoV using an infectious cDNA clone in tissue culture and in a mouse model system, and 3) structurally characterize the SARS-CoV -1 PRF signal using nuclease mapping, high-resolution NMR and calorimetric methodologies.