The proposed project will extend our molecular genetic analysis of the 5' noncoding regions (5'NCRs) of picornavirus RNAs to include biochemical assays as functional tests for RNA-protein interactions. We will utilize point mutations, deletions, and linker scanning mutations within predicted stem-loop structures in the poliovirus 5' NCR to address the role of sequence versus structure in the formation of functional complexes required for cap-independent translation. Our functional analysis for viable viruses containing 5' NCR lesions will include assays for viral-specific protein and RNA synthesis, complementation with genetically-marked viruses, determination of growth characteristics in human cells of neuronal origin, and RNA sequence analysis of pseudo-revertants. The need by picornavirus RNAs for specific complexes of cellular proteins distinct from those employed by cap-dependent translation mechanisms will be explored by binding and translation assays in the presence of fractionated cell extracts enriched in known and perhaps novel translation components. We propose a series of experiments employing U.V. crosslinking, primer extension footprinting, and chemical modification to define the specific nucleotide sequences in the 5'NCR of poliovirus RNA that are involved in the formation RNA-protein complexes. In addition, we will identify and isolate cellular proteins that bind to specific stem-loop structures in the poliovirus 5' NCR. The overall goal of the proposed studies is to determine the viral genetic and cellular polypeptide determinants in RNA-proteins interactions within the 5' NCR of genomic RNAs, information that ultimately will help to unravel the molecular mechanisms of cap-independent translation initiation utilized by picornaviruses. The health relatedness of this project stems from uncovering genetic and biochemical mechanisms employed by viral pathogens to subvert the cellular translation apparatus for dedicated synthesis of their own viral gene products. On a more general level, our studies will contribute to a basic understanding of how RNA-protein interactions are involved in translational regulation in eukaryotic cells.
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