Experiments proposed will continue efforts of the PI, to understand the molecular mechanisms underlying protein processing and RNA replication functions that occur during picornavirus infections of mammalian cells. Experimental approaches employ biochemical assays to measure capsid precursor processing, RNA replication and RNA-protein interactions. In the first AIM, functional domains of poliovirus 3CD protease that are involved in P1 capsid precursor cleavage or that bind 5' RNA sequences or interact with the cellular RNA binding protein PCBP2 will be identified. In the second AIM, HeLa cell cofactors that appear to include hsp70/hsc70 molecular chaperones will be purified and assayed for their role in facilitating P1 polyprotein cleavage. In the final, third AIM, protein and nucleic acid determinants needed for assembly of picornavirus replication complexes (RCs) will be defined. Specific experiments will attempt to identify the functional polypeptide components of the viral RCs, to define compensatory changes allowing replication of the genomes of human rhinovirus and poliovirus deleted in their entire 3' NCRs, and to identify cellular proteins binding to the 3' end of minus strand RNA. Results from these studies should reveal the nature of specific macromolecular interactions regulating viral and cellular gene expression and have the potential to identify molecular targets for antiviral therapy.
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