The Picornaviridae, a family of small positive-strand RNA viruses includes a large number of important human pathogens that cause a wide variety of diseases, including respiratory infections, meningitis, encephalitis, poliomyelitis, myocarditis and hepatitis. Viral RNAs with deletions in the capsid coding region of the viral genome act as replicons and are efficiently translated and replicated in the host cell. Replicons with most of the polyprotein coding sequence deleted can replicate in in vitro reactions containing viral proteins synthesized in trans from a non-replicating helper RNA. In this proposal, molecular genetic techniques are being used to investigate poliovirus RNA replication. A mutagenic analysis of structure-function relationships for viral proteins and RNA sequences, genetic complementation assays in vivo and in vitro, genetic recombination assays and the isolation and characterization of revertant viruses are being used to define the genetic elements that are required for the replication of the viral genome. Complementation assays have been developed that can be used in transfected cells and in RNA replication assays in vitro. Both conditional and nonviable mutations can be characterized in these assays. The recent demonstration that all of the viral proteins required for RNA replication can be synthesized in trans provides a powerful new approach for genetic analysis of RNA replication. The following specific aims are proposed to continue these studies : (1) Further characterize the cis and trans genetic elements in the viral genome using a mutagenic approach and complementation assays in vivo and in vitro, (2) develop RNA recombination assays for use in the in vitro replication assays, (3) identify cis-active sequences in the viral genome that are required for RNA replication, and (4) characterize RNA sequences and structures in the 3'NTR which are required for viral RNA replication, and test the proposed model for generating revertants in the 3'NTR.
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