Poliovirus has served as the prototype virus for studying the replication mechanisms of picornaviruses and other (+) strand RNA viruses. The Picornaviridae family of small RNA viruses includes a large number of important pathogens. Some of the diseases associated with these viruses include meningitis, encephalitis, poliomyelitis, myocarditis, respiratory infections and hepatitis. In this study, we are using molecular genetic techniques to study poliovirus RNA replication in vitro and in vivo. We have developed new experimental methods that can be used in combination with classic viral genetic procedures in virus-infected cells. Using HeLa S10 in vitro translation-RNA replication reactions and preinitiation RNA replication complexes, we are investigating the viral replication cycle in vitro including viral protein synthesis, the cre(2C)-dependent synthesis of VPgpUpU, the synthesis of (-) and (+) strand RNA and the formation of infectious virus. Genetic complementation and RNA recombination assays are powerful methods to study the activities associated with trans-acting viral proteins and the cis-acting sequences and structures in the viral RNA. Since all of the viral replication proteins can be provided in trans in cell-free reactions, we are using genetic complementation assays to identify specific viral proteins and viral protein precursors that are required for the assembly of functional RNA replication complexes. We are also using complementation assays to identify and characterize cis-acting RNA sequences that are required for the initiation of (-) and (+) strand RNA synthesis. During the course of our recent studies, we have developed an in vitro recombination assay and methods to specifically assay for (-) and (+) strand RNA synthesis. Our basic experimental plan is to use these methods to answer questions of fundamental importance regarding the replication of poliovirus and other picornaviruses. In this renewal application, we are requesting funding that will include the following specific aims. (1) Identify and characterize the cis-acting replication elements in poliovirus genomic RNA. (2) Use a genetic complementation analysis to characterize the viral replication proteins and their precursors regarding their role in viral RNA replication. (3) Investigate the mechanisms involved in viral RNA recombination using an in vitro recombination assay. (4) Characterize the role of the 3' NTR and associated poly(A) tail in viral RNA replication.