Enteroviruses cause a diverse spectrum of human diseases including conjunctivitis, myocarditits, aseptic meningitis, acute flaccid paralysis, and fatal systemic infections in neonates. Poliovirus, the prototypic enterovirus, is well characterized at the molecular level and still serves as the most appropriate virus for studies of RNA translation and replication. In this proposal, poliovirus mRNA translation and RNA replication will be studied in cell-free reactions capable of supporting the sequential translation and replication of poliovirus RNA. These reactions are advantageous because they support authentic translation and replication of poliovirus RNA while providing numerous technical advantages including the ability to synchronize viral mRNA translation and viral RNA replication. The interaction of cis-active RNA structures at the termini of poliovirus RNA will be examined. Temporally dynamic ribonucleoproteins form on poliovirus cis-active RNA structures to mediate and regulate the sequential steps of replication. Experiments will be performed to: 1) determine how the 5' cloverleaf RNA structure of poliovirus potentiates viral mRNA translation, 2) determine how translating ribosomes regulate, in part, the switch from viral mRNA translation to RNA replication, 3) determine how apparently distal cis-active RNA structures interact to regulate sequential steps of viral RNA translation and replication, and 4) determine the mechanisms behind the asymmetric replication of poliovirus RNA. These experiments will help elucidate the fundamental sequence of molecular interactions required for enterovirus RNA translation and replication. This information will provide for a better understanding of the mechanisms by which enteroviruses replicate. In particular, these studies will contribute substantial new information to support the popular new paradigm of 5'-3' RNA interactions in messenger ribonucleoprotein complexes and RNA replication complexes. The experiments directly test a hypothesis concerning the mechanism by which RNA replication machinery avoids ribosome-replicase collisions. Finally, the experiments test a new model that clearly explains the mechanisms controlling asymmetric RNA replication.
