Poliovirus is one of a large number of important pathogens in the Picornaviridae family of positive strand RNA viruses. The molecular mechanisms involved in poliovirus RNA replication will be examined in this study. It is now known that most of the noncapsid viral proteins are directly involved in viral RNA replication, but many of the activities associated with individual proteins and their specific functions during the viral RNA replication cycle remain unknown. Definitive studies to identify the function of these proteins and to characterize the mechanisms involved in the initiation of RNA replication and the covalent linkage of VPg to progeny RNA have been hampered by the lack of an experimental system to study the complete replication cycle of poliovirus RNA in vitro. In this study, we will utilize a newly developed HeLa S10 in vitro translation RNA replication system to investigate the molecular mechanisms involved in poliovirus RNA replication. The synthesis of labeled VPg-linked progeny RNA and infectious virus (>107 pfu/ml) in replication complexes formed in these reactions provides a complete (i.e., viral RNA input -> (-) strand RNA -> viral RNAprogeny - virus) and efficient system for biochemical analysis. This system reflects the molecular events that occur during viral RNA replication in vivo (e.g., guanidine - sensitive RNA replication) but provides direct access to these replicative processes which are shielded from experimental manipulation in infected cells. Thus, studies with the in vitro translation RNA replication system in combination with biochemical studies of individual proteins provides a powerful experimental approach for continuing studies on the molecular biology of poliovirus RNA replication. The overall strategy includes the following goals: 1) Characterize RNA replication complexes for the synthesis of replicative intermediate RNAs and virus. Determine the polarity of the progeny RNA synthesized in complexes and quantitate the initiation of VPg-linked nascent chains. 2) Use site specific mutations in viral proteins and protein specific antibodies to identify viral and cellular (e.g., human poly(A)- binding protein and terminal uridylyl transferase) proteins required for positive and negative strand RNA synthesis. 3) Characterize the initiation and elongation activities of recombinant 3Dpol on synthetic RNAs. Investigate the possible RNA helicase activity of 2C and quantitate the RNA binding properties of 3CD and 3Dpol. 4) Identify RNA sequences and structures at the 3' ends of positive and negative strand RNA which are required for authentic RNA replication in vitro. 5) Investigate the mechanisms involved in the initiation of viral RNA synthesis including the template priming: VPg- linkage model and the VPg-priming model.
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