The overall objective of this research proposal is to define at the molecular level the individual level the individual steps in the process of poliovirus replication, assembly and encapsidation. Previous studies have identified two viral proteins which have a central role in replication. The poliovirus RNA polymerase, 3Dpol, catalyzes the copying of the viral genome, while the genome linked viral protein, VPg, most probably in the form of a precursor, acts as a primer for replication. Recently, it has become apparent that certain proteins, including 3Dpol, have both cis and trans acting functions in replication, although the details of their action are unknown. The process of encapsidation of the RNA genome is linked to the replication process. The mechanism by which the individual capsid proteins interact with the viral RNA genomes is also unresolved. To further define the process of replication and encapsidation in poliovirus, the following specific aims are proposed: 1. To define the structural features of poliovirus 3Dpol and Vpg precursor required for enzymatic activity. Mutant 3Dpol genes will be expressed in E. coli and the enzymes tested for activity in vitro. The mutations will be subcloned into the infectious molecular clone of poliovirus to test for replication. Crystals of purified 3Dpol will be used for the determination of the three-dimensional structure. The function of a Vpg precursor, 2C3ABVPg, in replication will be determined by testing defined mutants. 2. To characterize the cis and trans functions of poliovirus proteins required for replication. The cis and trans functions of 3Dpol, including its precursor, 3CD, and Vpg in replication will be determined using a complements system based on poliovirus mini-replicons. 3. To identify specific amino acids of the capsid proteins required for interaction with the RNA genome, and characterize the encapsidation process. The individual amino acids which interact with the capsid proteins will be identified by mutagenesis. The mutants will be characterized in an assembly and encapsidation system developed by this laboratory. The long term goal of this proposal is to define the viral protein-protein and protein:nucleic acid interactions required for replication and encapsidation of the viral genome. This group of medically important viruses includes agents of the common cold and infectious hepatitis. Taken together, the results from the proposed experiments will provide fundamental information on the mechanism of replication and encapsidation of genome RNA which will lead to a more effective control of diseases associated with these viruses.
