The broad objective of this research is to elucidate the mechanism of poliovirus replication, with the first step being the definition of the viral and host cell proteins required for replication. Towards this goal, may laboratory has recently succeeded in the expression of enzymatically active poliovirus RNA dependent RNA polymerase (3 D pol) in E. coli. This result forms the specific aims of this proposal:
Specific Aim 1 : Express and Purify Enzymatically Active 3 D pol and VPg Precursors A poliovirus fusion gene was used consisting of the genome linked protein VPg, the viral proteinase, 3C pro and the 3 D pol gene to express the enzymatically active 3 D pol in E. coli. Experiments will determine if the 3 C pro is absolutely required for expression enzymatically active 3 D pol. The poliovirus replication genes will be expressed in eukaryotic cells using SV40 or vaccinia virus vectors.
Specific Aim 2 : Identify and Characterize Poliovirus Proteins Required for Replication The poliovirus proteins expressed in eukaryotic cells will be assayed for the capacity to replicate the poliovirus genome in vitro. Those viral proteins essential for replication, in addition 3 D pol, will be expressed in E. coli and tested with host cell proteins to completely define the requirement for eukaryotic cell proteins in poliovirus replication. To analyze the regulation of poliovirus replication in vivo, a mini replicon will be developed for poliovirus consisting of a truncated virus which encodes the viral proteins and the 5' and 3' RNA required for replication.
Specific Aim 3 : Define Regions of the Poliovirus Proteins Required for Replication. The expression of enzymatically active poliovirus replication proteins provides a system whereby for the first time, the important structural regions of these proteins can be identified. Mutant 3 D pol molecules will be generated by linker insertion, chemical or site specific mutagenesis, and tested for enzymatic activity in the E. coli and eukaryotic expression systems to define the regions essential for protein function in replication. The role of VPg in replication will be analyzed by generating mutant VPg molecules by site specific mutagenesis. The long term goal os this research then, is to understand the mechanism of replication of picornaviruses, as typified by poliovirus. This large group of medically important viruses includes agents of the common cold, infectious hepatitis and encephalomyocarditis. Studies on replication will provide clues into more effective control, and treatment of the disease caused by this group of viruses.
