The long term objective of this proposal is to determine the mechanism by which the single-stranded RNA genome of poliovirus, the prototype of prototype of picornaviruses, is replicated in infected cells. The hypothesis that poliovirus genome-linked protein, VPg (or a precursor to VPg, Pre-VPg) is used as a primer for initiating synthesis of poliovirus plus- and minus-strand RNAs will be tested. The virus-specific proteins 3Dpol (RNA-dependent RNA polymerase) and Pre-VPg and a host a cell protein (host factor, HF) involved in viral replication will be purified using various chromatographic techniques. Purified proteins and appropriate serological reagents will be used to study the mechanism of viral RNA replication in vitro. Specifically, the requirements for uridylylation of Pre-VPg will be examined using biochemical and serological techniques. Whether uridylylated Pre-VPg (Pre-Vpg-pU or Pre-VPg-pUpU) can be used as a protein-nucleotide primer for in vitro synthesis of poliovirus plus- and minus-strand RNAs will be determined. The protein-protein interaction of 3Dpol,HF and Pre-VPg will be studied using biochemical and serological techniques. The mechanism of viral RNA replication will be further studied using mutant polioviruses defective in RNA synthesis. The normal function of host factor in uninfected cells will be examined. Since a double-stranded RNA (dsRNA)-activated protein kinase activity which phosphorylates the alpha-subunit of eukaryotic initiation factor-2 (eIF-2) copurifies with host factor, the exact relationship between host factor and dsRNA-activated kinase will be examined using biochemical and serological techniques. Furthermore, the mechanism by which poliovirus prevents increased phosphorylation of eIF02lalpha despite activation of dsRNA- activated kinase in infected cells, will be examined. Elucidation of the mechanism of replication of poliovirus will certainly fill the remaining large gap in knowledge concerning replication of this medically important virus group which includes those inducing common cold, infectious hepatitis, encephalomyocarditis and foot-to-mouth disease in human and animals.
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