A host cell protein, host factor (HF) is required for the replication of the poliovirus genome. The goals of this research are to understand the role of HF in the replication of poliovirus, and to define the normal cellular function of HF. Recent studies from our laboratory have demonstrated that HF is a protein kinase which can phosphorylate itself and the alpha subunit of the eukaryotic protein synthesis initiation factor eIF-2. The objectives of this proposal are to: 1) Define the role of HF in poliovirus replication; 2) Ascertain the effects of HF on protein synthesis and 3) Analyze the regulation of the translation and transcription of HF in both normal and poliovirus infected cells. In order to further define the role of HF in poliovirus replication, ATP analogs will be used to determine if the kinase activity of HF is required for each round of poliovirus RNA synthesis. Phosphorylated HF will be isolated by biochemical means and assayed for the capacity to bind to the viral polymerase and stimulate poliovirus RNA synthesis. Finally, pulse chase experiments will be performed to determine the in vivo turnover time for phosphorylated HF before and during poliovirus infection. To further understand the role of HF in the regulation of protein synthesis, purified HF will be assayed for the capacity to inhibit in vitro protein synthesis. Experiments will also be performed to determine the relationship between HF and the interferon induced kinase, which has similar physical and biochemical characteristics. In order to analyze the translational regulation of HF, the biosynthesis of HF in both normal and poliovirus infected cells will be studied using metabolic labeling in combination with immunoprecipitation with anti-HF antibodies. HF specific cDNAs will be generated which can be used for structure-function studies and as probes for the expression of HF in different species and during different stages of development. The long term goal of this research, then, is to understand the dual functions of HF in picornavirus replication (as typified by poliovirus) and in the regulation of protein synthesis. The elucidation of the mechanism of picornavirus replication will help to complete our understanding of the replication of a large group of medically important viruses which include agents of the common cold, infectious hepatitis, encephalomyocarditis (human) and foot to mouth disease (bovine).
| Morrow, C D; Warren, B; Lentz, M R (1987) Expression of enzymatically active poliovirus RNA-dependent RNA polymerase in Escherichia coli. Proc Natl Acad Sci U S A 84:6050-4 |
