elF-2 Activity in Vaccinia Virus Infected Cells

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Eukaryotic initiation factor 2, eIF, is an essential protein synthesis initiation factor that is subject to regulation by reversible phosphorylation. As part of the cellular antiviral defense mechanism, an interferon-induced, dsRNA-dependent protein kinase,eIF-2.-PKds, becomes activated on viral infection and phosphorylates the alpha subunit eIF-2. This results in a limitation of functional eIF-2 available for protein synthesis with a concomitant reduction of protein synthesis. A number of animal viruses, including vaccinia virus, have evolved mechanisms to inhibit the activation of eIF-2.-PKds in interferon-treated cells. The ability of vaccinia virus to grow in interferon-treated cells stems in part from the production of a vaccinia early gene product, termed specific kinase inhibitory factor, SKIF, that prevents activation of eIF-2.-PKds. In addition vaccinia virus encodes a gene which has homology with the alpha subunit of eIF-2 and encodes a 10 kDa protein. Although the function of the eIF-2 alpha-like gene is not known, an eIF-2 alpha-like peptide of approximately 10 kDa accumulates early in vaccinia virus infection. The first aim of this proposal is to monitor the effect of vaccinia virus infection on the levels and activation state of elF-2.-PKds, as well as the phosphorylation state of elF-2. in vaccinia virus infected Hela cells. The reagents necessary to do this, antibody to elF-2.-PKds and elF-2. are available. The second aim of ths ptoposal is to investigate the function of pK2 and to determine whether its product has elF-2>.-like activity and whether it is involved in the ability of vaccinia virus to grow in interferon- treated cell. The K2 gene has been cloned into the expression vector, pTM1, which will enable us to synthesize radiolabelled PK2 in vitro, as well to transfect the gene into mammalian cells. The gene will be transferred to a bacterial expression vector to allow the production of milligram quantities of pK2 for biochemical analyses. The properties of the vaccinia encoded elF-2.-like gene product will be compared with those of cellular elF-2. with reference to its guanine nucleotide binding properties, its ability to enter preinitiation complexes, and its ability to function as a substrate for elF-2.-PKds.