This proposal concerns the regulation of mRNA stabilities by the virion host shutoff (vhs) protein of HSV. Early during lytic HSV infections, the vhs protein facilitates the shutoff of most cellular protein synthesis by inducing degradation of cellular mRNA. In addition, following the onset of viral gene expression, it causes rapid turnover of all kinetic classes of viral mRNA. The overall goal of the proposed experiments is to elucidate the mechanism of vhs-induced mRNA decay. The proposed studies are divided into four parts. Part I will involve the detailed genetic characterization of the vhs (UL41) protein. Site directed mutagenesis will be used to determine what amino acid differences between the vhs polypeptides of HSV-1 and HSV-2 account for the dramatically greater activity of the HSV-2 polypeptides. Other studies will test the importance of putative nuclease and RNA binding motifs within vhs. In Part II, the vhs protein will be purified and analyzed to determine if it has ribonuclease activity. Part III will investigate what factors are responsible for the specificity of the vhs-induced ribonuclease for mRNAs, and what determines the precise sites of mRNA cleavage. Finally, Part IV will investigate what interactions with other viral proteins are required for incorporation of newly synthesized progeny virions, and whether these interactions are also important for the regulation of vhs activity. The studies should provide important data concerning mechanisms that regulate host and viral gene expression during HSV infections. They should also add to the understanding of the biology of HSV and the mechanisms by which it causes disease.
