This proposal is concerned with the regulation of mRNA stability by the product of the virion host shutoff (vhs) gene of herpes simplex virus (HSV) . Early during lytic HSV infections the vhs protein shuts off most cellular protein synthesis by inducing degradation of cellular mRNAs in the cytoplasm. Subsequently, following the onset of viral gene expression, it causes rapid turnover of viral mRNAs belonging to all kinetic classes. The proposed studies are divided into four parts. Those described in Part I will delineate the overall characteristics of the pathway of vhs induced mRNA degradation in vivo and in vitro. Specifically, a series of RNAase protection experiments will determine whether vhs induced degradation of polyadenylated mRNAs occurs by 3' to 5' or 5' to 3' exonuclease digestion or by endonucleolytic cleavage of he message at multiple internal sites. These experiments will also detect degradation intermediates that are sufficiently long lived to accumulate to moderate levels. Other experiments will examine the size and rate of shortening of the poly(A) tail and determine whether deadenylation of mRNAs precedes vhs induced degradation of the body of the message. The studies described in Part II will involve fractionation of in vitro degradation extracts from HSV infected HeLa cells. The ultimate goal will be to identify and purify all of the molecules required for vhs induced mRNA degradation, to delineate the individual steps in the vhs degradation pathway, and to characterize the molecules responsible for catalyzing each of the steps. The experiments proposed in Part III will determine the sites of phosphorylation of the vhs polypeptide and determine the importance of phosphorylation to vhs function by site specific mutagenesis of the vhs gene. Finally, the experiments described in Part IV will involve detailed genetic characterization of the vhs gene to identify features and domains of the vhs polypeptide that are important to its message degradative activity. Chimeric vhs genes encoding domains from the vhs polypeptides of HSV-1, HSV-2, and varicella zoster virus will be constructed and analyzed to identify what domains of the different vhs polypeptides are responsible for their different activities. In addition, an extensive collection of nonsense and 6 base pair insertion mutants will be constructed and analyzed for host shutoff activity. Taken together, these studies should yield a detailed picture of the mechanism of vhs induced mRNA degradation.
