This proposal is concerned with the regulation of mRNA stability in cells infected with herpes simplex virus, type 1 (HSV-1). An early event during lytic HSV-1 infections is the shut-off of most cellular protein synthesis. This shut-off is initially caused by an as yet unidentified component of the infecting virion which induces disaggregation of cellular polyribosomes and degradation of pre-existing host mRNAs. Another early event is the expression of the alpha or immediate-early viral genes. Synthesis of the alpha polypeptides peaks at early times and then declines. Vhs 1 is a mutant of HSV-1, strain KOS, that is defective in the virion-associated host shut-off function responsible for the initial suppression of host protein synthesis and degradation of cellular mRNAs. Recent studies indicate that vhs 1 also carries a mutation that leads to the increased stabilities of a number of viral mRNAs. The experiments described in this application are divided into two parts. Those described in Part I will focus upon a genetic characterization of the vhs gene(s) defined by the mutant vhs 1.
The specific aims of these experiments will be: a) fine structure mapping of the mutation(s) carried by vhs 1 that affect host shut-off and the stabilities of viral mRNAs; b) determination of whether the multiple defects of vhs 1 are due to a mutation in a single gene or to genetically separable mutations in more than one gene; c) sequencing the vhs gene(s) and identification of their product(s); and d) in vitro mutagenesis of the vhs gene(s) and reintroduction of the mutations into virus to generate a collection of new vhs mutants. The experiments described in Part II will focus upon a characterization of the mRNAs that are the target of vhs induced degradation.
The specific aims of these studies will be a) to evaluate the role of the vhs gene product(s) in regulating the half lives of viral mRNAs belonging to different kinetic classes, and to assess the importance of controls of mRNA stability in the overall scheme of gene regulation in HSV-1 infected cells. This will involve measuring the half lives of selected viral mRNAs as well as their transcription rates and their rates of appearance in the cytoplasm.
Other aims will be b) to determine whether the vhs gene product(s) exhibit any target specificity in the induction of mRNA degradation, and if so, to characterize the features of selected mRNAs that render them more or less sensitive to degradation.
