This proposal is concerned with two types of translational control that occur in cells infected with herpes simplex virus (HSV). An early event during lytic HSV infections is the shut-off of most cellular protein synthesis. This shut-off is initially caused by a virion-associated host shut-off (vhs) function which induces disaggregation of cellular polysomes. Later during infection, translation of the alpha class of viral mRNAs is repressed through a mechanism apparently involving beta and/or gamma viral polypeptides. Virus mutants have been isolated that are simultaneously defective in both of these translational control functions. In the proposed studies these mutants will be used in a systematic analysis of the vhs and alpha shut-off functions. The goal is to elucidate the molecular mechanisms of translational control and to determine whether mutations that effect the two functions can be genetically separated. These studies should add to the understanding of translational control mechanisms in mammalian cells as well as yield information concerning the factors that determine whether the outcome of an HSV infection is a lytic or latent infection or the oncogenic transformation of the cell. Specific aims of the experiments examining the vhs function are: 1.) Comparison of the structural proteins of vhs mutant and wild type virions in an effort to identify the virion component that mediates the vhs function. 2.) Classification of the mutants into complementation groups. 3.) Analysis of the translatability in vitro of cellular mRNAs purified from infected cells following vhs induced host shut-off. 4.) Characterization of the free cytoplasmic ribonucleoprotein particles (RNPs) that contain cellular mRNAs released from polysomes as a result of the vhs function, and comparison of these RNPs to free and polysomal RNPs from mock infected and vhs mutant infected cells. 5.) Anlaysis of the effect of the vhs function upon the association of cellular mRNAs with the cytoskeleton. 6.) Comparison of in vitro translation extracts from mock infected, mutant infected and wild type infected cells. 7.) In vitro duplication of vhs induced host shut-off by addition of disrupted virions or virion components to cell extracts that are translating cellular mRNAs. The alpha translational shut-off function will be analyzed in a second series of experiments paralleling those just described for the vhs function. Other experiments will evaluate whether mutations effecting the vhs and alpha shut-off functions can be genetically separated.