Infections with Herpes Simplex Virus types 1 and 2 (HSV-1 and HSV-2) constitute a major health problem in the United States. This proposal aims to purify and characterize proteins present in HSV infected cells which are involved in the replication and expression of the viral DNA. We will continue our work on the major HSV DNA binding (ICP8). Affinity constants and stoichiometries of binding will be obtained for the interaction of ICP8 with single and double-stranded nucleic acids. We will also determine the rate of exchange of this protein between nucleic acids. The various functional domains of ICP8 will be mapped by means of specific modification of individual amino acid residues and by studies on the properties of ICP8 molecules containing temperature sensitive lesions in various locations. The effect of the presence of ICP8 on homologous (HSV) and heterologous (mammalian, yeast and procaryotic) DNA poloymerases will be determined. A DNA primase activity which has been identified in HSV-infected cells will be isolated and characterized. The purified primase activity will be used in an attempt to reconstruct the HSV replication apparatus. Z-DNA binding proteins and Z-DNA suequences in the HSV system will be identified and isolated. The Z-DNA binding proteins will be tested for binding specifi- city using cloned HSV restriction enzyme fragments. Z-DNA sequences present in HSV DNA will be tested for enhancer activity using a chloram- phenicol transacetylase (CAT) assay. Methodology used will include: fluorescence spectroscopy, filter binding, molecular cloning, DNA agarose/ cellulose chromatography and Z-DNA immunoaffinity assays.
