We wish to gain a comprehensive understanding of the regulation and function of the herpes simplex virus (HSV) DNA polymerase (pol) gene. HSV Pol is both an excellent model for eukaryotic replicative polymerases and an important target for antiviral drugs. HSV and other herpesviruses are important pathogens, especially in immunocompromised individuals such as AIDS patients and transplant and chemotherapy recipients. Drug resistance, due in part to pol mutations, is a growing problem. The mechanisms regulating the translational shut-off of pol gene expression and its importance to the virus will be analyzed through mutations that fail to down-regulate translation of pol mRNA. Studies of Pol function are facilitated by the availability of quantities of purified Pol from recombinant baculovirus-infected insect cells. The enzyme will be proteolytically cleaved into domains, which will be tested for various Pol activities. The structure of Pol and its domains will be examined by X-ray crystallography. Pol interactions with nucleic acids will be studied with spectroscopic, mobility shift and footprinting methods. Amino acids that bind deoxynucleoside substrates will be identified by affinity labeling. Genetic correlates to protein domains will be developed by analysis of mutations that exhibit intragenic complementation. The roles of conserved regions of Pol that are altered by acyclovir-resistance mutations will be investigated to determine which steps in the mechanism of acyclovir-triphosphate inhibition of Pol each mutation blocks. Amino acids hypothesized to be within active sites for the 3'-5' exonuclease and RNase H/5'3' exonuclease will be altered by site-specific mutagenesis and the importance of these residues for various Pol enzyme activities and for virus growth will be tested. A signal required for the localization of Pol to prereplicative sites in HSV-infected cells, which is dependent upon the viral single-stranded DNA binding protein, ICP8, will be sought by studies of mutations that prevent localization. The importance to the virus of this signal and of a domain that binds to the polymerase accessory protein, UL42, will be assessed by construction of HSV mutants. These studies should permit a detailed picture of the regulation and functional domains of Pol both in vitro and in infected cells and may uncover novel features of polymerases and new strategies for antiviral drug development.
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