Molecular Genetics of HSV DNA Polymerase Gene

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DESCRIPTION (provided by applicant): The long-term objective of this research is a detailed understanding of herpesvirus DNA polymerases. These enzymes, which include a catalytic subunit (Pol) and an accessory subunit that stimulates long-chain DNA synthesis, are prototype alpha-like DNA polymerases and excellent targets for antiviral drugs. New drugs are needed for treatment of herpesvirus infections, especially those caused by human cytomegalovirus (CMV) in immunosuppressed patients such as those with AIDS. Specific aim 1 is to determine mechanisms that regulate translation of HSV Pol and their importance to the virus. Mutational, RNA-binding, and translational analyses will test the hypotheses that a virion protein, US11, stimulates Pol translation early in infection, while inefficient translation later is beneficial to the virus. Specific aim 2 is to analyze the interaction of the accessory subunit, HSV UL42, with DNA that permits sliding despite high affinity binding and the implications of this interaction for processive DNA synthesis. The UL42-DNA interaction will be explored using mutational approaches, protein-DNA crosslinking, measurements of affinity, on, off, and sliding rates, and single molecule studies and will be correlated with effects on processivity. Specific aim 3 is to determine mechanisms by which mutant CMV Pols, especially those altered in exonuclease motifs, resist ganciclovir (GCV) action, by analyzinf Pol interactions with GCV-TP and GCV-containing DNA. Specific aim 4 is to investigate CMV Pol's interaction with its accessory subunit, UL44. Interacting residues will be defined genetically and tested for their importance in CMV DNA and viral replication to determine if this interaction is a valid drug target. If so, a high throughput screening assay will be used to discover new antiviral drugs. Specific aim 5 is to use X-ray crystallography and, if necessary, nuclear magnetic resonance, to solve the three dimensional structures of domains of HSV US11, and HSV and CMV DNA polymerase and/or domains thereof to gain basic information regarding polymerase functions and as a starting point for drug discovery.