MOLECULAR GENETICS OF HSV DNA POLYMERASE GENE

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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 in patients with AIDS, especially those caused by human cytomegalovirus (CMV). 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 functions of the small C-terminal domain of HSV Pol and to determine the mechanism by which the accessory subunit, UL42, enhances processivity despite stable UL42-DNA binding. Effects of mutations on DNA-binding by the C-terminal domain will be correlated with effects on Pol activity and virus replication. UL42 mechanisms will be examined using limited proteolysis and DNA footprinting. Specific aim 3 is to determine mechanisms by which mutant CMV Pols resist ganciclovir (GCV) action, by analyzing Pol interactions with GCV-TP and GCV-containing DNA. Specific aim 4 is to investigate CMV Pol's interaction with its accessory protein, UL44. Interacting residues will be defined genetically and tested for their importance in CMV DNA replication. This information will serve as a starting point for discovery of antiviral drugs. Specific aim 5 is to solve the three dimensional structures of domains of HSV Pol and UL42 and CMV Pol and UL44 using x-ray crystallography to gain basic information regarding polymerase functions and as a starting point for drug discovery.