The herpes simplex virus (HSV) DNA polymerase (pol) gene affords a unique model system amenable to molecular, pharmacological, and genetic approaches for studies of eukaryotic replicative polymerases. The overall aim of these studies is to understand the pol gene in detail in terms of its structure, expression and function. The availability of the DNA sequence of the pol gene permits the precise location of its RNA transcripts by S-1 nuclease and exonuclease VII mapping. The gene can now be cloned as a single stretch of DNA permitting its overexpression. This will facilitate isolation of antibodies to identify pol polypeptides in infected cells and to map them precisely onto the pol gene. The sequence predicts a number of features which may act to diminish pol expression. The role of these sequences and their importance to the virus will be assessed by mutational analysis; they may reflect general properties of polymerase genes. Polymerase function will be explored by mapping and sequencing mutations which differentially affect the deoxynucleoside triphosphate and pyrophosphate binding sites. These mutations cluster to define a substrate binding domain. Portions of the polymerase important for polymerase function will also be identified by complementation experiments with polymerases from HSV's relatives, Epstein-Barr virus and cytomegalovirus (CMV). Functional sites in the CMV pol gene will be identified by mutant isolation and mapping studies. Functional interactions of HSV polymerase with other HSV proteins will be investigated through mapping of altered drug sensitivity mutations to other loci and by mapping and sequencing regions of polymerase involved in such interactions. These studies of polymerase function will be related to studies of polymerase structure and its interaction with DNA using polymerase purified from an overexpression system. HSV and CMV are important human pathogens for which there are no known cures. Since HSV and CMV polymerase are targets for several licensed and promising antiviral drugs, information from these studies should aid drug development. Similarly, HSV polymerase provides a model for human Alpha-polymerase which is a potential target for antitumor drugs. Studies of drug resistance help elucidate drug mechanisms and predict properties of drug resistant mutants in the clinic. Thus, the proposed studies address issues regarding eukaryotic DNA replication and issues of direct health relevance.
Showing the most recent 10 out of 87 publications
