This proposal will study molecular events and gene products involved in genome replication, circularization, inversion and cleavage/packaging of herpesviruses with emphasis on a beta herpesvirus, human cytomegalovirus (CMV). Viral and host cell proteins that bind to or enzymatically act on the CMV a sequence, which is the cis-acting signal involved in genome circularization inversion and cleavage/packaging, will be isolated and their recognition sites will be determined. The genes for these viral proteins will be mapped on the viral genome. As a complementary approach to identify viral gene products involved in replication functions, the genes encoding viral nonstructural nuclear proteins will be mapped on the viral genome. Monoclonal and polyclonal antibodies specific for nuclear proteins will be generated in mice and viral genes will be identified via a lambda gtll library expressing random CMV open reading frames. Functional requirement for identified genes will be determined using insertional mutagenesis of the CMV genome. CMV insertion mutants will be constructed using E. coli beta galactosidase as a genetic marker. Gene products provided in trans from engineered retrovirus vectors to complement insertion mutations in essential CMV genes. These experiments will identify viral proteins that have specific functions in replication or have a nuclear localization and are nonstructural consistent with replication function. The genes encoding these proteins will be mapped on the viral genome and insertional-mutants will be derived. The mutants will enable an unambiguous determination of the functional significance of these gene products in replication. One area of herpesvirus replication will be studied using herpes simplex virus: the role of the HSV-1 DNA replication origin specific binding protein (ori bp) as a trans-acting function in DNA replication initiation. The ori bp gene will be mapped and conditionally lethal mutants will be derived to demonstrate the role of this gene product in HSV-1 DNA replication. The functional significance of the binding site for ori bp within oriS of HSV-1 will be assessed by site directed mutagenesis. OriS with one, two or zero ori bp sites will be compared for replication efficiency in competition assays. The role of the multiple DNA replication origins on the viral genome will be directly investigated by introducing modified versions oriS in place of existing origins. This research effort will provide a molecular understanding of the process and control of herpesvirus replication functions. This knowledge will impact directly, in the medical terms through the identification of targets for antiviral development. The understanding gained through the proposed research will enable characterization of more subtle viral functions, for example those involved in latency in future years.
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