Genetics of HSV DNA Replication

University Of Connecticut Sch Of Med/Dnt, Farmington CT

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Abstract

DNA replication is a complex process that is of critical importance to all living organisms. Despite considerable progress in the general area of both viral and cellular DNA replication during the last decade, there are significant holes in our knowledge which have far reaching implications in our understanding of normal and cancerous growth of cells, the development of antivird and iinticancer chemotiierapeutic strategies and at the most basic level, our understanding of how multiple proteins come together to form a large replication complex. This proposal seeks to further our understanding of several aspects of HSV-1 DNA replication using molecular genetic, viral genetic, biochemical, biophysical and cell biological approaches. In aims 1 and 2 of this proposal we plan to continue and expand our structure-function analysis of two HSV-1 helicases: helicase-primase (comprised of the UL5. UL8 and UL52 gene products, Aim 1) and the origin binding helicase (UL9, Aim 2). Helicases are emerging as essential components not only in DNA replication, recombination and transcription, but have recently been implicated in several disease processes some of which result in cancer. The mechanism of helicase action and communication with the rest of the replication machinery is very poorly understood for any helicase. Several aspects of helicase function will be addressed in Aims 1 and 2 includingmapping domains responsible for DNA binding and protein-protein interactions. In aim 3 we will analyze the formation of replication compartments in infected cells and probe the importance of preexisting cellular structures and cellular proteins. In particular, we will i ivestigate the role of the single strand binding protein (UL29, ICP8) and other viral proteins in the formation of biologically relevant subcomplexes of proteins. The HSV system provides an excellent model system for studies of DNA replication of other herpesviruses since functional homologues for the six replication fork proteins are present in every known herpesvirus si;ch as CMV, EBV and most likely the new herpesvirus KSHV (HHV8). Initiation of DNA replication in HSV has recently been implicatedas a key regulatory event in the balance between the lytic and latent infections in neuronal cells in vivo. Therefore, the study of DNA replication in HSV is of critical importance to viral pathogenesis and disease.