GENETICS OF HSV DNA REPLICATION

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Herpes simplex viruses are endemic in the population and are responsible for a variety of clinical diseases some of which are life threatening especially in immunocompromised individuals or in newborns. The herpes simplex virus type-1 (HSV-1) genome encodes seven viral genes that are necessary and sufficient for origin-dependent plasmid amplification in a transient transfection assay: UL30 and UL42 which specify a two subunit DNA polymerase; UL29, the single strand DNA binding protein; UL5, UL8 and UL52 which together make up a three protein helicase-primase complex and UL9 which specifies the HSV origin binding protein. UL5, 8, 9, and 52 which are the least abundant and least well studied of the replication proteins have been chosen for this study. The objective of this proposal is to carry out a detailed structure-function analysis of these four proteins in order to map domains responsible for catalytic activities, DNA binding (specific and nonspecific), sites for inter- and intramolecular interactions, nucleotide binding, and signals for intracellular localization. The first aim is to construct the necessary reagents to carry out all subsequent aims; these include constructs for expression of each protein in a variety of expression systems and the characterization of specific antisera. Aim 2 is to isolate a large number of point, insertion and deletion mutations in each gene. The third and fourth aims are to carry out in vivo and in vitro characterization of mutants isolated in aim 2 for overall ability to carry out DNA replication and for specific activities such as specific and non specific DNA binding, NTP binding, DNA-dependent ATPase, helicase and primase. The nuclear localization signals on each protein will be mapped by observing localization not only of wild-type and mutant forms of each protein but also of gene fusions to cytoplasmically located proteins. The fifth aim is to locate regions on UL5, UL8 and UL52 and UL9 responsible for protein-protein interactions using genetic and biochemical approaches. Potential interactions between these proteins and other members of the replication complex will also be pursued. Genetic methods include: interference by transdominant mutations, use of a novel two-hybrid system to identify interacting proteins, isolation of second site suppressor mutations and colocalization by immunofluorescence. Biochemical methods for detecting interactions include: affinity-based methods, coimmunoprecipitation and physical methods such as glycerol gradients and gel filtration. It is anticipated that a detailed structure-function analysis of these four replication proteins will not only facilitate our understanding of the mechanisms of their action within the replication complex but may also lead to the development of novel strategies for antiviral therapy.