This Program Project Grant application by four interactive investigators focuses on various aspects of the relationships between herpes simplex viruses (HSV) and the neurons and neuronal tissues of two experimental animals, the mouse and rat. The interaction of HSV with neuronal cells and tissues is reflected in (a) replication resulting in destruction of the central nervous system (neurovirulence), (b) latency, and (c) persistence of viral DNA in a non-reactivatible form in the brain stem or other CNS tissues. The specific objectives of the research proposed in this application are as follows: (i) identification of viral genes and non-coding sequences which enable the virus to access the central nervous system as a consequence of peripheral inoculation, (ii) identification of genes and sequences which enable the virus to replicate and destroy the central nervous system, (iii) identification of neurons in sensory ganglia of mice harboring latent virus, (iv) identification of the viral genes expressed constitutively and uniformly in all neurons harboring virus, (v) determination of the molecular basis for the failure of the viral genome harbored in a latent state in neurons to express all of the genes normally expressed during productive infection, (vi) analyses by deletion mutagenesis of the viral genome for genes or sequences essential for establishment and maintenance of the latent state, (vii) determination of the state of the viral genome during the latent state, (viii) determination of the state of the viral genome in the mouse brain stem from which infectious virus cannot be reactivated, and (ix) analyses of the viral genome for host factor dependent origins of DNA replication that enable the maintenance of the viral genomes in latently infected neurons. The experimental design of the proposed studies takes into account (i) familiarity of the investigators with experimental animal models, (ii) ability to express genes under their own or other appropriate promoters contained in viral genomes, (iii) experience in identification and mapping of viral origins and other cis-acting sites in the viral genome and (iv) application of molecular genetics and specifically, the construction of recombinant viral genomes with deletions or carrying at specified sites insertions of marker genes, genes driven by surrogate promoters, or appropriate cis- acting sites, for the purpose of testing specific hypotheses regarding the interaction of HSV with neuronal cells.
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