Herpes simplex virus, HSV, causes acute clinical infections and longterm latent infections characterized by periodic recurrences in a large portion of the population worldwide. It has long been recognized that the reservoir of HSV is the latently infected host, and that virus shed following reactivation from latency is the primary source of infection in spread to uninfected individuals. Little is known about the molecular mechanisms underlying latency or the factors that mediate the switch from acute, productive infection to latent infection and vice versa. Based on observations made during the past funding period, the studies proposed are designed to generate new information concerning the regulation of HSV gene expression during latency, focussing on the roles of individual viral and cellular factors in the reactivation process. The HSV immediate-early (IE) protein, ICP0, is a potent activator of all classes of HSV genes. Based on our observation that ICP0 is important for efficient reactivation from latency, efforts will be made to identify and fine map regions of the ICP0 molecule involved in reactivation. For this purpose, small insertion mutations will be introduced into ICP0 coding sequences by site-directed mutagenesis. The resulting mutant viruses will be tested for their ability to induce reactivation in vitro and in vivo in a mouse ocular model. The latency-associated transcripts or LATs are present in abundance in the nuclei of latently infected neurons and, like ICP0, are required for efficient reactivation from latency. Although no function has yet been assigned to the LATs, we have shown that the poly A-LATs are able to activate their own promoter as well as that of ICP0. Experiments are therefore proposed to identify LAT-responsive elements in the LAT and ICP0 promoters by site-directed mutagenesis and transient expression assays. Similar techniques will be used to identify regions of the LAT gene responsible for the observed activation. The relevance of the responsive elements and activation regions to latency will be tested as described for ICP0 mutants. Having recently identified a cellular function able to substitute for the transactivating function of ICP0 in productive infection, and able to activate HSV IE promoters in transient expression assays, efforts will be made to identify elements in IE promoters that respond to the cellular activating function(s) as described above for the LAT and ICP0 promoters. Because of their potential role as the primary """"""""activators"""""""" of viral gene expression in reactivation, efforts will be made to clone the genes specifying the activating function(s) by subtractive hybridization and to characterize them physically and biologically. Collectively, the proposed studies will generate new insights into the molecular mechanisms governing latency and reactivation, and, provide new approaches to the development of effective means for the prevention and cure of HSV-induced diseases.
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