Herpes simplex virus 1 (HSV-1) establishes life-long infections of the human host characterized by productive infection of actively cycling epithelial cells and fibroblasts, and nonproductive latent infections of noncycling sensory neurons. Latent infections are characterized by the reactivation of productive cycle viral gene expression and new virus synthesis following stress. During the early stages of productive infection, viral immediate-early (IE) proteins act together to create a nuclear environment that promotes viral gene expression. Available evidence indicates that the phosphorylation of IE proteins by cyclin dependent kinases (cdks), other cellular kinases and viral kinases is responsible for the timely activation of IE protein functions. The expression of kinase activities in cycling cells during productive infection, the absence of these activities in noncycling latently infected neurons, and their induction in noncycling neurons following stress-induced reactivation, has led to the hypothesis that productive infection, reactivation and the activities of IE proteins are associated with the activities of these cdks and other kinases whereas latency is associated with their absence. Among HSV IE proteins, ICP0, ICP22 and ppUS1.5 have been shown to affect and be affected by cdks and their cyclin partners. These proteins confer a significant growth advantage on HSV-1 and are required for the efficient establishment and reactivation of latency. This proposal will test the hypothesis that the phosphorylation of ICP0, ICP22 and ppUS1.5 by cdks and other kinases expressed differentially in nonneuronal and neuronal cells affects the functional capabilities of these proteins during productive infection, latency and reactivation. For this purpose we will i) identify the sites on ICP0, ICP22 and ppUS1.5 that are phosphorylated and the cdks and other kinases that phosphorylate these sites, ii) test the effects of phosphorylation at selected sites by specific kinases on the functions of ICP0, ICP22 and ppUS1.5 in cell culture and during productive infection and latency and iii) examine the molecular basis for the ability of ICP22 and/or ppUSl.5 and phosphorylation site mutants thereof to inhibit the functions of ICP0. The information obtained in these studies will provide new insight into the molecular mechanism underlying HSV- 1 productive infection and latency and lead to novel approaches to intervening in the life-cycle of this clinically important virus.
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