This proposal deals with the functional domains of the major regulatory protein of Herpes Simplex Virus (HSV), known as ICP4. The main function of ICP4 in virus infection is to activate the transcription of the 75 or so early and late genes, and allow the infection to proceed from the immediate early to early phase. ICP4 activates pol II transcription, in part, as a function of its association with DNA and TFIID, via TAF250. ICP4 also represses transcription by virtue of its ability to form a tripartite complex with TFIID and TFIIB on promoters containing an appropriately placed ICP4 binding site. The ICP4, LAT and Orf P (L/ST) promoters are examples of ICP4 repressed promoters. The repression of Orf P transcription by ICP4 is necessary for wild-type neurovirulence. In addition there is at least one region of ICP4 that is the target of several kinases and is required for viral growth in neurons, despite being dispensable in cell culture and in vitro systems, or in non-neuronal tissue in vivo. ICP4 is a 350 kd dimer assuming a very elongated conformation, making direct structural studies difficult. However, it has been possible to assign regions of the molecule that are important for repression, activation, DNA binding, interaction with TAF250, the ability to multimerize on DNA, and a function uniquely required for growth in ganglia. In addition, ICP4 possesses a domain with structural and functional similarity to a cellular co-activator, p15. p15 facilitates TFIID and TFIIA complex formation on promoters and regulates the activity of transcription activators as a function of its phosphorylation state. The region of ICP4 that is similar to 15 is involved in tripartite complex formation, growth in trigeminal ganglia and is the target of several kinases, including an ICP4 associated kinase. The goals of this application are to further define the regions of ICP4 that are important for these interactions and activities, and investigate the possibility that ICP4 may possess a domain, which like p15, regulates the activity of its transcriptional functions, particularly in neurons. Proposed are a combination of virus genetic studies, in vivo transcription and biochemical assays, and in vivo pathogenesis experiments, aimed at determining the regions of ICP4 that are important for these activities and how they affect the virus life cycle. All of the investigated activities are relevant from the standpoint of viral growth, both in vitro and in vivo. Therefore, a more precise structure-function correlation with respect to these aspects of ICP4 will further our understanding of HSV gene regulation in vitro and in vivo, and will aid in targeted antiviral approaches.
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