Herpes simplex virus (HSV) is a human pathogen, which causes a variety of diseases including; nonfatal cutaneous lesions, infections of the cornea, which may lead to blindness, and occasionally encephalitis, which can be fatal. While it is a rapidly replicating and cytotoxic virus in its lytic, or productive mode, it also has the capacity to establish a latent infection in the sensory ganglia of nerves that contact the primary site of productive infection. In this state, the virus is relatively silen from a transcriptional and cytotoxic standpoint, with the genome persisting in a potentially functional form for many years. Four regulatory proteins (ICP4, ICP0, ICP22, and ICP27) are expressed immediately following HSV infection. These immediate early (IE) proteins have profound effects on host cell metabolism and gene expression, and are involved in determining the fate of viral infection, and possibly the infected cell itself. The focus of this proposal is to determine the effects of the IE regulatory proteins on the transcriptional machinery of the cell. Two approaches have been developed to undertake these studies. This first is the application of reconstituted in vitro transcription to the HSV system. This involves the assembly of fractionated and purified cellular transcription factors, in vitro, along with proteins purified from HSV infected cells. This, and a method to isolate transcription initiation complexes and other biochemical approaches allows us to study mechanisms. The second is a system to isolate viral mutants that are simultaneously deficient in defined subsets of IE genes. This provides a means to examine how the expression of these genes affects transcription in infected cells, and also provides for the production and analysis of transcription factors from cells infected with the mutants to investigate specific IE protein induced changes. In this proposal, models for ICP4-activated and -repressed transcription will be tested by; (i) determining the minimal set of transcription factors required for activation, including initiator binding proteins, and (ii) examining the sequential assembly of activated and repressed transcription complexes as a function of ICP4. Data are also presented, which show that IE proteins other than ICP4, when expressed from mutant viruses, can affect pol II transcription during infection, and in vitro. We will determine: (iii) how different subsets of ICPs0, 22, and 27 expressed from the viral genome affect transcription in cells, and then (iv) determine how these proteins affect specific components of the cellular transcription machinery, and the assembly to transcription complexes.
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