Herpes simplex virus type 1 (HSV-1) undergoes two types of interaction with host cells, lytic infection and latent infection. During lytic growth, following primary exposure or reactivation, HSV-1 can cause a wide spectrum of human diseases. On the other hand, the virus can remain latent in the trigeminal ganglia of infected individuals for years. Little is known about the viral or cellular functions required for maintenance of the latent state or for reactivation. The regulation of HSV-1 gene expression during lytic infection in tissue culture is a complex and highly coordinated process. At least three classes of genes are expressed in a sequential fashion. The order of expression is mediated in part by the action of viral encoded trans-acting proteins on specific sequences in the promoter-regulatory domains of HSV-1 genes. To elucidate the mechanism of action of two of these trans-acting proteins, a detailed genetic and biochemical analysis will be performed on the alpha proteins, ICP27 and ICP0. ICP27 is required for late gene expression during infection. ICP0 also affects late gene expression although not to the extent that ICP27 does. In transfection assays, both proteins affect the expression of HSV-1 genes and heterologous genes but do so differently. ICP0 stimulates expression while ICP27 negatively regulates expression. To examine whether these proteins function by direct DNA interactions or indirectly through interactions with other transcription proteins, ICP27 and ICP0 will be purified. The specificity and affinity of binding to DNA will be analyzed. Protein-protein interactions will be investigated by immuno- affinity chromatography and protein affinity chromatography. The purified proteins will also be assayed for putative enzymatic functions by which they could modify other transcriptional proteins. To correlate activities of each protein with specific domains, a series of in-frame insertion mutations will be introduced into the ICP27 and ICP0 genes. Mutant proteins will be isolated and analyzed to determine which functions or activities are affected. In this way, a functional map of each protein can be derived.
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