The long-term goals of this research are to define the mechanisms by which the herpes simplex virus immediate early proteins move into the infected cell nucleus and regulate viral gene expression.  In this application we propose to continue our studies of HSV-1 IE proteins and to initiate studies on the mechanisms of gene regulation in HSV type 2.  Our first specific aim is to define the mechanism(s) by which HSV-1 ICP27 increases the levels of expression of viral early DNA replication proteins by measuring rates of transcription of the DNA replication genes by in vivo pulse labelling, by analyzing viral RNA transport, processing and stability if the effect is post-transcriptional, by mutational analysis of the gene to find the essential sequences, if the effect is post-transcriptional, and by mutational analysis of the promoter of one or more of these genes to determine the sequences needed for stimulation by ICP27, if the effect is transcriptional.  Our second specific aim is to define the mechanism(s) by which HSV-1 ICP27 increases the level of transcription of the viral late glycoprotein (gC) gene by determining if the increase in transcription is due to an increase in transcriptional elongation, by determining if ICP27 and ICP4 are associated with the RNA polymerase II holoenzyme at late times of infection, and by examining the intranuclear localization of viral and cellular proteins in the presence or absence of ICP27.  Our third specific aim is to determine the mechanisms of interaction of ICP4 with late transcriptional sites in replication compartments by definition of the ICP4 sequences needed for its association with replication compartments and by identifying cellular proteins interacting with ICP4 at late transcriptional sites in replication compartments by co- immunoprecipitation.  Our fourth aim is to define mechanisms of gene regulation in HSV type 2 by examining the role of HSV-2 ICP6, or the ribonucleotide reductase 1 subunit in E gene expression, by examining the role of the HSV-2 ICP27 in regulation of viral gene expression, by examining the role of the HSV-2 ICP4 serine-rich region in ICP4 function and determining if HSV-2 ICP4 has an associated kinase activity, and by comparing gene regulatory mechanisms in strains 186 and HG-52 to define the cause of the differences in gene expression and pathogenicity.  These studies should provide information that is important for designing antiviral strategies for HSV and for engineering HSV-2 strains for genital herpes vaccines.