The infected cell protein No. 0 (ICP0) is a 775 amino acid multifunctional protein that plays a very important role in the human biology of herpes simplex viruses 1 and 2 (HSV-1 and 2). ICP0 is encoded by 3 exons. ICP0 is encoded by 3 exons. The protein has been reported to play a major role in reactivation from latency and in productive infection it is essential for ythe manifestations of pathologies associated with HSV infection. In this application for renewed support we propose to continue the research conducted over the past 4 years. Specifically: (1) Recent studies from this laboratory have shown that HSV-1 ICP0 acts in in vitro assays as an ubiquitin ligase with rather unique properties: thus the ubiquitin- conjugating enzymes (E2) UbcH5a and UbcH6 interact with sequences encoded by exon 2 whereas the UbcH3 (cdc34) interacts with sequences encoded by exon 3. One target of the ubiquitin ligase is cdc34, a ubiquiting conjugating enzyme responsible for the turnover of D cyclins. The objectives of AIM 1 is to investigate the role of ICP0 as a ubiquitin ligase. Specifically, the objectives are to map and characterize the E3 ubiquitin ligase site in exon 3 and to define the cellular proteins that are the targets of this ICP0 ubiquitin ligase. (2) A comparison of wild-type virus with a mutant in which ICP0 has been replaced by a cDNA copy showed that in rabbit skin cells the onset of accumulation of early (?) and later (? and ?) proteins can be delayed by as much as 6 to 8 hrs although ultimately the cells synthesize equal amounts of key proteins and yield equivalent amounts of infectious virus. Yet the data clearly show that HSV-1 DNA has been delivered to the nucleus. The fact that none of the viral genes are expressed until several hours after infection and that the mutation maps to the transcribed domain of ICP0 raises the possibility that ICP0 plays a critical role in the initiation of expression of viral genes in a cell-type dependent manner. The objective of AIM 2 is to investigate the role of ICP0 at very early stages of infection. (3) We and other laboratories has assigned functions to the exon 2 and the carboxyl-terminal half of exon 3. A 200+ amino acid stretch in the amino terminal domain of ICP0 is uncharted and no functions have been ascribed to it. We have made a series of linker insertion mutants and have identified small regions of sequences conserved in HSV-1 and HSV-2. Linker insertions in two of the conserved sequences attenuate the virus. The objective of AIM 3 is to continue the investigation of the functions of the uncharted domains of ICP0. We expect that the proposed studies will help understand the role of ICP0 in the evolution of diseases caused by HSV-1 and HSV-2.