Herpesviruses are important pathogens of both humans and animals, and information on the molecular aspects of herpesvirus replication is useful in the diagnosis and treatment of these infections. Overall goals are to employ our models of equine herpesvirus 1 (EHV) cytocidal and persistent infection to understand herpesvirus gene regulation in terms of structure/function relationships of the viral regulatory proteins and to ascertain whether EHV regulatory polypeptides interact with cellular proteins to mediate the varied outcomes of infection. Aims for years #39 to #43 focus on the functions of EHV regulatory proteins with emphasis on the immediate-early protein (IEP): To characterize the transactivation domain (TAD) with aa#3-89 of the EHV IEP by assessing mutant forms of the TAD both as GAL4- fusion constructs and in the contest of the virus for transactivation ability. To use our panels of GST-EHV fusion proteins to identify the domains of EHV IEP, ICP22, ICP27 and ICP0 regulatory proteins that mediate their protein-protein interactions (by protein crosslinking assays) and enhance IEP binding to specific sequences within EHV regulatory proteins by the approaches of the two hybrid system, coimmunoprecipitation assays, and affinity chromatography. Mutant constructs and EHV-1 mutants that express specific domains of the auxiliary regulatory proteins, especially ICP22, will be used in transient transfection assays and experiments to monitor EHV gene programming, respectively, to define the roles of these proteins in EHV replication. Lastly, focused efforts will address the functions of the ICP22-ICP27 hybrid protein (HYB.) encoded by EHV DI particles (DIP) that mediate persistent infection. Since the HYB. retards expression of specific EHV promoters in initial transfection assays, our constructs, cell lines, and recombinant virus that express the HYB. will be employed to define viral promoters affected by the HYB. and ascertain whether HAB.. expression by a recombinant EHV alters viral gene programming, possibly mediates persistent infection, and is essential for DIP to establish persistent infection. If warranted by these data, the interaction of the HAB.. with EHV regulatory proteins and cellular factors will investigated by the approaches described above.