Information useful in the diagnosis and treatment of herpesviral infections has come from investigations of infected cell culture models of herpes biological outcomes--cytocidal infection, transformation, and persistent infection (Pers Inf). These investigations have yielded insight into viral gene programming required for cytocidal infection; little, however, is known about the molecular events of Pers Inf--be it latency or chronic virus production. Our goal is to employ Equine Herpesvirus Type 1 (EHV-1) infected cell systems to elucidate the structure, function, and programming of EHV 1 genes in cytocidal infection and eventually in Pers Inf. Since systems of EHV-1 cytocidal infection and Pers Inf are in hand, knowledge of the genomes of EHV-1 and its DI Particles (DIP) that establish Pers Inf is significant, and molecular reagents (clones, expression constructs, Mabs) are available, these investigations are timely. Importantly, EHV-1 has several unique features: The Inverted Repeat(IR) segment contains 6 genes of which 4 may be important in gene regulation; the Immediate Early (IR l) gene encodes four IE proteins; the 203K phosphoprotein being the major species; an IR2 Early gene (4.4 kb RNA; 130K protein) lies within the 3' end of the IE gene; gene IR3 is unique to EHV-1; the Early IR4 gene is an ICP22 homolog; gene IR6 is a Very Early gene. Interestingly, EHV-1 Pers Inf cells produce a unique 2.2 kb RNA antisense to the 3' portion of the IE gene, and the genome of the DIP that establish/ maintain the Pers Inf has a UNIQUE ORF generated from IR and L terminus sequences. Specific aims will concern the characterization of these newly discovered and sequenced IR genes with regard to the properties and synthesis of their RNA transcripts and gene products and their possible role(s) in gene regulation. Expression constructs that produce the various proteins, especially IR1,2,4 and 6, will be assayed for their ability to influence expression of IE, VE, Early (IR2 and IR4), delayed early (EHV-1 glycoprotein D) and late (IR3) genes by employing constructs of these regulatory/promoter sequences linked to a reporter gene (e.g.CAT) in transient expression assays and or cell lines that express the IE gene. Experiments to determine the origin and function(s) of the IE protein species will involve site directed mutagenesis, ATG removal, and hybrid-arrest translation. Importantly, our Mab's to 4 IE epitopes and antisera to IE peptides discriminate IE species. IR2 (truncated IE) and IR6 proteins will be tested for regulatory activity and their properties and synthesis determined by biochemical/ immunological analyses. Transcripts and proteins encoded by IR3,4, and 5 will be studied by similar approaches, with emphasis on IR4 function. Lastly, the expression and possible products of the 2.2kb antisense RNA and Unique DIP ORF will be examined by characterizing cDNAs of the antisense RNA and determining if the RNA, its products and those of Unique DIP ORF are associated with-Pers Inf.