The infected cell protein No.0 (ICP0) is the product of the alpha-0 genes herpes simplex viruses 1 and 2. The gene is expressed immediately after infection and it functions as an important promiscuous transactivator induced into cells by infection or transfection. While it merely enhances viral replication in dividing cells, alpha-0 is essential for productive infection in experimental animal systems, in non dividing cells, for the establishment of latency and for reactivation. The chief characteristics of ICP0 that form the basis of ongoing and proposed studies are as follows: (i) The gene contains two introns. Intron 1, the largest, contains 1 splice donor and 4 splice acceptor sites. RNAs extracted from infected cells form 4 populations predicted to make a family of proteins. (ii) Intron I RNA is nonpolyadenylated, transported into the cytoplasm and is more stable than the ICP0 mRNA. (iii) The 775-amino acid protein contains a zinc finger and a nuclear localization and a nucleotidylation signal sequence; it is also phosphorylated by the viral kinase. ICP0 has been shown to bind in a meaningful fashion to a ubiquitin-specific protease, to the translation elongation factor EF-1-delta and to bind and stabilize cyclin D3. Additional cellular proteins which appear to bind to ICP0 have been identified. These data indicate that ICP0 is an important multifunctional protein and suggest that the perceived function as a potent promiscuous transactivator reflects the sum total of the interactions of the protein and of the stable intron RNA with cellular and viral proteins. The proposed studies are to determine (i) the function of intron I of ICP0: verification of the existence of alternately spliced mRNAs and products of these mRNAs; (ii) the function of Intron I mRNA, (iii) the functions expressed by uncharted domains of ICP0; and (iv) the role of the ICP0 ligands in the biology of the viral infection. The experimental design involves identification of interactive ligands, identification of minimal ICP0 domains that interact with these ligands, and construction of recombinant viruses whose ICP0 will no longer interact with these ligands. The experimental design includes identification of the products of alternative splicing and characterization of the role of the ligands in infection.
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