RAZ, A TRANSDOMINANT REPRESSOR, MODULATES EBV REACTIVATION

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RAZ is a newly discovered EBV delayed immediate-early gene protein that rises by differential splicing of the bicistronic mRNAs for the BZLF-1 and BRLF-1 ORFs predicted to generate a chimeric protein composed of a portion of the amino terminus of the R protein and the carboxy terminus of the Z protein. Although the DNA-binding and dimerization of domains of Z are preserved, in vitro-made RAZ cannot bind to Z's cognate binding site (ZRE). Moreover upon heterodimerization with RAZ, Z can no longer bind to a ZRE. In transient transfection assays, RAZ stoichiometrically down-regulates Z- induced transactivation of early promoters. In biologic tests, RAZ diminishes viral reactivation of latent infection produced by Z. Since RAZ has structural and functional hallmarks of a transdominant transcriptional repressor protein, it may have a potential role in EBV latency or reactivation. In this next phase of work, we will first, study RAZ in cells in which latent EBV is reactivated by determining the time-course of the appearance of RAZ RNA and protein in the cell as well as quantitating the levels of the transcript and the protein. Second, we will study further the key biologic effect, namely, how RAZ attenuates viral reactivation. We will also examine whether RAZ is an encapsideated late protein that participates in primary infection. In an independent assessment of biologic function, er will verify RAZ's role in the viral replicative and latent cycles by creation of a RAZ knock-out viral mutant. Third, we will identify domains of RAZ and post-translational modifications, specifically, certain serine and tyrosine phosphorylations, that may be involved in the repressor function. Finally the possibility that RAZ may bind to DNA sites other than ZREs in latent and late promoters and heterodimerize will cellular b-Zip-like proteins or with other viral and cellular proteins that interact with Z will be examined in order to define the scope of RAZ function.