Triggering of the Epstein-Barr virus lytic cycle requires the expression of the viral gene product ZEBRA. Expression of ZEBRA, in conjunction with the product of the BRLF1 gene, results in the activation of a number of other viral genes, thereby initiating the lytic cascade. The major goal of this proposal is to understand the mechanisms involved in transcriptional activation by ZEBRA. We have completed an initial characterization of the regions of ZEBRA that are involved in transactivation, and have mapped one region that appears to be very important for activity. In addition, we have shown that DNA binding mutants of ZEBRA retain the ability to transactivate specific target promoters. Understanding these phenomenon will require further characterization of the regions of ZEBRA involved, and ultimately identification of the cellular factors through which these effects are mediated. Our approach will be as follows: (1) Further characterization of ZEBRA transactivation. (a) Fine mapping of critical regions/residues involved in mediating activation of transcription. (b) Assessing the possible role of ZEBRA DNA binding and dimerization domains in supporting """"""""activation""""""""; generation of GAL4/ZEBRA chimeras. (c) Comparison of amino acid sequence requirements for non-synergistic and homo-synergistic transactivation. (2) Further characterization of transactivation by ZEBRA DNA binding mutant, ZEBRAbm1. (a) Fine mapping of critical regions/residues involved in mediating activation of transcription. (b) Further characterization of cis elements within the BZLF1 promoter, Zp, which are involved in ZEBRAbm1 transactivation. (c) Characterization of the DNA binding properties of ZEBRA proteins expressed during the viral lytic cycle. (d) Assessing the influence of ZEBRAbm1 on expression of endogenous Zp. (3) Analysis of protein-protein interactions between ZEBRA and cellular factors. (a) In vitro approaches towards identifying ZEBRA associated protein factors. (b) In vivo approaches towards identifying ZEBRA associated protein factors.
