EBV BZLF1 GENE PRODUCT

EB病毒裂解周期的触发需要表达 病毒基因产物ZEBRA ZEBRA的表达，结合 BRLF 1基因的产物，导致许多其他 病毒基因，从而启动裂解级联反应。 这个项目的主要目标是 我们的建议是要了解参与转录的机制， 激活ZEBRA。 我们已经完成了对 ZEBRA的区域参与反式激活，并绘制了一个 这似乎是一个非常重要的活动。 另外我们 已经表明，ZEBRA的DNA结合突变体保留了 反式激活特定的靶启动子。 了解这些现象 将需要进一步表征涉及的ZEBRA区域， 最终确定细胞因子， 影响是中介的。 我们的方法如下： (1)ZEBRA反式激活的进一步表征。 (a)参与介导的关键区域/残基的精细作图 转录激活。 (b)评估ZEBRA DNA结合和二聚化的可能作用 支持“激活”的结构域; GAL 4/ZEBRA嵌合体的产生。 (c)比较非协同和非协同的氨基酸序列要求 同源协同反式激活。 (2)通过ZEBRA DNA结合进一步表征反式激活 突变体ZEBRAbm 1。 (a)参与介导的关键区域/残基的精细作图 转录激活。 (b)进一步表征BZLF 1启动子Zp内的顺式元件， 它们参与ZEBRAbm 1的反式激活。 (c)ZEBRA蛋白的DNA结合特性的表征 在病毒裂解周期中表达。 (d)评估ZEBRAbm 1对内源性Zp表达的影响。 (3)分析ZEBRA与细胞的蛋白质-蛋白质相互作用 因素 (a)ZEBRA相关蛋白的体外鉴定方法 因素 (b)鉴定ZEBRA相关蛋白的体内方法 因素

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.