CELLULAR REGULATION OF TYPE III EBV LATENCY

III 型 EBV 潜伏期的细胞调节

• 批准号: 6344696
• 负责人: JOSEPH S PAGANO
• 金额: $ 23.04万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-21 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6344696
• 关键词: DNA binding protein; DNA replication; Epstein Barr virus; SDS polyacrylamide gel electrophoresis; cell cycle; chimeric proteins; flow cytometry; gene expression; genetic promoter element; immunoprecipitation; latent virus infection; oncoproteins; phosphorylation; posttranslational modifications; protein structure function; site directed mutagenesis; tissue /cell culture; transcription factor; virus genetics; virus infection mechanism; virus protein; virus replication

In the most restricted form of EBV latency, Type I, only EBNA- l is expressed, and only the Q promoter (Qp) is used. EBNA- l is essential for replication of EBV episomes which is synchronized with the cell cycle. We have shown that expression of EBNA- l mRNA is synchronized with cell cycle with little RNA detected in Go and most expressed in S-phase. Here we propose to examine the more complex Type III latency state, in which all of the EBV latency proteins including the principal oncoprotein, LMP-1, are expressed, and promoter usage shifts to C/Wp and promoters for the LMP proteins. We will determine first whether expression of Type III latency genes is linked to cell cycle with a focus on EBNA-2- responsive viral genes (EBNA-l, -2,-3A, -3B, -3C and -LP, LMP-l and -2). We will test the prediction that expression of EBNA-l is also linked to cell cycle in Type III latency. Next we will study phosphorylation of selected Type III latency proteins in the context of cell cycle. Based on preliminary findings, we will verify that EBNA-2 as well as EBNA-LP are phosphorylated in a cell-cycle dependent manner and ask which amino acid residues are phosphorylated and when, and whether mutation of these residues disrupts linkage of phosphorylation of EBNA-2 and LP to cell cycle. Whether there is a consequent effect on LMP-1 will also be studied. Finally, we will determine the functional effects of phosphorylation on the implicated proteins. Little is known about the broader mechanisms whereby latent EBV infection states are regulated and maintained. Moreover, information on the effect of cell cycle on EBV genes is remarkably scant. This work should shed light not only on EBV latency, but also on the EBV transformation process, which may follow when Type III gene products are expressed.

在EBV潜伏期最有限的形式中，I型，仅表达EBNA-L，并且仅使用Q启动子（QP）。 EBNA-L对于复制与细胞周期同步的EBV发作至关重要。我们已经表明，EBNA-L mRNA的表达与细胞循环同步，在GO中检测到的RNA很少，并且在S期间表达最多。在这里，我们建议检查更复杂的III型潜伏期，其中所有EBV潜伏蛋白在内的所有EBV潜伏蛋白（包括主要的癌蛋白LMP-1）均表达，而LMP蛋白的启动子的使用转换向C/WP和启动子。我们将首先确定III型潜伏基因的表达是否与细胞周期有关，重点是EBNA-2-反应性病毒基因（EBNA-L，-2，-3a，-3a，-3b，-3b，-3c，-3c和-lp，LMP-L，LMP-L和-2）。我们将测试以下预测，即EBNA-L的表达也与III型潜伏期中的细胞周期有关。接下来，我们将研究细胞周期中选定的III型潜伏期蛋白的磷酸化。根据初步发现，我们将验证EBNA-2和EBNA-LP以细胞周期依赖性方式磷酸化，并询问哪些氨基酸残基被磷酸化以及何时磷酸化，以及这些残基突变是否会破坏EBNNA磷酸化的连接-2和LP到细胞周期。还会研究对LMP-1的影响。最后，我们将确定磷酸化对涉及蛋白的功能作用。关于潜在的EBV感染状态受到调节和维护的更广泛的机制知之甚少。此外，有关细胞周期对EBV基因的影响的信息非常少。这项工作不仅应该阐明EBV潜伏期，而且还应介绍EBV转换过程，当表达III型基因产物时可能会随之而来。