CELLULAR REGULATION OF TYPE III EBV LATENCY

III 型 EBV 潜伏期的细胞调节

• 批准号: 6493592
• 负责人: JOSEPH S PAGANO
• 金额: $ 43.42万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6493592
• 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.

在最受限制的EB病毒潜伏期形式I型中，只表达EBNA-L，并且只使用Q启动子(QP)。EBNA-L是EB病毒表型复制所必需的，与细胞周期同步。我们发现EBNA-L基因的表达与细胞周期同步，在GO中检测到少量的RNA，在S期表达最多。在这里，我们建议研究更复杂的III型潜伏期，在这种状态下，包括主要癌蛋白LMP-1在内的所有EBV潜伏期蛋白都得到表达，启动子的使用转移到C/WP和LMP蛋白的启动子。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应答病毒基因(EBNA-L、-2、-3A、-3B、-3C和-LP、LMP-L和-2)。我们将验证EBNA-L的表达与III型潜伏期细胞周期有关的预测。接下来，我们将在细胞周期的背景下研究选定的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以细胞周期依赖的方式被磷酸化，并询问哪些氨基酸残基被磷酸化，何时被磷酸化，以及这些残基的突变是否扰乱了EBNA-2和LP的磷酸化与细胞周期的联系。还将研究是否会对LMP-1产生后续影响。最后，我们将确定磷酸化对相关蛋白质的功能影响。对于潜伏的EBV感染状态被调节和维持的更广泛的机制，人们知之甚少。此外，关于细胞周期对EBV基因的影响的信息也非常少。这项工作不仅应该阐明EBV的潜伏期，而且还应该阐明EBV的转化过程，当III型基因产物表达时，EBV转化过程可能会随之而来。