CELLULAR REGULATION OF TYPE III EBV LATENCY

在EBV潜伏期最有限的I型中，只表达EBNA- 1，并且只使用Q启动子（Qp）。EBNA- 1对与细胞周期同步的EBV发作的复制至关重要。我们发现EBNA- l mRNA的表达与细胞周期同步，在Go中检测到的RNA很少，在s期表达最多。在这里，我们建议研究更复杂的III型潜伏期状态，在这种状态下，所有EBV潜伏期蛋白，包括主要的癌蛋白LMP-1，都被表达，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型潜伏期基因的表达是否与细胞周期有关，重点关注EBNA-2应答病毒基因（ebna - 1、-2、- 3a、- 3b、- 3c和- lp、lmp - 1和-2）。我们将验证ebna - 1的表达也与III型潜伏期的细胞周期有关的预测。接下来，我们将研究在细胞周期背景下选定的III型潜伏期蛋白的磷酸化。基于初步研究结果，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以及何时被磷酸化，以及这些残基的突变是否会破坏EBNA-2和LP磷酸化与细胞周期的联系。是否对LMP-1有后续影响也将被研究。最后，我们将确定磷酸化对相关蛋白的功能影响。关于EBV潜伏感染状态是如何调控和维持的更广泛的机制，我们知之甚少。此外，关于细胞周期对EBV基因的影响的信息非常少。这项研究不仅揭示了eb病毒的潜伏期，还揭示了III型基因产物表达后可能发生的eb病毒转化过程。

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.