CELLULAR REGULATION OF TYPE III EBV LATENCY

在EBV潜伏期的最受限制的形式（I型）中，仅表达EBNA-1，并且仅使用Q启动子（Qp）。EBNA-1对于EBV附加体的复制是必需的，其与细胞周期同步。我们已经表明EBNA-1 mRNA的表达与细胞周期同步，在Go中检测到很少的RNA，而在S期中大部分表达。在这里，我们建议研究更复杂的III型潜伏状态，其中所有的EBV潜伏蛋白，包括主要的癌蛋白，LMP-1，表达，启动子使用转移到C/Wp和启动子的LMP蛋白。我们将首先确定III型潜伏基因的表达是否与细胞周期相关，重点是EBNA-2应答性病毒基因（EBNA-1、-2、-3A、-3B、-3C和-1P、LMP-1和-2）。我们将测试EBNA-1的表达也与III型潜伏期的细胞周期相关的预测。接下来，我们将研究选定的III型潜伏蛋白在细胞周期中的磷酸化。基于初步发现，我们将验证EBNA-2以及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，何时被磷酸化，以及这些残基的突变是否破坏EBNA-2和LP的磷酸化与细胞周期的联系。还将研究是否对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。关于潜伏性EBV感染状态被调节和维持的更广泛的机制知之甚少。此外，关于细胞周期对EBV基因的影响的信息非常少。这项工作不仅应该阐明EBV潜伏期，而且还应该阐明EBV转化过程，这可能是III型基因产物表达时的结果。

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.