CELLULAR REGULATION OF TYPE III EBV LATENCY

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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.