The control of viral latency is a key issue in Epstein-Barr virus (EBV) biology. Overexpression of the immediate-early protein, BZLF1 (Z), is sufficient to disrupt viral latency. the cellular and viral regulation of this gene product is therefore crucial in determining whether virus infection is latent versus productive. The Z protein can be derived from either of two messages: a 1.0 kb message (transcribed from the BZLF1 promoter) making only Z protein, or a 2.8 kb bicistronic message (transcribed from the BRLF1 promoter) which can make both the Z and BRLF1 (R) immediate-early proteins. Disruption of viral latency could potentially be mediated through activation of either the BZLF1 or the BRLF1 promoter. In this grant we propose to identify the cellular transcription factors mediating disruption of viral latency through activation of either the BZLF1 or the BRLF1 promoter and to determine the biological role of the 1.0 kb message versus the 2.8 kb message as a source of Z protein. We have recently found that both the BZLF1 and BRLF1 promoters are bound by the cellular Yin Yang 1 (YY-1) protein as well as an oct-like protein. We have shown that the BRLF1 (but not the BZLF1) promoter is activated by the cellular transcription factor, Sp1. In addition, we have found that the BRLF1 promoter contains binding sites for the zif268 and WT1 transcription factors. In our first specific aim we will determine the exact binding sites of the various transcription factors binding to the BZLF1 and BRLF1 promoters and create site-directed mutations abolishing these sites. In the second specific aim we will examine the functional significance of each of the various transcription factors binding to the BZLF1 and BRLF1 promoters in different cell types. In the third specific aim we will examine the interaction of the Z and R immediate-early proteins with the cellular transcription factors shown to regulate BZLF1 and BRLF1 promoter function. In the fourth specific aim we will determine the role of the 1.0 kb versus the 2.8 kb message as a source of Z protein in various types of EBV infection using PCR analysis. In the final specific aim we will construct mutant viruses in which either the Z or the R proteins have been abolished, or in which the bicistronic nature of the 2.8 kb message has been destroyed, and determine the biological consequences of these mutations. These studies should help to establish the nature of the cellular factors contributing to viral latency and the role of the BZLF1 versus the BRLF1 promoter in the disruption of viral latency.
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