REGULATION OF EBV ORI-LYT BY CELLULAR AND VIRAL PROTEINS

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Epstein-Barr virus (EBV) infection has been associated with the development of several types of human malignancy. The horizontal transmission of EBV from host to host requires activation of the lytic origin of replication, ori-Lyt. In contrast, transformation of cells by EBV is generally associated with latent viral infection and may require that lytic replication be suppressed. Both of the EBV immediate-early transcriptional activators, BZLF1 (Z) and BRLF1 (R), are required for ori-Lyt replication. However, it is not known whether the role of Z and R is limited to transcriptional activation of ori-Lyt or whether one or both of these proteins also plays a more basic role in replication. In addition, the regulation of ori-Lyt replication by cellular factors has not been well studied. In this grant, we propose to study the regulation of ori-Lyt by viral and cellular factors. We hypothesize that the regulation of ori-Lyt replication plays a role in determining the efficiency of productive EBV infection in different cell types. We have recently shown that Z may interact directly with the viral polymerase processivity factor (BMRF1), suggesting a mechanism by which Z could direct the polymerase complex to ori-Lyt. We have shown that Z can also interact directly with p53 and that this interaction may inhibit replication. We have also obtained preliminary data suggesting that three cellular factors (Spl, NF-Y,and zif268) can bind to essential regions of ori-Lyt and potentially regulate its replicative function. We propose the following specific aims: 1) To examine the role of Z and R in ori-Lyt replication, we will compare the effect of deleting Z and R binding sites in ori-Lyt upon transcription versus replication and will attempt to create mutant Z and R proteins which can activate transcription but not support replication. 2) To determine the role of cellular transcription factors in ori-Lyt replication, we will use a variety of techniques to determine which proteins bind to the known essential sites of ori-Lyt in different cell types and will examine the effect of deleting specific transcription factor binding sites upon ori-Lyt replication versus transcription. 3) To examine the effect of p53 upon ori-Lyt replication, we will determine the location of p53 binding sites in ori-Lyt, examine the effect of mutating these sites in different cell types, and determine if mutant Z which no longer interacts with p53 is more (or less) efficient in supporting ori-Lyt replication. 4) We will construct mutations in the intact virus which eliminate the ability of Z to support ori-lyt replication (but still allow transactivation) and determine how these mutations affect the phenotype in vivo.