The long-term objective of the proposed research is to delineate the mechanisms that differentially regulate Epstein-Barr virus (EBV) gene expression in latently infected B lymphocytes. The potential of EBV to cause or contribute to the majority of its associated disease states relies on the maintenance of a latent infection. Recent data indicate that there are two states of EBV latency, gpI and gpIII. Establishment of latency depends on the actions of one or more of the nine viral proteins expressed in the gpIII state of latency. Based on the more restricted EBV gene expression in Burkitt lymphoma (gpI latency), we postulate viral gene expression is subsequently, or intermittently, restricted to the nuclear protein EBNA-1, necessary for replication of the viral genome, and to possibly as yet unknown proteins. Restricted expression would enable the production of progeny latently-infected cells and evasion of host immune surveillance. We propose the following aims to reach our long-term objectives: 1) Elucidate the mechanisms regulating transcription of the EBNA genes during gpI and gpIII latency; 2) Characterize the regulation of selective 3' processing and alternative splice-site selection in the maturation of the EBNA mRNAs; 3) Identify and characterize novel EBV genes expressed in gpI Burkitt lymphoma cells. During gpIII latency the 6 EBNA proteins are encoded by mRNAs derived by differential pre-mRNA processing of a common primary transcript. The central mechanism permitting EBNA-1 expression in the absence of the other EBNAs in gpI latency is a switch to a EBNA-1-specific promoter. By using a short-term expression assay in which the different EBNA promoters are linked to reporter genes, we will identify (under Aim 1) the mode of regulation of differential transcription of the EBNA genes in gpI and gpIII latently infected B cells. Subsequent characterization of positive and negative regulatory elements within these promoters will permit us to delineate the actual mechanisms regulating the shifts between gpI and gpIII latency.
In Aim 2 we will employ in vivo and in vitro RNA processing assays to examine the regulatory roles of alternative splicing and 3' processing in the differential expression of the EBNA mRNAs in gpIII latency, and exon skipping in the generation of the gpI EBNA-1 mRNA. Finally, through a process of characterization of mRNAs/cDNAs and identification of the proteins they encode, we will determine whether there are additional EBV genes expressed in gpI Burkitt cells that may be critical to this disease state (Aim 3).
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