The long-term goal of this proposal is to understand how Epstein-Barr virus (EBV) immortalizes B-lymphocytes. This study proposes to investigate the regulation of a pair of viral gene products, the EBER RNAs, as well as, two other genes which have similar promoter structures- the cellular 7SL RNA and the viral latent membrane protein (LMP). Both EBER and LMP genes are expressed during immortalization and may contribute to that process. While EBV is the etiological agent of infectious mononucleosis, its ability to immortalize B cells undoubtedly contributes to its involvement in the development of B cll lymphomas in immunosuppressed AIDS and organ transplant patients. EBV is also a cofactor in Burkitt's lymphoma and nasopharyngeal carcinoma. EBV is an important human pathogen and its association with these various neoplasms makes it one of the few human cancer viruses. During immortalization only a small portion of the EBV genome is expressed. While it is known which viral genes are expressed during immortalization, such as, the EBER and LMP genes, the regulation of these genes is not well understood. An investigation of this regulation will contribute to an understanding of how EBV immortalizes B cells. The EBER gene transcription unit is unusual because it contains both class II and III promoter elements. These elements include the Sp1 and activating transcription factor (ATF) promoter elements along with a TATA-like box. A study of this unusual gene structure will not only illuminate how these genes are regulated, but it will provide a unique opportunity to study the interaction between class II and III promoter elements.
The specific aims of this proposal are (1) to further characterize the proteins binding to the EBER transcription unit; specifically to determine whether the proteins binding the upstream ATF and TATA-like promoter elements are unique DNA binding proteins. If the proteins are unique they will be isolated by DNA affinity chromatography and a partial amino acid sequence determined along with antibodies produce against them. Using the antibodies or oligonucleotides derived from the partial amino acid sequence, a human B cell cDNA library will be screened and the subsequently isolated cDNA will be sequenced.
Specific aim (2) will be to investigate the function of the EBER TATA-like promoter element. Several hypotheses will be tested by mutating the TATA-like promoter element or putting it upstream of other genes.
Specific aim (3) will be to study the similarities between the promoters of the EBER, 7SL and LMP genes. Their possible coregulation during EBV immortalization will be studied by infecting isolated human B cells with purified EBV virus and following expression of the EBERs and 7SL RNAs by northern blotting analysis and the expression of the LMP and other viral proteins by western immunoblotting analysis. If the EBERs, 7SL and LMP gene products are coregulated, their promoters will be studied to determine whether they have similar functions and bind the same proteins by using the purified proteins isolated in specific aim (1).
Specific aim (4) is to study differences found in EBER transcription efficiency between HeLa and B cells. Initially the differences will be characterized in vivo by transfecting into the two cell lines both class II and III genes along with the EBER and 7SL genes to determine specificity. If the transcription differences are specific for the EBER and 7SL genes then a study will be undertaken to determine whether or not the upstream promoter elements are responsible by determining the effects of mutations in the various upstream promoter elements on the differences in transcription efficiency.
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