Epstein-Barr Virus (EBV) efficiently immortalizes human B cells in vitro and this requires expression of the viral proteins EBNA2 and EBNA-LP. EBNA2 is a transactivator of viral and cellular gene expression. EBNA-LP is a gene-specific coactivator of EBNA2, which up-regulates expression of the major viral oncoprotein LMP1. The broad objective of our lab is to understand the role of EBNA2 and EBNA-LP in modulating cellular processes that promote B cell immortalization.
Aim 1. Investigate the role of EBNA2 amino acid residues 1-58 in transcription activation and B cell immortalization. EBNA2 amino acid residues 1-58 have a dominant negative effect on full length EBNA2. Specific mutations within EBNA2 conserved regions (CR) 1 and 2, corresponding to residues 1-58, result in defective EBNA2 homo-oligomerization. We have obtained functionally-deficient mutants which are also unable to oligomerize. We will test these mutants in genetic complementation assays for their ability to maintain B cell immortalization. Functional assays will be performed in EBV-positive B cells to determine the ability of these mutant EBNA2 to induce LMP1. qRT-PCR will be used to test whether CR1 and/or CR2 mediate global or gene-specific EBNA2 activity.
Aim 2. Determine the mechanism of EBNA-LP-mediated displacement of SplOO from PML NBs, and how this contributes to EBNA2 coactivation. SplOO amino acid residues 3-152 mediate dimerization, PML NB localization, and interactions with EBNA-LP. To determine how EBNA-LP re-localizes SplOO out of PML NBs, consecutive 15 amino acid deletions have been introduced into SplOO between residues 1- 150. The mutants will be used in Co-IP and IF assays to define critical SplOO residues that mediate self- association, EBNA-LP binding and PML NB localization. The association of EBNA-LP, SplOO, and EBNA2, as well as specific modifications (e.g. methylation) on the LMP1 promoter will be determined by ChIP assays. Relevance: Small molecule inhibitors of EBNA2 function that target oligomerization may be a fruitful therapeutic approach for EBV-related cancers. We will clarify the mechanistic contributions of EBNA-LP in B cell immortalization, as well as the normal role of SplOO, especially as related to autoimmune diseases.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31CA126523-02
Application #
7317804
Study Section
Special Emphasis Panel (ZRG1-EMNR-E (29))
Program Officer
Bini, Alessandra M
Project Start
2007-01-01
Project End
2008-07-27
Budget Start
2008-01-01
Budget End
2008-07-27
Support Year
2
Fiscal Year
2008
Total Cost
$26,271
Indirect Cost
Name
Baylor College of Medicine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
051113330
City
Houston
State
TX
Country
United States
Zip Code
77030
Echendu, Chisaroka W; Ling, Paul D (2008) Regulation of Sp100A subnuclear localization and transcriptional function by EBNA-LP and interferon. J Interferon Cytokine Res 28:667-78