Contribution of RBP-Jk and BS69 to gene regulation by EBNA2 and EBNA3

Abstract

Epstein-Barr virus (EBV) is a large, ubiquitous human γ-herpesvirus. Infection of primary resting B lymphocytes with EBV forms proliferating lymphoblastoid cell lines (LCL). EBV nuclear antigens (EBNA) EBNA2, EBNA3A and 3C are required for efficient transformation of B lymphocytes. EBNA2 acts as a transcriptional activator of viral and cellular genes that cause cell proliferation by acting as an adapter molecule that binds to DNA-binding proteins. The best known of these cell proteins is RBP-Jκ, although other binding sites have also been proved to mediate EBNA2 functions. The superior B cell transformation ability of Type 1 (T1) EBV, in comparison to Type 2 (T2) EBV, is due to greater or more rapid induction of a small group of cell and viral genes. It appears that EBNA2 binds to sites near these genes by a different mechanism from the usual process, which involves PU.1 and IRF transcription factors and is not dependent on the usual mechanism involving cell protein RBP-Jκ. BS69 cell protein is a transcriptional repressor. It interacts with adenovirus E1A and EBNA2 through its MYND domain which recognizes a PXLXP peptide motif present in EBNA2. EBNA3A and EBNA3C in Type 1 EBV also have the same motif. The aims of this study were to create a system to identify the non RBPJ EBNA2 binding sites in cell genes using a lymphoma cell line lacking RBPJ gene (SM224.9 cells) and to test binding of Type 1 EBNA3A and EBNA3C fragments to BS69 by in vitro translation and pull-down assay to determine if there are any differences in BS69 binding to each. Overall, DG75 and SM224.9 cells EBNA2 expression was successful, although it was not possible to determine which cell genes EBNA2 can activate without using RBP-Jκ. The results are consistent through all the pull-down assays repeated, with the T1 EBNA3A and 3C having a distinct binding to BS69. It is noticeable there is a stronger binding of T1 EBNA3C to BS69. T1 EBNA3A is able to bind better to BS69 when comparing to the T2 EBNA3A and EBNA3C. In all the assays there is some unspecific binding to Rab11b, used as a negative control. The pull-down assays were repeated various times in order to achieve better conditions, adjusting the incubation times with the IVT product and changing the buffer washes, to try to reduce the non-specific binding (both to GST-Rab11b and the beads). Changing the conditions did not significantly reduce the unspecific binding, either to Rab11b or the beads.

Date of Award	27 Apr 2017
Original language	English
Awarding Institution	Universidade Católica Portuguesa
Supervisor	Paul Farrell (Supervisor)