Epstein--Barr virus (EBV) nuclear antigen 6 induces expression of the EBV latent membrane protein and an activated phenotype in Raji cells

Abstract

Epstein-Barr virus (EBV) nuclear antigen (EBNA) 6 (also known as 3c) is a latent nuclear protein with an Mr of about 160K which is invariably expressed in EBVimmortalized B cells. It includes a putative basic leucine zipper domain; as such it is a good candidate for a regulator of viral gene expression. More than 75 % of the EBNA 6 coding sequence is deleted from viral genomes carried in the Burkitt's lymphoma (BL) tumour-derived cell line, Raji. Thus although Raji cells express normal levels of the remaining five EBNAs and low levels of latent membrane protein (LMP), EBNA 6 protein is completely absent. In this study we have established Raji clones stably expressing EBNA 6 after cotransfection of an EBNA 6 gene under the control of the simian virus 40 early promoter with a selectable marker. Analysis of these clones has revealed that EBNA 6 induces a significant increase in the expression of LMP. In addition the cells have undergone a number of morphological and phenotypic changes consistent with blastactivation of normal B lymphocytes. The Raji cells expressing EBNA 6 show ruffling of the cell membrane and the development of a polarity defined by multiple villous (' spiky') projections at one end of the cell. This morphological change is associated with a dramatic increase in the expression of the cytoskeletal protein, vimentin. The EBV-associated B cell activation marker CD23 (blast 2) is induced to high levels although other activation markers such as CD30 and CD39 are unaffected. All these changes appear to be independent of the precise levels of EBNA 6 protein expressed. EBNA 2 has been shown previously to trans-activate the LMP gene and in the control Raji cells, EBNA 6-positive Raji cells and in B lymphoblastoid cells similar levels of EBNA 2 are expressed. Our findings are therefore most consistent with a model in which EBNA 6 either augments or complements the action of EBNA 2 in the induction of LMP and the cascade of gene expression which leads to B cell activation and immortalization by EBV.

Epstein–Barr virus encoded nuclear protein EBNA-3 binds XAP-2, a protein associated with Hepatitis B virus X antigen

“…This interaction mimics constitutive Notch mediated signalling (Strobl et al, 1997). The high molecular weight EBNAs (3, 4, 6 or as alternatively called 3a, 3b and 3c) are also able to bind RBP-Jk (Robertson et al, 1996) and depending on the promoter context, modify the initiation of transcription (Allday et al, 1993;Johannsen et al, 1996;Waltzer et al, 1996;Radkov et al, 1997;Krauer et al, 1998). EBNA-6 moreover has papilloma virus E7 like functions.…”

Epstein–Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines

“…Surprisingly, EBNA3A and EBNA3C are each uniquely important for initial and continued LCL growth, whereas EBNA3B is not required (11)(12)(13)(14)(15)(16), although EBNA3B also alters cell gene transcription (17,18). EBNA3C uniquely up-regulates LMP1 (19)(20)(21)(22)(23)(24), CD21(CR2), TCL1A, and ITGA4 expression and represses JAG1, NCALD, p16, BIM, and FLNA expression (14-16, 18, 19, 25, 26). Furthermore, EBNA3C amino acids 365-545 or 724-826 repress or activate transcription when tethered to DNA by the Gal4 DNA binding domain (27,28).…”

A Cyclin-Binding Motif within the Amino-Terminal Homology Domain of EBNA3C Binds Cyclin A and Modulates Cyclin A-Dependent Kinase Activity in Epstein-Barr Virus-Infected Cells

“…EBNA3C has been shown to play a complex regulatory role in the transcription of viral and cellular genes. EBNA3C antagonizes EBNA2-mediated transactivation by competing with EBNA2 for RBP-J binding (10,19,24), and, conversely, cooperates with EBNA2 in the upregulation of some promoters, like the EBV LMP1 promoter (2,3,33). Additionally, EBNA3C recruits both histone acetylase and deacetylase activities (12,20,29), in part through its association with the small acidic nuclear protein prothymosin-alpha (5).…”