Epstein—Barr virus (EBV) nuclear antigen (EBNA) 6 (also known as 3c) is a latent nuclear protein with an of about 160K which is invariably expressed in EBV-immortalized 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 blast-activation 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.


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