Functional aspects on the Epstein-Barr virus nuclear antigen 5

Abstract

The Epstein-Barr virus (EBV) infects resting B-cells. This leads to an EBV driven transformation of the resting B-cell into a cell with an activated B-cell phenotype that proliferates indefinitely in vitro. Epstein-Barr Nuclear antigen 5 (EBNA5) is one out of six EBV encoded protein involved in transformation of resting B-cell. In order to further characterise the role of EBNA5 in the transformation process, the effects of EBNA5 on the regulation of gene expression was studied in EBV negative and positive B-cell lines. Furthermore, novel cellular EBNA5 binding proteins were isolated by screening a two-hybrid B-cell library in yeast.The effects of EBNA5 on reporter gene activity in transient transfections were analysed and the results indicate two novel functions of EBNA5: (i) activator of transcription of the EBV C and W promoters, and (ii) repressor of gene expression by inhibiting pre-mRNA processing. EBNA5 was found to inhibit 3' end cleavage and polyadenylation of the pre-mRNA species transcribed from all of the promoters tested in the reporter plasmids. Dissection of the EBNA5 molecule suggests that the repetitive region of EBNA5 encoded by W is required and sufficient for suppression of polyadenylation and transcriptional activation of the C promoter.The effect of inhibitory levels of EBNA5 on the expression of cellular genes in a chromosomal context was analyzed in transient transfection experiments with an EBNA5 expression vector and using the DNA microarray technology. Under these circumstances, only 2 out of the 145 expressed genes in a panel of 588 genes were repressed. This shows that (i) EBNA5 can inhibit expression of genes also in a chromosomal environment, and (ii) a regulatory mechanism must exist in the cell that confers specificity to the selection of target genes for EBNA5. Interestingly, the pro-apoptotic protein BNIP3 was down regulated by EBNA5 suggesting that EBNA5 may inhibit apoptosis. Other experiments with cDNA arrays showed that EBNA5 can increase the expression of mRNA of the G2 to M phase cell cycle regulator CDC25C. Furthermore, the expression of CDC25C was shown to be strongly up regulated during infection of resting B-cells by EBV. This suggests that EBNA5 may facilitate G2 to M phase transition. HAX-1 was found to interact with EBNA5 in a yeast two-hybrid system screen of a B-cell cDNA library with EBNA5 as bait. HAX-1 has previously been shown to associate with HS1, a protein specifically expressed in cells of the hematopoietic lineage and thought to be involved in signal transduction in B-cells. Immunofluorescence experiments showed that the smallest EBNA5 species, composed of the unique Y domain and only one copy of the W repeat domain, like HAX-1 co-localises with the mitochondrial hsp60 protein in the B-cell cytoplasm.