Induction of p16INK4a Is the Major Barrier to Proliferation when Epstein-Barr Virus (EBV) Transforms Primary B Cells into Lymphoblastoid Cell Lines

To explore the role of p16ink4a as an intrinsic barrier to b cell transformation by ebv,we transformed primary b cells from an individual homozygous for a deletion in the cdkn2a locus encoding p16ink4a and p14arf. using recombinant ebv-bac viruses expressing conditional ebna3c (3cht),we developed a system that allows inactivation of ebna3c in lymphoblastoid cell lines (lcls) lacking active p16ink4a protein but expressing a functional 14arf-fusion protein (p14/p16). the ink4a locus is epigenetically repressed by ebna3c - in cooperation with ebna3a - despite the absence of functional p16ink4a. although inactivation of ebna3c in lcls from normal b cells leads to an increase in p16ink4a and growth arrest,ebna3c inactivation in the p16ink4a-null lcls has no impact on the rate of proliferation,establishing that the repression of ink4a is a major function of ebna3c in ebv-driven lcl proliferation. this conditional lcl system allowed us to use microarray analysis to identify and confirm genes regulated specifically by ebna3c,independently of proliferation changes modulated by the p16ink4a-rb-e2f axis. infections of normal primary b cells with recombinant ebv-bac virus from which ebna3c is deleted or with 3cht ebv in the absence of activating ligand 4-hydroxytamoxifen,revealed that ebna3c is necessary to overcome an ebv-driven increase in p16ink4a expression and concomitant block to proliferation 2-4 weeks post-infection. if cells are p16ink4a-null,functional ebna3c is dispensable for the outgrowth of lcls. © 2013 skalska et al.