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.为探究p16INK4a作为爱泼斯坦-巴尔病毒（Epstein-Barr virus, EBV）介导B细胞转化的内在屏障的作用，我们对一名携带CDKN2A基因座（编码p16INK4a与p14ARF）纯合缺失个体的原代B细胞进行转化。我们采用表达条件性EBNA3C（3CHT）的重组EBV-BAC病毒，构建了一套可在缺乏功能性p16INK4a蛋白但表达功能性p14ARF融合蛋白（p14/p16）的淋巴母细胞样细胞系（Lymphoblastoid cell lines, LCLs）中灭活EBNA3C的实验体系。尽管细胞本身缺乏功能性p16INK4a，INK4a基因座仍可通过EBNA3C与EBNA3A的协同作用发生表观遗传抑制。尽管在正常B细胞来源的LCLs中灭活EBNA3C会导致p16INK4a表达上调并引发生长停滞，但在p16INK4a缺失的LCLs中灭活EBNA3C并不会影响增殖速率，这证实INK4a基因座的抑制是EBNA3C在EBV驱动的LCL增殖过程中的核心功能。这套条件性LCL实验体系使我们能够通过微阵列分析，独立于p16INK4a-Rb-E2F通路介导的增殖变化，特异性地鉴定并验证受EBNA3C调控的基因。使用缺失EBNA3C的重组EBV-BAC病毒，或在缺乏激活配体4-羟基他莫昔芬的条件下以3CHT EBV感染正常原代B细胞，结果显示EBNA3C对于克服EBV感染后2~4周内p16INK4a表达上调及伴随的增殖阻滞是必需的。若细胞为p16INK4a缺失型，则功能性EBNA3C对于LCLs的增殖传代并非必需。