Mature human B cells infected by Epstein-Barr virus (EBV) become activated, grow, and proliferate. If the cells are infected ex vivo, they are transformed into continuously proliferating lymphoblastoid cell lines (LCLs) that carry EBV DNA as extra-chromosomal episomes, express 9 latency-associated EBV proteins, and phenotypically resemble antigen-activated B-blasts. In vivo similar B-blasts can differentiate to become memory B cells (MBC), in which EBV persistence is established. Three related latency-associated viral proteins EBNA3A, EBNA3B, and EBNA3C are transcription factors that regulate a multitude of cellular genes. EBNA3B is not necessary to establish LCLs, but EBNA3A and EBNA3C are required to sustain proliferation, in part, by repressing the expression of tumour suppressor genes. Here we show, using EBV-recombinants in which both EBNA3A and EBNA3C can be conditionally inactivated or using virus completely lacking the EBNA3 gene locus, that—after a phase of rapid proliferation—infected primary B cells express elevated levels of factors associated with plasma cell (PC) differentiation. These include the cyclin-dependent kinase inhibitor (CDKI) p18^INK4c, the master transcriptional regulator of PC differentiation B lymphocyte-induced maturation protein-1 (BLIMP-1), and the cell surface antigens CD38 and CD138/Syndecan-1. Chromatin immunoprecipitation sequencing (ChIP-seq) and chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) indicate that in LCLs inhibition of CDKN2C (p18^INK4c) and PRDM1 (BLIMP-1) transcription results from direct binding of EBNA3A and EBNA3C to regulatory elements at these loci, producing stable reprogramming. Consistent with the binding of EBNA3A and/or EBNA3C leading to irreversible epigenetic changes, cells become committed to a B-blast fate <12 days post-infection and are unable to de-repress p18^INK4c or BLIMP-1—in either newly infected cells or conditional LCLs—by inactivating EBNA3A and EBNA3C. In vitro, about 20 days after infection with EBV lacking functional EBNA3A and EBNA3C, cells develop a PC-like phenotype. Together, these data suggest that EBNA3A and EBNA3C have evolved to prevent differentiation to PCs after infection by EBV, thus favouring long-term latency in MBC and asymptomatic persistence.

被爱泼斯坦-巴尔病毒 (EBV) 感染的成熟人类 B 细胞被激活、生长和增殖。如果细胞在体外被感染，它们会转化为持续增殖的类淋巴母细胞系 (LCL)，这些细胞系携带 EBV DNA 作为染色体外附加体，表达 9 种潜伏相关 EBV 蛋白，并且表型类似于抗原激活的 B 母细胞。体内类似的 B 母细胞可以分化成为记忆 B 细胞 (MBC)，其中建立了 EBV 持久性。三种相关的潜伏相关病毒蛋白 EBNA3A、EBNA3B 和 EBNA3C 是调节大量细胞基因的转录因子。EBNA3B 不是建立 LCL 所必需的，但 EBNA3A 和 EBNA3C 是维持增殖所必需的，部分原因是通过抑制肿瘤抑制基因的表达。在这里我们展示，使用 EBNA3A 和 EBNA3C 都可以有条件地灭活的 EBV 重组体，或使用完全缺乏 EBNA3 基因位点的病毒，在快速增殖阶段之后，感染的原代 B 细胞表达与浆细胞 (PC) 分化相关的高水平因子. 这些包括细胞周期蛋白依赖性激酶抑制剂 (CDKI) p18^INK4c是 PC 分化 B 淋巴细胞诱导成熟蛋白-1 (BLIMP-1) 和细胞表面抗原 CD38 和 CD138/Syndecan-1 的主要转录调节因子。染色质免疫沉淀测序 (ChIP-seq) 和染色质免疫沉淀定量 PCR (ChIP-qPCR) 表明，在 LCL 中，CDKN2C (p18 ^INK4c ) 和PRDM1 (BLIMP-1) 转录的抑制是 EBNA3A 和 EBNA3C 与这些区域的调控元件直接结合的结果位点，产生稳定的重编程。与导致不可逆的表观遗传变化的 EBNA3A 和/或 EBNA3C 的结合一致，细胞在感染后 <12 天开始进入 B 母细胞命运，并且无法去抑制 p18 ^INK4c或 BLIMP-1——在新感染的细胞或条件性 LCL 中——通过使 EBNA3A 和 EBNA3C 失活。在体外，在感染缺乏功能性 EBNA3A 和 EBNA3C 的 EBV 后约 20 天，细胞会出现 PC 样表型。总之，这些数据表明 EBNA3A 和 EBNA3C 已经进化到可以防止在感染 EBV 后分化为 PC，从而有利于 MBC 的长期潜伏期和无症状持续存在。