Reagents, Antibodies and Kits.

Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues.

EB病毒（Epstein-Barr virus，EBV）在全球范围内持续感染95%的成年人，且与多种人类淋巴瘤相关，这些淋巴瘤表达病毒用以在B细胞区室中定植的特征性EBV潜伏程序。初次感染时，由6种EB病毒核抗原（Epstein-Barr Nuclear Antigens，EBNA）与2种潜伏膜蛋白（Latent Membrane Protein，LMP）抗原组成的EBV Ⅲ型潜伏程序，可将受感染的B细胞诱导进入生发中心（germinal center，GC）。目前其具体机制尚未完全阐明，生发中心微环境信号可触发EB病毒基因组切换至Ⅱ型潜伏程序，该程序由EBNA1、LMP1与LMP2A组成，在生发中心来源的霍奇金淋巴瘤中可被观测到。
为阐明EB病毒感染的B细胞遭遇微环境信号时，调控EBV潜伏重编程的通路与表观遗传机制，本研究表征了生发中心细胞因子对EBV潜伏蛋白表达及EBV表观基因组的影响。我们验证并拓展了此前聚焦于支持Ⅱ型潜伏转化的生发中心细胞因子效应的相关研究。作为生发中心应答的主要调控因子，滤泡辅助性T细胞细胞因子白细胞介素21（interleukin 21，IL-21），以及作用较弱的IL-4与IL-10，可过度诱导LMP1的表达，同时抑制EBNA的表达。而包括IL-15与IL-27在内的滤泡树突状细胞细胞因子，仅下调EBNA的表达，对LMP1的表达无影响。CRISPR编辑实验表明，STAT3与STAT5是细胞因子介导的EBNA沉默所必需的，该过程通过作用于EBV基因组C启动子的表观遗传效应实现。与之相反，STAT3却是LMP1启动子表观遗传重塑所必需的，该重塑包括激活型组蛋白染色质修饰的富集与抑制型多梳抑制复合体沉默修饰的缺失。综上，EB病毒已进化出劫持STAT信号通路的机制，以响应生发中心细胞因子信号，反向调控关键病毒基因组启动子的表观遗传状态。