A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection

Latent Epstein-Barr virus (EBV) infection contributes to both B-cell and epithelial-cell malignancies. However, whether lytic EBV infection also contributes to tumors is unclear, although the association between malaria infection and Burkitt lymphomas (BLs) may involve excessive lytic EBV replication. A particular variant of the viral promoter (Zp) that controls lytic EBV reactivation is over-represented, relative to its frequency in non-malignant tissue, in EBV-positive nasopharyngeal carcinomas and AIDS-related lymphomas. To date, no functional differences between the prototype Zp (Zp-P) and the cancer-associated variant (Zp-V3) have been identified. Here we show that a single nucleotide difference between the Zp-V3 and Zp-P promoters creates a binding site for the cellular transcription factor, NFATc1, in the Zp-V3 (but not Zp-P) variant, and greatly enhances Zp activity and lytic viral reactivation in response to NFATc1-inducing stimuli such as B-cell receptor activation and ionomycin. Furthermore, we demonstrate that restoring this NFATc1-motif to the Zp-P variant in the context of the intact EBV B95.8 strain genome greatly enhances lytic viral reactivation in response to the NFATc1-activating agent, ionomycin, and this effect is blocked by the NFAT inhibitory agent, cyclosporine, as well as NFATc1 siRNA. We also show that the Zp-V3 variant is over-represented in EBV-positive BLs and gastric cancers, and in EBV-transformed B-cell lines derived from EBV-infected breast milk of Kenyan mothers that had malaria during pregnancy. These results demonstrate that the Zp-V3 enhances EBV lytic reactivation to physiologically-relevant stimuli, and suggest that increased lytic infection may contribute to the increased prevalence of this variant in EBV-associated malignancies.潜伏性EB病毒（Epstein-Barr virus, EBV）感染可诱发B细胞及上皮细胞恶性肿瘤。目前学界对于裂解性EB病毒感染是否同样参与肿瘤发生仍存争议，尽管已有研究提示疟疾感染与伯基特淋巴瘤（Burkitt lymphomas, BLs）的关联可能涉及过度的EBV裂解性复制。调控EBV裂解性激活的病毒启动子（Zp）的特定变异体，在EBV阳性鼻咽癌与艾滋病相关淋巴瘤中的出现频率显著高于其在非恶性组织中的占比。迄今为止，尚未有研究证实原型Zp（Zp-P）与癌症相关变异体（Zp-V3）之间存在功能差异。本研究发现，Zp-V3与Zp-P启动子仅存在单个核苷酸差异，该差异可在Zp-V3（而非Zp-P）变异体中创建细胞转录因子NFATc1的结合位点，并能显著增强Zp活性，以及针对B细胞受体激活、离子霉素等NFATc1诱导刺激的裂解病毒再激活能力。此外，本研究证实，在完整的EBV B95.8毒株基因组背景下，向Zp-P变异体补回该NFATc1结合基序，可显著提升其对NFATc1激活剂离子霉素的裂解病毒再激活响应，且该效应可被NFAT抑制剂环孢素以及NFATc1小干扰RNA（siRNA）阻断。我们还观测到，Zp-V3变异体在EBV阳性BLs与胃癌中，以及从孕期罹患疟疾的肯尼亚母亲的EBV感染母乳中分离得到的EBV转化B细胞系内均呈现过度富集现象。上述结果表明，Zp-V3可增强EBV对生理相关刺激的裂解激活能力，提示裂解感染水平的提升可能是该变异体在EBV相关恶性肿瘤中患病率升高的潜在原因。