ISG15-Mediated Suppression of Type I Interferon Signaling Leads to Susceptibility to Epstein-Barr Virus Infection (Early_Infection_day7_BulkRNAseq)

Epstein-Barr Virus (EBV), the first identified human oncogenic virus, is linked to numerous malignancies, including gastric cancer, nasopharyngeal cancer and multiple B-cell Lymphomas. However, the host-virus interactions that regulate EBV-driven cell transformation and pathogenesis remain incompletely understood. Here, employing a Cas9–ribonucleoprotein (RNP)-based screen in primary B cells, we identified 11 host factors that promote and 35 that restrict EBV-driven outgrowth. Notably, ISG15, a ubiquitin-like, type I interferon-induced antiviral protein, exhibited a dependency role for growth in early stages of infection but not in LCLs. Mechanistically, we found that the regulation of free ISG15 levels greatly impacts the proliferation capacity of EBV-infected B-cells, by modulating the activation of type I IFN signaling. Finally, we identified rare germ-line variants in UBA7, the ISG15 E1 enzyme, in patients with EBV severe disorders, that impair the ISG15 conjugation process. This highlights a critical role of ISG15 in the control of EBV infections. Overall design: Bulk RNAseq of CD46- sorted from CD46-only knockout, and CD46 with ISG15 or STAT1 knockout EBV-infected cells, 7 days post infection.

EB病毒（Epstein-Barr Virus, EBV）是首个被鉴定的人类致癌病毒，与胃癌、鼻咽癌及多种B细胞淋巴瘤等恶性肿瘤密切相关。然而，调控EB病毒驱动的细胞转化与致病过程的宿主-病毒相互作用机制仍未完全阐明。本研究在原代B细胞中开展基于Cas9-核糖核蛋白（ribonucleoprotein, RNP）的筛选实验，鉴定出11个可促进EB病毒驱动的细胞增殖的宿主因子，以及35个可抑制该过程的宿主因子。值得注意的是，类泛素化I型干扰素诱导的抗病毒蛋白ISG15，在感染早期对细胞生长具有依赖作用，但在淋巴母细胞样细胞系（LCLs）中则无此效应。机制研究显示，游离ISG15的水平可通过调节I型干扰素信号通路的激活，显著影响EB病毒感染B细胞的增殖能力。最后，我们在罹患重症EB病毒感染相关疾病的患者中，发现了ISG15的E1连接酶UBA7的罕见生殖系变异，该变异会损害ISG15的缀合过程。这一发现凸显了ISG15在调控EB病毒感染中的关键作用。整体实验设计：于感染后7天，对经CD46分选的EB病毒感染细胞进行批量RNA测序，样本分为仅CD46基因敲除组，以及同时敲除CD46与ISG15或STAT1的实验组。