Data_Sheet_1_EGFR Activation Impairs Antiviral Activity of Interferon Signaling in Brain Microvascular Endothelial Cells During Japanese Encephalitis Virus Infection.PDF

The establishment of Japanese encephalitis virus (JEV) infection in brain microvascular endothelial cells (BMECs) is thought to be a critical step to induce viral encephalitis with compromised blood–brain barrier (BBB), and the mechanisms involved in this process are not completely understood. In this study, we found that epidermal growth factor receptor (EGFR) is related to JEV escape from interferon-related host innate immunity based on a STRING analysis of JEV-infected primary human brain microvascular endothelial cells (hBMECs) and mouse brain. At the early phase of the infection processes, JEV induced the phosphorylation of EGFR. In JEV-infected hBMECs, a rapid internalization of EGFR that co-localizes with the endosomal marker EEA1 occurred. Using specific inhibitors to block EGFR, reduced production of viral particles was observed. Similar results were also found in an EGFR-KO hBMEC cell line. Even though the process of viral infection in attachment and entry was not noticeably influenced, the induction of IFNs in EGFR-KO hBMECs was significantly increased, which may account for the decreased viral production. Further investigation demonstrated that EGFR downstream cascade ERK, but not STAT3, was involved in the antiviral effect of IFNs, and a lowered viral yield was observed by utilizing the specific inhibitor of ERK. Taken together, the results revealed that JEV induces EGFR activation, leading to a suppression of interferon signaling and promotion of viral replication, which could provide a potential target for future therapies for the JEV infection.

日本脑炎病毒（Japanese encephalitis virus, JEV）在脑微血管内皮细胞（brain microvascular endothelial cells, BMECs）中的感染定植被认为是诱发血脑屏障（blood–brain barrier, BBB）受损并引发病毒性脑炎的关键步骤，而该过程的具体分子机制尚未完全阐明。本研究通过对JEV感染的原代人脑微血管内皮细胞（primary human brain microvascular endothelial cells, hBMECs）及小鼠脑组织开展STRING分析，发现表皮生长因子受体（epidermal growth factor receptor, EGFR）与JEV逃避干扰素相关宿主先天免疫密切相关。在感染早期阶段，JEV可诱导EGFR发生磷酸化。在JEV感染的hBMECs中，EGFR快速内吞并与内体标志物EEA1发生共定位。使用特异性抑制剂阻断EGFR后，病毒粒子的产生量显著降低；在EGFR敲除（EGFR-KO）的hBMEC细胞系中也得到了一致的实验结果。尽管病毒的吸附与侵入过程未受明显影响，但EGFR-KO hBMECs中干扰素的诱导表达水平显著上调，这或许是病毒载量降低的核心原因。进一步研究表明，EGFR下游的ERK信号通路而非STAT3参与了干扰素的抗病毒效应，使用ERK特异性抑制剂同样可降低病毒滴度。综上，本研究结果揭示JEV可诱导EGFR激活，进而抑制干扰素信号通路并促进病毒复制，该发现可为未来JEV感染的防治提供潜在治疗靶点。