Zhu Yong-Zhe, Wu Da-Ge, Ren Hao, Xu Qing-Qiang, Zheng Kui-Cheng, Chen Wei, Chen Sheng-Lin, Qian Xi-Jing, Tao Qing-Yuan, Wang Yan, Zhao Ping, Qi Zhong-Tian
Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, Shanghai, China.
Cell Physiol Biochem. 2015;35(4):1347-59. doi: 10.1159/000373956. Epub 2015 Feb 12.
BACKGROUND/AIMS: Although it has been widely accepted that Enterovirus 71 (EV71) enters permissive cells via receptor-mediated endocytosis, the details of entry mechanism for EV71 still need more exploration. This study aimed to investigate the role of lipid rafts in the early stage of EV71 Infection.
The effect of cholesterol depletion or addition of exogenous cholesterol was detected by immunofluorescence assays and quantitative real-time PCR. Effects of cholesterol depletion on the association of EV71 with lipid rafts were determined by flow cytometry and co-immunoprecipitation assays. Localization and internalization of EV71 and its receptor were assayed by confocal microscpoy and sucrose gradient analysis. The impact of cholesterol on the activation of phosphoinositide 3'-kinase/Akt signaling pathway during initial virus infection was analyzed by Western-blotting.
Disruption of membrane cholesterol by a pharmacological agent resulted in a significant reduction in the infectivity of EV71. The inhibitory effect could be reversed by the addition of exogenous cholesterol. Cholesterol depletion post-infection did not affect EV71 infection. While virus bound equally to cholesterol-depleted cells, EV71 particles failed to be internalized by cholesterol-depleted cells. EV71 capsid protein co-localized with cholera toxin B, a lipid-raft-dependent internalization marker.
Lipid rafts play a critical role in virus endocytosis and in the activation of PI3K/Akt signaling pathway in the early stage of EV71 infection.
背景/目的：尽管肠道病毒71型（EV71）通过受体介导的内吞作用进入易感细胞已被广泛接受，但EV71进入机制的细节仍需更多探索。本研究旨在探讨脂筏在EV71感染早期的作用。
通过免疫荧光测定和定量实时PCR检测胆固醇耗竭或添加外源性胆固醇的效果。通过流式细胞术和共免疫沉淀测定确定胆固醇耗竭对EV71与脂筏结合的影响。通过共聚焦显微镜和蔗糖梯度分析检测EV71及其受体的定位和内化。通过蛋白质免疫印迹分析胆固醇对初始病毒感染期间磷酸肌醇3'-激酶/蛋白激酶B（PI3K/Akt）信号通路激活的影响。
用药物破坏膜胆固醇导致EV71的感染性显著降低。添加外源性胆固醇可逆转这种抑制作用。感染后胆固醇耗竭不影响EV71感染。虽然病毒与胆固醇耗竭的细胞结合相同，但胆固醇耗竭的细胞无法内化EV71颗粒。EV71衣壳蛋白与霍乱毒素B共定位，霍乱毒素B是一种依赖脂筏的内化标记物。
脂筏在EV71感染早期的病毒内吞作用和PI3K/Akt信号通路激活中起关键作用。
