The Essential Role of Clathrin-mediated Endocytosis in theInfectious Entry of Human Enterovirus 71
The Essential Role of Clathrin-mediated Endocytosis in theInfectious Entry of Human Enterovirus 71
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DOI：
10.1074/jbc.m110.168468
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发表时间：
2011-01-07
影响因子：
4.8
通讯作者：
Chu, Justin Jang Hann
中科院分区：
文献类型：
--
作者：
Hussain, Khairunnisa' Mohamed;Leong, Kim Lian Janet;Chu, Justin Jang Hann
目前对人肠道病毒71型（HEV71）感染进入宿主细胞的过程知之甚少，这可能是潜在的抗病毒靶向位点。在这项研究中，建立并验证了靶向小干扰RNA（siRNA）筛选平台试验，以鉴定和分析参与HEV71感染所必需的内吞作用、细胞骨架动力学和内体运输过程的关键细胞基因。通过充分表征的显性阴性突变体的表达、生物成像研究（双标记免疫荧光测定、透射电子显微镜分析）、基于次级siRNA的剂量依赖性研究和药物抑制测定进行筛选评价。HEV71感染性进入横纹肌肉瘤细胞被靶向网格蛋白介导的内吞作用（CME）相关基因的siRNA显著抑制，所述基因包括AP2A1、ARRB1、CLTC、CLTCL1、SYNJ1、ARPC 5、PAK 1、ROCK 1和WASF1。通过观察到HEV71颗粒和网格蛋白之间的强共定位以及在siRNA敲低CME相关基因后对HEV71感染的剂量依赖性抑制来验证CME的功能作用。通过显性阴性EPS15突变体的抑制和CME药物抑制剂的处理进一步证实了CME对HEV71的进入，在20 μ M氯丙嗪下观察到超过80%的抑制。此外，HEV71感染被证明是敏感的人基因在调节早期到晚期的内体运输以及内体酸性pH值的破坏。网格蛋白介导的内吞作用作为进入途径的敏感宿主细胞的HEV71感染有助于更好地了解HEV71的发病机制，并使未来的发展抗病毒策略对HEV71感染。
Little is currently known about the infectious entry process of human enterovirus 71 (HEV71) into host cells, which may represent potential anti-viral targeting sites. In this study a targeted small-interfering RNA (siRNA) screening platform assay was established and validated to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics, and endosomal trafficking essential for HEV71 infection. Screen evaluation was conducted via the expression of well characterized dominant-negative mutants, bioimaging studies (double-labeled immunofluorescence assays, transmission electron microscopy analysis), secondary siRNA-based dosage dependence studies, and drug inhibition assays. The infectious entry of HEV71 into rhabdomyosarcoma cells was shown to be significantly inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis (CME) that include AP2A1, ARRB1, CLTC, CLTCL1, SYNJ1, ARPC5, PAK1, ROCK1, and WASF1. The functional role of CME was verified by the observation of strong co-localization between HEV71 particles and clathrin as well as dose-dependent inhibition of HEV71 infection upon siRNA knockdown of CME-associated genes. HEV71 entry by CME was further confirmed via inhibition by dominant-negative EPS15 mutants and treatment of CME drug inhibitors, with more than 80% inhibition observed at 20 mu M chlorpromazine. Furthermore, HEV71 infection was shown to be sensitive to the disruption of human genes in regulating early to late endosomal trafficking as well as endosomal acidic pH. The identification of clathrin-mediated endocytosis as the entry pathway for HEV71 infection of susceptible host cells contributes to a better understanding of HEV71 pathogenesis and enables future development of anti-viral strategies against HEV71 infection.