Entry of Challenge Virus Standard (CVS)-11 into N2a cells via a clathrin-mediated, cholesterol-, dynamin-, pH-dependent endocytic pathway
Entry of Challenge Virus Standard (CVS)-11 into N2a cells via a clathrin-mediated, cholesterol-, dynamin-, pH-dependent endocytic pathway
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攻击病毒标准品 (CVS)-11 通过网格蛋白介导、胆固醇、动力、pH 依赖性内吞途径进入 N2a 细胞
DOI：
10.1186/s12985-019-1186-9
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发表时间：
2019-06-13
期刊：
影响因子：
4.8
通讯作者：
Zhang, Maolin
中科院分区：
文献类型：
--
作者：
Gao, Jie;Wang, Xinyu;Zhang, Maolin
兔病毒(Rabies Virus，RABV)是横纹病毒科溶血病毒属成员，是一种负链RNA病毒。由RABV引起的人畜共患病导致动物和人类的高死亡率。随着研究的深入，RABV进入细胞的机制尚不清楚。方法采用化学抑制剂和RNA干扰(RNAi)技术分析RABV的内化途径。用定量聚合酶链式反应和免疫印迹法检测病毒N蛋白的表达水平，用荧光显微镜观察病毒在细胞内的感染情况。氯丙嗪处理和敲除笼状蛋白重链(CHC)显著减少了病毒进入，这证明了笼状蛋白是必需的。同时，制霉菌素和在N2a细胞中击倒小凹蛋白-1(Cav1)对CVS-11的感染均无影响，提示小窝对CVS-11的内化是独立的。当用MCD提取细胞膜胆固醇时，对病毒感染有很强的影响。此外，通过使用特定的抑制剂DATORE和氯化铵，我们证实了动力蛋白和低pH环境是RABV感染的关键，这一点得到了共聚焦显微镜的证实。结论我们的结果表明CVS-11是通过笼蛋白介导的、胆固醇、pH、动力蛋白必需的和小凹非依赖性的内吞途径进入N2a细胞的。
BackgroundRabies virus (RABV), a member of Lyssavirus of Rhabdoviridae family, is a kind of negative-strand RNA virus. The zoonosis caused by RABV leads to high mortality in animals and humans. Though with the extensive investigation, the mechanisms of RABV entry into cells have not been well characterized.MethodsChemical inhibitors and RNA interference (RNAi) were used to analysis RABV internalization pathway. The expression level of viral N protein was examined by quantitative PCR and western blot, and the virus infection in the cells was visualized by fluorescence microscopy.ResultsWe firstly examined the endocytosis pathway of the challenge virus standard (CVS) -11 strain in N2a cells. Chlorpromazine treatment and knockdown of clathrin heavy chain (CHC) significantly reduced viral entry, which proved clathrin was required. Meanwhile neither nystatin nor knocking down caveolin-1 (Cav1) in N2a cells had an effect on CVS-11 infection, suggesting that caveolae was independent for CVS-11 internalization. And when cholesterol of cell membrane was extracted by MCD, viral infection was strongly impacted. Additionally by using the specific inhibitor dynasore and ammonium chloride, we verified that dynamin and a low-pH environment were crucial for RABV infection, which was confirmed by confocal microscopy.ConclusionsOur results demonstrated that CVS-11 entered N2a cells through a clathrin-mediated, cholesterol-, pH-, dynamin-required, and caveolae-independent endocytic pathway.