Porcine Hemagglutinating Encephalomyelitis Virus Enters Neuro-2a Cells via Clathrin-Mediated Endocytosis in a Rab5-, Cholesterol-, and pH-Dependent Manner.
Porcine Hemagglutinating Encephalomyelitis Virus Enters Neuro-2a Cells via Clathrin-Mediated Endocytosis in a Rab5-, Cholesterol-, and pH-Dependent Manner.
复制标题
猪血凝性脑脊髓炎病毒通过网格蛋白介导的内吞作用以 Rab5、胆固醇和 pH 依赖性方式进入 Neuro-2a 细胞
DOI：
10.1128/jvi.01083-17
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发表时间：
2017-12-01
影响因子：
5.4
通讯作者：
He W
中科院分区：
文献类型：
--
作者：
Li Z;Zhao K;Lan Y;Lv X;Hu S;Guan J;Lu H;Zhang J;Shi J;Yang Y;Song D;Gao F;He W
摘要猪血凝性脑脊髓炎病毒(PHEV)是一种侵袭仔猪中枢神经系统(CNS)的高神经毒力冠状病毒。虽然在理解PHEV的生物学方面已经取得了重要进展，但其生命周期的许多方面仍然不清楚。在这里，我们剖析了PHEV在小鼠神经母细胞瘤(Neuro-2a)细胞中进入和细胞内转运的分子机制。我们首先进行了一种薄片透射电子显微镜分析来表征PHEV的动力学，我们发现病毒的进入和转移是通过膜涂层介导的内吞和胞吞作用发生的。为了验证不同的内吞途径的作用，使用了系统的方法，包括药物抑制、RNA干扰、共聚焦显微镜分析、使用荧光标记的病毒颗粒和过表达显性负性(DN)突变体。对感染细胞的定量检测表明，PHEV通过网状蛋白介导的内吞作用(CME)进入细胞，低pH值、动力蛋白、胆固醇和Eps15参与了这一过程。有趣的是，PHEV的入侵导致了肌动蛋白的快速重排，这表明肌动蛋白细胞骨架的完整性和动力学与病毒的内吞作用呈正相关。我们接下来研究了内化的PHEV的运输，发现依赖Rab5和Rab7的途径是启动生产性感染所必需的。此外，GTPase激活分析表明，内源性Rab5是由PHEV激活的，对病毒的进展至关重要。我们的发现表明，PHEV劫持宿主的CME和内体系统进入神经细胞并在神经细胞内运输，为PHEV的发病机制提供了新的见解，并为抗病毒药物的设计提供了指导。猪血凝性脑脊髓炎病毒(PHEV)是一种非节段性、阳性、单链RNA冠状病毒，可侵袭中枢神经系统(CNS)并导致神经功能障碍。神经细胞是病毒发展的目标。然而，PHEV进入和贩运的详细机制仍不清楚。猪瘟病毒是猪血凝性脑脊髓炎的病原，该病是一种急性、高传染性疾病，可导致大量哺乳仔猪死亡，给中国造成巨大的经济损失。了解病毒进入途径不仅将促进我们对PHEV感染和发病机制的了解，而且还将为开发新的治疗策略开辟新的途径。因此，我们采用系统的方法剖析了PHEV在Neuro-2a细胞中的内化和细胞内转运机制。这是第一个描述PHEV通过笼蛋白介导的内吞进入神经细胞的过程的报告，这种内吞作用依赖于动力蛋白、胆固醇和pH，这需要Rab5和Rab7。
ABSTRACT Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurovirulent coronavirus that invades the central nervous system (CNS) in piglets. Although important progress has been made toward understanding the biology of PHEV, many aspects of its life cycle remain obscure. Here we dissected the molecular mechanism underlying cellular entry and intracellular trafficking of PHEV in mouse neuroblastoma (Neuro-2a) cells. We first performed a thin-section transmission electron microscopy (TEM) assay to characterize the kinetics of PHEV, and we found that viral entry and transfer occur via membranous coating-mediated endo- and exocytosis. To verify the roles of distinct endocytic pathways, systematic approaches were used, including pharmacological inhibition, RNA interference, confocal microscopy analysis, use of fluorescently labeled virus particles, and overexpression of a dominant negative (DN) mutant. Quantification of infected cells showed that PHEV enters cells by clathrin-mediated endocytosis (CME) and that low pH, dynamin, cholesterol, and Eps15 are indispensably involved in this process. Intriguingly, PHEV invasion leads to rapid actin rearrangement, suggesting that the intactness and dynamics of the actin cytoskeleton are positively correlated with viral endocytosis. We next investigated the trafficking of internalized PHEV and found that Rab5- and Rab7-dependent pathways are required for the initiation of a productive infection. Furthermore, a GTPase activation assay suggested that endogenous Rab5 is activated by PHEV and is crucial for viral progression. Our findings demonstrate that PHEV hijacks the CME and endosomal system of the host to enter and traffic within neural cells, providing new insights into PHEV pathogenesis and guidance for antiviral drug design. IMPORTANCE Porcine hemagglutinating encephalomyelitis virus (PHEV), a nonsegmented, positive-sense, single-stranded RNA coronavirus, invades the central nervous system (CNS) and causes neurological dysfunction. Neural cells are its targets for viral progression. However, the detailed mechanism underlying PHEV entry and trafficking remains unknown. PHEV is the etiological agent of porcine hemagglutinating encephalomyelitis, which is an acute and highly contagious disease that causes numerous deaths in suckling piglets and enormous economic losses in China. Understanding the viral entry pathway will not only advance our knowledge of PHEV infection and pathogenesis but also open new approaches to the development of novel therapeutic strategies. Therefore, we employed systematic approaches to dissect the internalization and intracellular trafficking mechanism of PHEV in Neuro-2a cells. This is the first report to describe the process of PHEV entry into nerve cells via clathrin-mediated endocytosis in a dynamin-, cholesterol-, and pH-dependent manner that requires Rab5 and Rab7.