Rabies Internalizes into Primary Peripheral Neurons via Clathrin Coated Pits and Requires Fusion at the Cell Body.
Rabies Internalizes into Primary Peripheral Neurons via Clathrin Coated Pits and Requires Fusion at the Cell Body.
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DOI：
10.1371/journal.ppat.1005753
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发表时间：
2016-07
期刊：
影响因子：
6.7
通讯作者：
Whelan SP
中科院分区：
文献类型：
--
作者：
Piccinotti S;Whelan SP
狂犬病病毒（RABV）的单一糖蛋白（G）决定了从受体结合到膜融合的所有病毒进入步骤。为了研究RABV进入培养的原代神经元细胞的摄取，我们产生了重组水泡性口炎病毒，其中G蛋白被替换为亲神经性RABV CVS-11株（rVSV CVS G）。使用微流控区室化培养，我们研究了单个病毒粒子进入背根神经节和腹侧脊髓的初级神经元末端的摄取。通过破坏远端神经突的内吞作用，我们证明了rVSV CVS G的摄取和感染依赖于发动蛋白。用荧光内吞标记物对单个病毒体摄取成像进一步鉴定了通过网格蛋白包被的小凹的内吞作用为主要的内化机制。透射电子显微镜照片也揭示了病毒颗粒的存在下，囊泡结构与不完全包被网格蛋白坑一致。这项工作将我们之前关于网格蛋白介导的RABV摄入上皮细胞的发现扩展到了参与体内狂犬病感染的两种神经元亚型。神经突或体隔室中的内体酸化的化学扰动进一步表明，感染的建立需要在细胞体处的病毒体的pH依赖性融合。这些发现将感染性与RABV的长程内体转运和质膜网格蛋白依赖性摄取的现有单颗粒证据相关联。狂犬病病毒是中枢神经系统（CNS）的一种通常致命且不可治愈的疾病的病原体。狂犬病的致命性需要病毒侵入大脑，这是通过从感染部位到中枢神经系统的神经传递完成的。使用周围神经元和化学物质的文化，扰乱特定的细胞进入途径，我们的特点狂犬病的吸收机制。使用高分辨率共聚焦显微镜，我们可视化的过程中的内化和感染的遗传编码的感染标记的表达建立个别病毒颗粒。我们表明，网格蛋白包被的坑介导的病毒内化到内吞囊泡运输病毒的细胞体。我们进一步证明，释放的病毒基因组核心的细胞体是需要有效地建立感染，并提供证据表明，一个子集的传入的病毒颗粒在非生产性网站在到达这个网站之前融合。本研究通过识别神经元中有效建立感染所需的进入机制和融合位点来扩展先前的知识。
The single glycoprotein (G) of rabies virus (RABV) dictates all viral entry steps from receptor engagement to membrane fusion. To study the uptake of RABV into primary neuronal cells in culture, we generated a recombinant vesicular stomatitis virus in which the G protein was replaced with that of the neurotropic RABV CVS-11 strain (rVSV CVS G). Using microfluidic compartmentalized culture, we examined the uptake of single virions into the termini of primary neurons of the dorsal root ganglion and ventral spinal cord. By pharmacologically disrupting endocytosis at the distal neurites, we demonstrate that rVSV CVS G uptake and infection are dependent on dynamin. Imaging of single virion uptake with fluorescent endocytic markers further identifies endocytosis via clathrin-coated pits as the predominant internalization mechanism. Transmission electron micrographs also reveal the presence of viral particles in vesicular structures consistent with incompletely coated clathrin pits. This work extends our previous findings of clathrin-mediated uptake of RABV into epithelial cells to two neuronal subtypes involved in rabies infection in vivo. Chemical perturbation of endosomal acidification in the neurite or somal compartment further shows that establishment of infection requires pH-dependent fusion of virions at the cell body. These findings correlate infectivity to existing single particle evidence of long-range endosomal transport of RABV and clathrin dependent uptake at the plasma membrane. Rabies virus is the causative agent of a generally fatal and incurable disease of the central nervous system (CNS). Rabies lethality requires that the virus invade the brain, a feat accomplished by neuronal transmission from the site of infection to the CNS. Using cultures of peripheral neurons and chemicals that perturb specific cellular entry pathways we characterize the mechanism of rabies uptake. Using high resolution confocal microscopy, we visualize individual viral particles in the process of internalization and the establishment of infection by expression of a genetically encoded marker for infection. We show that clathrin-coated pits mediate internalization of the virus into endocytic vesicles that transport the virus to the cell body. We further demonstrate that release of the viral genomic core at the cell body is required to efficiently establish infection, and provide evidence that a subset of incoming virus particles fuse at non-productive sites prior to arrival at this site. This study extends the prior knowledge by identifying the entry mechanism and the site of fusion required for effective establishment of infection in neurons.