Cellular mechanisms of alpha herpesvirus egress: live cell fluorescence microscopy of pseudorabies virus exocytosis.
Cellular mechanisms of alpha herpesvirus egress: live cell fluorescence microscopy of pseudorabies virus exocytosis.
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DOI：
10.1371/journal.ppat.1004535
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发表时间：
2014-12
期刊：
影响因子：
6.7
通讯作者：
Enquist LW
中科院分区：
文献类型：
--
作者：
Hogue IB;Bosse JB;Hu JR;Thiberge SY;Enquist LW
新组装的疱疹病毒颗粒从受感染细胞中排出是一个高度动态的过程，涉及宿主分泌途径与病毒成分的协同作用。为了阐明α疱疹病毒流出的位置、动力学和分子机制，我们开发了一种活细胞荧光显微镜方法，以可视化伪狂犬病病毒（PRV）颗粒在非极化上皮细胞中的最终运输和胞吐作用。该方法基于全内反射荧光 (TIRF) 显微镜，选择性地对质膜附近的荧光病毒颗粒进行成像，并利用病毒编码的 pH 敏感探针来可视化颗粒胞吐作用的精确时刻和位置。我们进行了单粒子跟踪和均方位移分析来表征粒子运动，并对一组细胞蛋白质进行成像以识别那些在空间和动态上与病毒胞吐作用相关的蛋白质。根据我们的数据，单个病毒颗粒在小的酸化分泌囊泡内进入质膜。 Rab GTPases、Rab6a、Rab8a 和 Rab11a 是质膜定向分泌途径的关键调节因子，存在于病毒分泌囊泡上。这些囊泡直接快速、定向运输到胞吐位点，该位点最常见于 LL5β 斑块附近，LL5β 是将微管锚定到质膜的复合物的一部分。囊泡紧密地停靠在胞吐作用的位点几秒钟，然后发生膜融合，使病毒颗粒在质膜上移动一小段距离。胞吐作用后，颗粒仍然紧密地限制在细胞外表面。根据细胞生物学和α疱疹病毒文献的最新报告，结合我们关于病毒出口的空间和动态数据，我们提出了一个综合模型，将介导α疱疹病毒出口的细胞内运输途径和胞吐机制联系在一起。伪狂犬病病毒属于甲型疱疹病毒，是一种重要的兽医病原体，与人水痘带状疱疹病毒和单纯疱疹病毒有关。新的α疱疹病毒颗粒在受感染的细胞内组装，并且必须利用细胞机制从受感染的细胞中排出。这些病毒颗粒如何在受感染的细胞内运输并分泌到细胞表面尚不清楚。特别是，使用以前的方法不容易观察到这个过程如何随着时间的推移而展开。在这项研究中，我们开发了一种新方法来观察这个出口过程。使用这种方法，我们描述了病毒颗粒如何移动：单个病毒颗粒移动到细胞表面，直接到达出口位点，在那里它们暂停几秒钟，然后穿过细胞。我们鉴定了参与该过程的几种细胞蛋白。退出后，病毒颗粒仍然粘附在细胞外表面。最后，我们将我们的观察结果与其他最近的研究联系起来，提出一个关于α疱疹病毒颗粒如何从受感染细胞中排出的综合模型。
Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF) microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement analysis to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically associated with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biological and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress. Pseudorabies virus, an alpha herpesvirus, is an important veterinary pathogen, and related to human varicella-zoster virus and herpes simplex viruses. New alpha herpesvirus particles are assembled inside an infected cell, and must exit from the infected cell by taking advantage of cellular mechanisms. How these virus particles are transported inside the infected cell and secreted at the cell surface is not understood in great detail. In particular, how this process unfolds over time is not easily observed using previous methods. In this study, we developed a new method to observe this egress process. Using this method, we described how virus particles move on their way out: individual virus particles travel to the cell surface, directly to the exit site, where they pause for several seconds before crossing out of the cell. We identified several cellular proteins that are involved in this process. After exiting, virus particles remained stuck to the outer cell surface. Finally, we draw connections between our observations and other recent studies to propose an integrated model of how alpha herpesvirus particles exit from infected cells.
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