Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization.
Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization.
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DOI：
10.1371/journal.ppat.1000394
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发表时间：
2009-04
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：
Cureton DK;Massol RH;Saffarian S;Kirchhausen TL;Whelan SP
许多通过网格蛋白依赖的内吞作用进入细胞的病毒明显比典型的网格蛋白包被小泡的尺寸大。病毒利用网格蛋白机制实现高效内化的机制仍不确定。在此我们研究了网格蛋白包被小泡在水疱性口炎病毒（VSV）进入细胞过程中是如何容纳该病毒的。通过对单个病毒颗粒内化进入表达荧光网格蛋白和衔接分子的细胞进行高分辨率成像，我们表明VSV通过部分网格蛋白包被的小泡进入细胞。我们发现，平均而言，含病毒的小泡比常规小泡含有更多的网格蛋白和网格蛋白衔接分子，但这种增加不足以使小泡完全被包被。我们进一步表明，含病毒的小泡依赖肌动蛋白机制实现内化。具体而言，我们发现肌动蛋白机制的成分被招募到含病毒的小泡中，并且肌动蛋白聚合的化学抑制会将病毒颗粒捕获在质膜处的小泡中。通过对多个独立的病毒内化事件进行分析，我们表明VSV诱导网格蛋白成核以实现其摄取，而不是依赖于通过形成网格蛋白包被凹陷的随机捕获。这项工作为病毒内化过程以及网格蛋白依赖的内吞机制对非常规货物的摄取提供了新的机制性见解。
网格蛋白依赖的内吞作用占质膜摄取的大部分。然而，许多通过内吞途径感染细胞的病毒比典型的网格蛋白包被小泡的尺寸大。我们以水疱性口炎病毒为研究对象，确定了这种货物是如何进入细胞的。我们提供的证据表明，VSV在与质膜结合后，通过网格蛋白依赖的内吞机制诱导自身的摄取。含有病毒的网格蛋白包被小泡与内化常规的网格蛋白依赖货物（如低密度脂蛋白和转铁蛋白）的小泡不同。具体而言，我们表明VSV颗粒是由仅部分被网格蛋白包被的小泡内化的，而不是像常规小泡那样有完整的包被。我们表明网格蛋白依赖的内吞衔接蛋白AP - 2是进入所必需的。最后，我们首次表明肌动蛋白被招募到含病毒的凹陷中，并且颗粒内化依赖于肌动蛋白的功能。我们的工作为VSV进入提供了新的机制性见解，这可能与理解其他病毒的网格蛋白依赖摄取直接相关。
Many viruses that enter cells by clathrin-dependent endocytosis are significantly larger than the dimensions of a typical clathrin-coated vesicle. The mechanisms by which viruses co-opt the clathrin machinery for efficient internalization remain uncertain. Here we examined how clathrin-coated vesicles accommodate vesicular stomatitis virus (VSV) during its entry into cells. Using high-resolution imaging of the internalization of single viral particles into cells expressing fluorescent clathrin and adaptor molecules, we show that VSV enters cells through partially clathrin-coated vesicles. We found that on average, virus-containing vesicles contain more clathrin and clathrin adaptor molecules than conventional vesicles, but this increase is insufficient to permit full coating of the vesicle. We further show that virus-containing vesicles depend upon the actin machinery for their internalization. Specifically, we found that components of the actin machinery are recruited to virus-containing vesicles, and chemical inhibition of actin polymerization trapped viral particles in vesicles at the plasma membrane. By analysis of multiple independent virus internalization events, we show that VSV induces the nucleation of clathrin for its uptake, rather than depending upon random capture by formation of a clathrin-coated pit. This work provides new mechanistic insights into the process of virus internalization as well as uptake of unconventional cargo by the clathrin-dependent endocytic machinery. Clathrin-dependent endocytosis accounts for the majority of uptake from the plasma membrane. However, many viruses that infect cells through an endocytic route are larger than the dimensions of a typical clathrin-coated vesicle. Working with vesicular stomatitis virus, we determined how this cargo enters cells. We present evidence that VSV induces its own uptake by the clathrin-dependent endocytic machinery following binding to the plasma membrane. The clathrin-coated vesicles that contain virus differ from vesicles that internalize conventional clathrin dependent cargo such as LDL and transferrin. Specifically, we show that VSV particles are internalized by vesicles that are only partially coated with clathrin, rather than the complete coat found on conventional vesicles. We show that the clathrin-dependent endocytic adaptor AP-2 is required for entry. Finally, we show for the first time that actin is recruited to virus-containing pits and that particle internalization depends upon actin function. Our work provides new mechanistic insights into VSV entry that may be directly relevant in understanding the clathrin dependent uptake of other viruses.
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