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Assembly of endocytic machinery around individual influenza viruses during viral entry

自然结构和分子生物学体积11，，，，pages567–573（2004）引用本文

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Most viruses enter cells via receptor-mediated endocytosis. However, the entry mechanisms used by many of them remain unclear. Also largely unknown is the way in which viruses are targeted to cellular endocytic machinery. We have studied the entry mechanisms of influenza viruses by tracking the interaction of single viruses with cellular endocytic structures in real time using fluorescence microscopy. Our results show that influenza can exploit clathrin-mediated and clathrin- and caveolin-independent endocytic pathways in parallel, both pathways leading to viral fusion with similar efficiency. Remarkably, viruses taking the clathrin-mediated pathway enter cells via the从头formation of clathrin-coated pits (CCPs) at viral-binding sites. CCP formation at these sites is much faster than elsewhere on the cell surface, suggesting a virus-induced CCP formation mechanism that may be commonly exploited by many other types of viruses.

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图1：表达EYFP-clathrin的BS-C-1细胞中网格蛋白涂层结构的荧光图像。
Figure 2: Internalization of influenza viruses through different pathways.
图3：形成的CCP和CCV的动力学从头在病毒结合部位。
Figure 4: The effect of neuraminidase (NA) inhibitors on the endocytosis of influenza viruses.
图5：内吞作用后成功融合的病毒的时间轨迹。

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Acknowledgements

我们感谢J.H.Keen（Thomas Jefferson University）和A. Helenius（瑞士联邦技术学院）分别赋予了GFP-Clathrin-LCA和Caveolin-1-EGFP质粒的礼物。这项工作得到了Searle奖学金，贝克曼青年研究员奖，美国海军研究办公室和美国国家科学基金会（授予X.Z.）的部分支持。M.J.R.是美国国家科学基金会前研究员。

作者信息

作者注意

  1. Michael J Rust and Melike Lakadamyali: These authors contributed equally to this work.

Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, 02138, Massachusetts, USA

    Michael J Rust，Melike Lakadamyali，Feng Zhang和Xiaowei Zhuang

作者
  1. Michael J Rust
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  2. Melike Lakadamyali
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  3. Feng Zhang
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  4. 小王Zhuang
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Corresponding author

对应小王Zhuang。

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这authors declare no competing financial interests.

补充信息

Supplementary Video 1

视频是在以下成像条件下拍摄的。为了视频1-5，EYFP-clathrin或Caveolin-1-EGFP的激发激光器每1.5 s打开0.5 s，而DID标记的病毒的激发激光始终处于打开状态。每个帧的相机集成时间为0.5 s。所有相机像素均单独读取。为了Videos 6and7，，，，the excitation laser for the EYFP-clathrin was turned on for 0.5 s every 1.5 s while that for the DiD-viruses was turned on for 0.5 s every 1s. The pixels were binned in a 2x2 fashion.Videos 6and7因此，不允许准确确定CCP在病毒结合位点是否形成或关闭病毒结合位点，而仅仅是为了表明依赖性依赖性和非依赖性内吞作用都会导致病毒融合。所有视频均通过减去由细胞质EYFP-CLATHRIN或Caveolin-1-EGFP产生的低空间频率背景信号来处理。这些视频被显着压缩，压缩会损害视频质量。随着病毒添加到细胞中在situthe binding of viruses to cells is highly asynchronous. Thus some viruses appear to move only slowly while the circled viruses show the specified behavior of three-stage movement. These slow-moving viruses in the field of view are either in stage I or in stage III during the exhibited time window. (MP4 1242 kb)

在活细胞中，一部双色的电影结构（绿色）和标记的病毒（红色）的双色电影。

Supplementary Video 2

A dual-color movie of EGFP-tagged caveolin structures (green) and DiD-labeled viruses (red) in a live cell. (MP4 1395 kb)

Supplementary Video 3

通过CCP的流感病毒的内在化。这从头formation of a CCP (green) around the virus (red, in white circles), the gradual increase of clathrin intensity, and the rapid disappearance of the clathrin signal immediately before the virus exhibit a rapid, unidirectional movement towards the perinuclear region (stage II movement). (MP4 2335 kb)

Supplementary Video 4

通过CCP的流感病毒的内在化。这从头formation of a CCP (green) around the virus (red, in white circles), the gradual increase of clathrin intensity, and the rapid disappearance of the clathrin signal immediately before the virus exhibit a rapid, unidirectional movement towards the perinuclear region (stage II movement). (MP4 2919 kb)

Supplementary Video 5

这在ternalization of an influenza virus without association with a CCP. The virus (red, in a white circle) did not associate with a CCP before its stage-II movement inside the cell. (MP4 2995 kb)

Supplementary Video 6

这在ternalization and fusion of an influenza virus (red, in a white circle) after association with a CCP (green). Fusion is indicated by a dramatic increase of the DiD signal (red). (MP4 2785 kb)

Supplementary Video 7

流感病毒的内在化和融合（红色，白色圆圈）与CCP相关。（MP4 4321 KB）

Supplementary Fig. 1 (PDF 190 kb)

Supplementary Fig. 2 (PDF 250 kb)

补充图3（PDF 127 KB）

补充图4（PDF 138 kb）

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Rust, M., Lakadamyali, M., Zhang, F.等。病毒进入过程中单个流感病毒周围的内吞机械组装。Nat Struct Mol Biol11，，，，567–573（2004）。https://doi.org/10.1038/nsmb769

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