Abstract
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 de novo 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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Acknowledgements
We thank J.H. Keen (Thomas Jefferson University) and A. Helenius (Swiss Federal Institute of Technology) for their gifts of GFP-clathrin-LCa and Caveolin-1-EGFP plasmids, respectively. This work is supported in part by a Searle Scholarship, a Beckman Young Investigator award, the US Office of Naval Research and the US National Science Foundation (to X.Z.). M.J.R. is a US National Science Foundation pre-doctoral fellow.
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Supplementary Video 1
Videos were taken under the following imaging condition. For Videos 1-5, the excitation laser for the EYFP-clathrin or Caveolin-1-EGFP was turned on for 0.5 s every 1.5 s while that for the DiD-labeled viruses was always on. The camera integration time for each frame is 0.5 s. All camera pixels were read out individually. For Videos 6 and 7, 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 6 and 7 thus do not allow an accurate determination of whether CCPs form at or off the virus-binding sites, but are only meant to show that both clathrin-dependent and -independent endocytosis can lead to viral fusion. All videos are processed by subtracting the low-spatial frequency background signal generated by cytoplasmic EYFP-clathrin or Caveolin-1-EGFP. The videos are compressed significantly and compression compromises video quality. As the viruses were added to the cells in situ the 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)
A dual-color movie of EYFP-tagged clathrin structures (green) and DiD-labeled viruses (red) in a live cell.
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
The internalization of influenza viruses via CCPs. The de novo 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
The internalization of influenza viruses via CCPs. The de novo 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
The internalization 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
The internalization 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
The internalization and fusion of an influenza virus (red, in a white circle) without association with a CCP. (MP4 4321 kb)
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Rust, M., Lakadamyali, M., Zhang, F. et al. Assembly of endocytic machinery around individual influenza viruses during viral entry. Nat Struct Mol Biol 11, 567–573 (2004). https://doi.org/10.1038/nsmb769
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DOI: https://doi.org/10.1038/nsmb769
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