Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Assembly of endocytic machinery around individual influenza viruses during viral entry

Nature Structural & Molecular Biology volume 11, pages 567–573 (2004)Cite this article

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Fluorescence images of clathrin-coated structures in BS-C-1 cells expressing EYFP-clathrin.
Figure 2: Internalization of influenza viruses through different pathways.
Figure 3: Dynamics of the CCPs and CCVs formed de novo at the virus-binding sites.
Figure 4: The effect of neuraminidase (NA) inhibitors on the endocytosis of influenza viruses.
Figure 5: Time trajectories of viruses that successfully fused after endocytosis.

Similar content being viewed by others

References

  1. Matlin, K.S., Reggio, H., Helenius, A. & Simons, K. Infectious entry pathway of influenza-virus in a canine kidney-cell line. J. Cell Biol. 91, 601–613 (1981).

    Article  CAS  PubMed  Google Scholar 

  2. Doxsey, S.J., Brodsky, F.M., Blank, G.S. & Helenius, A. Inhibition of endocytosis by anti-clathrin antibody. Cell 50, 453–463 (1987).

    Article  CAS  PubMed  Google Scholar 

  3. Anderson, H.A., Chen, Y.Z. & Norkin, L.C. Bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae. Mol. Biol. Cell 7, 1825–1834 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Carbone, R. et al. Eps15 and eps15R are essential components of the endocytic pathway. Cancer Res. 57, 5498–5504 (1997).

    CAS  PubMed  Google Scholar 

  5. Stang, E., Kartenbeck, J. & Parton, R.G. Major histocompatibility complex class I molecules mediate association of SV40 with caveolae. Mol. Biol. Cell 8, 47–57 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. De Tulleo, L. & Kirchhausen, T. The clathrin endocytic pathway in viral infection. EMBO J. 17, 4585–4593 (1998).

    Article  CAS  Google Scholar 

  7. Pelkmans, L., Kartenbeck, J. & Helenius, A. Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat. Cell Biol. 3, 473–483 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Sieczkarski, S.B. & Whittaker, G.R. Influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis. J. Virol. 76, 10455–10464 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pelkmans, L. & Helenius, A. Insider information: what viruses tell us about endocytosis. Curr. Opin. Cell Biol. 15, 414–422 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Nichols, B.J. & Lippincott-Schwartz, J. Endocytosis without clathrin coats. Trends Cell Biol. 11, 406–412 (2001).

    Article  CAS  PubMed  Google Scholar 

  11. Conner, S.D. & Schmid, S.L. Regulated portals of entry into cells. Nature 422, 37–44 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Brodsky, F.M., Chen, C.-Y., Knuehl, C., Towler, M.C. & Wakeham, D.E. Biological basket weaving: formation and function of clathrin-coated vesicles. Annu. Rev. Cell Dev. Biol. 17, 517–568 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Kirchhausen, T.Clathrin. Annu. Rev. Biochem. 69, 699–727 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Nabi, I.R. & Le, P.U. Caveolae/raft-dependent endocytosis. J. Cell Biol. 161, 673–677 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Scott, M.G.H., Benmerah, A., Muntaner, O. & Marullo, S. Recruitment of activated G protein–coupled receptors to pre-existing clathrin coated pits in living cells. J. Biol. Chem. 277, 3552–3559 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Santini, F., Gaidarov, I. & Keen, J.H. G protein–couple dreceptor/arrestin3 modulation of the endocytic machinery. J. Cell Biol. 156, 665–676 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Gaidarov, I., Santini, F., Warren, R.A. & Keen, J.H. Spatial control of coated-pits dynamics in living cells. Nat. Cell Biol. 1, 1–7 (1999).

    Article  CAS  PubMed  Google Scholar 

  18. Skehel, J.J. & Wiley, D.C. Receptor binding and membrane fusion in viral entry: the influenza hemagglutinin. Annu. Rev. Biochem. 69, 531–569 (2000).

    Article  CAS  PubMed  Google Scholar 

  19. White, J., Helenius, A. & Gething, M.-J. Haemagglutinin of influenza virus expressed from a cloned gene promote membrane fusion. Nature 300, 658–659 (1982).

    Article  CAS  PubMed  Google Scholar 

  20. Yoshimura, A. & Ohnishi, S. Uncoating of influenza-virus in endosomes. J. Virol. 51, 497–504 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Martin, K. & Helenius, A. Transport of incoming influenza-virus nucleocapsids into the nucleus. J. Virol. 65, 232–244 (1990).

    Google Scholar 

  22. Lamb, R.A. & Krug, R.M. Orthomyxoviridae: the viruses and their replication. in Fields Virology (eds. Knipe, D.M. & Howley, P.M.) 1487–1531 (Lippincott Williams and Wilkins, Philadelphia, 2001).

    Google Scholar 

  23. Klasse, P.J., Bron, R. & Marsh, M. Mechanisms of enveloped virus entry into animal cells. Adv. Drug. Deliv. Rev. 34, 65–91 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Helenius, A., Kartenbeck, J., Simons, K. & Fries, E. On the entry of Semliki Forest viruses into BHK-21 cells. J. Cell Biol. 84, 404–420 (1980).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Marrifield, C.J., Feldman, M.E., Wan, L. & Almers, W. Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits. Nat. Cell Biol. 4, 691–698 (2002).

    Article  Google Scholar 

  26. Volonte, D., Galbiati, F. & Lisanti, M.P. Visualization of caveolin-1, a caveolar marker protein, in living cells using green fluorescent protein (GFP) chimeras: the subcellular distribution of caveolin-1 is modulated by cell-cell contact. FEBS Lett. 445, 431–439 (1999).

    Article  CAS  PubMed  Google Scholar 

  27. Wu, X. et al. Clathrin exchange during clathrin-mediated endocytosis. J. Cell Biol. 155, 291–300 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Rappoport, J.Z. & Simon, S.M. Clathrin-mediated endocytosis during cell migration. J. Cell Sci. 116, 847–855 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Pelkmans, L., Punterner, D. & Helenius, A. Local actin polymerization and dynamin recruitment in SV-40–induced internalization of caveolae. Science 296, 535–539 (2002).

    Article  CAS  PubMed  Google Scholar 

  30. Thomsen, P., Roepstorff, K., Stahlhut, M. & Deurs, B.V. Caveolar are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol. Biol. Cell 13, 238–250 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Schnitzer, J.E., Oh, P., Pinney, E. & Allard, J. Filipin-sensitive caveolae-mediated transport in endothelium-reduced transcytosis, scavenger endocytosis, and capillary-permeability of select macromolecules. J. Cell Biol. 127, 1217–1232 (1994).

    Article  CAS  PubMed  Google Scholar 

  32. Georgi, A., Mottola-Hartshorn, C., Warner, W., Fields, B. & Chen, L.B. Detection of individual fluorescently labelled reovirions in living cells. Proc. Natl. Acad. Sci. USA 87, 6579–6583 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Suomalainen, M. et al. Microtubule-dependent plus- and minus end–directed motilities are competing processes for nuclear targeting of adenovirus. J. Cell Biol. 144, 657–672 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Seisenberger, G. et al. Real-time single-molecule imaging of the infection pathway of an adeno-associated virus. Science 294, 1929–1932 (2001).

    Article  CAS  PubMed  Google Scholar 

  35. McDonald, D. et al. Visualization of the intracellular behavior of HIV in living cells. J. Cell Biol. 159, 441–452 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lakadamyali, M., Rust, M.J., Babcock, H.P. & Zhuang, X. Visualizing infection of individual influenza viruses. Proc. Natl. Acad. Sci. USA 100, 9280–9285 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wagner, R., Matrosovich, M. & Klenk, H.-D. Functional balance between haemagglutinin and neuraminidase in influenza virus infections. Rev. Med. Virol. 12, 159–166 (2002).

    Article  PubMed  Google Scholar 

  38. Babu, Y.S. et al. BCX-1812 (RWJ-270201): discovery of a novel, highly potent, orally active, and selective influenza neuraminidase inhibitor through structure-based drug design. J. Med. Chem. 43, 3482–3486 (2000).

    Article  CAS  PubMed  Google Scholar 

  39. Sidwell, R.W. et al. In vivo influenza virus–inhibitory effects of the cyclopentane neuraminidase inhibitor RWJ-270201. Antimicrob. Agents Chemother. 45, 749–757 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Rothberg, K.G., Ying, Y.S., Kamen, B.A. & Anderson, R.G. Cholesterol controls the clustering of the glycophospholipid-anchored membrane-receptor for 5-methyltetrahydrofolate. J. Cell Biol. 111, 2931–2938 (1990).

    Article  CAS  PubMed  Google Scholar 

  41. Naslavsky, N., Weigert, R. & Donaldson, J.G. Convergence of non-clathrin-and clathrin-derived endosomes involves Arf6 inactivation and changes in phosphoinositides. Mol. Biol. Cell 14, 417–431 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Sieczkarski, S.B. & Whittaker, G.R. Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses. Traffic 4, 333–343 (2003).

    Article  CAS  PubMed  Google Scholar 

  43. Connolly, J.L., Green, S.A. & Greene, L.A. Pit formation and rapid changes in surface morphology of sympathetic neurons in response to nerve growth factors. J. Cell Biol. 90, 176–180 (1981).

    Article  CAS  PubMed  Google Scholar 

  44. Wilde, A. et al. EGF receptor signaling stimulates SRC kinase phosphorylation of clathrin, influencing clathrin redistribution and EGF uptake. Cell 96, 677–687 (1999).

    Article  CAS  PubMed  Google Scholar 

  45. Grimes, M.L. et al. Endocytosis of activated TrkA: evidence that nerve growth factor induces formation of signaling endosomes. J. Neurosci. 16, 7950–7964 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Beattie, E.C., Howe, C.L., Wilde, A., Brodsky, F.M. & Mobley, W.C. NGF signals through TrkA to increase clathrin at the plasma membrane and enhance clathrin-mediated membrane trafficking. J. Neurosci. 20, 7325–7333 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Peter, B.J. et al. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495–499 (2004).

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

Author notes

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

Authors and Affiliations

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

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

Authors
  1. Michael J Rust
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Melike Lakadamyali
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Feng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiaowei Zhuang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiaowei Zhuang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

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)

Supplementary Fig. 1 (PDF 190 kb)

Supplementary Fig. 2 (PDF 250 kb)

Supplementary Fig. 3 (PDF 127 kb)

Supplementary Fig. 4 (PDF 138 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb769

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing