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⇱ SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway | Cell Research

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Abstract

While severe acute respiratory syndrome coronavirus (SARS-CoV) was initially thought to enter cells through direct fusion with the plasma membrane, more recent evidence suggests that virus entry may also involve endocytosis. We have found that SARS-CoV enters cells via pH- and receptor-dependent endocytosis. Treatment of cells with either SARS-CoV spike protein or spike-bearing pseudoviruses resulted in the translocation of angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV, from the cell surface to endosomes. In addition, the spike-bearing pseudoviruses and early endosome antigen 1 were found to colocalize in endosomes. Further analyses using specific endocytic pathway inhibitors and dominant-negative Eps15 as well as caveolin-1 colocalization study suggested that virus entry was mediated by a clathrin- and caveolae-independent mechanism. Moreover, cholesterol- and sphingolipid-rich lipid raft microdomains in the plasma membrane, which have been shown to act as platforms for many physiological signaling pathways, were shown to be involved in virus entry. Endocytic entry of SARS-CoV may expand the cellular range of SARS-CoV infection, and our findings here contribute to the understanding of SARS-CoV pathogenesis, providing new information for anti-viral drug research.

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Abbreviations

(ACE2):

angiotensin converting enzyme 2

(CPZ):

chlorpromazine

(CTB):

cholera toxin subunit B

(EEA1):

early endosome antigen 1

(HEK293E):

human embryonic kidney 293E

(MβCD):

methyl-β-cyclodextrin

(SARS):

severe acute respiratory syndrome

(SARS-CoV):

severe acute respiratory syndrome coronavirus

References

  1. Drosten C, Gunther S, Preiser W, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 2003; 348:1967–1976.

    Article  CAS  PubMed  Google Scholar 

  2. Ksiazek TG, Erdman D, Goldsmith CS, et al. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 2003; 348:1953–1966.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  4. Sieczkarski SB, Whittaker GR . Dissecting virus entry via endocytosis. J Gen Virol 2002; 83:1535–1545.

    Article  CAS  PubMed  Google Scholar 

  5. Fackler OT, Peterlin BM . Endocytic entry of HIV-1. Curr Biol 2000; 10:1005–1008.

    Article  CAS  PubMed  Google Scholar 

  6. Matlin KS, Reggio H, Helenius A, Simons K . Infectious entry pathway of influenza virus in a canine kidney cell line. J Cell Biol 1981; 91:601–613.

    Article  CAS  PubMed  Google Scholar 

  7. Sieczkarski SB, Whittaker GR . Influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis. J Virol 2002; 76:10455–10464.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Nunes-Correia I, Eulalio A, Nir S, Pedroso de Lima MC . Caveolae as an additional route for influenza virus endocytosis in MDCK cells. Cell Mol Biol Lett 2004; 9:47–60.

    PubMed  Google Scholar 

  9. Ng ML, Tan SH, See EE, Ooi EE, Ling AE . Early events of SARS coronavirus infection in vero cells. J Med Virol 2003; 71:323–331.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Qinfen Z, Jinming C, Xiaojun H, et al. The life cycle of SARS coronavirus in Vero E6 cells. J Med Virol 2004; 73:332–337.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Simmons G, Reeves JD, Rennekamp AJ, Amberg SM, Piefer AJ, Bates P . Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry. Proc Natl Acad Sci USA 2004; 101:4240–4245.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yang ZY, Huang Y, Ganesh L, et al. pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN. J Virol 2004; 78:5642–5650.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Huang IC, Bosch BJ, Li F, et al. SARS coronavirus, but not human coronavirus NL63, utilizes cathepsin L to infect ACE2-expressing cells. J Biol Chem 2006; 281:3198–3203.

    Article  CAS  PubMed  Google Scholar 

  14. Simmons G, Gosalia DN, Rennekamp AJ, Reeves JD, Diamond SL, Bates P . Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proc Natl Acad Sci USA 2005; 102:11876–11881.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Moore MJ, Dorfman T, Li W, et al. Retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2. J Virol 2004; 78:10628–10635.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kuba K, Imai Y, Rao S, et al. A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat Med 2005; 11:875–879.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ray RB, Basu A, Steele R, et al. Ebola virus glycoprotein-mediated anoikis of primary human cardiac microvascular endothelial cells. Virology 2004; 321:181–188.

    Article  CAS  PubMed  Google Scholar 

  18. Blanchard E, Belouzard S, Goueslain L, et al. Hepatitis C virus entry depends on clathrin-mediated endocytosis. J Virol 2006; 80:6964–6972.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Codran A, Royer C, Jaeck D, et al. Entry of hepatitis C virus pseudotypes into primary human hepatocytes by clathrin-dependent endocytosis. J Gen Virol 2006; 87:2583–2593.

    Article  CAS  PubMed  Google Scholar 

  20. Empig CJ, Goldsmith MA . Association of the caveola vesicular system with cellular entry by filoviruses. J Virol 2002; 76:5266–5270.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Dautry-Varsat A . Receptor-mediated endocytosis: the intracellular journey of transferrin and its receptor. Biochimie 1986; 68:375–381.

    Article  CAS  PubMed  Google Scholar 

  22. Dautry-Varsat A, Ciechanover A, Lodish HF . pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci USA 1983; 80:2258–2262.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Harding C, Heuser J, Stahl P . Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J Cell Biol 1983; 97:329–339.

    Article  CAS  PubMed  Google Scholar 

  24. Wang LH, Rothberg KG, Anderson RG . Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J Cell Biol 1993; 123:1107–1117.

    Article  CAS  PubMed  Google Scholar 

  25. Benmerah A, Bayrou M, Cerf-Bensussan N, Dautry-Varsat A . Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J Cell Sci 1999; 112 Part 9:1303–1311.

    CAS  PubMed  Google Scholar 

  26. Benmerah A, Poupon V, Cerf-Bensussan N, Dautry-Varsat A . Mapping of Eps15 domains involved in its targeting to clathrin-coated pits. J Biol Chem 2000; 275:3288–3295.

    Article  CAS  PubMed  Google Scholar 

  27. Stan RV . Structure of caveolae. Biochim Biophys Acta 2005; 1746:334–348.

    Article  CAS  PubMed  Google Scholar 

  28. Orlandi PA, Fishman PH . Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains. J Cell Biol 1998; 141:905–915.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Sanchez-San Martin C, Lopez T, Arias CF, Lopez S . Characterization of rotavirus cell entry. J Virol 2004; 78:2310–2318.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lanzrein M, Schlegel A, Kempf C . Entry and uncoating of enveloped viruses. Biochem J 1994; 302 (Part 2):313–320.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Cantin C, Holguera J, Ferreira L, Villar E, Munoz-Barroso I . Newcastle disease virus may enter cells by caveolae-mediated endocytosis. J Gen Virol 2007; 88:559–569.

    Article  CAS  PubMed  Google Scholar 

  32. Li GM, Li YG, Yamate M, Li SM, Ikuta K . Lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle. Microbes Infect 2007; 9:96–102.

    Article  CAS  PubMed  Google Scholar 

  33. Cinatl J Jr, Hoever G, Morgenstern B, et al. Infection of cultured intestinal epithelial cells with severe acute respiratory syndrome coronavirus. Cell Mol Life Sci 2004; 61:2100–2112.

    Article  CAS  PubMed  Google Scholar 

  34. Mossel EC, Huang C, Narayanan K, Makino S, Tesh RB, Peters CJ . Exogenous ACE2 expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication. J Virol 2005; 79:3846–3850.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Mirre C, Monlauzeur L, Garcia M, Delgrossi MH, Le Bivic A . Detergent-resistant membrane microdomains from Caco-2 cells do not contain caveolin. Am J Physiol 1996; 271:C887–C894.

    Article  CAS  PubMed  Google Scholar 

  36. Inoue Y, Tanaka N, Tanaka Y, et al. Clathrin-dependent entry of SARS coronavirus into target cells expressing cytoplasmic tail-deleted ACE2. J Virol 2007; 81:8722–8729.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hamming I, Timens W, Bulthuis ML, Lely AT, Navis GJ, van Goor H . Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol 2004; 203:631–637.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Popik W, Alce TM, Au WC . Human immunodeficiency virus type 1 uses lipid raft-colocalized CD4 and chemokine receptors for productive entry into CD4(+) T cells. J Virol 2002; 76:4709–4722.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Manes S, del Real G, Martinez AC . Pathogens: raft hijackers. Nat Rev 2003; 3:557–568.

    CAS  Google Scholar 

  40. Shigeta S, Yamase T . Current status of anti-SARS agents. Antivir Chem Chemother 2005; 16:23–31.

    Article  CAS  PubMed  Google Scholar 

  41. Kliger Y, Levanon EY, Gerber D . From genome to antivirals: SARS as a test tube. Drug Disc Today 2005; 10:345–352.

    Article  CAS  Google Scholar 

  42. Negre D, Mangeot PE, Duisit G, et al. Characterization of novel safe lentiviral vectors derived from simian immunodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cells. Gene Ther 2000; 7:1613–1623.

    Article  CAS  PubMed  Google Scholar 

  43. Kohler G, Milstein C . Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256:495–497.

    Article  CAS  PubMed  Google Scholar 

  44. Kee SH, Cho EJ, Song JW, Park KS, Baek LJ, Song KJ . Effects of endocytosis inhibitory drugs on rubella virus entry into VeroE6 cells. Microbiol Immunol 2004; 48:823–829.

    Article  CAS  PubMed  Google Scholar 

  45. Meertens L, Bertaux C, Dragic T . Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles. J Virol 2006; 80:11571–11578.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Dr James Kappler for editing. We thank Drs Brian Seed and Michael Farzan from Harvard Medical School, Dr Alexandre Benmerah (through Dr Du Feng, Tsinghua University, China) from Institut Pasteur for providing materials and reagents. We thank Ju Xiangwu from Shenyang Pharmaceutical University, China for the statistic analysis. This work was funded by National Natural Science Foundation of China (30421003, 30528002, 30625013, and 30623009) and Ministry of Science and Technology of China (2006AA02Z152 and 2005CB523000). Y. Z. was supported by NNSFC 30600020.

Author information

Authors and Affiliations

  1. National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Peking Union Medical College, Tsinghua University and Chinese Academy of Medical Sciences, Beijing, 100005, China

    Hongliang Wang, Peng Yang, Kangtai Liu, Feng Guo, Yanli Zhang & Chengyu Jiang

  2. Department of Immunology, National Jewish Medical and Research Center, Denver, 80206, CO, USA

    Gongyi Zhang

Corresponding author

Correspondence to Chengyu Jiang.

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Wang, H., Yang, P., Liu, K. et al. SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway. Cell Res 18, 290–301 (2008). https://doi.org/10.1038/cr.2008.15

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  • DOI: https://doi.org/10.1038/cr.2008.15

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