The SARS coronavirus (SARS-CoV) spike is the largest known viral spike molecule, and shares a similar function with all class 1 viral fusion proteins. Previous structural studies of membrane fusion proteins have largely used crystallography of static molecular fragments, in isolation of their transmembrane domains. In this study we have produced purified, irradiated SARS-CoV virions that retain their morphology, and are fusogenic in cell culture. We used cryo-electron microscopy and image processing to investigate conformational changes that occur in the entire spike of intact virions when they bind to the viral receptor, angiotensin-converting enzyme 2 (ACE2). We have shown that ACE2 binding results in structural changes that appear to be the initial step in viral membrane fusion, and precisely localized the receptor-binding and fusion core domains within the entire spike. Furthermore, our results show that receptor binding and subsequent membrane fusion are distinct steps, and that each spike can bind up to three ACE2 molecules. The SARS-CoV spike provides an ideal model system to study receptor binding and membrane fusion in the native state, employing cryo-electron microscopy and single-particle image analysis.
References
[1]
Dutch RE, Jardetzky TS, Lamb RA (2000) Virus membrane fusion proteins: biological machines that undergo a metamorphosis. Biosci Rep 20: 597–612.
[2]
Hakansson-McReynolds S, Jiang S, Rong L, Caffrey M (2006) Solution structure of the severe acute respiratory syndrome-coronavirus heptad repeat 2 domain in the prefusion state. J Biol Chem 281: 11965–11971.
[3]
Xu Y, Lou Z, Liu Y, Pang H, Tien P, et al. (2004) Crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core. J Biol Chem 279: 49414–49419.
[4]
Skehel JJ, Wiley DC (2000) Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69: 531–569.
[5]
Kobe B, Center RJ, Kemp BE, Poumbourios P (1999) Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins. Proc Natl Acad Sci U S A 96: 4319–4324.
[6]
Weissenhorn W, Dessen A, Harrison SC, Skehel JJ, Wiley DC (1997) Atomic structure of the ectodomain from HIV-1 gp41. Nature 387: 426–430.
[7]
Malashkevich VN, Schneider BJ, McNally ML, Milhollen MA, Pang JX, et al. (1999) Core structure of the envelope glycoprotein GP2 from Ebola virus at 1.9-A resolution. Proc Natl Acad Sci U S A 96: 2662–2667.
[8]
Weissenhorn W, Carfi A, Lee KH, Skehel JJ, Wiley DC (1998) Crystal structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain. Mol Cell 2: 605–616.
[9]
Chen L, Gorman JJ, McKimm-Breschkin J, Lawrence LJ, Tulloch PA, et al. (2001) The structure of the fusion glycoprotein of Newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion. Structure 9: 255–266.
[10]
Zhao X, Singh M, Malashkevich VN, Kim PS (2000) Structural characterization of the human respiratory syncytial virus fusion protein core. Proc Natl Acad Sci U S A 97: 14172–14177.
[11]
Yin HS, Paterson RG, Wen X, Lamb RA, Jardetzky TS (2005) Structure of the uncleaved ectodomain of the paramyxovirus (hPIV3) fusion protein. Proc Natl Acad Sci U S A 102: 9288–9293.
[12]
Yin HS, Wen X, Paterson RG, Lamb RA, Jardetzky TS (2006) Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation. Nature 439: 38–44.
[13]
Hernandez LD, Peters RJ, Delos SE, Young JA, Agard DA, et al. (1997) Activation of a retroviral membrane fusion protein: soluble receptor-induced liposome binding of the ALSV envelope glycoprotein. J Cell Biol 139: 1455–1464.
[14]
Damico RL, Crane J, Bates P (1998) Receptor-triggered membrane association of a model retroviral glycoprotein. Proc Natl Acad Sci U S A 95: 2580–2585.
[15]
Fenouillet E, Barbouche R, Jones IM (2007) Cell Entry by Enveloped Viruses: Redox Considerations for HIV and SARS-Coronavirus. Antioxid Redox Signal 9: 1009–34.
[16]
Simmons G, Gosalia DN, Rennekamp AJ, Reeves JD, Diamond SL, et al. (2005) Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proc Natl Acad Sci U S A 102: 11876–11881.
[17]
Baker KA, Dutch RE, Lamb RA, Jardetzky TS (1999) Structural basis for paramyxovirus-mediated membrane fusion. Mol Cell 3: 309–319.
[18]
Colman PM, Lawrence MC (2003) The structural biology of type I viral membrane fusion. Nat Rev Mol Cell Biol 4: 309–319.
Hofmann H, Pohlmann S (2004) Cellular entry of the SARS coronavirus. Trends Microbiol 12: 466–472.
[21]
Beniac DR, Andonov A, Grudeski E, Booth TF (2006) Architecture of the SARS coronavirus prefusion spike. Nat Struct Mol Biol 13: 751–752.
[22]
Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, et al. (2003) Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426: 450–454.
[23]
Earp LJ, Delos SE, Park HE, White JM (2005) The many mechanisms of viral membrane fusion proteins. Curr Top Microbiol Immunol 285: 25–66.
[24]
Li F, Li W, Farzan M, Harrison SC (2005) Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309: 1864–1868.
[25]
Supekar VM, Bruckmann C, Ingallinella P, Bianchi E, Pessi A, et al. (2004) Structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus S2 fusion protein. Proc Natl Acad Sci U S A 101: 17958–17963.
[26]
Duquerroy S, Vigouroux A, Rottier PJ, Rey FA, Bosch BJ (2005) Central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the SARS coronavirus spike glycoprotein. Virology 335: 276–285.
[27]
Prabakaran P, Gan J, Feng Y, Zhu Z, Choudhry V, et al. (2006) Structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody. J Biol Chem 281: 15829–15836.
[28]
Hwang WC, Lin Y, Santelli E, Sui J, Jaroszewski L, et al. (2006) Structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80R. J Biol Chem 281: 34610–34616.
[29]
Booth TF, Kournikakis B, Bastien N, Ho J, Kobasa D, et al. (2005) Detection of airborne severe acute respiratory syndrome (SARS) coronavirus and environmental contamination in SARS outbreak units. J Infect Dis 191: 1472–1477.
[30]
Rao PV, Gallagher TM (1998) Intracellular complexes of viral spike and cellular receptor accumulate during cytopathic murine coronavirus infections. J Virol 72: 3278–3288.
[31]
Collins AR, Knobler RL, Powell H, Buchmeier MJ (1982) Monoclonal antibodies to murine hepatitis virus-4 (strain JHM) define the viral glycoprotein responsible for attachment and cell–cell fusion. Virology 119: 358–371.
[32]
Dimitrov DS (2003) The secret life of ACE2 as a receptor for the SARS virus. Cell 115: 652–653.
[33]
Xiao X, Chakraborti S, Dimitrov AS, Gramatikoff K, Dimitrov DS (2003) The SARS-CoV S glycoprotein: expression and functional characterization. Biochem Biophys Res Commun 312: 1159–1164.
[34]
Petit CM, Chouljenko VN, Iyer A, Colgrove R, Farzan M, et al. (2007) Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion. Virology 360: 264–274.
[35]
Frank J, Radermacher M, Penczek P, Zhu J, Li Y, et al. (1996) SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 116: 190–199.
[36]
Penczek PA, Grassucci RA, Frank J (1994) The ribosome at improved resolution: new techniques for merging and orientation refinement in 3D cryo-electron microscopy of biological particles. Ultramicroscopy 53: 251–270.
[37]
Wriggers W, Milligan RA, McCammon JA (1999) Situs: A package for docking crystal structures into low-resolution maps from electron microscopy. J Struct Biol 125: 185–195.
[38]
Kemble GW, Bodian DL, Rose J, Wilson IA, White JM (1992) Intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin. J Virol 66: 4940–4950.
[39]
Sauter NK, Hanson JE, Glick GD, Brown JH, Crowther RL, et al. (1992) Binding of influenza virus hemagglutinin to analogs of its cell-surface receptor, sialic acid: analysis by proton nuclear magnetic resonance spectroscopy and X-ray crystallography. Biochemistry 31: 9609–9621.
[40]
Bullough PA, Hughson FM, Skehel JJ, Wiley DC (1994) Structure of influenza haemagglutinin at the pH of membrane fusion. Nature 371: 37–43.
[41]
Moya A, Holmes EC, Gonzalez-Candelas F (2004) The population genetics and evolutionary epidemiology of RNA viruses. Nat Rev Microbiol 2: 279–288.
[42]
Li W, Zhang C, Sui J, Kuhn JH, Moore MJ, et al. (2005) Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. EMBO J 24: 1634–1643.
[43]
Li W, Wong SK, Li F, Kuhn JH, Huang IC, et al. (2006) Animal origins of the severe acute respiratory syndrome coronavirus: insight from ACE2-S-protein interactions. J Virol 80: 4211–4219.
[44]
Wentworth DE, Holmes KV (2001) Molecular determinants of species specificity in the coronavirus receptor aminopeptidase N (CD13): influence of N-linked glycosylation. J Virol 75: 9741–9752.
[45]
Ludtke SJ, Baldwin PR, Chiu W (1999) EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128: 82–97.
[46]
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, et al. (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25: 1605–1612.
