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:

Molecular mechanism of respiratory syncytial virus fusion inhibitors

Nature Chemical Biology volume 12, pages 87–93 (2016)Cite this article

Subjects

Abstract

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors.

Access through your institution
Buy or subscribe

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

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Inhibitors bind to a three-fold-symmetric cavity in prefusion RSV F.
Figure 2: Inhibitors tether hydrophobic residues in two structurally labile regions.
Figure 3: RSV F rearrangements required for inhibitor binding are prevented by the D489Y resistance mutation.
Figure 4: Inhibitors stabilize prefusion RSV F.
Figure 5: Effects of inhibitor-escape mutations on cell-cell fusion activity and viral fitness.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Nair, H. et al. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet 375, 1545–1555 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Lozano, R. et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380, 2095–2128 (2012).

    Article  PubMed  Google Scholar 

  3. Falsey, A.R., Hennessey, P.A., Formica, M.A., Cox, C. & Walsh, E.E. Respiratory syncytial virus infection in elderly and high-risk adults. N. Engl. J. Med. 352, 1749–1759 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Anderson, L.J. et al. Strategic priorities for respiratory syncytial virus (RSV) vaccine development. Vaccine 31 (suppl. 2): B209–B215 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  5. The IMpact-RSV Study Group. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. Pediatrics 102, 531–537 (1998).

  6. Smart, K.A., Lanctôt, K.L. & Paes, B.A. The cost effectiveness of palivizumab: a systematic review of the evidence. J. Med. Econ. 13, 453–463 (2010).

    Article  PubMed  Google Scholar 

  7. McLellan, J.S., Ray, W.C. & Peeples, M.E. Structure and function of respiratory syncytial virus surface glycoproteins. Curr. Top. Microbiol. Immunol. 372, 83–104 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Karron, R.A. et al. Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant. Proc. Natl. Acad. Sci. USA 94, 13961–13966 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Walsh, E.E. & Hruska, J. Monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion protein. J. Virol. 47, 171–177 (1983).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. McLellan, J.S. et al. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science 340, 1113–1117 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Liljeroos, L., Krzyzaniak, M.A., Helenius, A. & Butcher, S.J. Architecture of respiratory syncytial virus revealed by electron cryotomography. Proc. Natl. Acad. Sci. USA 110, 11133–11138 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yunus, A.S. et al. Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation. Virology 396, 226–237 (2010).

    Article  CAS  PubMed  Google Scholar 

  13. Lamb, R.A. & Jardetzky, T.S. Structural basis of viral invasion: lessons from paramyxovirus F. Curr. Opin. Struct. Biol. 17, 427–436 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. McLellan, J.S., Yang, Y., Graham, B.S. & Kwong, P.D. Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. J. Virol. 85, 7788–7796 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zhao, X., Singh, M., Malashkevich, V.N. & Kim, P.S. Structural characterization of the human respiratory syncytial virus fusion protein core. Proc. Natl. Acad. Sci. USA 97, 14172–14177 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Cianci, C. et al. Orally active fusion inhibitor of respiratory syncytial virus. Antimicrob. Agents Chemother. 48, 413–422 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Andries, K. et al. Substituted benzimidazoles with nanomolar activity against respiratory syncytial virus. Antiviral Res. 60, 209–219 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Douglas, J.L. et al. Inhibition of respiratory syncytial virus fusion by the small molecule VP-14637 via specific interactions with F protein. J. Virol. 77, 5054–5064 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bonfanti, J.F. et al. Selection of a respiratory syncytial virus fusion inhibitor clinical candidate. 2. Discovery of a morpholinopropylaminobenzimidazole derivative (TMC353121). J. Med. Chem. 51, 875–896 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Roymans, D. & Koul, A. in Human Respiratory Syncytial Virus Infection (ed. Resch, B.) 197–234 (InTech, 2011).

  21. DeVincenzo, J.P. et al. Oral GS-5806 activity in a respiratory syncytial virus challenge study. N. Engl. J. Med. 371, 711–722 (2014).

    Article  PubMed  Google Scholar 

  22. Roymans, D. et al. Binding of a potent small-molecule inhibitor of six-helix bundle formation requires interactions with both heptad-repeats of the RSV fusion protein. Proc. Natl. Acad. Sci. USA 107, 308–313 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Douglas, J.L. et al. Small molecules VP-14637 and JNJ-2408068 inhibit respiratory syncytial virus fusion by similar mechanisms. Antimicrob. Agents Chemother. 49, 2460–2466 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cianci, C. et al. Targeting a binding pocket within the trimer-of-hairpins: small-molecule inhibition of viral fusion. Proc. Natl. Acad. Sci. USA 101, 15046–15051 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Yan, D. et al. Cross-resistance mechanism of respiratory syncytial virus against structurally diverse entry inhibitors. Proc. Natl. Acad. Sci. USA 111, E3441–E3449 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Hallak, L.K., Collins, P.L., Knudson, W. & Peeples, M.E. Iduronic acid-containing glycosaminoglycans on target cells are required for efficient respiratory syncytial virus infection. Virology 271, 264–275 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Morton, C.J. et al. Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay. Virology 311, 275–288 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Krarup, A. et al. A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism. Nat. Commun. 6, 8143 (2015).

    Article  PubMed  Google Scholar 

  29. Mackman, R.L. et al. Discovery of an oral respiratory syncytial virus (RSV) fusion inhibitor (GS-5806) and clinical proof of concept in a human RSV challenge study. J. Med. Chem. 58, 1630–1643 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Samuel, D. et al. GS-5806 inhibits pre- to post-fusion conformational changes of the RSV fusion protein. Antimicrob. Agents Chemother. 59, 7109–7112 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hotard, A.L. et al. Identification of residues in the human respiratory syncytial virus fusion protein that modulate fusion activity and pathogenesis. J. Virol. 89, 512–522 (2015).

    Article  PubMed  Google Scholar 

  32. Madani, N. et al. Small-molecule CD4 mimics interact with a highly conserved pocket on HIV-1 gp120. Structure 16, 1689–1701 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. LaLonde, J.M. et al. Structure-based design, synthesis, and characterization of dual hotspot small-molecule HIV-1 entry inhibitors. J. Med. Chem. 55, 4382–4396 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Acharya, P. et al. Structure-based identification and neutralization mechanism of tyrosine sulfate mimetics that inhibit HIV-1 entry. ACS Chem. Biol. 6, 1069–1077 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lin, P.F. et al. A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding. Proc. Natl. Acad. Sci. USA 100, 11013–11018 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Herschhorn, A. et al. A broad HIV-1 inhibitor blocks envelope glycoprotein transitions critical for entry. Nat. Chem. Biol. 10, 845–852 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Si, Z. et al. Small-molecule inhibitors of HIV-1 entry block receptor-induced conformational changes in the viral envelope glycoproteins. Proc. Natl. Acad. Sci. USA 101, 5036–5041 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Russell, R.J. et al. Structure of influenza hemagglutinin in complex with an inhibitor of membrane fusion. Proc. Natl. Acad. Sci. USA 105, 17736–17741 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Luo, G. et al. Molecular mechanism underlying the action of a novel fusion inhibitor of influenza A virus. J. Virol. 71, 4062–4070 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Bodian, D.L. et al. Inhibition of the fusion-inducing conformational change of influenza hemagglutinin by benzoquinones and hydroquinones. Biochemistry 32, 2967–2978 (1993).

    Article  CAS  PubMed  Google Scholar 

  41. Leneva, I.A., Russell, R.J., Boriskin, Y.S. & Hay, A.J. Characteristics of arbidol-resistant mutants of influenza virus: implications for the mechanism of anti-influenza action of arbidol. Antiviral Res. 81, 132–140 (2009).

    Article  CAS  PubMed  Google Scholar 

  42. Battye, T.G., Kontogiannis, L., Johnson, O., Powell, H.R. & Leslie, A.G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Evans, P.R. & Murshudov, G.N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  46. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Terwilliger, T.C., Adams, P.D., Moriarty, N.W. & Cohn, J.D. Ligand identification using electron-density map correlations. Acta Crystallogr. D Biol. Crystallogr. 63, 101–107 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Branigan, P.J. et al. Use of a novel cell-based fusion reporter assay to explore the host range of human respiratory syncytial virus F protein. Virol. J. 2, 54 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Cornelissen, F., Cik, M. & Gustin, E. Phaedra, a protocol-driven system for analysis and validation of high-content imaging and flow cytometry. J. Biomol. Screen. 17, 496–506 (2012).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank members of the McLellan lab and the Janssen Infectious Disease & Vaccines labs for comments on the manuscript, A. Draffan and C. Morton at Biota for BTA-9881 and BMS-433771, and E. Shipman for assistance with protein expression and purification. We thank P. Raboisson, S. Vendeville and J.-F. Bonfanti for design and assistance during synthesis of compounds, D. Wuyts for help in generating high-content imaging data, M. Van Ginderen and N. Verheyen for identification of mutant viruses, and I. Bisschop and R. Voorzaat for technical support. We thank C. Ralston, D. Bryant and members of the Berkeley Center for Structural Biology for help with X-ray diffraction data collection. The Berkeley Center for Structural Biology is supported in part by the US National Institutes of Health, National Institute of General Medical Studies, and Howard Hughes Medical Institute. Results shown in this report were also derived from work performed at Argonne National Laboratory, Structural Biology Center (SBC) at the Advanced Photon Source. We thank S. Ginell, J. Lazarz, M. Ficner-Radford and the beamline scientists for data collection support at SBC 19-ID. Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. This work was supported by the Janssen Infectious Diseases & Vaccines group, NIH grant AI 095684 (M.E.P.), and the Charles H. Hood Foundation, Inc., Boston, Massachusetts, USA (J.S.M.).

Author information

Authors and Affiliations

  1. Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA

    Michael B Battles & Jason S McLellan

  2. Janssen Infectious Diseases and Vaccines, Leiden, the Netherlands

    Johannes P Langedijk & Polina Furmanova-Hollenstein

  3. Center for Vaccines & Immunity, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, USA

    Supranee Chaiwatpongsakorn, Heather M Costello & Mark E Peeples

  4. Respiratory Infections Research, Janssen Infectious Diseases & Vaccines BVBA, Beerse, Belgium

    Leen Kwanten, Luc Vranckx, Anil Koul & Dirk Roymans

  5. Discovery Sciences, Janssen Pharmaceutica NV, Beerse, Belgium

    Paul Vink & Steffen Jaensch

  6. Medicinal Chemistry Department, Janssen Infectious Diseases & Vaccines BVBA, Beerse, Belgium

    Tim H M Jonckers

  7. Computational Chemistry, Janssen R&D LLC, Spring House, Pennsylvania, USA

    Eric Arnoult

  8. Department of Pediatrics, The Ohio State University College of Medicine, Columbus, Ohio, USA

    Mark E Peeples

Authors
  1. Michael B Battles
    View author publications

    Search author on:PubMed Google Scholar

  2. Johannes P Langedijk
    View author publications

    Search author on:PubMed Google Scholar

  3. Polina Furmanova-Hollenstein
    View author publications

    Search author on:PubMed Google Scholar

  4. Supranee Chaiwatpongsakorn
    View author publications

    Search author on:PubMed Google Scholar

  5. Heather M Costello
    View author publications

    Search author on:PubMed Google Scholar

  6. Leen Kwanten
    View author publications

    Search author on:PubMed Google Scholar

  7. Luc Vranckx
    View author publications

    Search author on:PubMed Google Scholar

  8. Paul Vink
    View author publications

    Search author on:PubMed Google Scholar

  9. Steffen Jaensch
    View author publications

    Search author on:PubMed Google Scholar

  10. Tim H M Jonckers
    View author publications

    Search author on:PubMed Google Scholar

  11. Anil Koul
    View author publications

    Search author on:PubMed Google Scholar

  12. Eric Arnoult
    View author publications

    Search author on:PubMed Google Scholar

  13. Mark E Peeples
    View author publications

    Search author on:PubMed Google Scholar

  14. Dirk Roymans
    View author publications

    Search author on:PubMed Google Scholar

  15. Jason S McLellan
    View author publications

    Search author on:PubMed Google Scholar

Contributions

M.B.B. crystallized inhibitor complexes, processed and refined X-ray data, and performed ITC experiments; P.F.-H. performed the triggering assay; S.C. and H.M.C. performed the cell-cell fusion assays; L.K. measured antiviral activity of compounds against wild-type and escape viruses; L.V. performed the time-lapse, high-content imaging of RSV-infected cell cultures; D.R., P.V. and S.J. designed the time-lapse, high-content imaging of RSV-infected cell cultures assay, developed software scripts for endpoint quantification and analyzed the data; T.H.M.J. contributed to the design, synthesis and selection of JNJ-49153390. D.R. and A.K. designed antiviral assays and assisted with data analysis; E.A. generated 2D ligand-interaction diagrams and docked GS-5806 into the JNJ-49155390-bound RSV F structure; J.P.L., M.E.P., D.R., and J.S.M. designed the study, analyzed data and, along with M.B.B., wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Jason S McLellan.

Ethics declarations

Competing interests

These studies were funded in part by Janssen. All authors except M.B.B., S.C., H.M.C., M.E.P. and J.S.M. are employees at Janssen. T.H.M.J. is an inventor on patent WO2012080446.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–8 and Supplementary Tables 1 and 2. (PDF 14137 kb)

Conformational rearrangements in RSV F required for inhibitor binding. (MPG 6918 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Battles, M., Langedijk, J., Furmanova-Hollenstein, P. et al. Molecular mechanism of respiratory syncytial virus fusion inhibitors. Nat Chem Biol 12, 87–93 (2016). https://doi.org/10.1038/nchembio.1982

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/nchembio.1982

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