The hepatitis B virus preS1 domain hijacks host trafficking proteins by motif mimicry

Abstract

Hepatitis B virus (HBV) is an infectious, potentially lethal human pathogen. However, there are no effective therapies for chronic HBV infections. Antiviral development is hampered by the lack of high-resolution structures for essential HBV protein-protein interactions. The interaction between preS1, an HBV surface-protein domain, and its human binding partner, γ2-adaptin, subverts the membrane-trafficking apparatus to mediate virion export. This interaction is a putative drug target. We report here atomic-resolution descriptions of the binding thermodynamics and structural biology of the interaction between preS1 and the EAR domain of γ2-adaptin. NMR, protein engineering, X-ray crystallography and MS showed that preS1 contains multiple γ2-EAR–binding motifs that mimic the membrane-trafficking motifs (and binding modes) of host proteins. These motifs localize together to a relatively rigid, functionally important region of preS1, an intrinsically disordered protein. The preS1–γ2-EAR interaction was relatively weak and efficiently outcompeted by a synthetic peptide. Our data provide the structural road map for developing peptidomimetic antivirals targeting the γ2-EAR–preS1 interaction.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Domain structure and topology for HBV surface proteins and γ2-adaptin.
Figure 2: Mapping the γ2-EAR–preS1 interaction using NMR spectroscopy and ITC.
Figure 3: CSPs induced in γ2-EAR amide resonances by binding to preS1 and preS1-derived peptides.
Figure 4: Crystal structures of wild-type γ2-EAR in complex with peptide ligands.
Figure 5: Backbone dynamics and conformational diversity of preS1.
Figure 6: Models of γ2-adaptin interactions leading to envelopment of HBV nucleocapsids.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. 1

    Beck, J. & Nassal, M. Hepatitis B virus replication. World J. Gastroenterol. 13, 48–64 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Bruss, V. Envelopment of the hepatitis B virus nucleocapsid. Virus Res. 106, 199–209 (2004).

    CAS  PubMed  Google Scholar 

  3. 3

    Nassal, M. Hepatitis B virus replication: novel roles for virus-host interactions. Intervirology 42, 100–116 (1999).

    CAS  PubMed  Google Scholar 

  4. 4

    Steven, A.C. et al. Structure, assembly, and antigenicity of hepatitis B virus capsid proteins. Adv. Virus Res. 64, 125–164 (2005).

    CAS  PubMed  Google Scholar 

  5. 5

    Glebe, D. & Urban, S. Viral and cellular determinants involved in hepadnaviral entry. World J. Gastroenterol. 13, 22–38 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Roseman, A.M., Berriman, J.A., Wynne, S.A., Butler, P.J.G. & Crowther, R.A. A structural model for maturation of the hepatitis B virus core. Proc. Natl. Acad. Sci. USA 102, 15821–15826 (2005).

    CAS  PubMed  Google Scholar 

  7. 7

    Seitz, S., Urban, S., Antoni, C. & Böttcher, B. Cryo-electron microscopy of hepatitis B virions reveals variability in envelope capsid interactions. EMBO J. 26, 4160–4167 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Wynne, S.A., Crowther, R.A. & Leslie, A.G. The crystal structure of the human hepatitis B virus capsid. Mol. Cell 3, 771–780 (1999).

    CAS  PubMed  Google Scholar 

  9. 9

    Awe, K., Lambert, C. & Prange, R. Mammalian BiP controls posttranslational ER translocation of the hepatitis B virus large envelope protein. FEBS Lett. 582, 3179–3184 (2008).

    CAS  PubMed  Google Scholar 

  10. 10

    Yan, H. et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 2012, e00049 (2012).

    Google Scholar 

  11. 11

    Prange, R. Host factors involved in hepatitis B virus maturation, assembly, and egress. Med. Microbiol. Immunol. (Berl.) 201, 449–461 (2012).

    CAS  Google Scholar 

  12. 12

    Hartmann-Stühler, C. & Prange, R. Hepatitis B virus large envelope protein interacts with γ2-adaptin, a clathrin adaptor-related protein. J. Virol. 75, 5343–5351 (2001).

    PubMed  PubMed Central  Google Scholar 

  13. 13

    Döring, T., Gotthardt, K., Stieler, J. & Prange, R. γ2-adaptin is functioning in the late endosomal sorting pathway and interacts with ESCRT-I and -III subunits. Biochim. Biophys. Acta 1803, 1252–1264 (2010).

    PubMed  Google Scholar 

  14. 14

    Boehm, M. & Bonifacino, J.S. Adaptins: the final recount. Mol. Biol. Cell 12, 2907–2920 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Lewin, D.A. et al. Cloning, expression, and localization of a novel γ-adaptin–like molecule. FEBS Lett. 435, 263–268 (1998).

    CAS  PubMed  Google Scholar 

  16. 16

    Rost, M., Döring, T. & Prange, R. γ2-Adaptin, a ubiquitin-interacting adaptor, is a substrate to coupled ubiquitination by the ubiquitin ligase Nedd4 and functions in the endosomal pathway. J. Biol. Chem. 283, 32119–32130 (2008).

    CAS  PubMed  Google Scholar 

  17. 17

    Takatsu, H., Sakurai, M., Shin, H.W., Murakami, K. & Nakayama, K. Identification and characterization of novel clathrin adaptor-related proteins. J. Biol. Chem. 273, 24693–24700 (1998).

    CAS  PubMed  Google Scholar 

  18. 18

    Zizioli, D. et al. Early embryonic death of mice deficient in γ-adaptin. J. Biol. Chem. 274, 5385–5390 (1999).

    CAS  PubMed  Google Scholar 

  19. 19

    Lambert, C., Döring, T. & Prange, R. Hepatitis B virus maturation is sensitive to functional inhibition of ESCRT-III, Vps4, and γ2-adaptin. J. Virol. 81, 9050–9060 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Rost, M. et al. γ-adaptin, a novel ubiquitin-interacting adaptor, and Nedd4 ubiquitin ligase control hepatitis B virus maturation. J. Biol. Chem. 281, 29297–29308 (2006).

    CAS  PubMed  Google Scholar 

  21. 21

    Chi, S.W., Kim, D., Lee, S., Chang, I. & Han, K. Pre-structured motifs in the natively unstructured preS1 surface antigen of hepatitis B virus. Protein Sci. 16, 2108–2117 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Tompa, P. & Fuxreiter, M. Fuzzy complexes: polymorphism and structural disorder in protein-protein interactions. Trends Biochem. Sci. 33, 2–8 (2008).

    CAS  PubMed  Google Scholar 

  23. 23

    Dunker, A.K. et al. The unfoldomics decade: an update on intrinsically disordered proteins. BMC Genomics 9 (suppl. 2), S1 (2008).

    PubMed  PubMed Central  Google Scholar 

  24. 24

    Mattera, R., Ritter, B., Sidhu, S.S., McPherson, P.S. & Bonifacino, J.S. Definition of the consensus motif recognized by γ-adaptin ear domains. J. Biol. Chem. 279, 8018–8028 (2004).

    CAS  PubMed  Google Scholar 

  25. 25

    Kent, H.M., McMahon, H.T., Evans, P.R., Benmerah, A. & Owen, D.J. γ-adaptin appendage domain: structure and binding site for Eps15 and γ-synergin. Structure 10, 1139–1148 (2002).

    CAS  PubMed  Google Scholar 

  26. 26

    Miller, G.J., Mattera, R., Bonifacino, J.S. & Hurley, J.H. Recognition of accessory protein motifs by the γ-adaptin ear domain of GGA3. Nat. Struct. Biol. 10, 599–606 (2003).

    CAS  PubMed  Google Scholar 

  27. 27

    Wishart, D.S. & Sykes, B.D. Chemical shifts as a tool for structure determination. Methods Enzymol. 239, 363–392 (1994).

    CAS  PubMed  Google Scholar 

  28. 28

    Turnbull, W.B. & Daranas, A.H. On the value of c: can low affinity systems be studied by isothermal titration calorimetry? J. Am. Chem. Soc. 125, 14859–14866 (2003).

    CAS  PubMed  Google Scholar 

  29. 29

    Inoue, M. et al. Molecular basis for autoregulatory interaction between GAE domain and hinge region of GGA1. Traffic 8, 904–913 (2007).

    CAS  PubMed  Google Scholar 

  30. 30

    Mills, I.G. et al. EpsinR: an AP1/clathrin interacting protein involved in vesicle trafficking. J. Cell Biol. 160, 213–222 (2003).

    PubMed  PubMed Central  Google Scholar 

  31. 31

    Yamada, Y., Shiba, T., Kato, R. & Nakayama, K. Structure determination of GGA-GAE and γ1-ear in complex with peptides: crystallization of low-affinity complexes in membrane traffic. Acta Crystallogr. D Biol. Crystallogr. 61, 731–736 (2005).

    PubMed  Google Scholar 

  32. 32

    Mészáros, B., Simon, I. & Dosztányi, Z. Prediction of protein binding regions in disordered proteins. PLoS Comput. Biol. 5, e1000376 (2009).

    PubMed  PubMed Central  Google Scholar 

  33. 33

    Mooney, C., Pollastri, G., Shields, D.C. & Haslam, N.J. Prediction of short linear protein binding regions. J. Mol. Biol. 415, 193–204 (2012).

    CAS  PubMed  Google Scholar 

  34. 34

    Ferguson, N., Sharpe, T.D., Johnson, C.M., Schartau, P.J. & Fersht, A.R. Analysis of “downhill” protein folding. Nature 445, E14–E15 (2007).

    CAS  PubMed  Google Scholar 

  35. 35

    Reddy, T. & Rainey, J.K. Interpretation of biomolecular NMR spin relaxation parameters. Biochem. Cell Biol. 88, 131–142 (2010).

    CAS  PubMed  Google Scholar 

  36. 36

    Smith, D.P. et al. Deciphering drift time measurements from travelling wave ion mobility spectrometry–mass spectrometry studies. Eur. J. Mass Spectrom. (Chichester, Eng.) 15, 113–130 (2009).

    CAS  Google Scholar 

  37. 37

    Smith, D.P., Giles, K., Bateman, R.H., Radford, S.E. & Ashcroft, A.E. Monitoring copopulated conformational states during protein folding events using electrospray ionization–ion mobility spectrometry–mass spectrometry. J. Am. Soc. Mass Spectrom. 18, 2180–2190 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Clemmer, D.E., Hudgins, R.R. & Jarrold, M.F. Naked protein conformations: cytochrome c in the gas phase. J. Am. Chem. Soc. 117, 10141–10142 (1995).

    CAS  Google Scholar 

  39. 39

    Ruotolo, B.T. & Robinson, C.V. Aspects of native proteins are retained in vacuum. Curr. Opin. Chem. Biol. 10, 402–408 (2006).

    CAS  PubMed  Google Scholar 

  40. 40

    Saikusa, K. et al. Characterisation of an intrinsically disordered protein complex of Swi5-Sfr1 by ion mobility mass spectrometry and small-angle X-ray scattering. Analyst 138, 1441–1449 (2013).

    CAS  PubMed  Google Scholar 

  41. 41

    Pagel, K., Natan, E., Hall, Z., Fersht, A.R. & Robinson, C.V. Intrinsically disordered p53 and its complexes populate compact conformations in the gas phase. Angew. Chem. Int. Edn. Engl. 52, 361–365 (2013).

    CAS  Google Scholar 

  42. 42

    Davey, N.E., Travé, G. & Gibson, T.J. How viruses hijack cell regulation. Trends Biochem. Sci. 36, 159–169 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Maritzen, T. et al. A novel subtype of AP-1-binding motif within the palmitoylated trans-Golgi network/endosomal accessory protein Gadkin/γ-BAR. J. Biol. Chem. 285, 4074–4086 (2010).

    CAS  PubMed  Google Scholar 

  44. 44

    Schulze, A., Schieck, A., Ni, Y., Mier, W. & Urban, S. Fine mapping of pre-S sequence requirements for hepatitis B virus large envelope protein–mediated receptor interaction. J. Virol. 84, 1989–2000 (2010).

    CAS  PubMed  Google Scholar 

  45. 45

    Le Seyec, J., Chouteau, P., Cannie, I., Gripon, P. & Guguen-Guillouzo, C. Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain. J. Virol. 73, 2052–2057 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Bruss, V. & Thomssen, R. Mapping a region of the large envelope protein required for hepatitis B virion maturation. J. Virol. 68, 1643–1650 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Böttcher, B. et al. Peptides that block hepatitis B virus assembly: analysis by cryomicroscopy, mutagenesis and transfection. EMBO J. 17, 6839–6845 (1998).

    PubMed  PubMed Central  Google Scholar 

  48. 48

    Freund, S.M.V., Johnson, C.M., Jaulent, A.M. & Ferguson, N. Moving towards high-resolution descriptions of the molecular interactions and structural rearrangements of the human hepatitis B core protein. J. Mol. Biol. 384, 1301–1313 (2008).

    CAS  PubMed  Google Scholar 

  49. 49

    Nogi, T. et al. Structural basis for the accessory protein recruitment by the γ-adaptin ear domain. Nat. Struct. Biol. 9, 527–531 (2002).

    CAS  PubMed  Google Scholar 

  50. 50

    Sattler, M., Schleucher, J. & Griesinger, C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Magn. Reson. Spectrosc. 34, 93–158 (1999).

    CAS  Google Scholar 

  51. 51

    Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    Google Scholar 

  53. 53

    Long, F., Vagin, A.A., Young, P. & Murshudov, G.N. BALBES: a molecular-replacement pipeline. Acta Crystallogr. D Biol. Crystallogr. 64, 125–132 (2008).

    CAS  PubMed  Google Scholar 

  54. 54

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

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Langer, G., Cohen, S.X., Lamzin, V.S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc. 3, 1171–1179 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    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 

  58. 58

    Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

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

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Ruotolo, B.T., Benesch, J.L.P., Sandercock, A.M., Hyung, S.-J. & Robinson, C.V. Ion mobility–mass spectrometry analysis of large protein complexes. Nat. Protoc. 3, 1139–1152 (2008).

    CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank K. Nakayama (University of Kyoto) for the γ2-adaptin clone; J. Lyons (Trinity College Dublin) and Deutsches Elektronen-Synchrotron–European Molecular Biology Laboratory beamline scientists for assistance with X-ray data collection; N. Moran (Royal College of Surgeons in Ireland) for access to biophysical instrumentation; M. Caffrey (Trinity College Dublin) for access to X-ray apparatus; and D. Shields and C. Mooney for bioinformatics help. This work was supported by Science Foundation Ireland (SFI)–President of Ireland Young Researcher Award (09/YI/B1682 to N.F.), Stokes Lecturer Award (07/SK/B1224a to N.F.), Royal Irish Academy–Royal Society Exchange award (IE111031 to N.F. and A.E.A.), SFI grant (07/IN.1/B1836) and US National Institutes of Health grants (GM75915, P50GM073210 U54GM094599) to M. Caffrey (funding V.E.P. and X-ray generator). D.A.S. was funded by an Engineering and Physical Sciences Research Council PhD Studentship. The Synapt mass spectrometer was purchased with Biotechnology and Biological Sciences Research Council UK funds (BB/E012558/1).

Author information

Affiliations

Authors

Contributions

M.C.J. purified proteins, performed ITC and NMR experiments, determined crystal structures, analyzed data and wrote the paper; J.V. purified proteins, performed ITC and NMR experiments, analyzed data and wrote the paper; G.J.P.R. performed and analyzed NMR experiments and wrote the paper; J.M. performed NMR experiments; D.A.S. performed and analyzed MS experiments and wrote the paper; V.E.P. determined crystal structures and wrote the paper; C.M.J. performed and analyzed calorimetry experiments; A.E.A. designed and interpreted MS experiments and wrote the paper; S.M.V.F. performed and analyzed NMR experiments and wrote the paper; N.F. directed research, designed and interpreted experiments and wrote the paper.

Corresponding author

Correspondence to Neil Ferguson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–6 and Supplementary Tables 1–2. (PDF 4090 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Jürgens, M., Vörös, J., Rautureau, G. et al. The hepatitis B virus preS1 domain hijacks host trafficking proteins by motif mimicry. Nat Chem Biol 9, 540–547 (2013). https://doi.org/10.1038/nchembio.1294

Download citation

Further reading

Search

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