Structural basis for receptor recognition by New World hemorrhagic fever arenaviruses

Journal name:
Nature Structural & Molecular Biology
Volume:
17,
Pages:
438–444
Year published:
DOI:
doi:10.1038/nsmb.1772
Received
Accepted
Published online

Abstract

New World hemorrhagic fever arenaviruses are rodent-borne agents that cause severe human disease. The GP1 subunit of the surface glycoprotein mediates cell attachment through transferrin receptor 1 (TfR1). We report the structure of Machupo virus (MACV) GP1 bound with human TfR1. Atomic details of the GP1-TfR1 interface clarify the importance of TfR1 residues implicated in New World arenavirus host specificity. Analysis of sequence variation among New World arenavirus GP1s and their host-species receptors, in light of the molecular structure, indicates determinants of viral zoonotic transmission. Infectivities of pseudoviruses in cells expressing mutated TfR1 confirm that contacts at the tip of the TfR1 apical domain determine the capacity of human TfR1 to mediate infection by particular New World arenaviruses. We propose that New World arenaviruses that are pathogenic to humans fortuitously acquired affinity for human TfR1 during adaptation to TfR1 of their natural hosts.

At a glance

Figures

  1. Structure of the MACV GP1-human TfR1 complex.
    Figure 1: Structure of the MACV GP1–human TfR1 complex.

    (a) The butterfly-shaped TfR1 dimer is shown oriented with the cell surface at the bottom. One TfR1 protomer is colored according to the TfR1 domains: the apical domain is green, the protease-like domain is red and the helical domain is yellow. The other protomer is in cyan. MACV GP1, shown in purple, interacts with the TfR1 apical domain. The N-linked glycans are in gray. (b) Enlargement of the TfR1 apical domain with bound MACV GP1. Four N-linked glycans are on surfaces of the viral glycoprotein not involved in binding to TfR1. The N and C termini of MACV GP1 and the αII-2 helix and βII-2 strand of TfR1 are labeled.

  2. MACV GP1.
    Figure 2: MACV GP1.

    (a) Domain organization of the MACV envelope glycoprotein (GP). GP is processed during its maturation into three noncovalently associated subunits: the stable signal peptide (SSP), GP1 and GP2. Sites of potential N-linked glycosylation are shown as tree diagrams. The triangle indicates the subtilisin/kexin isozyme 1 (SKI-1) cleavage site. TM, transmembrane segment. (b) Sequence alignment of MACV GP1 residues 80–250 with the GP1 proteins of NW clade B arenaviruses JUNV, TCRV, GTOV, AMAV and SABV. Secondary structure elements are shown for Residues are colored according to the ClustalX color scheme. GP1 residues that interact with hTfR1 are labeled with asterisks colored according to the interaction motifs shown in Figure 3; green for motif 1, magenta for motif 2, red for motif 3, yellow for motif 4 and blue for motif 5. (c) Ribbon diagram of The first N-acetylglucosamine is shown for glycosylation sites at Asn95, Asn137, Asn166 and Asn178. The four disulfide bonds that rigidify the GP1 core are in pink. The conserved Cys92-Cys237 and Cys135-Cys164 disulfide bridges are labeled with asterisks. The three loops that protrude from the GP1 core to contact TfR1 (L3, L7 and L10) are in yellow. Note that L10 is much shorter in the other viruses and will not contact TfR1.

  3. Five interaction motifs in the MACV GP1-human TfR1 interface.
    Figure 3: Five interaction motifs in the MACV GP1-human TfR1 interface.

    (a) Motif 1, centered on Tyr211 on the βII-2 strand in the receptor. (b) Motif 2, centered on TfR1 Asn348, which caps the C terminus of αII-2 helix. (c) Motif 3, centered on TfR1 Val210 in the βII-2 strand of the receptor, on the side opposite Tyr211. A segment unique to MACV GP1 is in red, with the Cys220-Cys229 disulfide bond in yellow. (d) Motif 4, at the lateral base of the TfR1 apical domain. An extensive network of polar interactions radiates from TfR1 Lys344. (e) Motif 5 at the lateral tip of the apical domain, centered on TfR1 Leu209. TfR1 residues Asp204 and Arg208, also shown, have been implicated previously in the host specificity of NW arenaviruses18, 23. Asn95 is a conserved glycosylation site in New World arenavirus GP1s. The anchor residues for each motif are labeled with an asterisk. Hydrogen bonds are indicated as dashed lines.

  4. Conservation of the GP1-TfR1 interface.
    Figure 4: Conservation of the GP1-TfR1 interface.

    (a) Ribbon diagram of MACV GP1, as seen from the apical domain of TfR1 in the complex. GP1 residues that contact TfR1 are colored according to the interaction motifs shown in Figure 3, using the color scheme outlined in the caption to Figure 2b. (b) List of MACV GP1 residues that contact TfR1, colored according to the five interaction motifs. Residues found in analogous positions for the New World clade B arenaviruses JUNV, TCRV, GTOV, AMAV and SABV (based on the sequence alignment shown in Fig. 2b) are also shown. (c) Side-view ribbon diagram of the TfR1 apical domain. TfR1 residues that contact MACV GP1 are colored according to the five interaction motifs as in a. (d) List of hTfR1 residues that contact MACV GP1, colored according to the five interaction motifs. Residues found in analogous positions in C. callosus (MACV host), C. musculinus (JUNV host), A. jamaicensis (TCRV host), Z. brevicauda (GTOV host) and N. spinosus (AMAV host) TfR1 are listed based on sequence alignment (see Supplementary Fig. 3).

  5. Importance of the tip of the apical domain in determining the role of human TfR1 as a receptor for pathogenic New World arenaviruses.
    Figure 5: Importance of the tip of the apical domain in determining the role of human TfR1 as a receptor for pathogenic New World arenaviruses.

    (a) CHO cells were transfected with vector alone (mock) or plasmids encoding hTfR1 (human), or hTfR1 with the Arg208Gly mutation (R208G); 48 h later, the cells were treated with an anti-Flag antibody to measure cell surface expression of receptors (left), or with immunoglobulin (Ig) fusion proteins containing the truncated GP1 subunits (GP1Δ) of MACV, JUNV or GTOV or the receptor binding domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein (right). Association of these proteins with cells was measured by flow cytometry. (b) CHO cells were transfected with the indicated plasmids, and cell surface expression of the receptors (left) determined 48 h later with an anti-Flag antibody as in (a). Aliquots of these cells were infected in parallel with MACV, JUNV, GTOV or vesicular stomatitis virus (VSV) pseudoviruses expressing GFP. Infection levels were assessed 48 h later by measuring GFP expression by flow cytometry. Error bars indicate s.d.

Accession codes

Primary accessions

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Referenced accessions

Protein Data Bank

References

  1. Oldstone, M.B. Arenaviruses. I. The epidemiology molecular and cell biology of arenaviruses. Introduction. Curr. Top. Microbiol. Immunol. 262, VXII (2002).
  2. Bowen, M.D., Peters, C.J. & Nichol, S.T. The phylogeny of New World (Tacaribe complex) arenaviruses. Virology 219, 285290 (1996).
  3. Clegg, J.C. Molecular phylogeny of the arenaviruses. Curr. Top. Microbiol. Immunol. 262, 124 (2002).
  4. Emonet, S., Lemasson, J.J., Gonzalez, J.P., de Lamballerie, X. & Charrel, R.N. Phylogeny and evolution of old world arenaviruses. Virology 350, 251257 (2006).
  5. Charrel, R.N. & de Lamballerie, X. Arenaviruses other than Lassa virus. Antiviral Res. 57, 89100 (2003).
  6. Lisieux, T. et al. New arenavirus isolated in Brazil. Lancet 343, 391392 (1994).
  7. Tesh, R.B., Jahrling, P.B., Salas, R. & Shope, R.E. Description of Guanarito virus (Arenaviridae: Arenavirus), the etiologic agent of Venezuelan hemorrhagic fever. Am. J. Trop. Med. Hyg. 50, 452459 (1994).
  8. Delgado, S. et al. Chapare virus, a newly discovered arenavirus isolated from a fatal hemorrhagic fever case in Bolivia. PLoS Pathog. 4, e1000047 (2008).
  9. Buchmeier, M.J. Arenaviruses: protein structure and function. Curr. Top. Microbiol. Immunol. 262, 159173 (2002).
  10. Eichler, R. et al. Identification of Lassa virus glycoprotein signal peptide as a trans-acting maturation factor. EMBO Rep. 4, 10841088 (2003).
  11. Saunders, A.A. et al. Mapping the landscape of the lymphocytic choriomeningitis virus stable signal peptide reveals novel functional domains. J. Virol. 81, 56495657 (2007).
  12. York, J. & Nunberg, J.H. Role of the stable signal peptide of Junin arenavirus envelope glycoprotein in pH-dependent membrane fusion. J. Virol. 80, 77757780 (2006).
  13. York, J. & Nunberg, J.H. Distinct requirements for signal peptidase processing and function in the stable signal peptide subunit of the Junin virus envelope glycoprotein. Virology 359, 7281 (2007).
  14. Harrison, S.C. Viral membrane fusion. Nat. Struct. Mol. Biol. 15, 690698 (2008).
  15. Rojek, J.M. & Kunz, S. Cell entry by human pathogenic arenaviruses. Cell. Microbiol. 10, 828835 (2008).
  16. York, J., Agnihothram, S.S., Romanowski, V. & Nunberg, J.H. Genetic analysis of heptad-repeat regions in the G2 fusion subunit of the Junin arenavirus envelope glycoprotein. Virology 343, 267274 (2005).
  17. Radoshitzky, S.R. et al. Transferrin receptor 1 is a cellular receptor for New World haemorrhagic fever arenaviruses. Nature 446, 9296 (2007).
  18. Radoshitzky, S.R. et al. Receptor determinants of zoonotic transmission of New World hemorrhagic fever arenaviruses. Proc. Natl. Acad. Sci. USA 105, 26642669 (2008).
  19. Flanagan, M.L. et al. New World clade B arenaviruses can use transferrin receptor 1 (TfR1)-dependent and independent entry pathways, and glycoproteins from human pathogenic strains are associated with the use of TfR1. J. Virol. 82, 938948 (2008).
  20. Aisen, P. Transferrin receptor 1. Int. J. Biochem. Cell Biol. 36, 21372143 (2004).
  21. Lawrence, C.M. et al. Crystal structure of the ectodomain of human transferrin receptor. Science 286, 779782 (1999).
  22. Cheng, Y., Zak, O., Aisen, P., Harrison, S.C. & Walz, T. Structure of the human transferrin receptor-transferrin complex. Cell 116, 565576 (2004).
  23. Abraham, J. et al. Host-species transferrin receptor 1 orthologs are cellular receptors for nonpathogenic new world clade B arenaviruses. PLoS Pathog. 5, e1000358 (2009).
  24. Bowen, M.D., Peters, C.J. & Nichol, S.T. Phylogenetic analysis of the Arenaviridae: patterns of virus evolution and evidence for cospeciation between arenaviruses and their rodent hosts. Mol. Phylogenet. Evol. 8, 301316 (1997).
  25. Bennett, M.J., Lebron, J.A. & Bjorkman, P.J. Crystal structure of the hereditary haemochromatosis protein HFE complexed with transferrin receptor. Nature 403, 4653 (2000).
  26. Bowden, T.A. et al. Unusual molecular architecture of the machupo virus attachment glycoprotein. J. Virol. 83, 82598265 (2009).
  27. Mothes, W., Boerger, A.L., Narayan, S., Cunningham, J.M. & Young, J.A. Retroviral entry mediated by receptor priming and low pH triggering of an envelope glycoprotein. Cell 103, 679689 (2000).
  28. Smith, J.G., Mothes, W., Blacklow, S.C. & Cunningham, J.M. The mature avian leukosis virus subgroup A envelope glycoprotein is metastable, and refolding induced by the synergistic effects of receptor binding and low pH is coupled to infection. J. Virol. 78, 14031410 (2004).
  29. Strong, M. et al. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis . Proc. Natl. Acad. Sci. USA 103, 80608065 (2006).
  30. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658674 (2007).
  31. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 21262132 (2004).
  32. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 19481954 (2002).
  33. Brunger, A.T. Version 1.2 of the Crystallography and NMR system. Nat. Protoc. 2, 27282733 (2007).
  34. DeLano, W.L. The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, California, USA, 2002).
  35. Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 46734680 (1994).
  36. Clamp, M., Cuff, J., Searle, S.M. & Barton, G.J. The Jalview Java alignment editor. Bioinformatics 20, 426427 (2004).
  37. Waterhouse, A.M., Procter, J.B., Martin, D.M., Clamp, M. & Barton, G.J. Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 11891191 (2009).
  38. Li, W. et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450454 (2003).

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Author information

Affiliations

  1. Laboratory of Molecular Medicine, Harvard Medical School, Boston, Massachusetts, USA.

    • Jonathan Abraham &
    • Stephen C Harrison
  2. Department of Medicine, Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • Jonathan Abraham &
    • Hyeryun Choe
  3. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

    • Kevin D Corbett &
    • Stephen C Harrison
  4. Department of Microbiology and Molecular Genetics, Harvard Medical School, New England Primate Center, Southborough, Massachusetts, USA.

    • Michael Farzan
  5. Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA.

    • Stephen C Harrison

Contributions

J.A. designed and performed the experiments, analyzed the data and wrote the paper; K.D.C. assisted with data collection, molecular replacement, re-interpretation of the unliganded TfR1 structures and edited the paper; M.F. and H.C. assisted with data analysis and interpretation and edited the paper; S.C.H. helped design experiments, advised on model building and interpretation and participated in writing and editing the paper.

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The authors declare no competing financial interests.

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