Abstract
Arenaviruses exist worldwide and can cause hemorrhagic fever and neurologic disease. A single glycoprotein expressed on the viral surface mediates entry into target cells. This glycoprotein, termed GPC, contains a membrane-associated signal peptide, a receptor-binding subunit termed GP1 and a fusion-mediating subunit termed GP2. Although GPC is a critical target of antibodies and vaccines, the structure of the metastable GP1–GP2 prefusion complex has remained elusive for all arenaviruses. Here we describe the crystal structure of the fully glycosylated prefusion GP1–GP2 complex of the prototypic arenavirus LCMV at 3.5 Å. This structure reveals the conformational changes that the arenavirus glycoprotein must undergo to cause fusion and illustrates the fusion regions and potential oligomeric states.
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Acknowledgements
The authors wish to acknowledge the Viral Hemorrhagic Fever Research Consortium and US National Institutes of Health grant 1U19AI109762-01 (E.O.S., J.E.R. and R.F.G.), US National Institutes of Health grant R21 AI116112 (E.O.S.), an Investigators in Pathogenesis of Infectious Diseases award from the Burroughs Wellcome Fund (E.O.S.) and US National Institutes of Health grants AI009484 (M.B.O.) and A1099699 (M.B.O.) for funding; X. Dai for assistance with data processing; M. Buchmeier (University of California, Irvine) for the anti-LCMV GP1 (KL25) antibody; S. Whelan (Harvard Medical School) for LAMP1-knockout cells; and C. Corbaci for assistance in creating figures. The authors also acknowledge beamlines 11-1, 12-1 and 12-2 of the Stanford Synchrotron Radiation Lightsource; 5.0.2, 5.0.3 and 8.2.2 of the Advanced Light Source; 19-ID, 23-ID-B and 23-ID-D of the Advanced Photon Source; and SOLEIL Proxima 1 (Gif-sur-Yvette, France) for data collection. This is manuscript #29106 from The Scripps Research Institute.
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K.M.H. built and refined the model, cloned the constructs for biochemical characterization, performed the receptor binding experiments, analyzed the data and wrote the manuscript; S.I. cloned the constructs for crystallization, produced and crystallized the recombinant GP ectodomains, collected diffraction data at room temperature, phased the data and determined the structure; B.M.S. grew the recombinant viruses, designed and performed the receptor binding experiments and analyzed the data; P.L. collected diffraction data on frozen crystals and built and refined the model; M.A.Z. cloned the constructs and produced recombinant protein; J.E.R. and R.F.G. generated and produced antibodies used throughout the studies; F.A.R. supervised the initial work toward structure determination; M.B.O. designed experiments and analyzed the data; and E.O.S. analyzed the data and wrote the manuscript. K.M.H. and S.I. contributed equally to the study. All authors commented on the manuscript.
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Supplementary Figure 1 Expression, purification and model building of LCMV GPe.
(a) Schematic diagram of LCMV GPC and the expression constructs used for crystallization. The sites for the nine predicted N-linked glycosylations are shown as “Y”. Ek designates the enterokinase consensus site and strepII is the affinity purification tag. (b) SEC-MALS-RI analysis of LCMV GP demonstrates soluble GPe is a dimer while GPeFib is trimeric. Solid traces are a measure of the relative light scattered for each sample. Analysis of the SEC-MALS-RI biophysical results allows the determination of the absolute molar mass, as well as the protein and glycan composition of the eluting sample. These are shown as blue and green connected dots under the main peak for each sample. (c) Trypsin-digestion of LCMV GPeFib results in a dimer-like elution profile. GPeFib was treated with trypsin and purified by SEC (blue trace). The resulting protein elutes at approximately the same volume as GPe with the strepII removed by enterokinase digestion (green trace). (d) Electron density map showing the sulfur anomalous signal and glycan density. The GPe dimer is colored according to the scheme in Figure 4. Methionine and cysteine residues are shown in ball-and-stick with the electron density for the anomalous sulfur signal colored yellow and contoured to 3σ. Asn residues and attached glycans are shown in ball-and-stick with the 2Fo-Fc electron density colored light blue and contoured to 1σ.
Supplementary Figure 2 Structural alignment of the GP1 subdomain of LCMV and LASV.
(a) The β-sheet face of LCMV (faded into the distance) is largely conserved in LASV GP1 (PDB: 4ZJF, Cohen-Dvashi, et al., J Virol 89, 7584-92, 2015), while the helix-loop face and termini are not. The locations of the N- and C-termini of each GP1 are shown as spheres. The LASV GP1 structure contains residues 77-237 and is illustrated in green, with the terminal residues (77 and 243) noted with spheres. The equivalent residues in LCMV numbering (H83 and G243) and the N and C termini of the LCMV structure (residues 63 and 261) are all noted with blue spheres. The histidine triad in LASV, critical for LAMP1 binding, includes residues H92, H93, and H230, which are illustrated in green ball-and-stick. The equivalent residues in LCMV, H99, H98, and H238 are illustrated in blue ball-and-stick. (b) Different orientation of GP1. The relative positions of secondary structural elements in the helix-loop face differ between LASV and LCMV. Note the alternate orientations of α2 and α3 and the break of the single α2 in LASV into helices α1 and α2 in LCMV. Residues implicated in αDG binding are shown in ball-and-stick. S153 and H155 (Y155 for high-affinity strains) are located in loop1, which is structurally conserved between LCMV and LASV. LASV binds to αDG with medium affinity and has N148 and Y150. L260 is located near the GP1-GP2 junction of LCMV and was not contained in the construct used for structure determination of LASV GP1. LASV numbering and secondary structural elements associated with LASV GP1 are donated with “Las”.
Supplementary Figure 3 Sequence conservation of arenavirus glycoproteins.
(a) Sequence alignment of the Old World arenaviruses LCMV (WE strains) and LASV with two pathogenic, TfR1-binding New World arenaviruses (Machupo virus (MACV) and Junin virus (JUNV)). Secondary structure elements shown above the alignment correspond to those seen in the structure of LCMV GP (Robert and Gouet, Nucleic Acids Res, 42, W320-4, 2014). Identical residues are highlighted in red boxes, similar residues are highlighted in white boxes and dissimilar residues are colored black. The cleavage sites between the SSP and GP1 and GP1 and GP2 are denoted with arrows. Cysteine bond pairs are numbered in black. Residues involved in high-affinity binding to αDG are denoted with asterisks. (b) Ribbon diagram of LCMV GP1 colored according to the sequence alignment to LASV GP1. Identical residues are colored red, similar residues are colored yellow and dissimilar residues are colored white. (c) Ribbon diagram of GP2 colored according to the scheme in (b). The GP2 subunit of Old and New World arenaviruses share ~75% sequence similarity and ~55% sequence identity.
Supplementary Figure 4 Fusion peptides and loops of viral glycoproteins.
The fusion peptides and loops of the pre-fusion conformation of class I influenza virus HA (PDB: 1RUZ, Gamblin, et al., Science, 303, 1838-42, 2004), class I parainfluenza virus 5 F (PDB: 4GIP, Welch, et al., Proc Natl Acad Sci USA, 109, 16672-7, 2012), class I EBOV GP (PDB: 3CSY, Lee, et al., Nature, 454, 177-82, 2008), class II TBEV E (PDB: 1SVB, Rey, et al., Nature, 375, 291-8, 1995) and class III VSV G (PDB: 2J6J, Roche, et al., Science, 315, 843-8, 2007) are shown alongside the pre-fusion conformation of LCMV GP. Each of the viral glycoproteins shown here has either an N-terminal fusion peptide (Flu HA and PIV5) or an internal fusion loop (EBOV GP, TBEV E and VSV G), while LCMV GP apparently has both. LCMV has a bipartite fusion region that includes an N-terminal fusion peptide as well as an internal fusion loop with a helix at its center.
Supplementary Figure 5 Biochemical analysis of the dimeric interface.
(a) H136R and S143R, which lie at the dimeric interface were mutated to arginines to prevent the formation of the dimer. (b) GPe proteins bearing either point mutation were produced in S2 cells and analyzed by SEC-MALS. The stoichiometry for each protein was calculated based upon the observed mass from MALS and the expected mass based on the protein sequence. H136R-GPe elutes as a primarily monomeric species. S143R-GPe elutes as a mixture of monomer and dimer. (c) Wild-type (WT) or mutant GPC was transiently expressed in 293T cells and analyzed for processing of the GPC into the GP1 and GP2 subunits.
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Supplementary Figures 1–5 and Supplementary Table 1 (PDF 10543 kb)
Supplementary Data Set 1
Subunit requirements for immunoprecipitation of α-dystroglycan (PDF 714 kb)
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Hastie, K., Igonet, S., Sullivan, B. et al. Crystal structure of the prefusion surface glycoprotein of the prototypic arenavirus LCMV. Nat Struct Mol Biol 23, 513–521 (2016). https://doi.org/10.1038/nsmb.3210
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DOI: https://doi.org/10.1038/nsmb.3210
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