Molecular organization and dynamics of the fusion protein Gc at the hantavirus surface
- Fundación Ciencia & Vida, Chile
- Institut Pasteur, CNRS UMR 3569, France
Figures
Figure 1 with 1 supplement
A cysteine residue engineered at the crystallographic Gc dimer interface cross-links spikes on viral particles.
(a) X-ray structure of the crystallographic Hantaan virus Gc dimer (upper panel, PDB: 5LJY, Guardado-Calvo et al., 2016), displayed alongside the crystallographic alphavirus E1 dimer (PDB:2ALA, Rouss…
Figure 1—figure supplement 1
Conservation of Gc:Gc dimer interface contacts.
Multiple sequence alignment of Gc from seven representative hantaviruses. Strictly conserved and highly similar residues are highlighted in red or brown background, respectively. Each host is noted …
Figure 2
The covalent Gc:Gc dimer disulfide bond G187C increases Gn/Gc spike stability.
(a–b) Representative BN-PAGE and western blotting of ANDV wild type (a) and G187C mutant spikes (b). The spikes were extracted from VLPs by Triton X-100 and treated at the indicated temperatures of …
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Figure 2—source data 1
Original blots and data points for Figure 2c.
- https://doi.org/10.7554/eLife.46028.006
Figure 3 with 2 supplements
The inter-spike Gc:Gc dimer interface affect VLP release and hantavirus Gn/Gc spike stability.
a) VLP assembly and release of ANDV Gc mutants including Ala-substitutions, chemically similar mutations and opposed charge substitutions quantified from western blot analysis using anti-Gc antibody …
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Figure 3—source data 1
Data points for graphs of Figure 3a and b.
- https://doi.org/10.7554/eLife.46028.008
Figure 3—figure supplement 1
Characterization of the expression yields, trafficking and VLP assembly of ANDV Gc mutants.
Mutants include Ala-substitutions (a, d), chemically similar mutations (b), and opposed charge substitutions (c). Western blot analysis using anti-Gc or anti-β-actin antibodies of different …
Figure 3—figure supplement 2
Thermal stability of the Gn/Gc spike complex from Gc:Gc interface mutants.
(a–d) Native western blot of spikes including Gc mutants E26Q (a), D28A (b), R301Q (c) and H303F (d) treated at the indicated temperatures and extracted from mutant VLPs by Triton X-100. To detect …
Figure 4 with 1 supplement
The Gc:Gc dimer contacts modulate low pH fusion activation.
(a-b) Fusion activity of cells expressing ANDV wild type Gn/Gc (WT) or wild type Gn/mutant Gc at different pHs. Syncytia formation was induced by lowering the pH to 5.5 or 4.5 and was quantified by …
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Figure 4—source data 1
Data points for graphs of Figure 4a, b and c.
- https://doi.org/10.7554/eLife.46028.013
Figure 4—figure supplement 1
Acid-induced activation and membrane interaction of WT and mutant VLPs assessed by a VLP-liposome coflotation assay.
VLPs including wt spikes (a) or Gc mutants D28A (b) or H303F (c) were incubated with DPH-labeled liposomes at different pHs at 37°C. Fractions of the step gradient sedimentation were examined for …
Figure 5 with 1 supplement
Temperature-induced Gc fusion loop exposure.
(a-b) Liposome coflotation assay to visualize temperature-induced membrane interaction of ANDV wild type (WT) VLPs (a) or ANDV VLPs bearing the Gc fusion loop mutant W115A/F250A (b). VLPs were …
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Figure 5—source data 1
Data points for graphs of Figure 5c and d.
- https://doi.org/10.7554/eLife.46028.016
Figure 5—figure supplement 1
Temperature-induced Gc fusion loop exposure of VLPs bearing the Gc 187C mutant measured by a VLP-liposome coflotation assay.
The mutant VLPs were incubated with DPH-labeled liposomes at pH 7.4 at different temperatures and subsequently subjected to flotation in a step gradient. The different fractions were examined for …
Figure 6
The open spike conformation does not induce viral infection and membrane fusion.
(a-b) Infectivity of ANDV after temperature treatment and recovery at 37°C (a) or 4°C (b) during adsorption to cells for 1 h. Infection was quantified by flow cytometry 16 h later using anti-ANDV …
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Figure 6—source data 1
Data points for graphs and statistics of Figure 6a–e.
- https://doi.org/10.7554/eLife.46028.018
Figure 7
Hantavirus Gn/Gc spike dynamics and Gc conformational changes.
Diagrammatic representation of ‘breathing’ spikes. (a) The Gn ectodomain is represented in gray and Gc in colors: red for domain I; yellow domain II; blue domain III; stem and transmembrane (TM) …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Add. inform. |
|---|---|---|---|---|
| Strain, strain background (Andes virus) | Andes virus isolate CHI-7913 | Galeno et al., 2002 | ||
| Cell line (Homo sapiens) | 293FT | Thermo Fisher Scientific | Cat.:R700-07; RRID:CVCL_6911 | |
| Cell line (Cercopithecus aethiops) | Vero 76 (Vero E6) | American Type Culture Collection (ATCC) | CRL 1587; RRID:CVCL_0603 | |
| Recombinant DNA reagent | pI.18/GPC from ANDV | Cifuentes-Muñoz et al., 2010 | ||
| Recombinant DNA reagent | pWRG/PUU-M(s2) | Hooper et al., 2006 | ||
| Recombinant DNA reagent | pcDNA/Sin Nombre virus-GP | Kleinfelter et al., 2015 | ||
| Biological sample | L-α-phosphatidylcholine (egg, chicken) | Avanti Polar Lipids | Cat.:840051C | |
| Biological sample | L-α-phosphatidyl-ethanolamine (egg, chicken) | Avanti Polar Lipids | Cat.:840021C | |
| Biological sample | Sphingomyelin (Brain, Porcine) | Avanti Polar Lipids | Cat.:860062C | |
| Biological sample | Cholesterol (ovine wool) | Avanti Polar Lipids | Cat.:700000P | |
| Biological sample | Gel Filtration Standard | Biorad | Cat.:1511901 | |
| Biological sample | NativeMark Unstained Protein Standard | Thermo Fisher Scientific | Cat.:LC0725 | |
| Biological sample | PageRuler Prestained Protein Ladder | Thermo Fisher Scientific | Cat.:26616 | |
| Antibody (monoclonal) | Mouse anti-ANDV nucleoprotein clone 7B3/F6 | Tischler et al., 2008 | 1:2000 dilution | |
| Antibody (monoclonal) | Mouse anti-Gc clone 2H4/F6 | Godoy et al., 2009 | 1:2500 dilution | |
| Antibody (monoclonal) | Mouse anti-Gn clone 6B9/F5 | Cifuentes-Muñoz et al., 2010 | 1:2500 dilution | |
| Antibody (monoclonal) | Mouse anti-β-actin | Sigma | Cat:A2228; RRID:AB_476697 | 1:2500 dilution |
| Antibody (oligoclonal) | Goat anti-mouse immunoglobulin Alexa 555 conjugate | Thermo Fisher Scientific | Cat:A28180; RRID:AB_2536164 | 1:500 dilution |
| Antibody (polyclonal) | Goat anti-mouse IgG (H + L) horseradish peroxidase conjugate | Thermo Fisher Scientific | Cat:31430; RRID:AB_228307 | 1:5000 dilution |
| Antibody (oligoclonal) | Goat anti-mouse IgG (H + L) Alexa555 conjugate | Thermo Fisher Scientific | Cat:A28175; RRID:AB_2536161 | 1:500 dilution |
| Recombinant DNA reagent | Mutant constructs of pI.18/GPC | This paper, produced by GenScript, Piscataway, NJ. | ||
| Commercial assay or kit | Cell surface protein isolation kit | Pierce | Cat:89881 | |
| Commercial assay or kit | 1,6-diphenyl-1,3,5-hexatriene (DPH) | Sigma-Aldrich | Cat.:D208000 | |
| Commercial assay or kit | Octadecyl Rhodamine B Chloride (R18) | Thermo Fisher Scientific | Cat.:O246 | |
| Commercial assay or kit | 5-chloromethyl-fluorescein diacetate (Cell Tracker green CMFDA) | Thermo Fisher Scientific | Cat.:C7025 | |
| Commercial assay or kit | Lipofectamine 2000 | Thermo Fisher Scientific | Cat.:11668019 | |
| Software, algorithm | GraphPad Prism, version 6, and SPSS software (SPSS, Inc) | GraphPad Software | ||
| Software, algorithm | SigmaPlot 12.0 | Systat Software | ||
| Other (Hantaan virus) | Gc ectodomain structure from Hantaan virus | Guardado-Calvo et al., 2016 | PDB: 5LJY | |
| Other (Semliki Forest virus) | E1 ectodomain structure from Semliki Forest virus | Roussel et al., 2006 | PDB: 2ALA |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.46028.020
