Abstract
Viruses sense environmental cues such as pH to engage in membrane interactions for cell entry during infection, but how nonenveloped viruses sense pH is largely undefined. Here, we report both high- and low-pH structures of bluetongue virus (BTV), which enters cells via a two-stage endosomal process. The receptor-binding protein VP2 possesses a zinc finger that may function to maintain VP2 in a metastable state and a conserved His866, which senses early-endosomal pH. The membrane-penetration protein VP5 has three domains: dagger, unfurling and anchoring. Notably, the β-meander motif of the anchoring domain contains a histidine cluster that can sense late-endosomal pH and also possesses four putative membrane-interaction elements. Exposing BTV to low pH detaches VP2 and dramatically refolds the dagger and unfurling domains of VP5. Our biochemical and structure-guided-mutagenesis studies support these coordinated pH-sensing mechanisms.
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Acknowledgements
We thank S. Shivakoti and J. Sun for assistance in cell culture and virus isolation, M. Boyce for advice in sample purification and I. Jones, L. Nguyen and L. Wang for reading the manuscript. This project was supported in part by grants from the US National Institutes of Health (AI094386 to Z.H.Z.) and the Wellcome Trust UK (to P.R.). We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines supported by UCLA and by instrumentation grants from the US National Institutes of Health (1S10OD018111) and the US National Science Foundation (DBI-1338135).
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Z.H.Z., P.R. and X.Z. designed the experiments. A.P. expressed proteins and performed the mutagenesis and biochemical experiments. C.C.C. and X.Y. grew and isolated viruses. X.Z. collected and analyzed the cryoEM data and built the atomic models. Z.H.Z., X.Z. and P.R. interpreted the structures and wrote the paper.
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Integrated supplementary information
Supplementary Figure 1 CryoEM imaging, reconstruction and validation.
(a) Representative cryoEM DED image of BTV virions. (b) Fourier shell correlation coefficients (FSC) as a function of spatial frequency between maps from half datasets. (c-d) Representative regions of the virion reconstructions showing matching of side-chain features of VP3 (c) and VP7 (d) between the cryoEM density (grey) and x-ray models (ribbons). (e-f) CryoEM reconstructions of the BTV virion at pH3.4 (e) and pH5.5 (f) conditions, both low-pass filtered to 25Å resolution, showing similar structural features. The radial color scheme is the same as that in the Figure 1a.
Supplementary Figure 2 Sequences and secondary structures of VP2 and VP5.
Sequences and secondary structures of VP2 (a) and VP5 (b). α-Helices are marked by cylinders, β-strands by arrows, loops by thin lines. The flexible tip domain (P191-K405) of VP2 are indicated by a dashed line in (a).
Supplementary Figure 3 Structure of VP5.
(a) Conservation of VP5. The bar indicates the color scale of the conservation from most conserved (1, blue) to no conservation (0, white). (b) The internal surface of the β-meander core shown as a ribbon, with the side chains represented as sticks. (c) Hydrophilicity surface of the β-meander core of the VP5 anchoring domain. Gray: hydrophobic; red: oxygen atoms; blue: nitrogen atoms. (d) Possible transmembrane β-strands of VP5 anchoring domain. BOCTOPUS server predicts six transmembrane β-strands in the anchoring domain. HMM: Hidden Markov model; SVM: Support Vector Machines.
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Supplementary Text and Figures
Supplementary Figures 1–3 (PDF 721 kb)
Structure of the BTV virion
Radially colored surface representation of the cryoEM reconstruction of the BTV virion at 3.5 Å resolution (MP4 10643 kb)
Structure of VP2
Surface representation of the density map of a VP2 monomer colored by domains: body (red), hairpin (green), and hub (blue). At the relatively high density threshold used, the flexible tip domain is invisible (MP4 10845 kb)
Zinc finger of VP2
Part of VP2 density map (mesh) containing the zinc-finger motif. The atomic model is superimposed into the map as sticks (MP4 5684 kb)
Structure of VP5
Surface representation of the density map of a VP5 monomer colored by domains: dagger (blue), unfurling (red) and anchoring (green). The density of the stem helix α3 in the unfurling domain is marked cyan as in Figure 3 (MP4 7201 kb)
Density map of a helix in VP5
Validation of the cryoEM map. Density map of the α17 helix of the VP5 is shown as semi-transparent surface (cyan) and is superimposed with the atomic model (sticks). Note that the side chains of amino acid residues match the densities of the cryoEM map (MP4 7227 kb)
Histidine cluster of VP5
The histidine cluster located at the interface between the anchoring and unfurling domains of VP5. Density map is shown as semi-transparent surface (grey) and is superimposed with the atomic model (ribbons and sticks) (MP4 5639 kb)
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Zhang, X., Patel, A., Celma, C. et al. Atomic model of a nonenveloped virus reveals pH sensors for a coordinated process of cell entry. Nat Struct Mol Biol 23, 74–80 (2016). https://doi.org/10.1038/nsmb.3134
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DOI: https://doi.org/10.1038/nsmb.3134
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