Abstract
Venezuelan equine encephalitis virus (VEEV) is an enveloped RNA virus that causes encephalitis and potentially mortality in infected humans and equines1. At present, no vaccines or drugs are available that prevent or cure diseases caused by VEEV. Low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3) was recently identified as a receptor for the entry of VEEV into host cells2. Here we present the cryo-electron microscopy structure of the LDLRAD3 extracellular domain 1 (LDLRAD3-D1) in complex with VEEV virus-like particles at a resolution of 3.0 Å. LDLRAD3-D1 has a cork-like structure and is inserted into clefts formed between adjacent VEEV E2–E1 heterodimers in the viral-surface trimer spikes through hydrophobic and polar contacts. Mutagenesis studies of LDLRAD3-D1 identified residues that are involved in the key interactions with VEEV. Of note, some of the LDLRAD3-D1 mutants showed a significantly increased binding affinity for VEEV, suggesting that LDLRAD3-D1 may serve as a potential scaffold for the development of inhibitors of VEEV entry. Our structures provide insights into alphavirus assembly and the binding of receptors to alphaviruses, which may guide the development of therapeutic countermeasures against alphaviruses.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The atomic coordinates and electron microscopy maps have been deposited into the PDB (http://www.pdb.org) and the Electron Microscopy Data Bank (EMDB), respectively. The deposited data include the cryo-EM maps and PDB coordinates for an asymmetric unit (EMD-31567, 7FFF), the twofold block (EMD-31568, 7FFL) and the fivefold block (EMD-31569, 7FFN) of the VEEV VLP–LDLRAD3-D1 complex and an asymmetric unit (EMD-31566, 7FFE), the twofold block (EMD-31571, 7FFQ) and the fivefold block (EMD-31570, 7FFO) of VEEV VLP. All other relevant data are available from the corresponding authors upon reasonable request.
References
Taylor, K. G. & Paessler, S. Pathogenesis of Venezuelan equine encephalitis. Vet. Microbiol. 167, 145–150 (2013).
Ma, H. et al. LDLRAD3 is a receptor for Venezuelan equine encephalitis virus. Nature 588, 308–314 (2020).
Strauss, J. H. & Strauss, E. G. The alphaviruses: gene expression, replication, and evolution. Microbiol. Rev. 58, 491–562 (1994).
Suhrbier, A., Jaffar-Bandjee, M. C. & Gasque, P. Arthritogenic alphaviruses—an overview. Nat. Rev. Rheumatol. 8, 420–429 (2012).
Carossino, M., Thiry, E., de la Grandière, A. & Barrandeguy, M. E. Novel vaccination approaches against equine alphavirus encephalitides. Vaccine 32, 311–319 (2014).
Zacks, M. A. & Paessler, S. Encephalitic alphaviruses. Vet. Microbiol. 140, 281–286 (2010).
Sharma, A. & Knollmann-Ritschel, B. Current understanding of the molecular basis of Venezuelan equine encephalitis virus pathogenesis and vaccine development. Viruses 11, 164 (2019).
Bronze, M. S., Huycke, M. M., Machado, L. J., Voskuhl, G. W. & Greenfield, R. A. Viral agents as biological weapons and agents of bioterrorism. Am. J. Med. Sci. 323, 316–325 (2002).
Lescar, J. et al. The fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH. Cell 105, 137–148 (2001).
Li, L., Jose, J., Xiang, Y., Kuhn, R. J. & Rossmann, M. G. Structural changes of envelope proteins during alphavirus fusion. Nature 468, 705–708 (2010).
Roussel, A. et al. Structure and interactions at the viral surface of the envelope protein E1 of Semliki Forest virus. Structure 14, 75–86 (2006).
Voss, J. E. et al. Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography. Nature 468, 709–712 (2010).
Chen, L. et al. Implication for alphavirus host-cell entry and assembly indicated by a 3.5 Å resolution cryo-EM structure. Nat. Commun. 9, 5326 (2018).
Cheng, R. H. et al. Nucleocapsid and glycoprotein organization in an enveloped virus. Cell 80, 621–630 (1995).
Hasan, S. S. et al. Cryo-EM structures of eastern equine encephalitis virus reveal mechanisms of virus disassembly and antibody neutralization. Cell Rep. 25, 3136–3147 (2018).
Kostyuchenko, V. A. et al. The structure of Barmah Forest virus as revealed by cryo-electron microscopy at a 6-angstrom resolution has detailed transmembrane protein architecture and interactions. J. Virol. 85, 9327–9333 (2011).
Pletnev, S. V. et al. Locations of carbohydrate sites on alphavirus glycoproteins show that E1 forms an icosahedral scaffold. Cell 105, 127–136 (2001).
Zhang, R. et al. 4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus. EMBO J. 30, 3854–3863 (2011).
Sun, S. et al. Structural analyses at pseudo atomic resolution of Chikungunya virus and antibodies show mechanisms of neutralization. Elife 2, e00435 (2013).
Zhang, W. et al. Aura virus structure suggests that the T=4 organization is a fundamental property of viral structural proteins. J. Virol. 76, 7239–7246 (2002).
Jose, J., Snyder, J. E. & Kuhn, R. J. A structural and functional perspective of alphavirus replication and assembly. Future Microbiol. 4, 837–856 (2009).
Holmes, A. C., Basore, K., Fremont, D. H. & Diamond, M. S. A molecular understanding of alphavirus entry. PLoS Pathog. 16, e1008876 (2020).
Vancini, R., Hernandez, R. & Brown, D. Alphavirus entry into host cells. Prog. Mol. Biol. Transl. Sci. 129, 33–62 (2015).
Zhang, R. et al. Mxra8 is a receptor for multiple arthritogenic alphaviruses. Nature 557, 570–574 (2018).
Basore, K. et al. Cryo-EM structure of chikungunya virus in complex with the Mxra8 receptor. Cell 177, 1725–1737 (2019).
Song, H. et al. Molecular basis of arthritogenic alphavirus receptor MXRA8 binding to chikungunya virus envelope protein. Cell 177, 1714–1724 (2019).
Fass, D., Blacklow, S., Kim, P. S. & Berger, J. M. Molecular basis of familial hypercholesterolaemia from structure of LDL receptor module. Nature 388, 691–693 (1997).
Ivanova, L. & Schlesinger, M. J. Site-directed mutations in the Sindbis virus E2 glycoprotein identify palmitoylation sites and affect virus budding. J. Virol. 67, 2546–2551 (1993).
Ko, S. Y. et al. A virus-like particle vaccine prevents equine encephalitis virus infection in nonhuman primates. Sci. Transl. Med. 11, eaav3113 (2019).
Jiang, L. et al. Potent neutralization of MERS-CoV by human neutralizing monoclonal antibodies to the viral spike glycoprotein. Sci. Transl. Med. 6, 234ra259 (2014).
Wu, C., Huang, X., Cheng, J., Zhu, D. & Zhang, X. High-quality, high-throughput cryo-electron microscopy data collection via beam tilt and astigmatism-free beam-image shift. J. Struct. Biol. 208, 107396 (2019).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7, e42166 (2018).
Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).
Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).
Acknowledgements
We thank the Tsinghua University Branch of the China National Center for Protein Sciences (Beijing) for providing the facility support; L. Q. Zhang for providing the vectors; L. Kong for cryo-EM data storage and back-up; and D. J. Zhu and M. J. Zhao for discussions of cryo-EM data processing. Cryo-EM data collection was carried out at the Center for Biological Imaging, Core Facilities for Protein Science at the Institute of Biophysics (IBP), Chinese Academy of Sciences (CAS). We thank X. J. Huang, B. L. Zhu, X. J. Li, L. H. Chen, F. Sun and other staff members at the Center for Biological Imaging (IBP, CAS). This work was supported by funds from the Ministry of Science and Technology of China (grants 2017YFA0504700 and 2016YFA0501100); the Spring Breeze Fund of Tsinghua University; the National Natural Science Foundation of China (grants 31925023, 21827810, 31861143027, 31930069 and 31470721); the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB37040101); the Key Research Program of Frontier Sciences at the Chinese Academy of Sciences (ZDBS-LY-SM003); the Beijing Frontier Research Center for Biological Structure; and the Beijing Advanced Innovation Center for Structure Biology to Y.X. and X.Z. J.M. is supported by the Youth Innovation Promotion Association of CAS (no. 2019094) and the Beijing Nova Program (no. Z201100006820033).
Author information
Authors and Affiliations
Contributions
B.M. prepared the VEEV VLPs and developed the protein expression and purification procedures for LDLRAD3-D1, performed the mutagenesis and BLI analysis and helped with the cryo-EM grid and manuscript preparation. C.H. prepared the cryo-EM grids, collected the cryo-EM data and calculated the structures. J.M. built the atomic model and helped with the cryo-EM grid preparation, cryo-EM data collection and manuscript preparation. Y.X. oversaw the preparation of the VEEV VLPs and LDLRAD3-D1 and the mutagenesis assay. X.Z oversaw the cryo-EM structure determination. Y.X. and X.Z. initiated the project, planned and analysed experiments, supervised the research and wrote the manuscript with input from all co-authors.
Corresponding authors
Ethics declarations
Competing interests
A patent application applied by Tsinghua University and the Institute of Biophysics, Chinese Academy of Sciences with Y.X., X.Z., B.M. and C.H. listed as the inventors on the LDLRAD3-D1 core and the LDLRAD3-D1 mutants is pending.
Additional information
Peer review information Nature thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Preparation of the VEEV VLP and LDLRAD3-D1.
a, Left: A schematic diagram showing the VEEV polyprotein and cleavage sites that result in the VEEV structural proteins that assemble into VLPs. Middle: Negative staining electron microscopy analysis of the purified VEEV VLPs. Right: SDS-PAGE analysis of the purified VEEV VLPs. b, Left: Schematic diagrams of the domain organization of LDLRAD3 and the construct used to produce the recombinant LDLRAD3-D1-Fc fusion protein. Right: SDS-PAGE analysis of the purified LDLRAD3-D1-Fc recombinant protein under reducing and non-reducing conditions.
Extended Data Fig. 2 Cryo-EM reconstruction of VEEV VLP and the complex of VEEV VLP with LDLRAD3-D1.
a, A typical cryo-EM image of the VEEV VLP complexed with LDLRAD3-D1. b, A reconstructed map of the VEEV VLP complexed with LDLRAD3-D1 at a resolution of 4.1 Å shows the q3 E1/E2 trimeric spikes surrounding the icosahedral 5-fold axes, the i3 E1/E2 trimeric spikes sitting on the icosahedral 3-fold positions, and the positions of the four E1-E2-E3-C assemblies (1, 2, 3 and 4) in the asymmetric unit. c, Diagrams showing the positions of the 5-fold and 2-fold blocks used for the block-based subregion reconstructions of the VEEV VLP. The two blocks were refined and reconstructed separately and cover the complete icosahedral asymmetric unit. d, Fourier shell coefficient curves calculated between the odd and even half maps show that the map resolutions of the two reconstructed blocks are 3.03 and 3.06 Å, respectively. The threshold used was 0.143. e, The local resolution maps of the two reconstructed blocks calculated with the software Resmap38. f, The work flow used for the cryo-EM data processing and block-based reconstructions of VEEV VLP and the complex of VEEV VLP with LDLRAD3-D1.
Extended Data Fig. 3 Representative densities of VEEV VLP and the bound LDLRAD3-D1.
a–d, The density maps are contoured at 0.025 e− per Å3.
Extended Data Fig. 4 Structural comparisons between the receptor-bound and receptor-free VEEV E proteins, overall shape of LDLRAD3-D1 and the low-pH-treated VEEV VLP with the bound LDLRAD3-D1.
a, Ribbon diagrams showing the LDLRAD3-D1 bound and free VEEV E2 structures. LDLRAD3-D1 and VEEV E2 are shown in ribbons and coloured pink and green, respectively. Upon binding LDLRAD3-D1, conformational changes were observed at residues H96 and H156 of E2. In addition, the E2 loop that involves residues 57-64 becomes ordered in the complex structure owing to the establishment of the salt bridges between R64 of E2 and D54 and D50 of LDLRAD3-D1. b, Ribbon and schematic diagrams showing the elliptical cylinder shape of LDLRAD3-D1. The ribbon of LDLRAD3-D1 is coloured blue through the rainbow spectrum to red (from N to C). c, Cryo-EM images of the low pH treated VEEV VLP complexed with LDLRAD3-D1 showing the aggregated particles, which is an indication for the exposure of the fusion loop.
Extended Data Fig. 5 Sequence alignments of LDLRAD3 with its orthologues and other members of the LDL receptor family.
a, Sequence alignments of the human LDLRAD3 domain 1 and the LDL-receptor class A domains of human LRP1, LRP2, VLDLR, LRP5, LRP1B, LRP4, LRP12, LRP10, LRP6, LDLR, and LRP8. Conserved residues are boxed and coloured red. Completely conserved residues are shown in white on a red background. Triangles indicate residues of LDLRAD3-D1 shown to be critical in the interactions with VEEV VLP. b, Sequence alignments of the mouse (Mus musculus), horse (Equus caballus), human (Homo sapiens), chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), cattle (Bos taurus), pig (Sus scrofa), dog (Canis lupus familiaris), chicken (Gallus gallus), duck (Oxyura jamaicensis), and turkey (Meleagris gallopavo) LDLRAD3 ectodomains. Conserved residues are boxed and coloured red. Completely conserved residues are shown in white on a red background. Triangles indicate the residues of LDLRAD3-D1 shown to be critical in the interactions with VEEV VLP. Stars indicate the D-x-S-D-E calcium binding motif of LDLRAD3.
Extended Data Fig. 6 BLI analyses of the binding of LDLRAD3-D1 or its mutants to VEEV VLP.
BLI analysis showing the representative binding and disassociation curves of LDLRAD3-D1 and mutants N30A, I31A, N34A, N39A, R41A, R41N, R41L, W47G, W47F, W47I, D50G, D54G, F56G, and D57G to VEEV VLP. The KD value displayed for each sample is the mean value of two independent measurements.
Extended Data Fig. 7 Sequence alignments of the E1 proteins from different alphaviruses.
Sequence alignments of the E1 proteins from VEEV (AAB02517.1), WEEV (UniProt ID: P13897), EEEV (UniProt ID: P08768), ONNV (UniProt ID: O90369), SINV (UniProt ID: P03316), CHIKV (UniProt ID: Q5XXP3), MAYV (UniProt ID: Q8QZ72), and RRV (UniProt ID: P08491). Conserved residues are boxed and coloured red. Completely conserved residues are shown in white on a red background. Diamonds underneath the alignments indicate residues involved in the interactions between the VEEV E1 and E2. Red diamonds indicate residues that participate in the formation of hydrogen bonds at the VEEV E1-E2 interfaces. Triangles indicate residues of VEEV VLP involved in the interactions with LDLRAD3-D1. Red triangles indicate residues of VEEV VLP that participate in the formation of hydrogen bonds with LDLRAD3-D1. Rectangles indicate residues of CHIKV VLP that participate in the interactions with the receptor MXRA8. Red rectangles indicate residues of CHIKV VLP involved in the formation of hydrogen bonds with MXRA8. Stars indicate residues involved in the interactions between the VEEV capsid protein and E1. Red stars indicate residues of VEEV E1 that participate in the formation of hydrogen bonds with VEEV capsid protein.
Extended Data Fig. 8 Sequence alignments of the E2 proteins from different alphaviruses.
Sequence alignments of the E2 proteins from VEEV (AAB02517.1), WEEV (UniProt ID: P13897), EEEV (UniProt ID: P08768), ONNV (UniProt ID: O90369), SINV (UniProt ID: P03316), CHIKV (UniProt ID: Q5XXP3), MAYV (UniProt ID: Q8QZ72), and RRV (UniProt ID: P08491). Conserved residues are boxed and coloured red. Completely conserved residues are shown in white on a red background. Diamonds indicate residues of VEEV VLP involved in the interactions between E1 and E2. Red diamonds indicate residues of VEEV VLP that participate in the formation of hydrogen bonds at the VEEV E1-E2 interfaces. Triangles indicate residues of VEEV VLP that participate in the interactions with LDLRAD3-D1. Red triangles indicate residues of VEEV VLP that participate in the formation of hydrogen bonds with LDLRAD3-D1. Rectangles indicate residues of CHIKV VLP that participate in the interactions with the receptor MXRA8. Red rectangles indicate residues of CHIKV VLP that participate in the formation of hydrogen bonds with MXRA8. Stars indicate residues involved in the interactions between the VEEV capsid protein and E2. Red stars indicate residues of VEEV E2 that participate in the formation of hydrogen bonds with VEEV capsid protein.
Extended Data Fig. 9 Sequence alignments of the capsid proteins from different alphaviruses.
Sequence alignments of the capsid proteins from VEEV (AAB02517.1), WEEV (UniProt ID: P13897), EEEV (UniProt ID: P08768), ONNV (UniProt ID: O90369), SINV (UniProt ID: P03316), CHIKV (UniProt ID: Q5XXP3), MAYV (UniProt ID: Q8QZ72), and RRV (UniProt ID: P08491). Conserved residues are boxed and coloured red. Completely conserved residues are shown in white on a red background. Diamonds underneath the alignments indicate residues involved in the interactions between the VEEV capsid and E1. Red diamonds indicate residues that participate in the formation of hydrogen bonds at the VEEV capsid-E1 interfaces. Triangles underneath the alignments indicate residues involved in the interactions between the VEEV capsid and E2. Red triangles indicate residues that participate in the formation of hydrogen bonds at the VEEV capsid-E2 interfaces.
Supplementary information
Supplementary Information
This file contains Supplementary Figure 1 and Supplementary Tables 1–3
Rights and permissions
About this article
Cite this article
Ma, B., Huang, C., Ma, J. et al. Structure of Venezuelan equine encephalitis virus with its receptor LDLRAD3. Nature 598, 677–681 (2021). https://doi.org/10.1038/s41586-021-03909-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-021-03909-1
This article is cited by
-
Post-exposure intranasal IFNα suppresses replication and neuroinvasion of Venezuelan Equine Encephalitis virus within olfactory sensory neurons
Journal of Neuroinflammation (2024)
-
LDLR is used as a cell entry receptor by multiple alphaviruses
Nature Communications (2024)
-
The low-density lipoprotein receptor promotes infection of multiple encephalitic alphaviruses
Nature Communications (2024)
-
A molecular understanding of alphavirus entry and antibody protection
Nature Reviews Microbiology (2023)
-
Identification of human and mosquito receptors for alphaviruses
Nature (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
