Abstract
Ebola viral protein 35 (VP35), encoded by the highly pathogenic Ebola virus, facilitates host immune evasion by antagonizing antiviral signaling pathways, including those initiated by RIG-I–like receptors. Here we report the crystal structure of the Ebola VP35 interferon inhibitory domain (IID) bound to short double-stranded RNA (dsRNA), which together with in vivo results reveals how VP35-dsRNA interactions contribute to immune evasion. Conserved basic residues in VP35 IID recognize the dsRNA backbone, whereas the dsRNA blunt ends are 'end-capped' by a pocket of hydrophobic residues that mimic RIG-I–like receptor recognition of blunt-end dsRNA. Residues critical for RNA binding are also important for interferon inhibition in vivo but not for viral polymerase cofactor function of VP35. These results suggest that simultaneous recognition of dsRNA backbone and blunt ends provides a mechanism by which Ebola VP35 antagonizes host dsRNA sensors and immune responses.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 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
References
Bosio, C.M. et al. Ebola and Marburg viruses replicate in monocyte-derived dendritic cells without inducing the production of cytokines and full maturation. J. Infect. Dis. 188, 1630–1638 (2003).
Mahanty, S. et al. Protection from lethal infection is determined by innate immune responses in a mouse model of Ebola virus infection. Virology 312, 415–424 (2003).
Bray, M. & Geisbert, T.W. Ebola virus: the role of macrophages and dendritic cells in the pathogenesis of Ebola hemorrhagic fever. Int. J. Biochem. Cell Biol. 37, 1560–1566 (2005).
Baize, S. et al. Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients. Nat. Med. 5, 423–426 (1999).
Feldmann, H., Wahl-Jensen, V., Jones, S.M. & Stroher, U. Ebola virus ecology: a continuing mystery. Trends Microbiol. 12, 433–437 (2004).
Matsukura, S. et al. Role of RIG-I, MDA-5, and PKR on the expression of inflammatory chemokines induced by synthetic dsRNA in airway epithelial cells. Int. Arch. Allergy Immunol. 143 Suppl 1, 80–83 (2007).
Hausmann, S., Marq, J.B., Tapparel, C., Kolakofsky, D. & Garcin, D. RIG-I and dsRNA-induced IFN-β activation. PLoS One 3, e3965 (2008).
Saito, T. & Gale, M. Jr. Principles of intracellular viral recognition. Curr. Opin. Immunol. 19, 17–23 (2007).
Spiropoulou, C.F. et al. RIG-I activation inhibits ebolavirus replication. Virology 392, 11–15 (2009).
Schlee, M. et al. Approaching the RNA ligand for RIG-I? Immunol. Rev. 227, 66–74 (2009).
Schlee, M. et al. Recognition of 5′ triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus. Immunity 31, 25–34 (2009).
Yoneyama, M. et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5, 730–737 (2004).
Schmidt, A. et al. 5′-triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I. Proc. Natl. Acad. Sci. USA 106, 12067–12072 (2009).
Li, X. et al. Structural basis of double-stranded RNA recognition by the RIG-I like receptor MDA5. Arch. Biochem. Biophys. 488, 23–33 (2009).
Basler, C.F. et al. The Ebola virus VP35 protein inhibits activation of interferon regulatory factor 3. J. Virol. 77, 7945–7956 (2003).
Basler, C.F. et al. The Ebola virus VP35 protein functions as a type I IFN antagonist. Proc. Natl. Acad. Sci. USA 97, 12289–12294 (2000).
Cardenas, W.B. et al. Ebola virus VP35 protein binds double-stranded RNA and inhibits α/β interferon production induced by RIG-I signaling. J. Virol. 80, 5168–5178 (2006).
Habjan, M. et al. Processing of genome 5′ termini as a strategy of negative-strand RNA viruses to avoid RIG-I-dependent interferon induction. PLoS One 3, e2032 (2008).
Hartman, A.L. et al. Inhibition of IRF-3 activation by VP35 is critical for the high level of virulence of ebola virus. J. Virol. 82, 2699–2704 (2008).
Hartman, A.L., Dover, J.E., Towner, J.S. & Nichol, S.T. Reverse genetic generation of recombinant Zaire Ebola viruses containing disrupted IRF-3 inhibitory domains results in attenuated virus growth in vitro and higher levels of IRF-3 activation without inhibiting viral transcription or replication. J. Virol. 80, 6430–6440 (2006).
Hartman, A.L., Ling, L., Nichol, S.T. & Hibberd, M.L. Whole-genome expression profiling reveals that inhibition of host innate immune response pathways by Ebola virus can be reversed by a single amino acid change in the VP35 protein. J. Virol. 82, 5348–5358 (2008).
Hartman, A.L., Towner, J.S. & Nichol, S.T. A C-terminal basic amino acid motif of Zaire ebolavirus VP35 is essential for type I interferon antagonism and displays high identity with the RNA-binding domain of another interferon antagonist, the NS1 protein of influenza A virus. Virology 328, 177–184 (2004).
Kash, J.C. et al. Global suppression of the host antiviral response by Ebola- and Marburgviruses: increased antagonism of the type I interferon response is associated with enhanced virulence. J. Virol. 80, 3009–3020 (2006).
Haasnoot, J. et al. The Ebola virus VP35 protein is a suppressor of RNA silencing. PLoS Pathog. 3, e86 (2007).
Schumann, M., Gantke, T. & Muhlberger, E. Ebola virus VP35 antagonizes PKR activity through its C-terminal interferon inhibitory domain. J. Virol. 83, 8993–8997 (2009).
Huang, Y., Xu, L., Sun, Y. & Nabel, G.J. The assembly of Ebola virus nucleocapsid requires virion-associated proteins 35 and 24 and posttranslational modification of nucleoprotein. Mol. Cell 10, 307–316 (2002).
Shi, W. et al. A filovirus-unique region of Ebola virus nucleoprotein confers aberrant migration and mediates its incorporation into virions. J. Virol. 82, 6190–6199 (2008).
Johnson, R.F., McCarthy, S.E., Godlewski, P.J. & Harty, R.N. Ebola virus VP35–VP40 interaction is sufficient for packaging 3E–5E minigenome RNA into virus-like particles. J. Virol. 80, 5135–5144 (2006).
Noda, T. et al. Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP. J. Virol. 76, 4855–4865 (2002).
Reid, S.P., Cardenas, W.B. & Basler, C.F. Homo-oligomerization facilitates the interferon-antagonist activity of the ebolavirus VP35 protein. Virology 341, 179–189 (2005).
Basler, C.F. & Amarasinghe, G.K. Evasion of interferon responses by Ebola and Marburg viruses. J. Interferon Cytokine Res. 29, 511–520 (2009).
Leung, D.W. et al. Structure of the Ebola VP35 interferon inhibitory domain. Proc. Natl. Acad. Sci. USA 106, 411–416 (2009).
Prins, K.C. et al. Mutations abrogating VP35 interaction with dsRNA render Ebola virus avirulent in guinea pigs. J. Virol. (in the press).
Li, X. et al. The RIG-I-like receptor LGP2 recognizes the termini of double-stranded RNA. J. Biol. Chem. 284, 13881–13891 (2009).
Lawrence, M.C. & Colman, P.M. Shape complementarity at protein/protein interfaces. J. Mol. Biol. 234, 946–950 (1993).
Muhlberger, E., Weik, M., Volchkov, V.E., Klenk, H.D. & Becker, S. Comparison of the transcription and replication strategies of marburg virus and Ebola virus by using artificial replication systems. J. Virol. 73, 2333–2342 (1999).
Pippig, D.A. et al. The regulatory domain of the RIG-I family ATPase LGP2 senses double-stranded RNA. Nucleic Acids Res. 37, 2014–2025 (2009).
Chang, T.H. et al. Ebola Zaire virus blocks type I interferon production by exploiting the host SUMO modification machinery. PLoS Pathog. 5, e1000493 (2009).
Prins, K.C., Cardenas, W.B. & Basler, C.F. Ebola virus protein VP35 impairs the function of interferon regulatory factor-activating kinases IKKepsilon and TBK-1. J. Virol. 83, 3069–3077 (2009).
Kato, H. et al. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441, 101–105 (2006).
Saito, T. & Gale, M. Jr. Differential recognition of double-stranded RNA by RIG-I-like receptors in antiviral immunity. J. Exp. Med. 205, 1523–1527 (2008).
Nallagatla, S.R. et al. 5′-triphosphate-dependent activation of PKR by RNAs with short stem-loops. Science 318, 1455–1458 (2007).
Takahasi, K. et al. Solution structures of cytosolic RNA sensor MDA5 and LGP2 C-terminal domains: identification of the RNA recognition loop in RIG-I-like receptors. J. Biol. Chem. 284, 17465–17474 (2009).
Murali, A. et al. Structure and function of LGP2, a DEX(D/H) helicase that regulates the innate immunity response. J. Biol. Chem. 283, 15825–15833 (2008).
Leung, D.W. et al. Expression, purification, crystallization and preliminary X-ray studies of the Ebola VP35 interferon inhibitory domain. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 65, 163–165 (2009).
McKenna, S.A. et al. Purification and characterization of transcribed RNAs using gel filtration chromatography. Nat. Protoc. 2, 3270–3277 (2007).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscilation mode. Methods Enzymol. 276, 307–326 (1997).
Pflugrath, J.W. The finer things in X-ray diffraction data collection. Acta Crystallogr. D Biol. Crystallogr. 55, 1718–1725 (1999).
Collaborative Computational Project. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).
Vagin, A.A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022 (1997).
Read, R.J. Pushing the boundaries of molecular replacement with maximum likelihood. Acta Crystallogr. D Biol. Crystallogr. 57, 1373–1382 (2001).
Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).
Lamzin, V.S. & Wilson, K.S. Automated refinement of protein models. Acta Crystallogr. D Biol. Crystallogr. 49, 129–147 (1993).
Perrakis, A., Sixma, T.K., Wilson, K.S. & Lamzin, V.S. wARP: improvement and extension of crystallographic phases by weighted averaging of multiple-refined dummy atomic models. Acta Crystallogr. D Biol. Crystallogr. 53, 448–455 (1997).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Painter, J. & Merritt, E.A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D Biol. Crystallogr. 62, 439–450 (2006).
Davis, I.W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).
Lee, B. & Richards, F.M. The interpretation of protein structures: estimation of static accessibility. J. Mol. Biol. 55, 379–400 (1971).
DeLano, W.L. The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, California, USA, 2002).
Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).
Johnson, B.A. Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol. Biol. 278, 313–352 (2004).
Acknowledgements
We thank the Iowa State University Biotechnology Facilities and J. Hoy, N. Pohl and D.B. Fulton for providing access to instrumentation and support. We also thank M. Nilsen-Hamilton and M. Shogren-Knaak for discussions, J. Binning, C. Brown and T. Wang for reading the manuscript, L. Tantral and D. Peterson for lab assistance and S. Ginnell, N. Duke, F. Rotella, M. Cuff and J. Lazarz at Advanced Photon Source Sector 19. Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the US Department of Energy under contract DE-AC02-06CH11357. This work is supported by US National Institutes of Health grants (1F32AI084324 to D.W.L., R01GM053163 to Z.O., R01NS010546 to R.B.H., R01AI059536 and AI057158 (Northeast Biodefense Center-Lipkin) to C.F.B. and R01AI081914 to G.K.A.), a Midwest Regional Center of Excellence Developmental grant (U54AI057160-Virgin(PI) to G.K.A.) and the Roy J. Carver Charitable Trust (09-3271 to G.K.A.).
Author information
Authors and Affiliations
Contributions
D.W.L., C.F.B. and G.K.A. designed research; D.W.L., K.C.P., D.M.B., M.F., J.M.T., P.R., J.C.N., L.A.H., Z.O., R.B.H., C.F.B. and G.K.A. performed research and analyzed data; D.W.L., K.C.P., C.F.B. and G.K.A. wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
Iowa State University has submitted a provisional patent application on the “Crystal Structure of Ebola VP35 Protein and Methods of Use.”
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–10 and Supplementary Table 1 (PDF 599 kb)
Rights and permissions
About this article
Cite this article
Leung, D., Prins, K., Borek, D. et al. Structural basis for dsRNA recognition and interferon antagonism by Ebola VP35. Nat Struct Mol Biol 17, 165–172 (2010). https://doi.org/10.1038/nsmb.1765
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.1765
This article is cited by
-
Spatial and functional arrangement of Ebola virus polymerase inside phase-separated viral factories
Nature Communications (2023)
-
A cryptic pocket in Ebola VP35 allosterically controls RNA binding
Nature Communications (2022)
-
Ebola virus VP35 hijacks the PKA-CREB1 pathway for replication and pathogenesis by AKIP1 association
Nature Communications (2022)
-
Crystal structure of human LC8 bound to a peptide from Ebola virus VP35
Journal of Microbiology (2021)
-
Ebola virus disease
Nature Reviews Disease Primers (2020)