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Atomic structure of the translation regulatory protein NS1 of bluetongue virus


Bluetongue virus (BTV) non-structural protein 1 (NS1) regulates viral protein synthesis and exists as tubular and non-tubular forms in infected cells, but how tubules assemble and how protein synthesis is regulated are unknown. Here, we report near-atomic resolution structures of two NS1 tubular forms determined by cryo-electron microscopy. The two tubular forms are different helical assemblies of the same NS1 monomer, consisting of an amino-terminal foot, a head and body domains connected to an extended carboxy-terminal arm, which wraps atop the head domain of another NS1 subunit through hydrophobic interactions. Deletion of the C terminus prevents tubule formation but not viral replication, suggesting an active non-tubular form. Two zinc-finger-like motifs are present in each NS1 monomer, and tubules are disrupted by divalent cation chelation and restored by cation addition, including Zn2+, suggesting a regulatory role of divalent cations in tubule formation. In vitro luciferase assays show that the NS1 non-tubular form upregulates BTV mRNA translation, whereas zinc-finger disruption decreases viral mRNA translation, tubule formation and virus replication, confirming a functional role for the zinc-fingers. Thus, the non-tubular form of NS1 is sufficient for viral protein synthesis and infectious virus replication, and the regulatory mechanism involved operates through divalent cation-dependent conversion between the non-tubular and tubular forms.

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Fig. 1: NS1 tubules are dynamic.
Fig. 2: Structure of the NS1 monomer.
Fig. 3: C-terminal arm deletion mutations demonstrate that the non-tubular form of NS1 is the functional form.
Fig. 4: NS1 contains two putative zinc-finger motifs, which are important for virus replication and tubule formation.
Fig. 5: Inter-layer and intra-layer interactions in NS1 tubules.

Data availability

The atomic models and cryoEM density map that support the findings of this study have been deposited in the Protein Data Bank and Electron Microscopy Data Bank with accession numbers 6N9Y and EMD-0383, respectively.


  1. 1.

    Walsh, D. & Mohr, I. Viral subversion of the host protein synthesis machinery. Nat. Rev. Microbiol. 9, 860–875 (2011).

    CAS  Article  Google Scholar 

  2. 2.

    Piron, M., Vende, P., Cohen, J. & Poncet, D. Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F. EMBO J. 17, 5811–5821 (1998).

    CAS  Article  Google Scholar 

  3. 3.

    Piron, M., Delaunay, T., Grosclaude, J. & Poncet, D. Identification of the RNA-binding, dimerization, and eIF4GI-binding domains of rotavirus nonstructural protein NSP3. J. Virol. 73, 5411–5421 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Boyce, M., Celma, C. P. & Roy, P. Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis. Virol. J. 9, 178 (2012).

    CAS  Article  Google Scholar 

  5. 5.

    Vitour, D., Lindenbaum, P., Vende, P., Becker, M. M. & Poncet, D. RoXaN, a novel cellular protein containing TPR, LD, and zinc finger motifs, forms a ternary complex with eukaryotic initiation factor 4G and rotavirus NSP3. J. Virol. 78, 3851–3862 (2004).

    CAS  Article  Google Scholar 

  6. 6.

    Harb, M. et al. Nuclear localization of cytoplasmic poly(A)-binding protein upon rotavirus infection involves the interaction of NSP3 with eIF4G and RoXaN. J. Virol. 82, 11283–11293 (2008).

    CAS  Article  Google Scholar 

  7. 7.

    Deo, R. C., Groft, C. M., Rajashankar, K. R. & Burley, S. K. Recognition of the rotavirus mRNA 3′ consensus by an asymmetric NSP3 homodimer. Cell 108, 71–81 (2002).

    CAS  Article  Google Scholar 

  8. 8.

    Groft, C. M. & Burley, S. K. Recognition of eIF4G by rotavirus NSP3 reveals a basis for mRNA circularization. Mol. Cell 9, 1273–1283 (2002).

    CAS  Article  Google Scholar 

  9. 9.

    Hewat, E. A., Booth, T. F., Wade, R. H. & Roy, P. 3-D reconstruction of bluetongue virus tubules using cryoelectron microscopy. J. Struct. Biol. 108, 35–48 (1992).

    CAS  Article  Google Scholar 

  10. 10.

    Urakawa, T. & Roy, P. Bluetongue virus tubules made in insect cells by recombinant baculoviruses: expression of the NS1 gene of bluetongue virus serotype 10. J. Virol. 62, 3919–3927 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Odom, T. W., Huang, J. L. & Lieber, C. M. Single-walled carbon nanotubes: from fundamental studies to new device concepts. Ann. N. Y. Acad. Sci. 960, 203–215 (2002).

    CAS  Article  Google Scholar 

  12. 12.

    Monastyrskaya, K., Gould, E. A. & Roy, P. Characterization and modification of the carboxy-terminal sequences of bluetongue virus type 10 NS1 protein in relation to tubule formation and location of an antigenic epitope in the vicinity of the carboxy terminus of the protein. J. Virol. 69, 2831–2841 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Boyce, M., Celma, C. C. & Roy, P. Development of reverse genetics systems for bluetongue virus: recovery of infectious virus from synthetic RNA transcripts. J. Virol. 82, 8339–8348 (2008).

    CAS  Article  Google Scholar 

  14. 14.

    Monastyrskaya, K., Booth, T., Nel, L. & Roy, P. Mutation of either of 2 cysteine residues or deletion of the amino or carboxy-terminus of nonstructural protein Ns1 of bluetongue virus abrogates virus-specified tubule formation in insect cells. J. Virol. 68, 2169–2178 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Mikhailov, M., Monastyrskaya, K., Bakker, T. & Roy, P. A new form of particulate single and multiple immunogen delivery system based on recombinant bluetongue virus-derived tubules. Virology 217, 323–331 (1996).

    CAS  Article  Google Scholar 

  16. 16.

    Ghosh, M. K., Borca, M. V. & Roy, P. Virus-derived tubular structure displaying foreign sequences on the surface elicit CD4+ Th cell and protective humoral responses. Virology 302, 383–392 (2002).

    CAS  Article  Google Scholar 

  17. 17.

    Murphy, A. & Roy, P. Manipulation of the bluetongue virus tubules for immunogen delivery. Future Microbiol. 3, 351–359 (2008).

    CAS  Article  Google Scholar 

  18. 18.

    Owens, R. J., Limn, C. & Roy, P. Role of an arbovirus nonstructural protein in cellular pathogenesis and virus release. J. Virol. 78, 6649–6656 (2004).

    CAS  Article  Google Scholar 

  19. 19.

    Mortola, E., Noad, R. & Roy, P. Bluetongue virus outer capsid proteins are sufficient to trigger apoptosis in mammalian cells. J. Virol. 78, 2875–2883 (2004).

    CAS  Article  Google Scholar 

  20. 20.

    Hall, T. M. Multiple modes of RNA recognition by zinc finger proteins. Curr. Opin. Struct. Biol. 15, 367–373 (2005).

    CAS  Article  Google Scholar 

  21. 21.

    Laity, J. H., Lee, B. M. & Wright, P. E. Zinc finger proteins: new insights into structural and functional diversity. Curr. Opin. Struct. Biol. 11, 39–46 (2001).

    CAS  Article  Google Scholar 

  22. 22.

    Attar, N. et al. The histone H3–H4 tetramer is a copper reductase enzyme. Preprint at (2018).

  23. 23.

    Yu, X., Jiang, J., Sun, J. & Zhou, Z. H. A putative ATPase mediates RNA transcription and capping in a dsRNA virus. eLife 4, e07901 (2015).

    Article  Google Scholar 

  24. 24.

    Carragher, B. et al. Leginon: an automated system for acquisition of images from vitreous ice specimens. J. Struct. Biol. 132, 33–45 (2000).

    CAS  Article  Google Scholar 

  25. 25.

    Ge, P. & Zhou, Z. H. Hydrogen-bonding networks and RNA bases revealed by cryo electron microscopy suggest a triggering mechanism for calcium switches. Proc. Natl Acad. Sci. USA 108, 9637–9642 (2011).

    CAS  Article  Google Scholar 

  26. 26.

    Zhang, X., Jin, L., Fang, Q., Hui, W. H. & Zhou, Z. H. 3.3 Å cryo-EM structure of a nonenveloped virus reveals a priming mechanism for cell entry. Cell 141, 472–482 (2010).

    CAS  Article  Google Scholar 

  27. 27.

    Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semi-automated software for high resolution single particle reconstructions. J. Struct. Biol. 128, 82–97 (1999).

    CAS  Article  Google Scholar 

  28. 28.

    Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

    Article  Google Scholar 

  29. 29.

    Diaz, R., Rice, W. J. & Stokes, D. L. Fourier–Bessel reconstruction of helical assemblies. Methods Enzymol. 482, 131–165 (2010).

    CAS  Article  Google Scholar 

  30. 30.

    Egelman, E. H. The iterative helical real space reconstruction method: surmounting the problems posed by real polymers. J. Struct. Biol. 157, 83–94 (2007).

    CAS  Article  Google Scholar 

  31. 31.

    Ge, P. et al. Atomic structures of a bactericidal contractile nanotube in its pre- and postcontraction states. Nat. Struct. Mol. Biol. 22, 377–382 (2015).

    CAS  Article  Google Scholar 

  32. 32.

    Clemens, D. L., Ge, P., Lee, B. Y., Horwitz, M. A. & Zhou, Z. H. Atomic structure of T6SS reveals interlaced array essential to function. Cell 160, 940–951 (2015).

    CAS  Article  Google Scholar 

  33. 33.

    Bartesaghi, A. et al. 2.2 Å resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor. Science 348, 1147–1151 (2015).

    CAS  Article  Google Scholar 

  34. 34.

    Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  Article  Google Scholar 

  35. 35.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  36. 36.

    Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  Article  Google Scholar 

  38. 38.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  Article  Google Scholar 

  39. 39.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  Article  Google Scholar 

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We thank M. Turmaine for his advice and support for imaging at the UCL EM facility and C. Celma (LSHTM) for advising in the BTV reverse genetics method. This project is supported partly by grants from the US NIH (AI094386 to Z.H.Z.) and The Wellcome Trust, UK (100218, Investigator Award to P.R.). We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines supported by UCLA and grants from the NIH (1S10OD018111 and 1U24 GM116792) and the National Science Foundation (DBI-1338135 and DMR-1548924). This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the National Science Foundation grant number ACI-1548562 (Comet cluster at the San Diego Supercomputing Center through allocation MCB140140).

Author information




Z.H.Z., P.R. and P.G. designed the experiments. M.B. purified the wild-type NS1 tubules. X.Z. recorded some of the cryoEM data. P.G. recorded the cryoEM data and determined the structure. M.L. and J.J. built the atomic models. A.K. expressed proteins, performed the mutagenesis and biochemical experiments, reverse genetics, virology and fluorescence microscopy analyses. M.L., Z.H.Z., P.R., P.G. and A.K. interpreted the data and wrote the paper.

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Correspondence to Z. Hong Zhou or Polly Roy.

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Kerviel, A., Ge, P., Lai, M. et al. Atomic structure of the translation regulatory protein NS1 of bluetongue virus. Nat Microbiol 4, 837–845 (2019).

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