Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Dual modes of RNA-silencing suppression by Flock House virus protein B2

Abstract

As a counter-defense against antiviral RNA silencing during infection, the insect Flock House virus (FHV) expresses the silencing suppressor protein B2. Biochemical experiments show that B2 binds to double-stranded RNA (dsRNA) without regard to length and inhibits cleavage of dsRNA by Dicer in vitro. A cocrystal structure reveals that a B2 dimer forms a four-helix bundle that binds to one face of an A-form RNA duplex independently of sequence. These results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHV small interfering RNAs into the RNA-induced silencing complex.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Biochemical analysis of B2 dsRNA-binding specificity and Dicer inhibition.
Figure 2: Overall view of the B2–dsRNA complex.
Figure 3: Characterization of the RNA-protein interface of B2.
Figure 4: Model of B2 bound to long dsRNA, in two orientations.
Figure 5: Diagram of the RNA-silencing pathway.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Li, H., Li, W.X. & Ding, S.W. Induction and suppression of RNA silencing by an animal virus. Science 296, 1319–1321 (2002).

    CAS  PubMed  Google Scholar 

  2. Friesen, P.D. & Rueckert, R.R. Black beetle virus: messenger for protein B is subgenomic viral RNA. J. Virol. 42, 986–995 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Lu, R. et al. Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans. Nature 436, 1040–1043 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Li, W.X. et al. Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc. Natl. Acad. Sci. USA 101, 1350–1355 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Dasgupta, R., Garcia, B.H. II & Goodman, R.M. Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes. Proc. Natl. Acad. Sci. USA 98, 4910–4915 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Sullivan, C.S. & Ganem, D. A virus-encoded inhibitor that blocks RNA interference in mammalian cells. J. Virol. 79, 7371–7379 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Voinnet, O. Induction and suppression of RNA silencing: insights from viral infections. Nat. Rev. Genet. 6, 206–220 (2005).

    CAS  PubMed  Google Scholar 

  8. Lakatos, L., Szittya, G., Silhavy, D. & Burgyan, J. Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses. EMBO J. 23, 876–884 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Molnar, A. et al. Plant virus-derived small interfering RNAs originate predominantly from highly structured single-stranded viral RNAs. J. Virol. 79, 7812–7818 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Vargason, J.M., Szittya, G., Burgyan, J. & Tanaka Hall, T.M. Size selective recognition of siRNA by an RNA silencing suppressor. Cell 115, 799–811 (2003).

    CAS  PubMed  Google Scholar 

  11. Ye, K., Malinina, L. & Patel, D.J. Recognition of small interfering RNA by a viral suppressor of RNA silencing. Nature 426, 874–878 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Johnson, K.L., Price, B.D., Eckerle, L.D. & Ball, L.A. Nodamura virus nonstructural protein B2 can enhance viral RNA accumulation in both mammalian and insect cells. J. Virol. 78, 6698–6704 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Crick, F.H.C. The packing of alpha-helices: simple coiled-coils. Acta Crystallogr. 6, 689–697 (1953).

    CAS  Google Scholar 

  14. Harris, N.L., Presnell, S.R. & Cohen, F.E. Four helix bundle diversity in globular proteins. J. Mol. Biol. 236, 1356–1368 (1994).

    CAS  PubMed  Google Scholar 

  15. Lupas, A., Van Dyke, M. & Stock, J. Predicting coiled coils from protein sequences. Science 252, 1162–1164 (1991).

    CAS  PubMed  Google Scholar 

  16. Lupas, A. Prediction and analysis of coiled-coil structures. Methods Enzymol. 266, 513–525 (1996).

    CAS  PubMed  Google Scholar 

  17. Holm, L. & Sander, C. Dali/FSSP classification of three-dimensional protein folds. Nucleic Acids Res. 25, 231–234 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Banner, D.W., Kokkinidis, M. & Tsernoglou, D. Structure of the ColE1 rop protein at 1.7 A resolution. J. Mol. Biol. 196, 657–675 (1987).

    CAS  PubMed  Google Scholar 

  19. Tomizawa, J. & Som, T. Control of ColE1 plasmid replication: enhancement of binding of RNA I to the primer transcript by the Rom protein. Cell 38, 871–878 (1984).

    CAS  PubMed  Google Scholar 

  20. Predki, P.F., Nayak, L.M., Gottlieb, M.B. & Regan, L. Dissecting RNA-protein interactions: RNA-RNA recognition by Rop. Cell 80, 41–50 (1995).

    CAS  PubMed  Google Scholar 

  21. Comolli, L.R., Pelton, J.G. & Tinoco, I., Jr. Mapping of a protein-RNA kissing hairpin interface: Rom and Tar-Tar*. Nucleic Acids Res. 26, 4688–4695 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Christ, D. & Winter, G. Identification of functional similarities between proteins using directed evolution. Proc. Natl. Acad. Sci. USA 100, 13202–13206 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Johnson, K.N., Johnson, K.L., Dasgupta, R., Gratsch, T. & Ball, L.A. Comparisons among the larger genome segments of six nodaviruses and their encoded RNA replicases. J. Gen. Virol. 82, 1855–1866 (2001).

    CAS  PubMed  Google Scholar 

  24. Ryter, J.M. & Schultz, S.C. Molecular basis of double-stranded RNA-protein interactions: structure of a dsRNA-binding domain complexed with dsRNA. EMBO J. 17, 7505–7513 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Lichner, Z., Silhavy, D. & Burgyan, J. Double-stranded RNA-binding proteins could suppress RNA interference-mediated antiviral defences. J. Gen. Virol. 84, 975–980 (2003).

    CAS  PubMed  Google Scholar 

  26. Brandt, T.A. & Jacobs, B.L. Both carboxy- and amino-terminal domains of the vaccinia virus interferon resistance gene, E3L, are required for pathogenesis in a mouse model. J. Virol. 75, 850–856 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Chien, C.Y. et al. Biophysical characterization of the complex between double-stranded RNA and the N-terminal domain of the NS1 protein from influenza A virus: evidence for a novel RNA-binding mode. Biochemistry 43, 1950–1962 (2004).

    CAS  PubMed  Google Scholar 

  28. Doublie, S. Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530 (1997).

    CAS  PubMed  Google Scholar 

  29. Recht, M.I. & Williamson, J.R. Central domain assembly: thermodynamics and kinetics of S6 and S18 binding to an S15-RNA complex. J. Mol. Biol. 313, 35–48 (2001).

    CAS  PubMed  Google Scholar 

  30. Leslie, A.G.W. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography No. 26 (1992).

    Google Scholar 

  31. Holton, J. & Alber, T. Automated protein crystal structure determination using ELVES. Proc. Natl. Acad. Sci. USA 101, 1537–1542 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Collaborative Computational Project Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  33. Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Terwilliger, T. Maximum likelihood density modification. Acta Crystallogr. D Biol. Crystallogr. 56, 965–972 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    CAS  PubMed  Google Scholar 

  36. McRee, D.E. XtalView/Xfit—A versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999).

    CAS  PubMed  Google Scholar 

  37. Lavery, R. & Sklenar, H. The definition of generalized helicoidal parameters and of axis curvature for irregular nucleic acids. J. Biomol. Struct. Dyn. 6, 63–91 (1988).

    CAS  PubMed  Google Scholar 

  38. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

    CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the US National Institutes of Health (grants GM-53320 to J.R.W. and GM-53491 to A.S.), the Skaggs Institute for Chemical Biology and an Achievement Rewards for College Scientists Foundation fellowship to J.A.C. The authors wish to thank E. Sperling for assistance with protein purification, M. Hennig for assistance with NMR spectroscopy and X. Dai, S. Nguyen, S. Ryder, R. Stanfield and R. Williams for helpful discussions.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to James R Williamson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Electrophoretic mobility shift of MBP-B2 with various nucleic acid constructs. (PDF 954 kb)

Supplementary Fig. 2

Experimental electron density map of the B2–dsRNA complex from SeMet MAD phasing (PDF 2970 kb)

Supplementary Fig. 3

Packing of α1 with core of B2 dimer (PDF 1731 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chao, J., Lee, J., Chapados, B. et al. Dual modes of RNA-silencing suppression by Flock House virus protein B2. Nat Struct Mol Biol 12, 952–957 (2005). https://doi.org/10.1038/nsmb1005

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1005

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing