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Reovirus polymerase λ3 localized by cryo-electron microscopy of virions at a resolution of 7.6 Å

Nature Structural & Molecular Biology volume 10, pages 1011–1018 (2003)Cite this article

Abstract

Reovirus is an icosahedral, double-stranded (ds) RNA virus that uses viral polymerases packaged within the viral core to transcribe its ten distinct plus-strand RNAs. To localize these polymerases, the structure of the reovirion was refined to a resolution of 7.6 Å by cryo-electron microscopy (cryo-EM) and three-dimensional (3D) image reconstruction. X-ray crystal models of reovirus proteins, including polymerase λ3, were then fitted into the density map. Each copy of λ3 was found anchored to the inner surface of the icosahedral core shell, making major contacts with three molecules of shell protein λ1 and overlapping, but not centering on, a five-fold axis. The overlap explains why only one copy of λ3 is bound per vertex. λ3 is furthermore oriented with its transcript exit channel facing a small channel through the λ1 shell, suggesting how the nascent RNA is passed into the large external cavity of the pentameric capping enzyme complex formed by protein λ2.

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Figure 1: Assessment of the resolution limit in reovirion reconstruction.
Figure 2: Reovirion cryo-EM reconstructions.
Figure 3: Fits of λ1 and λ3 X-ray models into reovirion cryo-EM map.
Figure 4: Location of λ3 inside reovirus λ1 shell.
Figure 5: Stereo view of λ3 X-ray ribbon model with α-helices color-coded to signify position and correspondence with features in the cryo-EM map.
Figure 6: Space-filling, cutaway view of the reovirus core, showing a proposed exit pathway for newly synthesized plus-strand (+) RNA transcripts leading from λ3 through the λ1 shell to the λ2 cavity.

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References

  1. Nibert, M.L., Schiff, L.A. & Fields, B.N. Reoviruses and their replication. in Fields Virology (eds. Fields, B.N., Knipe, D.M. & Howley, P.M.) 1679–1728 (Raven, Philadelphia, 2001).

    Google Scholar 

  2. Dryden, K.A. et al. Early steps in reovirus infection are associated with dramatic changes in supramolecular structure and protein conformation: analysis of virions and subviral particles by cryoelectron microscopy and image reconstruction. J. Cell Biol. 122, 1023–1041 (1993).

    Article  CAS  Google Scholar 

  3. Liemann, S., Chandran, K., Baker, T.S., Nibert, M.L. & Harrison, S.C. Structure of the reovirus membrane-penetration protein, μ1, in a complex with its protector protein, σ3. Cell 108, 283–295 (2002).

    Article  CAS  Google Scholar 

  4. Furlong, D.B., Nibert, M.L. & Fields, B.N. Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles. J. Virol. 62, 246–256 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Strong, J.E., Leone, G., Duncan, R., Sharma, R.K. & Lee, P.W.K. Biochemical and biophysical characterization of the reovirus cell attachment protein σ1: evidence that it is a homotrimer. Virology 184, 23–32 (1991).

    Article  CAS  Google Scholar 

  6. Larson, S.M., Antczak, J.B. & Joklik, W.K. Reovirus exists in the form of 13 particle species that differ in their content of protein σ1. Virology 201, 303–311 (1994).

    Article  CAS  Google Scholar 

  7. Chappell, J.D., Porta, A.E., Dermody, T.S. & Stehle, T. Crystal structure of reovirus attachment protein σ1 reveals evolutionary relationship to adenovirus fiber. EMBO J. 21, 1–11 (2002).

    Article  CAS  Google Scholar 

  8. Sturzenbecker, L.J., Nibert, M., Furlong, D. & Fields, B.N. Intracellular digestion of reovirus particles requires a low pH and is an essential step in the viral infectious cycle. J. Virol. 61, 2351–2361 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Chandran, K., Farsetta, D.L. & Nibert, M.L. Strategy for nonenveloped virus entry: a hydrophobic conformer of reovirus penetration protein μ1 mediates membrane disruption. J. Virol. 76, 9920–9933 (2002).

    Article  CAS  Google Scholar 

  10. Chandran, K., Parker, J.S.L., Ehrlich, M., Kirchhausen, Y. & Nibert, M.L. The δ region of outer-capsid protein μ1 undergoes conformational change and release from reovirus particles during cell entry. J. Virol. (in the press).

  11. Reinisch, K.M., Nibert, M.L. & Harrison, S.C. Structure of the reovirus core at 3.6 Å resolution. Nature 404, 960–967 (2000).

    Article  CAS  Google Scholar 

  12. Samuel, C.E. Reoviruses and the interferon system. Curr. Top. Microbiol. Immunol. 233, 125–145 (1998).

    CAS  PubMed  Google Scholar 

  13. Cullen, B.R. RNA interference: antiviral defense and genetic tool. Nat. Immunol. 3, 597–599 (2002).

    Article  CAS  Google Scholar 

  14. Coombs, K.M. Stoichiometry of reovirus structural proteins in virus, ISVP, and core particles. Virology 243, 218–228 (1998).

    Article  CAS  Google Scholar 

  15. Dryden, K.A. et al. Internal structures containing transcriptase-related proteins in top component particles of mammalian orthoreovirus. Virology 245, 33–46 (1998).

    Article  CAS  Google Scholar 

  16. Drayna, D. & Fields, B.N. Activation and characterization of the reovirus transcriptase: genetic analysis. J. Virol. 41, 110–118 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Starnes, M.C. & Joklik, W.K. Reovirus protein λ3 is a poly(C)-dependent poly(G) polymerase. Virology 193, 356–366 (1993).

    Article  CAS  Google Scholar 

  18. Tao, Y., Farsetta, D.L., Nibert, M.L. & Harrison, S.C. RNA synthesis in a cage—structural studies of reovirus polymerase λ3. Cell 111, 733–745 (2002).

    Article  CAS  Google Scholar 

  19. Yin, P., Cheang, M. & Coombs, K.M. The M1 gene is associated with differences in the temperature optimum of the transcriptase activity in reovirus core particles. J. Virol. 70, 1223–1227 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Noble, S. & Nibert, M.L. Core protein μ2 is a second determinant of nucleoside triphosphatase activities by reovirus cores. J. Virol. 71, 7728–7735 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Noble, S. & Nibert, M.L. Characterization of an ATPase activity in reovirus cores and its genetic association with core-shell protein λ1. J. Virol. 71, 2182–2191 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Bisaillon, M., Bergeron, J. & Lemay, G. Characterization of the nucleoside triphosphate phosphohydrolase and helicase activities of the reovirus λ1 protein. J. Biol. Chem. 272, 18298–18303 (1997).

    Article  CAS  Google Scholar 

  23. Bisaillon, M. & Lemay, G. Characterization of the reovirus λ1 protein RNA 5′-triphosphatase activity. J. Biol. Chem. 272, 29954–29957 (1997).

    Article  CAS  Google Scholar 

  24. Furuichi, Y., Muthukrishnan, S., Tomasz, J. & Shatkin, A.J. Caps in eukaryotic mRNAs: mechanism of formation of reovirus mRNA 5′-terminal m7GpppGm-C. Prog. Nucleic Acid Res. Mol. Biol. 19, 3–20 (1976).

    Article  CAS  Google Scholar 

  25. Cleveland, D.R., Zarbl, H. & Millward, S. Reovirus guanylyltransferase is L2 gene product λ2. J. Virol. 60, 307–311 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Mao, Z.X. & Joklik, W.K. Isolation and enzymatic characterization of protein λ2, the reovirus guanylyltransferase. Virology 185, 377–386 (1991).

    Article  CAS  Google Scholar 

  27. Fausnaugh, J. & Shatkin, A.J. Active site localization in a viral mRNA capping enzyme. J. Biol. Chem. 265, 7669–7672 (1990).

    CAS  PubMed  Google Scholar 

  28. Luongo, C.L., Contreras, C.M., Farsetta, D.L. & Nibert, M.L. Binding site for S-adenosyl-L-methionine in a central region of mammalian reovirus λ2 protein. Evidence for activities in mRNA cap methylation. J. Biol. Chem. 273, 23773–23780 (1998).

    Article  CAS  Google Scholar 

  29. Luongo, C.L., Reinisch, K.M., Harrison, S.C. & Nibert, M.L. Identification of the guanylyltransferase region and active site in reovirus mRNA capping protein λ2. J. Biol. Chem. 275, 2804–2810 (2000).

    Article  CAS  Google Scholar 

  30. Gillies, S., Bullivant, S. & Bellamy, A.R. Viral RNA polymerases: electron microscopy of reovirus reaction cores. Science 174, 694–696 (1971).

    Article  CAS  Google Scholar 

  31. Bartlett, N.M., Gillies, S.C., Bullivant, S. & Bellamy, A.R. Electron microscopy study of reovirus reaction cores. J. Virol. 14, 315–326 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Yeager, M., Weiner, S. & Coombs, K.M. Transcriptionally active reovirus core particles visualized by electron cryo-microscopy and image reconstruction. Biophys. J. 70, A116 (1996).

    Google Scholar 

  33. Furuichi, Y. & Shatkin, A.J. Viral and cellular mRNA capping: past and prospects. Adv. Virus Res. 55, 135–184 (2000).

    Article  CAS  Google Scholar 

  34. Shuman, S. Structure, mechanism, and evolution of the mRNA capping apparatus. Prog. Nucleic Acid Res. Mol. Biol. 66, 1–40 (2001).

    CAS  PubMed  Google Scholar 

  35. Prasad, B.V.V. et al. Visualization of ordered genomic RNA and localization of transcriptional complexes in rotavirus. Nature 382, 471–473 (1996).

    Article  CAS  Google Scholar 

  36. Pesavento, J.B., Lawton, J.A., Estes, M.K. & Prasad, B.V.V. The reversible condensation and expansion of the rotavirus genome. Proc. Natl. Acad. Sci. USA 98, 1381–1386 (2001).

    Article  CAS  Google Scholar 

  37. Gouet, P. et al. The highly ordered double-stranded RNA genome of bluetongue virus revealed by crystallography. Cell 97, 481–490 (1999).

    Article  CAS  Google Scholar 

  38. Grimes, J.M. et al. The atomic structure of the bluetongue virus core. Nature 395, 470–478 (1998).

    Article  CAS  Google Scholar 

  39. Zhang, H. et al. Visualization of protein-RNA interactions in cytoplasmic polyhedrosis virus. J. Virol. 73, 1624–1629 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Baker, T.S., Olson, N.H. & Fuller, S.D. Adding the third dimension to virus life cycles: three-dimensional reconstruction of icosahedral viruses from cryo-electron micrographs. Microbiol. Mol. Biol. Rev. 63, 862–922 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Rossmann, M.G., Bernal, R. & Pletnev, S.V. Combining electron microscopic with X-ray crystallographic structures. J. Struct. Biol. 136, 190–200 (2001).

    Article  CAS  Google Scholar 

  42. Chacón, P. & Wriggers, W. Multi-resolution contour-based fitting of macromolecular structures. J. Mol. Biol. 317, 375–384 (2002).

    Article  Google Scholar 

  43. Farsetta, D.L., Chandran, K. & Nibert, M.L. Transcriptional activities of reovirus RNA polymerase in recoated cores. Initiation and elongation are regulated by separate mechanisms. J. Biol. Chem. 275, 39693–39701 (2000).

    Article  CAS  Google Scholar 

  44. Joklik, W.K. The reovirus particle. In The Reoviridae (ed. Joklik, W.K.) 9–78 (Plenum, New York, 1983).

    Chapter  Google Scholar 

  45. Shatkin, A.J. & Kozak, M. Biochemical aspects of reovirus transcription and translation. In The Reoviridae (ed. Joklik, W.K.) 79–106 (Plenum, New York, 1983).

    Chapter  Google Scholar 

  46. Cheetham, G.M. & Steitz, T.A. Insights into transcription: structure and function of single-subunit DNA-dependent RNA polymerases. Curr. Opin. Struct. Biol. 10, 117–123 (2000).

    Article  CAS  Google Scholar 

  47. Spencer, S.M., Sgro, J.-Y., Dryden, K.A., Baker, T.S. & Nibert, M.L. IRIS explorer software for radial-depth cueing reovirus particles and other macromolecular structures determined by cryoelectron microscopy and image reconstruction. J. Struct. Biol. 120, 11–21 (1997).

    Article  CAS  Google Scholar 

  48. Luongo, C.L. et al. Loss of activities for mRNA synthesis accompanies loss of λ2 spikes from reovirus cores: an effect of λ2 on λ1 shell structure. Virology 296, 24–38 (2002).

    Article  CAS  Google Scholar 

  49. Diprose, J.M. et al. Translocation portals for the substrates and products of a viral transcription complex: the bluetongue virus core. EMBO J. 20, 7229–7239 (2001).

    Article  CAS  Google Scholar 

  50. Lawton, J.A., Estes, M.K. & Prasad, B.V.V. Three-dimensional visualization of mRNA release from actively transcribing rotavirus particles. Nat. Struct. Biol. 4, 118–121 (1997).

    Article  CAS  Google Scholar 

  51. Le Blois, H., French, T., Mertens, P.P., Burroughs, J.N. & Roy, P. The expressed VP4 protein of bluetongue virus binds GTP and is the candidate guanylyl transferase of the virus. Virology 189, 757–761 (1992).

    Article  CAS  Google Scholar 

  52. Baker, T.S. & Cheng, R.H. A model-based approach for determining orientations of biological macromolecules imaged by cryoelectron microscopy. J. Struct. Biol. 116, 120–130 (1996).

    Article  CAS  Google Scholar 

  53. Bowman, V.D. et al. An antibody to the putative aphid recognition site on cucumber mosaic virus recognizes pentons but not hexons. J. Virol. 76, 12250–12258 (2002).

    Article  CAS  Google Scholar 

  54. van Heel, M. et al. Single-particle electron cryo-microscopy: towards atomic resolution. Q. Rev. Biophys. 33, 371–424 (2000).

    Article  Google Scholar 

  55. Esnouf, R.M. An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. J. Mol. Graph. Model. 15, 132–134 (1997).

    Article  CAS  Google Scholar 

  56. Merritt, E.A. & Bacon, D.J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  58. Grimes, J.M. et al. An atomic model of the outer layer of the bluetongue virus core derived from X-ray crystallography and electron cryomicroscopy. Structure 5, 885–893 (1997).

    Article  CAS  Google Scholar 

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Acknowledgements

We are especially grateful to S. Harrison and Y. Tao for providing the coordinates of the λ3 crystal structure before publication and also for discussions and comments on the manuscript. We also thank W. Zhang, C. Xiao, R. Ashmore, J. Johnson, A. McGough, R. Bernal, M. Sherman, M. Rossmann, P. Chacón and B. Bahlke for helpful discussions; V. Bowman, A. Simpson, P. Leiman, Y. Tao and J. Zhiu for assistance with figures; K. Reinisch and S. Liemann for providing crystal coordinates and discussion; and L. Szpankowski for digitizing micrographs. Work was supported in part by grants from the US National Institutes of Health to T.S.B. and M.L.N., a shared equipment grant from the US National Science Foundation to T.S.B., a Keck Foundation award to the Purdue Structural Biology group for purchase of the CM300 FEG microscope and a Purdue University reinvestment grant to the Structural Biology group. S.B.W. was additionally supported by the Purdue Biophysics Training Grant and a Purdue Research Foundation Fellowship.

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  1. Department of Biological Sciences, Purdue University, West Lafayette, 47907, Indiana, USA

    Xing Zhang, Stephen B Walker, Paul R Chipman & Timothy S Baker

  2. Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Max L Nibert

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Zhang, X., Walker, S., Chipman, P. et al. Reovirus polymerase λ3 localized by cryo-electron microscopy of virions at a resolution of 7.6 Å. Nat Struct Mol Biol 10, 1011–1018 (2003). https://doi.org/10.1038/nsb1009

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