Abstract

Viruses are molecular machines sustained through a life cycle that requires replication within host cells. Throughout the infectious cycle, viral and cellular components interact to advance the multistep process required to produce progeny virions. Despite progress made in understanding the virus–host protein interactome, much remains to be discovered about the cellular factors that function during infection, especially those operating at terminal steps in replication. In an RNA interference screen, we identified the eukaryotic chaperonin T-complex protein-1 (TCP-1) ring complex (TRiC; also called CCT for chaperonin containing TCP-1) as a cellular factor required for late events in the replication of mammalian reovirus. We discovered that TRiC functions in reovirus replication through a mechanism that involves folding the viral σ3 major outer-capsid protein into a form capable of assembling onto virus particles. TRiC also complexes with homologous capsid proteins of closely related viruses. Our data define a critical function for TRiC in the viral assembly process and raise the possibility that this mechanism is conserved in related non-enveloped viruses. These results also provide insight into TRiC protein substrates and establish a rationale for the development of small-molecule inhibitors of TRiC as potential antiviral therapeutics.

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References

  1. 1.

    Bouziat, R. et al. Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease. Science 356, 44–50 (2017).

  2. 2.

    Dermody, T. S., Parker, J. S. & Sherry, B. in Fields Virology, Vol. 2 (eds. D. M. Knipe & P. M. Howley) pp. 1304–1346 (Lippincott Williams & Wilkins, Philadelphia, 2013).

  3. 3.

    Barton, E. S. et al. Junction adhesion molecule is a receptor for reovirus. Cell 104, 441–451 (2001).

  4. 4.

    Konopka-Anstadt, J. L. et al. The Nogo receptor NgR1 mediates infection by mammalian reovirus. Cell Host Microbe 15, 681–691 (2014).

  5. 5.

    Maginnis, M. S. et al. NPXY motifs in the β1 integrin cytoplasmic tail are required for functional reovirus entry. J. Virol. 82, 3181–3191 (2008).

  6. 6.

    Ebert, D. H., Deussing, J., Peters, C. & Dermody, T. S. Cathepsin L and cathepsin B mediate reovirus disassembly in murine fibroblast cells. J. Biol. Chem. 277, 24609–24617 (2002).

  7. 7.

    Leitner, A. et al. The molecular architecture of the eukaryotic chaperonin TRiC/CCT. Structure 20, 814–825 (2012).

  8. 8.

    Bigotti, M. G. & Clarke, A. R. Chaperonins: the hunt for the group II mechanism. Arch. Biochem. Biophys. 474, 331–339 (2008).

  9. 9.

    Hartl, F. U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324–332 (2011).

  10. 10.

    Spiess, C., Meyer, A. S., Reissmann, S. & Frydman, J. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets. Trends Cell Biol. 14, 598–604 (2004).

  11. 11.

    Yam, A. Y. et al. Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly made proteins with complex topologies. Nat. Struct. Mol. Biol. 15, 1255–1262 (2008).

  12. 12.

    Frydman, J. et al. Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits. EMBO J. 11, 4767–4778 (1992).

  13. 13.

    Gao, Y., Thomas, J. O., Chow, R. L., Lee, G. H. & Cowan, N. J. A cytoplasmic chaperonin that catalyzes beta-actin folding. Cell 69, 1043–1050 (1992).

  14. 14.

    Gao, Y., Vainberg, I. E., Chow, R. L. & Cowan, N. J. Two cofactors and cytoplasmic chaperonin are required for the folding of alpha- and beta-tubulin. Mol. Cell Biol. 13, 2478–2485 (1993).

  15. 15.

    Hong, S. et al. Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC. J. Virol. 75, 2526–2534 (2001).

  16. 16.

    Lingappa, J. R. et al. A eukaryotic cytosolic chaperonin is associated with a high molecular weight intermediate in the assembly of hepatitis B virus capsid, a multimeric particle. J. Cell Biol. 125, 99–111 (1994).

  17. 17.

    Kashuba, E., Pokrovskaja, K., Klein, G. & Szekely, L. Epstein-Barr virus-encoded nuclear protein EBNA-3 interacts with the epsilon-subunit of the T-complex protein 1 chaperonin complex. J. Hum. Virol. 2, 33–37 (1999).

  18. 18.

    Inoue, Y. et al. Chaperonin TRiC/CCT participates in replication of hepatitis C virus genome via interaction with the viral NS5B protein. Virology 410, 38–47 (2011).

  19. 19.

    Zhang, J. et al. Cellular chaperonin CCTγ contributes to rabies virus replication during infection. J. Virol. 87, 7608–7621 (2013).

  20. 20.

    Fislova, T., Thomas, B., Graef, K. M. & Fodor, E. Association of the influenza virus RNA polymerase subunit PB2 with the host chaperonin CCT. J. Virol. 84, 8691–8699 (2010).

  21. 21.

    Solignat, M., Gay, B., Higgs, S., Briant, L. & Devaux, C. Replication cycle of chikungunya: a re-emerging arbovirus. Virology 393, 183–197 (2009).

  22. 22.

    Fernandez de Castro, I. et al. Reovirus forms neo-organelles for progeny particle assembly within reorganized cell membranes. mBio 5, e00931-13 (2014).

  23. 23.

    Thulasiraman, V., Yang, C. F. & Frydman, J. In vivo newly translated polypeptides are sequestered in a protected folding environment. EMBO J. 18, 85–95 (1999).

  24. 24.

    Shing, M. & Coombs, K. M. Assembly of the reovirus outer capsid requires μ1/σ3 interactions which are prevented by misfolded σ3 protein in temperature-sensitive mutant tsG453. Virus Res. 46, 19–29 (1996).

  25. 25.

    Chandran, K. et al. In vitro recoating of reovirus cores with baculovirus-expressed outer-capsid proteins μ1 and σ3. J. Virol. 73, 3941–3950 (1999).

  26. 26.

    Kasembeli, M. et al. Modulation of STAT3 folding and function by TRiC/CCT chaperonin. PLoS Biol. 12, e1001844 (2014).

  27. 27.

    Freund, A. et al. Proteostatic control of telomerase function through TRiC-mediated folding of TCAB1. Cell 159, 1389–1403 (2014).

  28. 28.

    Tian, G., Vainberg, I. E., Tap, W. D., Lewis, S. A. & Cowan, N. J. Specificity in chaperonin-mediated protein folding. Nature 375, 250–253 (1995).

  29. 29.

    Feldman, D. E., Thulasiraman, V., Ferreyra, R. G. & Frydman, J. Formation of the VHL-elongin BC tumor suppressor complex is mediated by the chaperonin TRiC. Mol. Cell 4, 1051–1061 (1999).

  30. 30.

    Miyata, Y., Shibata, T., Aoshima, M., Tsubata, T. & Nishida, E. The molecular chaperone TRiC/CCT binds to the Trp-Asp 40 (WD40) repeat protein WDR68 and promotes its folding, protein kinase DYRK1A binding, and nuclear accumulation. J. Biol. Chem. 289, 33320–33332 (2014).

  31. 31.

    Virgin, H. W. IV, Mann, M. A., Fields, B. N. & Tyler, K. L. Monoclonal antibodies to reovirus reveal structure/function relationships between capsid proteins and genetics of susceptibility to antibody action. J. Virol. 65, 6772–6781 (1991).

  32. 32.

    Olland, A. M., Jané-Valbuena, J., Schiff, L. A., Nibert, M. L. & Harrison, S. C. Structure of the reovirus outer capsid and dsRNA-binding protein σ3 at 1.8 Å resolution. EMBO J. 20, 979–989 (2001).

  33. 33.

    Miller, J. E. & Samuel, C. E. Proteolytic cleavage of the reovirus σ3 protein results in enhanced double-stranded RNA-binding activity: identification of a repeated basic amino acid motif within the C-terminal binding region. J. Virol. 66, 5347–5356 (1992).

  34. 34.

    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).

  35. 35.

    Martin, J. et al. Chaperonin-mediated protein folding at the surface of groEL through a ‘molten globule’-like intermediate. Nature 352, 36–42 (1991).

  36. 36.

    Frydman, J., Nimmesgern, E., Ohtsuka, K. & Hartl, F. U. Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature 370, 111–117 (1994).

  37. 37.

    Jané-Valbuena, J. et al. Reovirus virion-like particles obtained by recoating infectious subvirion particles with baculovirus-expressed σ3 protein: an approach for analyzing σ3 functions during virus entry. J. Virol. 73, 2963–2973 (1999).

  38. 38.

    Joachimiak, L. A., Walzthoeni, T., Liu, C. W., Aebersold, R. & Frydman, J. The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT. Cell 159, 1042–1055 (2014).

  39. 39.

    Georgescauld, F. et al. GroEL/ES chaperonin modulates the mechanism and accelerates the rate of TIM-barrel domain folding. Cell 157, 922–934 (2014).

  40. 40.

    Tian, G. & Cowan, N. J. Tubulin-specific chaperones: components of a molecular machine that assembles the α/β heterodimer. Methods Cell Biol. 115, 155–171 (2013).

  41. 41.

    Plimpton, R. L. et al. Structures of the Gβ-CCT and PhLP1-Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly. Proc. Natl Acad. Sci. USA 112, 2413–2418 (2015).

  42. 42.

    Spiess, C., Miller, E. J., McClellan, A. J. & Frydman, J. Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins. Mol. Cell 24, 25–37 (2006).

  43. 43.

    Leroux, M. R. & Hartl, F. U. Protein folding: versatility of the cytosolic chaperonin TRiC/CCT. Curr. Biol. 10, R260–R264 (2000).

  44. 44.

    Feldman, D. E., Spiess, C., Howard, D. E. & Frydman, J. Tumorigenic mutations in VHL disrupt folding in vivo by interfering with chaperonin binding. Mol. Cell 12, 1213–1224 (2003).

  45. 45.

    Attoui, H. et al. Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus (genus Aquareovirus, family Reoviridae). J. Gen. Virol. 83, 1941–1951 (2002).

  46. 46.

    Stins, M. F., Gilles, F. & Kim, K. S. Selective expression of adhesion molecules on human brain microvascular endothelial cells. J. Neuroimmunol. 76, 81–90 (1997).

  47. 47.

    Mainou, B. A. & Dermody, T. S. Transport to late endosomes is required for efficient reovirus infection. J. Virol. 86, 8346–8358 (2012).

  48. 48.

    Virgin, H. Wt, Bassel-Duby, R., Fields, B. N. & Tyler, K. L. Antibody protects against lethal infection with the neurally spreading reovirus type 3 (Dearing). J. Virol. 62, 4594–4604 (1988).

  49. 49.

    Kobayashi, T. et al. A plasmid-based reverse genetics system for animal double-stranded RNA viruses. Cell Host Microbe 1, 147–157 (2007).

  50. 50.

    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).

  51. 51.

    Parker, J. S., Broering, T. J., Kim, J., Higgins, D. E. & Nibert, M. L. Reovirus core protein μ2 determines the filamentous morphology of viral inclusion bodies by interacting with and stabilizing microtubules. J. Virol. 76, 4483–4496 (2002).

  52. 52.

    Mainou, B. A. et al. Reovirus cell entry requires functional microtubules. m Bio 4, e00405-13 (2013).

  53. 53.

    Becker, M. M. et al. Reovirus σNS protein is required for nucleation of viral assembly complexes and formation of viral inclusions. J. Virol. 75, 1459–1475 (2001).

  54. 54.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

  55. 55.

    Mainou, B. A. & Dermody, T. S. Src kinase mediates productive endocytic sorting of reovirus during cell entry. J. Virol. 85, 3203–3213 (2011).

  56. 56.

    Becker, M. M., Peters, T. R. & Dermody, T. S. Reovirus σNS and μNS proteins form cytoplasmic inclusion structures in the absence of viral infection. J. Virol. 77, 5948–5963 (2003).

  57. 57.

    Barton, E. S., Connolly, J. L., Forrest, J. C., Chappell, J. D. & Dermody, T. S. Utilization of sialic acid as a coreceptor enhances reovirus attachment by multistep adhesion strengthening. J. Biol. Chem. 276, 2200–2211 (2001).

  58. 58.

    Fontana, J., Lopez-Montero, N., Elliott, R. M., Fernandez, J. J. & Risco, C. The unique architecture of Bunyamwera virus factories around the Golgi complex. Cell Microbiol. 10, 2012–2028 (2008).

  59. 59.

    Hurbain, I. & Sachse, M. The future is cold: cryo-preparation methods for transmission electron microscopy of cells. Biol. Cell 103, 405–420 (2011).

  60. 60.

    Szklarczyk, D. et al. STRINGv10: protein–protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 43, D447–D452 (2015).

  61. 61.

    Sourisseau, M. et al. Characterization of reemerging chikungunya virus. PLoS Pathog. 3, e89 (2007).

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Acknowledgements

This work was supported in part by Public Health Service awards AI032539, AI122563, GM007347, UL1TR000445, and the Vanderbilt Lamb Center for Pediatric Research. The RNAi screen was performed in the Vanderbilt high-throughput screening facility, which is an institutionally supported core. Confocal images were captured in the cell imaging core at the Rangos Research Center at Children’s Hospital of Pittsburgh of UPMC. The authors thank P. Aravamudhan, J. Brown, B. Mainou, L. Silva, D. Sutherland and G. Taylor of the Dermody lab for essential discussions and critically editing the manuscript.

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Affiliations

  1. Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA

    • Jonathan J. Knowlton
    • , Alison W. Ashbrook
    •  & Paula F. Zamora
  2. Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    • Jonathan J. Knowlton
    • , Paula F. Zamora
    •  & Terence S. Dermody
  3. National Center for Biotechnology, Spanish National Research Council, CNB-CSIC, Madrid, Spain

    • Isabel Fernández de Castro
    •  & Cristina Risco
  4. Department of Biology, Stanford University, Palo Alto, CA, USA

    • Daniel R. Gestaut
    •  & Judith Frydman
  5. Department of Biochemistry, Institute of Chemical Biology, High-Throughput Screening Facility, Vanderbilt University School of Medicine, Nashville, TN, USA

    • Joshua A. Bauer
  6. Department of Microbiology and Immunology, Center for Microbial Pathogenesis and Host Responses, University of Arkansas for Medical Sciences, Little Rock, AR, USA

    • J. Craig Forrest
  7. Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    • Terence S. Dermody

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Author contributions

J.J.K. conceived, designed experiments and performed experiments, analysed data, contributed materials/analysis tools and wrote the paper. T.S.D. conceived and designed experiments, analysed the data and wrote the paper. I.F.C., A.W.A. and P.F.Z. conceived, designed experiments and performed experiments, and analysed data. J.A.B. conceived and designed experiments and performed experiments. D.R.G, J.F. and C.R. conceived and designed experiments, analysed data, and contributed materials/analysis tools. J.C.F. analysed data. All authors reviewed, critiqued and provided comments on the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Terence S. Dermody.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–14 and Supplementary Tables 1–3.

  2. Life Sciences Reporting Summary

  3. Supplementary Dataset 1

    Compiled RNA-interference screen raw data from three independent replicates.

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https://doi.org/10.1038/s41564-018-0122-x

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