Letter | Published:

Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function

Nature volume 530, pages 358361 (18 February 2016) | Download Citation

Abstract

Retroviral integrase catalyses the integration of viral DNA into host target DNA, which is an essential step in the life cycle of all retroviruses1. Previous structural characterization of integrase–viral DNA complexes, or intasomes, from the spumavirus prototype foamy virus revealed a functional integrase tetramer2,3,4,5, and it is generally believed that intasomes derived from other retroviral genera use tetrameric integrase6,7,8,9. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Here, using single-particle cryo-electron microscopy and X-ray crystallography, we determine an unexpected octameric integrase architecture for the intasome of the betaretrovirus mouse mammary tumour virus. The structure is composed of two core integrase dimers, which interact with the viral DNA ends and structurally mimic the integrase tetramer of prototype foamy virus, and two flanking integrase dimers that engage the core structure via their integrase carboxy-terminal domains. Contrary to the belief that tetrameric integrase components are sufficient to catalyse integration, the flanking integrase dimers were necessary for mouse mammary tumour virus integrase activity. The integrase octamer solves a conundrum for betaretroviruses as well as alpharetroviruses by providing critical carboxy-terminal domains to the intasome core that cannot be provided in cis because of evolutionarily restrictive catalytic core domain–carboxy-terminal domain linker regions. The octameric architecture of the intasome of mouse mammary tumour virus provides new insight into the structural basis of retroviral DNA integration.

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Accessions

Primary accessions

Electron Microscopy Data Bank

Data deposits

Coordinates of cryo-EM density maps for the full and core intasome datasets have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-6440 and EMD-6441, respectively. X-ray diffraction data and the resulting INCCD, INNTD–CCD and INCTD structures have been deposited in the Protein Data Bank (PDB) under accession numbers 5CZ1, 5CZ2 and 5D7U, respectively. The core intasome structure has been deposited in the Protein Data Bank under accession number 3JCA.

References

  1. 1.

    & HIV DNA integration. Cold Spring Harb. Perspect. Med. 2, a006890 (2012)

  2. 2.

    , , , & Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 464, 232–236 (2010)

  3. 3.

    , & The mechanism of retroviral integration from X-ray structures of its key intermediates. Nature 468, 326–329 (2010)

  4. 4.

    , & 3′-Processing and strand transfer catalysed by retroviral integrase in crystallo. EMBO J. 31, 3020–3028 (2012)

  5. 5.

    et al. Structural basis for retroviral integration into nucleosomes. Nature 523, 366–369 (2015)

  6. 6.

    , , & Crystal structure of an active two-domain derivative of Rous sarcoma virus integrase. J. Mol. Biol. 296, 535–548 (2000)

  7. 7.

    , , & Structure of a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein. EMBO J. 20, 7333–7343 (2001)

  8. 8.

    et al. Functional oligomeric state of avian sarcoma virus integrase. J. Biol. Chem. 278, 1323–1327 (2003)

  9. 9.

    , , & Retroviral DNA integration: reaction pathway and critical intermediates. EMBO J. 25, 1295–1304 (2006)

  10. 10.

    et al. A novel co-crystal structure affords the design of gain-of-function lentiviral integrase mutants in the presence of modified PSIP1/LEDGF/p75. PLoS Pathog. 5, e1000259 (2009)

  11. 11.

    & Nucleotide sequences at host-proviral junctions for mouse mammary tumour virus. Nature 289, 253–258 (1981)

  12. 12.

    , & Structure of the termini of DNA intermediates in the integration of retroviral DNA: dependence on IN function and terminal DNA sequence. Cell 58, 47–54 (1989)

  13. 13.

    , & The IN protein of Moloney murine leukemia virus processes the viral DNA ends and accomplishes their integration in vitro. Cell 62, 829–837 (1990)

  14. 14.

    & A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in vitro. Proc. Natl Acad. Sci. USA 91, 7316–7320 (1994)

  15. 15.

    et al. Atomic-accuracy models from 4.5-Å cryo-electron microscopy data with density-guided iterative local refinement. Nature Methods 12, 361–365 (2015)

  16. 16.

    et al. Structure of the type VI secretion system contractile sheath. Cell 160, 952–962 (2015)

  17. 17.

    et al. De novo protein structure determination from near-atomic-resolution cryo-EM maps. Nature Methods 12, 335–338 (2015)

  18. 18.

    , , & Complementation between HIV integrase proteins mutated in different domains. EMBO J. 12, 3261–3267 (1993)

  19. 19.

    , & Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex. EMBO J. 12, 3269–3275 (1993)

  20. 20.

    & Assembly and catalysis of concerted two-end integration events by Moloney murine leukemia virus integrase. J. Virol. 75, 9561–9570 (2001)

  21. 21.

    & Division of labor within human immunodeficiency virus integrase complexes: determinants of catalysis and target DNA capture. J. Virol. 79, 15376–15387 (2005)

  22. 22.

    , , & Division of labor among monomers within the Mu transposase tetramer. Cell 74, 723–733 (1993)

  23. 23.

    , , & Critical contacts between HIV-1 integrase and viral DNA identified by structure-based analysis and photo-crosslinking. EMBO J. 16, 6849–6859 (1997)

  24. 24.

    , , , & Crystal structure of the Rous sarcoma virus intasome. Nature (this issue)

  25. 25.

    et al. HIV-1 integrase crosslinked oligomers are active in vitro. Nucleic Acids Res. 33, 977–986 (2005)

  26. 26.

    , , & Molecular interactions between HIV-1 integrase and the two viral DNA ends within the synaptic complex that mediates concerted integration. J. Mol. Biol. 389, 183–198 (2009)

  27. 27.

    , , , & Zn2+ promotes the self-association of human immunodeficiency virus type-1 integrase in vitro. Biochemistry 36, 173–180 (1997)

  28. 28.

    & Photo-cross-linking studies suggest a model for the architecture of an active human immunodeficiency virus type 1 integrase-DNA complex. Biochemistry 37, 6667–6678 (1998)

  29. 29.

    , , , & Key determinants of target DNA recognition by retroviral intasomes. Retrovirology 12, 39 (2015)

  30. 30.

    & Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro. J. Virol. 66, 6361–6369 (1992)

  31. 31.

    , , , & Biochemical characterization of novel retroviral integrase proteins. PLoS ONE 8, e76638 (2013)

  32. 32.

    LEDGF/p75 interacts with divergent lentiviral integrases and modulates their enzymatic activity in vitro. Nucleic Acids Res. 35, 113–124 (2007)

  33. 33.

    et al. UltraScan-III version 2.2: a comprehensive data analysis software package for analytical ultracentrifugation experiments (2014)

  34. 34.

    , , & in Analytical Ultracentrifugation in Biochemistry and Polymer Science (eds , & ) 90–125 (Royal Society of Chemistry, 1992)

  35. 35.

    in Current Protocols in Protein Science (eds et al.) Ch. 7, Unit 7.13, 7.13.1–7.13.24 (Wiley, 2010)

  36. 36.

    , & A two-dimensional spectrum analysis for sedimentation velocity experiments of mixtures with heterogeneity in molecular weight and shape. Eur. Biophys. J. 39, 405–414 (2010)

  37. 37.

    & Direct sedimentation analysis of interference optical data in analytical ultracentrifugation. Biophys. J. 76, 2288–2296 (1999)

  38. 38.

    & in Proceedings of the 9th Annual Conference on Genetic and Evolutionary Computation 361–368 (Association for Computing Machinery, 2007)

  39. 39.

    & Monte Carlo analysis of sedimentation experiments. Colloid Polym. Sci. 286, 129–137 (2008)

  40. 40.

    XDS. Acta Crystallogr. D 66, 125–132 (2010)

  41. 41.

    & How good are my data and what is the resolution? Acta Crystallogr. D 69, 1204–1214 (2013)

  42. 42.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

  43. 43.

    et al. High-resolution structure of the catalytic domain of avian sarcoma virus integrase. J. Mol. Biol. 253, 333–346 (1995)

  44. 44.

    et al. Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: a model for viral DNA binding. Proc. Natl Acad. Sci. USA 97, 8233–8238 (2000)

  45. 45.

    , & ARP/wARP and automatic interpretation of protein electron density maps. Methods Enzymol. 374, 229–244 (2003)

  46. 46.

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

  47. 47.

    et al. The Phenix software for automated determination of macromolecular structures. Methods 55, 94–106 (2011)

  48. 48.

    , & Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

  49. 49.

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

  50. 50.

    et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005)

  51. 51.

    et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature Methods 10, 584–590 (2013)

  52. 52.

    et al. Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 envelope trimer. Science 342, 1484–1490 (2013)

  53. 53.

    & Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. eLife 4, e06980 (2015)

  54. 54.

    et al. Appion: an integrated, database-driven pipeline to facilitate EM image processing. J. Struct. Biol. 166, 95–102 (2009)

  55. 55.

    et al. A clustering approach to multireference alignment of single-particle projections in electron microscopy. J. Struct. Biol. 171, 197–206 (2010)

  56. 56.

    RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

  57. 57.

    , , & Optimod – an automated approach for constructing and optimizing initial models for single-particle electron microscopy. J. Struct. Biol. 184, 417–426 (2013)

  58. 58.

    FREALIGN: high-resolution refinement of single particle structures. J. Struct. Biol. 157, 117–125 (2007)

  59. 59.

    , , & Likelihood-based classification of cryo-EM images using FREALIGN. J. Struct. Biol. 183, 377–388 (2013)

  60. 60.

    , , & Cryo-EM model validation using independent map reconstructions. Protein Sci. 22, 865–868 (2013)

  61. 61.

    & Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014)

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Acknowledgements

We acknowledge support from US National Institutes of Health (NIH) grants R01 AI070042 (to A.N.E.), NIH P50 GM103368 and the Leona M. and Harry B. Helmsley Charitable Trust grant number 2012-PG-MED002 (to D.L., both funding sources provided equal support), NIH P50 GM082251 (to P.C.), NIH P30 AI060354 (Harvard University Center for AIDS Research), and US National Science Foundation grants NSF-ACI-1339649 and TG-MCB070039 (to B.D.). B.D. acknowledges support from San Antonio Cancer Institute grant CA054174 for the Center for Analytical Ultracentrifugation of Macromolecular Assemblies at the University of Texas Health Science Center at San Antonio. Molecular graphics and analyses were performed with the USCF Chimera package (supported by NIH P41 GM103331). CryoEM data collection was in part facilitated by the National Resource for Automated Molecular Microscopy (9 P41 GM103310). We thank B. Anderson and J.-C. Ducom at The Scripps Research Institute for help with EM data collection and network infrastructure, J. Fitzpatrick and F. Dwyer for computational support at The Salk Institute, V. Pye for help with X-ray structure refinement and the staff of BM14 (European Synchrotron Radiation Facility, Grenoble, France) and I03 (Diamond Light Source, Oxfordshire, UK) beamlines for assistance with data collection.

Author information

Affiliations

  1. Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, 450 Brookline Avenue, Boston, Massachusetts 02215, USA

    • Allison Ballandras-Colas
    • , Tamaria G. Dewdney
    •  & Alan N. Engelman
  2. Laboratory of Genetics and Helmsley Center for Genomic Medicine, The Salk Institute for Biological Studies, 10010 N Torrey Pines Road, La Jolla, California 92037, USA

    • Monica Brown
    •  & Dmitry Lyumkis
  3. Clare Hall Laboratories, The Francis Crick Institute, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3LD, UK

    • Nicola J. Cook
    •  & Peter Cherepanov
  4. Department of Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229, USA

    • Borries Demeler
  5. Division of Medicine, Imperial College London, St. Mary’s Campus, Norfolk Place, London W2 1PG, UK

    • Peter Cherepanov

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Contributions

A.B.-C. and A.N.E. discovered how to assemble MMTV intasomes; A.B.-C. and T.G.D. expressed and purified MMTV IN proteins for biochemical analysis; A.B.-C. assembled intasomes, characterized their biochemistry, supplied them for cryo-EM and centrifugation analyses, and performed IN activity assays; M.B. and D.L. performed EM work, collected cryo-EM data and determined the structure; D.L. modelled the intasome structure; B.D. collected and analysed the sedimentation velocity data; N.J.C. and P.C. expressed and purified INCCD, INNTD–CCD and INCTD/212–266 constructs, established crystallization conditions and determined these structures.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Dmitry Lyumkis or Alan N. Engelman.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figure 1 – source data gels for Figures 1c and 4a.

Videos

  1. 1.

    Catalytic core domain in segmented electron density map

    Catalytic core domain in segmented electron density map

  2. 2.

    N-terminal domain in segmented electron density map

    N-terminal domain in segmented electron density map

  3. 3.

    C-terminal domain in segmented electron density map

    C-terminal domain in segmented electron density map.

  4. 4.

    Viral DNA in segmented electron density map

    Viral DNA in segmented electron density map.

  5. 5.

    MMTV intasome structure in electron density map with close up views of NTD1-CCD1, CCD1-CTD1 and CCD2-CTD2 linker regions

    MMTV intasome structure in electron density map with close up views of NTD1-CCD1, CCD1-CTD1 and CCD2-CTD2 linker regions

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DOI

https://doi.org/10.1038/nature16955

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