Structure of the immature retroviral capsid at 8 Å resolution by cryo-electron microscopy

Abstract

The assembly of retroviruses such as HIV-1 is driven by oligomerization of their major structural protein, Gag. Gag is a multidomain polyprotein including three conserved folded domains: MA (matrix), CA (capsid) and NC (nucleocapsid)1. Assembly of an infectious virion proceeds in two stages2. In the first stage, Gag oligomerization into a hexameric protein lattice leads to the formation of an incomplete, roughly spherical protein shell that buds through the plasma membrane of the infected cell to release an enveloped immature virus particle. In the second stage, cleavage of Gag by the viral protease leads to rearrangement of the particle interior, converting the non-infectious immature virus particle into a mature infectious virion. The immature Gag shell acts as the pivotal intermediate in assembly and is a potential target for anti-retroviral drugs both in inhibiting virus assembly and in disrupting virus maturation3. However, detailed structural information on the immature Gag shell has not previously been available. For this reason it is unclear what protein conformations and interfaces mediate the interactions between domains and therefore the assembly of retrovirus particles, and what structural transitions are associated with retrovirus maturation. Here we solve the structure of the immature retroviral Gag shell from Mason–Pfizer monkey virus by combining cryo-electron microscopy and tomography. The 8-Å resolution structure permits the derivation of a pseudo-atomic model of CA in the immature retrovirus, which defines the protein interfaces mediating retrovirus assembly. We show that transition of an immature retrovirus into its mature infectious form involves marked rotations and translations of CA domains, that the roles of the amino-terminal and carboxy-terminal domains of CA in assembling the immature and mature hexameric lattices are exchanged, and that the CA interactions that stabilize the immature and mature viruses are almost completely distinct.

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Figure 1: Cryo-EM reconstruction of M-PMV CANC tubes.
Figure 2: Fitting of known atomic structures into the cryo-EM map.
Figure 3: Comparison of the arrangement of CA domains in the mature and immature retrovirus lattices.
Figure 4: Modularity of protein interfaces.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Cryo-EM structural data have been deposited at the EMDB under accession numbers EMD-2089 and EMD-2090, and at the Protein Data Bank under accession numbers 4ard and 4arg.

References

  1. 1

    Göttlinger, H. G. The HIV-1 assembly machine. AIDS 15 (Suppl. 5). S13–S20 (2001)

    Article  Google Scholar 

  2. 2

    Briggs, J. A. & Kräusslich, H. G. The molecular architecture of HIV. J. Mol. Biol. 410, 491–500 (2011)

    CAS  Article  Google Scholar 

  3. 3

    Waheed, A. A. & Freed, E. O. HIV type 1 Gag as a target for antiviral therapy. AIDS Res. Hum. Retroviruses 28, 54–75 (2012)

    CAS  Article  Google Scholar 

  4. 4

    Gross, I., Hohenberg, H., Huckhagel, C. & Kräusslich, H. G. N-terminal extension of human immunodeficiency virus capsid protein converts the in vitro assembly phenotype from tubular to spherical particles. J. Virol. 72, 4798–4810 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    von Schwedler, U. K. et al. Proteolytic refolding of the HIV-1 capsid protein amino-terminus facilitates viral core assembly. EMBO J. 17, 1555–1568 (1998)

    CAS  Article  Google Scholar 

  6. 6

    Johnson, M. C., Scobie, H. M., Ma, Y. M. & Vogt, V. M. Nucleic acid-independent retrovirus assembly can be driven by dimerization. J. Virol. 76, 11177–11185 (2002)

    CAS  Article  Google Scholar 

  7. 7

    Accola, M. A., Strack, B. & Göttlinger, H. G. Efficient particle production by minimal Gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain. J. Virol. 74, 5395–5402 (2000)

    CAS  Article  Google Scholar 

  8. 8

    Yeager, M., Wilson-Kubalek, E. M., Weiner, S. G., Brown, P. O. & Rein, A. Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: implications for retroviral assembly mechanisms. Proc. Natl Acad. Sci. USA 95, 7299–7304 (1998)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Li, S., Hill, C. P., Sundquist, W. I. & Finch, J. T. Image reconstructions of helical assemblies of the HIV-1 CA protein. Nature 407, 409–413 (2000)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Briggs, J. A., Wilk, T., Welker, R., Kräusslich, H. G. & Fuller, S. D. Structural organization of authentic, mature HIV-1 virions and cores. EMBO J. 22, 1707–1715 (2003)

    CAS  Article  Google Scholar 

  11. 11

    Briggs, J. A. et al. The stoichiometry of Gag protein in HIV-1. Nature Struct. Mol. Biol. 11, 672–675 (2004)

    CAS  Article  Google Scholar 

  12. 12

    Ganser-Pornillos, B. K., Cheng, A. & Yeager, M. Structure of full-length HIV-1 CA: a model for the mature capsid lattice. Cell 131, 70–79 (2007)

    CAS  Article  Google Scholar 

  13. 13

    Pornillos, O. et al. X-ray structures of the hexameric building block of the HIV capsid. Cell 137, 1282–1292 (2009)

    Article  Google Scholar 

  14. 14

    Pornillos, O., Ganser-Pornillos, B. K. & Yeager, M. Atomic-level modelling of the HIV capsid. Nature 469, 424–427 (2011)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Cardone, G., Purdy, J. G., Cheng, N., Craven, R. C. & Steven, A. C. Visualization of a missing link in retrovirus capsid assembly. Nature 457, 694–698 (2009)

    ADS  CAS  Article  Google Scholar 

  16. 16

    Briggs, J. A. et al. Structure and assembly of immature HIV. Proc. Natl Acad. Sci. USA 106, 11090–11095 (2009)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Wright, E. R. et al. Electron cryotomography of immature HIV-1 virions reveals the structure of the CA and SP1 Gag shells. EMBO J. 26, 2218–2226 (2007)

    CAS  Article  Google Scholar 

  18. 18

    de Marco, A. et al. Conserved and variable features of Gag structure and arrangement in immature retrovirus particles. J. Virol. 84, 11729–11736 (2010)

    CAS  Article  Google Scholar 

  19. 19

    Ulbrich, P. et al. Distinct roles for nucleic acid in in vitro assembly of purified Mason-Pfizer monkey virus CANC proteins. J. Virol. 80, 7089–7099 (2006)

    CAS  Article  Google Scholar 

  20. 20

    Sachse, C. et al. High-resolution electron microscopy of helical specimens: a fresh look at tobacco mosaic virus. J. Mol. Biol. 371, 812–835 (2007)

    CAS  Article  Google Scholar 

  21. 21

    Egelman, E. H. Reconstruction of helical filaments and tubes. Methods Enzymol. 482, 167–183 (2010)

    CAS  Article  Google Scholar 

  22. 22

    Macek, P. et al. NMR structure of the N-terminal domain of capsid protein from the Mason-Pfizer monkey virus. J. Mol. Biol. 392, 100–114 (2009)

    CAS  Article  Google Scholar 

  23. 23

    de Marco, A. et al. Structural analysis of HIV-1 maturation using cryo-electron tomography. PLoS Pathog. 6, e1001215 (2010)

    Article  Google Scholar 

  24. 24

    Lanman, J. et al. Key interactions in HIV-1 maturation identified by hydrogen–deuterium exchange. Nature Struct. Mol. Biol. 11, 676–677 (2004)

    CAS  Article  Google Scholar 

  25. 25

    Ternois, F., Sticht, J., Duquerroy, S., Kräusslich, H. G. & Rey, F. A. The HIV-1 capsid protein C-terminal domain in complex with a virus assembly inhibitor. Nature Struct. Mol. Biol. 12, 678–682 (2005)

    CAS  Article  Google Scholar 

  26. 26

    Bartonova, V. et al. Residues in the HIV-1 capsid assembly inhibitor binding site are essential for maintaining the assembly-competent quaternary structure of the capsid protein. J. Biol. Chem. 283, 32024–32033 (2008)

    CAS  Article  Google Scholar 

  27. 27

    Chu, H. H., Chang, Y. F. & Wang, C. T. Mutations in the alpha-helix directly C-terminal to the major homology region of human immunodeficiency virus type 1 capsid protein disrupt Gag multimerization and markedly impair virus particle production. J. Biomed. Sci. 13, 645–656 (2006)

    CAS  Article  Google Scholar 

  28. 28

    von Schwedler, U. K., Stray, K. M., Garrus, J. E. & Sundquist, W. I. Functional surfaces of the human immunodeficiency virus type 1 capsid protein. J. Virol. 77, 5439–5450 (2003)

    CAS  Article  Google Scholar 

  29. 29

    Yu, I. M. et al. Structure of the immature dengue virus at low pH primes proteolytic maturation. Science 319, 1834–1837 (2008)

    ADS  CAS  Article  Google Scholar 

  30. 30

    Conway, J. F. et al. Virus maturation involving large subunit rotations and local refolding. Science 292, 744–748 (2001)

    ADS  CAS  Article  Google Scholar 

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Acknowledgements

This study was technically supported by the use of the European Molecular Biology Laboratory Information Technology Service unit. This work was partly funded by a grant from the Deutsche Forschungsgemeinschaft within SPP1175 to J.A.G.B. and by grants P302/12/1895 and 204/09/1388 from the Czech Science foundation to T.R. and M.R.

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T.A.M.B., P.U., M.R., T.R. and J.A.G.B. designed the research. T.A.M.B. and P.U. prepared samples for electron microscopy. T.A.M.B. and J.D.R. collected cryo-EM data. T.A.M.B., J.D.R., A.D.M. and J.A.G.B. analysed cryo-ET data. C.S. supported helical image-processing techniques. T.A.M.B. and J.A.G.B. developed and applied the variable-symmetry helical reconstruction methodology. T.A.M.B., N.D., P.U., M.R., C.S., T.R. and J.A.G.B. analysed fitted pseudo-atomic models. T.A.M.B. and J.A.G.B. wrote the paper with support from all the authors.

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Correspondence to John A. G. Briggs.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-8, Supplementary Methods, Supplementary Tables 1-2, legends for Supplementary Movies 1-2 and Supplementary References. (PDF 3688 kb)

Supplementary Movie 1

In this movie we see the structure of the M-PMV CANC tubes at sub-nanometre resolution - see Supplementary Information file for full legend. (MOV 16313 kb)

Supplementary Movie 2

In this movie we see the comparison of the immature and mature retroviral lattices - see Supplementary Information file for full legend. (MOV 5283 kb)

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Bharat, T., Davey, N., Ulbrich, P. et al. Structure of the immature retroviral capsid at 8 Å resolution by cryo-electron microscopy. Nature 487, 385–389 (2012). https://doi.org/10.1038/nature11169

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