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.

  • Letter
  • Published:

Structural basis of lentiviral subversion of a cellular protein degradation pathway

Nature volume 505, pages 234–238 (2014)Cite this article

Subjects

Abstract

Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells1,2 as well as resting CD4+ T cells3,4 by reducing the cellular deoxynucleoside 5′-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function5,6. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation7,8. The molecular mechanism by which these viral proteins are able to usurp the host cell’s ubiquitination machinery to destroy the cell’s protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell’s normal protein degradation pathway to inactivate the cellular viral defence system.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: The SAMHD1-CtD–Vpxsm–DCAF1-CtD complex.
Figure 2: Intermolecular interfaces.
Figure 3: SAMHD1 C-terminal region.
Figure 4: Species specificity of the SAMHD1–Vpx interaction.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates of DCAF1-CtD–Vpxsm–SAMHD1-CtD have been deposited in the PDB under accession 4CC9.

References

  1. Laguette, N. et al. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature 474, 654–657 (2011)

    Article  CAS  Google Scholar 

  2. Hrecka, K. et al. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature 474, 658–661 (2011)

    Article  CAS  Google Scholar 

  3. Baldauf, H. M. et al. SAMHD1 restricts HIV-1 infection in resting CD4+ T cells. Nature Med. 18, 1682–1689 (2012)

    Article  CAS  Google Scholar 

  4. Descours, B. et al. SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4+ T-cells. Retrovirology 9, 87 (2012)

    Article  CAS  Google Scholar 

  5. Goldstone, D. C. et al. HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase. Nature 480, 379–382 (2011)

    Article  ADS  CAS  Google Scholar 

  6. Lahouassa, H. et al. SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates. Nature Immunol. 13, 223–228 (2012)

    Article  CAS  Google Scholar 

  7. Lim, E. S. et al. The ability of primate lentiviruses to degrade the monocyte restriction factor SAMHD1 preceded the birth of the viral accessory protein Vpx. Cell Host Microbe 11, 194–204 (2012)

    Article  CAS  Google Scholar 

  8. Ahn, J. et al. HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1. J. Biol. Chem. 287, 12550–12558 (2012)

    Article  CAS  Google Scholar 

  9. Morellet, N., Bouaziz, S., Petitjean, P. & Roques, B. P. NMR structure of the HIV-1 regulatory protein VPR. J. Mol. Biol. 327, 215–227 (2003)

    Article  CAS  Google Scholar 

  10. Wei, W. et al. A novel DCAF1-binding motif required for Vpx-mediated degradation of nuclear SAMHD1 and Vpr-induced G2 arrest. Cell. Microbiol. 14, 1745–1756 (2012)

    Article  CAS  Google Scholar 

  11. Srivastava, S. et al. Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection. PLoS Pathog. 4, e1000059 (2008)

    Article  Google Scholar 

  12. Bergamaschi, A. et al. The human immunodeficiency virus type 2 Vpx protein usurps the CUL4A–DDB1 DCAF1 ubiquitin ligase to overcome a postentry block in macrophage infection. J. Virol. 83, 4854–4860 (2009)

    Article  CAS  Google Scholar 

  13. DeLucia, M., Mehrens, J., Wu, Y. & Ahn, J. HIV-2 and SIVmac accessory virulence factor Vpx down-regulates SAMHD1 enzyme catalysis prior to proteasome-dependent degradation. J. Biol. Chem. 288, 19116–19126 (2013)

    Article  CAS  Google Scholar 

  14. Bennett, E. J., Bence, N. F., Jayakumar, R. & Kopito, R. R. Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation. Mol. Cell 17, 351–365 (2005)

    Article  CAS  Google Scholar 

  15. Scrima, A. et al. Structural basis of UV DNA-damage recognition by the DDB1–DDB2 complex. Cell 135, 1213–1223 (2008)

    Article  CAS  Google Scholar 

  16. Fischer, E. S. et al. The molecular basis of CRL4DDB2/CSA ubiquitin ligase architecture, targeting, and activation. Cell 147, 1024–1039 (2011)

    Article  CAS  Google Scholar 

  17. Yan, J. et al. Tetramerization of SAMHD1 is required for biological activity and inhibition of HIV infection. J. Biol. Chem. 288, 10406–10417 (2013)

    Article  CAS  Google Scholar 

  18. Angers, S. et al. Molecular architecture and assembly of the DDB1–CUL4A ubiquitin ligase machinery. Nature 443, 590–593 (2006)

    Article  ADS  CAS  Google Scholar 

  19. Li, T., Robert, E. I., van Breugel, P. C., Strubin, M. & Zheng, N. A promiscuous α-helical motif anchors viral hijackers and substrate receptors to the CUL4–DDB1 ubiquitin ligase machinery. Nature Struct. Mol. Biol. 17, 105–111 (2010)

    Article  Google Scholar 

  20. Fregoso, O. I. et al. Evolutionary toggling of Vpx/Vpr specificity results in divergent recognition of the restriction factor SAMHD1. PLoS Pathog. 9, e1003496 (2013)

    Article  CAS  Google Scholar 

  21. Wen, X., Duus, K. M., Friedrich, T. D. & de Noronha, C. M. The HIV1 protein Vpr acts to promote G2 cell cycle arrest by engaging a DDB1 and Cullin4A-containing ubiquitin ligase complex using VprBP/DCAF1 as an adaptor. J. Biol. Chem. 282, 27046–27057 (2007)

    Article  CAS  Google Scholar 

  22. Schröfelbauer, B., Hakata, Y. & Landau, N. R. HIV-1 Vpr function is mediated by interaction with the damage-specific DNA-binding protein DDB1. Proc. Natl Acad. Sci. USA 104, 4130–4135 (2007)

    Article  ADS  Google Scholar 

  23. Le Rouzic, E. et al. HIV1 Vpr arrests the cell cycle by recruiting DCAF1/VprBP, a receptor of the Cul4–DDB1 ubiquitin ligase. Cell Cycle 6, 182–188 (2007)

    Article  CAS  Google Scholar 

  24. Hrecka, K. et al. Lentiviral Vpr usurps Cul4–DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle. Proc. Natl Acad. Sci. USA 104, 11778–11783 (2007)

    Article  ADS  CAS  Google Scholar 

  25. Barnitz, R. A., Chaigne-Delalande, B., Bolton, D. L. & Lenardo, M. J. Exposed hydrophobic residues in human immunodeficiency virus type 1 Vpr helix-1 are important for cell cycle arrest and cell death. PLoS ONE 6, e24924 (2011)

    Article  ADS  CAS  Google Scholar 

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

    Article  Google Scholar 

  27. Zhao, Y., Chapman, D. A. & Jones, I. M. Improving baculovirus recombination. Nucleic Acids Res. 31, e6 (2003)

    Article  Google Scholar 

  28. Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D 66, 133–144 (2010)

    Article  CAS  Google Scholar 

  29. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008)

    Article  ADS  CAS  Google Scholar 

  30. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007)

    CAS  Google Scholar 

  31. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006)

    Article  Google Scholar 

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

    Article  Google Scholar 

  33. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012)

    Article  CAS  Google Scholar 

  34. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D 60, 2256–2268 (2004)

    Article  CAS  Google Scholar 

  35. Yap, M. W., Nisole, S., Lynch, C. & Stoye, J. P. Trim5α protein restricts both HIV-1 and murine leukemia virus. Proc. Natl Acad. Sci. USA 101, 10786–10791 (2004)

    Article  ADS  CAS  Google Scholar 

  36. Bock, M., Bishop, K. N., Towers, G. & Stoye, J. P. Use of a transient assay for studying the genetic determinants of Fv1 restriction. J. Virol. 74, 7422–7430 (2000)

    Article  CAS  Google Scholar 

  37. Groom, H. C. et al. Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome. Retrovirology 7, 10 (2010)

    Article  Google Scholar 

  38. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996)

    Article  ADS  CAS  Google Scholar 

  39. Bainbridge, J. W. et al. In vivo gene transfer to the mouse eye using an HIV-based lentiviral vector; efficient long-term transduction of corneal endothelium and retinal pigment epithelium. Gene Ther. 8, 1665–1668 (2001)

    Article  CAS  Google Scholar 

  40. Sundström, C. & Nilsson, K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int. J. Cancer 17, 565–577 (1976)

    Article  Google Scholar 

  41. Gallois-Montbrun, S. et al. Antiviral protein APOBEC3G localizes to ribonucleoprotein complexes found in P bodies and stress granules. J. Virol. 81, 2165–2178 (2007)

    Article  CAS  Google Scholar 

  42. Nègre, D. et al. Characterization of novel safe lentiviral vectors derived from simian immunodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cells. Gene Ther. 7, 1613–1623 (2000)

    Article  Google Scholar 

Download references

Acknowledgements

We thank L. Haire and R. Ogrodowicz for help with crystallization, I. Jones for the provision of the modified A. californica baculovirus bacmid, BAC10:1629KO, S. Smerdon and S. Gamblin for comments on the manuscript. We gratefully acknowledge the Diamond Light Source for synchrotron access (grant no. 7707). This work was supported by the UK Medical Research Council, file references U117565647 (I.A.T.), U117592729 (K.N.B.) and U117512710 (J.P.S.); the Wellcome Trust, ref. 084955 (K.N.B.); and by an EMBO long-term fellowship co-funded by the European Commission Marie Curie Actions (EMBOCOFUND2010, GA-2010-267146) (D.S.).

Author information

Authors and Affiliations

  1. Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK,

    David Schwefel, Evangelos Christodoulou, Philip A. Walker & Ian A. Taylor

  2. Division of Virology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK,

    Harriet C. T. Groom, Virginie C. Boucherit, Jonathan P. Stoye & Kate N. Bishop

Authors
  1. David Schwefel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Harriet C. T. Groom
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Virginie C. Boucherit
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Evangelos Christodoulou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Philip A. Walker
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jonathan P. Stoye
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kate N. Bishop
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ian A. Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.S., H.C.T.G., V.C.B., E.C. and P.A.W. performed experiments. D.S., H.C.T.G., V.C.B., E.C., P.A.W., J.P.S., K.N.B. and I.A.T. contributed to experimental design, data analysis and manuscript writing.

Corresponding author

Correspondence to Ian A. Taylor.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Experimental electron density.

Experimental electron density observed after solvent flattening for DCAF1, Vpxsm and SAMHD1 is shown as light blue wireframe, contoured at 1σ. The backbone Cα traces of the final refined protein structure are shown in green ribbon representation.

Extended Data Figure 2 Model of the hijacked CUL4A–DDB1–ROC1 E3 ubiquitin ligase complex.

Vpxsm (blue) bound to the substrate-specificity module DCAF1-CtD (grey) is shown in cartoon representation as in Fig. 1c. SAMHD1-CtD is shown as red spheres. The DDB1 adaptor is shown as green cartoon. The inset to the right shows the superposition of the DDB2 helical hairpin (H-box, orange), which inserts into the binding groove created by DDB1 β-propellers 1 and 3 (BPA, BPC), and the N terminus of DCAF1-CtD presented in this study. The CUL4A scaffold is represented as an orange semi-transparent surface, the ROC1 RING module as purple spheres. Owing to the conformational freedom of the DDB1–CUL4A connection, the two most extreme conformations of CUL4A with respect to DDB1 available in the PDB were modelled. See Methods for modelling procedures and PDB accessions. The model clearly shows that in both extreme CUL4 conformations, the ROC1 RING finger (purple spheres) is well positioned to reach the SAMHD1 protein, which would be attached at the N-terminal end of the SAMHD1-CtD. The SAMHD1 globular fold is probably mobile with respect to the fixed position of SAMHD1-CtD owing to the flexibility of the sequence stretch between SAMHD1 residues 583 (the last ordered residue of PDB accession 3U1N5) and 606 (the first ordered residue of SAMHD1-CtD presented here).

Extended Data Figure 3 Analogous mechanism of restriction factor counteraction in SIV/HIV-2 and HIV-1 Vpx and Vpr.

SAMHD1 provides a potent post-entry block against immunodeficiency viruses in non-cycling cells. Its dNTP-triphosphohydrolase activity lowers the cellular dNTP pool, preventing viral reverse transcription. HIV-2/SIVs use their Vpx and Vpr accessory proteins to modify the host cell’s CUL4A–DDB1–DCAF1 ubiquitin ligase specificity towards SAMHD1, resulting in its proteasomal degradation and ultimately raising dNTP levels, making the cells permissive to viral replication. Sequence similarity and comparative functional analysis suggest that the ancestral HIV-1 accessory protein Vpr uses a similar mechanism to exploit the CUL4A–DDB1–DCAF1 system to induce proteasomal degradation of an as yet undiscovered cellular factor whose absence causes cell cycle arrest in the G2 phase, promoting viral replication and pathogenesis in vivo.

Extended Data Table 1 Data collection and refinement statistics
Full size table
Extended Data Table 2 Intermolecular interactions
Full size table
Extended Data Table 3 Vpx and Vpr mutations
Full size table

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schwefel, D., Groom, H., Boucherit, V. et al. Structural basis of lentiviral subversion of a cellular protein degradation pathway. Nature 505, 234–238 (2014). https://doi.org/10.1038/nature12815

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12815

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced 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