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Structural basis of lentiviral subversion of a cellular protein degradation pathway

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

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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.

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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.

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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.

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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.).

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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

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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.

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Correspondence to Ian A. Taylor.

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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

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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

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