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Structural insights into PPP2R5A degradation by HIV-1 Vif

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Abstract

HIV-1 Vif recruits host cullin-RING-E3 ubiquitin ligase and CBFβ to degrade the cellular APOBEC3 antiviral proteins through diverse interactions. Recent evidence has shown that Vif also degrades the regulatory subunits PPP2R5(A–E) of cellular protein phosphatase 2A to induce G2/M cell cycle arrest. As PPP2R5 proteins bear no functional or structural resemblance to A3s, it is unclear how Vif can recognize different sets of proteins. Here we report the cryogenic-electron microscopy structure of PPP2R5A in complex with HIV-1 Vif–CBFβ–elongin B–elongin C at 3.58 Å resolution. The structure shows PPP2R5A binds across the Vif molecule, with biochemical and cellular studies confirming a distinct Vif–PPP2R5A interface that partially overlaps with those for A3s. Vif also blocks a canonical PPP2R5A substrate-binding site, indicating that it suppresses the phosphatase activities through both degradation-dependent and degradation-independent mechanisms. Our work identifies critical Vif motifs regulating the recognition of diverse A3 and PPP2R5A substrates, whereby disruption of these host–virus protein interactions could serve as potential targets for HIV-1 therapeutics.

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Fig. 1: The overall structure of HIV-1 Vif in complex with PPP2R5A and host E3 ligase components.
Fig. 2: The observed HIV-1 Vif–PPP2R5A-binding interface.
Fig. 3: The detailed Vif–PPP2R5A interface.
Fig. 4: Comparison of Vif-mediated recruitment of PPP2R5A and A3 proteins.
Fig. 5: Complete models of Vif-mediated inhibition of PP2A through the Cul5 E3 ligase.

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Data availability

The model of the PPP2R5A–VCBC complex has been deposited in the Protein Data Bank (PDB) with accession code PDB 8SZK. The cryo-EM map of the PPP2R5A–VCBC complex has been deposited in the Electron Microscopy Data Bank (EMDB) with accession code EMD-40919. The structures used for model building of the PPP2R5A–VCBC complex and making figures could be downloaded from the PDB: VCBC (extracted from PDB 4N9F), PPP2R5A (extracted from PDB 6NTS), A3F (PDB 6NIL), A3G (PDB 8CX0) and Cul5CTD/Rbx2 (PDB 3DPL). Source data are provided with this paper.

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Acknowledgements

We thank G. Hu, J. Kaminsky and L. Wang at the Brookhaven Laboratory Cryo-EM facility for assistance with data collection. We thank other Xiong laboratory members for discussions. This work was supported by National Institutes of Health (NIH) grant no. R37AI116313 (Y.X.). This work was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research and by the Innovation Award, Office of AIDS Research, NIH (V.K.P.).

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Author notes

  1. These authors contributed equally: Yingxia Hu, Krista A. Delviks-Frankenberry.

Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Yingxia Hu, Chunxiang Wu, Fidel Arizaga & Yong Xiong

  2. Viral Mutation Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA

    Krista A. Delviks-Frankenberry & Vinay K. Pathak

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  1. Yingxia Hu
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Contributions

Y.X., V.K.P., Y.H. and K.A.D.-F. designed the experiments. Y.H. and C.W. performed the biophysical and biochemical experiments. K.A.D.-F. performed the virological experiments. Data were analyzed by Y.X., V.K.P., Y.H., K.A.D.-F. and C.W. F.A. contributed to experiments and discussions. Y.X., V.K.P., Y.H. and K.A.D.-F. wrote the paper.

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Correspondence to Vinay K. Pathak or Yong Xiong.

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Nature Structural & Molecular Biology thanks Christopher Hill and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 The screening and optimization of PPP2R5A complex with HIV-1 Vif/CBFβ/Cul5 E3 ligase.

a, Size exclusion chromatography (SEC) profiles and SDS-PAGE analysis of individual PPP2R5A, Vif/CBFβ/Cul5 E3 and their complexes (repeated twice independently). The cryo-EM reconstructions (surface with fitted models in cartoon representation) of the fully assembled Vif/CBFβ/Cul5 E3/PPP2R5A showed no presence of PPP2R5A. b, The SEC profile and SDS-PAGE analysis of PPP2R5A/VCBC complex crosslinked by Bissulfosuccinimidyl suberate (BS3). The large scale crosslinking of the complex has been repeated 7 times independently. The OD280 and OD260 are shown in blue and red, respectively. The protein bands were detected by Coomassie blue stain.

Source data

Extended Data Fig. 2 The superposition of the VCBC complex structure with and without A3F (left) or A3G (right) bound.

The Vif flexible loop that shows the largest local conformational changes upon A3F or A3G binding is highlighted by dashed circles and with details illustrated in insets. PDBs used for the superpositions: A3FCTD alone: 3WUS; Vif/CBFβ: 4N9F; Vif/CBFβ/A3FCTD: 6NIL; VCBC/A3G: 8CX1. The individual Vif/CBFβ structures are shown in gray, the A3F or A3G bound Vif/CBFβ are shown in magenta/cyan.

Extended Data Fig. 3 Conservation of PPP2R5 family protein sequences at the Vif interface.

a, Alignment of PPP2R5A (amino acids 82 to 397) to PPP2R5B-E with the amino acids at the PPP2R5A:Vif interface and corresponding conserved amino acids in PPP2R5B-E highlighted in cyan (patch 3), yellow (patch 1), and magenta (patch 2). NCBI reference protein sequence (NP accession numbers): PPP2R5A (NP_006234); PPPP2R5B (NP_006235); PPP2R5C (NP_001339842); PPP2R5D (NP_006236); PPP2R5E (NP_006237). b, Structural modeling of PPP2R5-containing PP2As and their complexes with the Vif/CBFβ/Cul5 E3 ligase. Left: top, overlay of PPP2R5A (gray, PDB ID 6nts) and PPP2R5C (colored, PDB 2npp)-containing PP2A complex; bottom, overlay of different PPP2R5 family members (PPP2R5A, gray, PDB 6nts; PPP2R5B, purple, AlphaFold model; PPP2R5C, yellow, PDB 2npp; PPP2R5D, green, AlphaFold model; PPP2R5E, blue, AlphaFold model). Right: structural model of the HIV-1 Vif recruitment of PPP2R5C-containing PP2A onto the Cul5 E3 ligase complex.

Extended Data Fig. 4 Mutational validation of the PPP2R5A-Vif interactions by western blot analysis.

The in vitro binding assay was performed using MBP-tagged Vif/CBFβ/EloB/EloC variants to pull down SUMO-PPP2R5A variants. The SUMO-PPP2R5A bands were recognized by anti-SUMO antibody, and the His-CBFβ bands were detected by Anti-His antibody (upper panel). The ratio of band intensities (SUMO-PPP2R5A/His-CBFβ) was quantified by mean ± sem; n = 2 biologically independent samples for R127E:WT, WT:E106K, R127E:E106K, n = 3 biologically independent samples for WT:Y294A, 273031AAA:WT, 273031AAA:Y294A, R33D:WT, WT:D205R/E251K, R33D:D205R/E251K, WT:Y373A, H43A/Y44A:WT, H43A/Y44A:Y373A, n = 4 biologically independent samples for K22E:WT, R23E:WT, K22E/R23E:WT, n = 7 biologically independent samples for WT:WT, with individual data points shown as dots (lower panel).

Source data

Extended Data Fig. 5 The observed structure differs from that predicted based on mutational analysis52.

The observed (upper panel) and computational predicted (lower panel) structures are shown in three different views to demonstrate the differences between the observed and predicted binding modes of PPP2R5A.

Extended Data Fig. 6 Structural analysis of Vif/CBFβ binding interfaces for different host targets.

Comparison of the distinct Vif/CBFβ interfaces (marked with ovals of different colors) for A3FCTD (bottom left), A3C (bottom middle, PDB 3VOW), A3DCTD (bottom right, homology model built from A3FCTD [PDB 3WUS]), A3G (middle left, [PDBs 8E40 and 8CX0]), A3H hapII (middle right, PDB 6BBO), and PPP2R5A (top). Critical residues observed are highlighted in red at the CBFβ-A3F/G interface and in yellow at the Vif-A3/PPP2R5A interfaces. A summary of the diverse locations of the Vif/CBFβ-A3/PPP2R5A interfaces is illustrated at middle center, with ovals of corresponding colors. Reproduced from: Fig. 3, Multifaceted HIV-1 Vif interactions with human E3 ubiquitin ligase and APOBEC3s, Yong Xiong, The FEBS Journal 288 3407–3417, Copyright © [2020], Federation of European Biochemical Societies, Wiley.

Extended Data Fig. 7 Cryo-EM image processing workflow for the Vif/CBFβ/EloB/EloC/PPP2R5A complex.

Tilted and untilted particles were combined and first cleaned by 3D and 2D classifications, then fractions of particles in two dominated views were discarded randomly to alleviate the preferred orientation issue. The remaining particles were further cleaned up by iterative heterogeneous refinement, and a final round of 3D classification identified a class of 500,511 particles with more balanced orientation distribution which was able to generate a 3D reconstruction of good quality. The resolution of the final reconstruction was further improved by non-uniform 3D refinement, local refinement and DeepEMhancer72.

Extended Data Fig. 8 Resolving the preferred orientation of the crosslinked PPP2R5A/VCBC complex.

a, An example of the raw image of the complex (left) and the particle orientation distribution of the untilted dataset (right). b, Top 2D class averages of the combined untilted and tilted datasets. From a total of 516,465 particles in the classes showing preferred orientations (boxed in red), 456,465 particles (~88%) were removed and excluded randomly from the subsequent analysis. c, The final particle set showed a more balanced orientation distribution.

Extended Data Fig. 9 Quality of the cryo-EM reconstruction of the crosslinked PPP2R5A/VCBC complex.

a, The Fourier shell correlation (FSC) curves of the cryo-EM reconstruction. b, Local resolution estimate of the cryo-EM map. c, The model-to-map FSC.

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Unprocessed blue stain gels.

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Statistical source data.

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Unprocessed western blots.

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Unprocessed western blots and gels.

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Unprocessed gels.

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Unprocessed western blots.

Source Data Extended Data Fig. 4

Statistical source data.

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Hu, Y., Delviks-Frankenberry, K.A., Wu, C. et al. Structural insights into PPP2R5A degradation by HIV-1 Vif. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01314-6

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