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Vpu modulates DNA repair to suppress innate sensing and hyper-integration of HIV-1

Nature Microbiology volume 5, pages 1247–1261 (2020)Cite this article

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Abstract

To avoid innate sensing and immune control, human immunodeficiency virus type 1 (HIV-1) has to prevent the accumulation of viral complementary DNA species. Here, we show that the late HIV-1 accessory protein Vpu hijacks DNA repair mechanisms to promote degradation of nuclear viral cDNA in cells that are already productively infected. Vpu achieves this by interacting with RanBP2–RanGAP1*SUMO1–Ubc9 SUMO E3-ligase complexes at the nuclear pore to reprogramme promyelocytic leukaemia protein nuclear bodies and reduce SUMOylation of Bloom syndrome protein, unleashing end degradation of viral cDNA. Concomitantly, Vpu inhibits RAD52-mediated homologous repair of viral cDNA, preventing the generation of dead-end circular forms of single copies of the long terminal repeat and permitting sustained nucleolytic attack. Our results identify Vpu as a key modulator of the DNA repair machinery. We show that Bloom syndrome protein eliminates nuclear HIV-1 cDNA and thereby suppresses immune sensing and proviral hyper-integration. Therapeutic targeting of DNA repair may facilitate the induction of antiviral immunity and suppress proviral integration replenishing latent HIV reservoirs.

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Fig. 1: Proviral HIV-1 gene expression modulates homologous repair.
Fig. 2: Viral proteins involved in HIV-1-modulated homologous repair.
Fig. 3: HIV-1 regulates BLM-mediated DNA-end processing.
Fig. 4: Effects of Vpu on SUMOylation and PML-NBs.
Fig. 5: Interaction of Vpu with RanBP2–RanGAP1*SUMO1–Ubc9 SUMO E3-ligase complexes.
Fig. 6: Modulation of viral DNA species and superinfection by Vpu, RAD52 and BLM.

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Data availability

The data that support the finding of this study are available from the corresponding author on request. Complete western blot images in the manuscript are provided in the source data. Correspondence and requests for materials should be addressed to F.K. Source data are provided with this paper.

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Acknowledgements

We thank D. Schloesser for technical help with the PLA experiments, V. Lallemand-Breitenbach for providing us with the antibody recognizing PML isoforms I–IV, M. Lusic for support and helpful discussions, and J. M. Stark for giving us the EJ5SceGFP construct for NHEJ measurements. The Quadro P6000 used for this research project was made available by the NVIDIA Corporation. L.W. was supported by grants from the German Research Foundation (grant no. DFG WI 3099/7, B05 in CRC 1279), European–German Space Agency (ESA/DLR) and German Ministry of Economy (BMWi, A0-10-IBER-2 funding grant no. 50WB1225). F.K. was supported by the DFG (grant no. KI 548/11-1; CRC 1279) and an Advanced ERC investigator grant (Anti-Virome). D.S., K.M.J.S. and F.K. are funded by the DFG priority program grant no. SPP1923 and T.G.H. is supported by CRC 1361. K.M.J.S. was supported by a Marie Skłodowska-Curie Individual Fellowship from the European Union’s Framework Program for Research and Innovation Horizon 2020 (2014–2020) under grant agreement no. 794803 (‘VIAR’) and the DFG (grant no. SP1600/4-1).

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Author notes

  1. Thomas G. Hofmann

    Present address: Institute of Toxicology, University of Mainz, Mainz, Germany

  2. These authors contributed equally: Lisa Wiesmüller, Frank Kirchhoff.

Authors and Affiliations

  1. Division of Gynecological Oncology, Department of Obstetrics and Gynecology, Ulm University, Ulm, Germany

    Meta Volcic & Lisa Wiesmüller

  2. Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany

    Meta Volcic, Konstantin M. J. Sparrer, Lennart Koepke, Dominik Hotter, Daniel Sauter, Christina M. Stürzel, Nathalie J. Arhel & Frank Kirchhoff

  3. Institute of Virology, Ulm University, Ulm, Germany

    Myriam Scherer & Thomas Stamminger

  4. Department of Epigenetics, Deutsches Krebsforschungszentrum, Heidelberg, Germany

    Thomas G. Hofmann

  5. Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé (CPBS) CNRS FRE3689, Montpellier University, Montpellier, France

    Nathalie J. Arhel

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Contributions

M.V. performed and analysed most of the experiments. K.M.J.S., L.K., D.H. and D.S. contributed to additional assay work, image analysis and experimental expertise. C.M.S. performed the cloning. M.S. and T.S. contributed to the assays to analyse PML. N.J.A. contributed expertise and T.G.H. contributed materials and expertise. M.V., K.M.J.S., L.W. and F.K. wrote the manuscript. M.V., L.W. and F.K. supervised the study.

Corresponding authors

Correspondence to Lisa Wiesmüller or Frank Kirchhoff.

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Extended data

Extended Data Fig. 1 Primary DSB repair data and controls.

Primary DSB repair data and Role of DNA damage sensors. a, Primary FACS data for EGFP-based evaluation of DSB repair as in Fig. 1d. Evaluation of DSB repair frequency described in Methods section. b-c, Apoptosis induction and cell cycle distribution in Jurkat cells transfected with pHIV-1-NL4-3-env*-IRES-mCherry (HIV-1), pCMV-HA-I-SceI plus repair reporter plasmid HR-EGFP/5´EGFP. In parallel, split samples were treated with caspase inhibitor zVAD-fmk. Cells were fixed 48 h post-transfection with ethanol/acetone and stained with propidium iodide for DNA content analysis. Bars represent means ± SEM, n=2 biologically independent experiments in triplicates, *p=0.0313. In c, samples in the same cell cycle phase were statistically analyzed. d, Extrachromosomal homologous repair frequency (Hom. repair freq.) in WTK1 cells transfected with pHIV-1-NL4-3-env*-IRES-mCherry, pCMV-HA-I-SceI plus repair plasmid HR-EGFP/3´EGFP for detection of HR and SSA (construct on top). Split samples were treated with zVAD-fmk. Bars represent means ± SEM, n=3 biologically independent experiments in triplicates, *p=0.0391. Control samples transfected with control plasmid set to 100 % (absolute mean value: 2 %). e-f, Apoptosis induction and cell cycle distribution in WTK1(HR/3′) cells 48 h after transfection with HIV-1 plus pCMV-HA-I-SceI. Bars represent means ± SEM, n=2 biologically independent experiments in duplicates (f) or triplicates (g). In (f) samples in the same cell cycle phase were statistically analyzed. g, Intrachromosomal homologous repair frequency in KMV(Δ/3′) cells transfected with HIV-1 plus pCMV-HA-I-SceI. Split samples were treated with and without Hygromycin B (60 µg/ml) 4 h post-transfection. Bars represent means ± SEM, n=2 biologically independent experiments in triplicates, *p=0.0313, (absolute mean value: 0.2 %). Note the schematic presentation of the chromosomally integrated repair construct ΔEGFP/3´EGFP in KMV(Δ/3′) with a Hygromycin resistance cassette (blue bar) on top. h-l, Homologous repair frequency in WTK1(HR/3′) cells co-transfected with pHIV-1-NL4-3-env*-IRES-mCherry, pCMV-HA-I-SceI and shRNA to downregulate ATM (a), ATR (b), Nibrin (c), CtIP (d) and Ku70 (e) and corresponding empty vectors in the controls. Bars represent means ± SEM, n=2 biologically independent experiments in triplicates, *p=0.0313 (absolute mean value: 0.04%). Lower panels show Western blots demonstrating efficiency of knockdowns. Two sided Wilcoxon matched-pairs test in b, d, g, h-l.

Source data

Extended Data Fig. 2 Vpu expression in different experimental settings and controls to exclude potentially confounding factors.

Vpu expression in different experimental settings and controls to exclude potentially confounding factors. a, Vpu titration. Evaluation of DSB repair frequencies in WTK1(HR/3′) cells transfected with increasing amounts of NL4-3 Vpu-AU1 expression plasmid. Bars represent means ± SEM, n=2 biologically independent experiments in triplicates, *p=0.0313. Lower panels show Western blot demonstrating expression efficiency. b, Expression of WITO Vpu-AU1 in WTK1(HR/3′) cells transduced with pHIV-1-NL4-3 WITO Vpu-AU1 compared to cells transfected with pCG-WITO Vpu-AU1 expression vector or the pHIV-1-NL4-3 WITO Vpu-AU1 proviral construct. In the proviral construct vpu and env were separated and the NL-4-3 vpu replaced by an AU-1-tagged WITO vpu. 24 h post-transfection samples were analyzed by Western blot. c, Apoptosis and cell cycle analysis in WTK1(HR/3′) cells after transfection with pCG-NL4-3 Vpu-AU1 expression plasmid. Bars represent means ± SEM, n=2 biologically independent experiments in triplicates, statistical analysis was calculated for comparisons between different samples in the same cell cycle phase. d, Infection rates. Graph presenting mean values ± SD of p24 stained in CD4+ T cells from 3 donors shown in Fig. 3d. Right, primary flow cytometry data. Percentages of p24 positive cells in the live cell population (SSC/FSC gate) were calculated. e, Homologous repair, apoptosis and cell cycle in primary HIV-1 transfected CD4+ T cells. Cells were transfected with infectious (env+) HIV-1 CH058 wt, vpr* or vpu* constructs, together with repair reporter and meganuclease I-SceI expression plasmid. 48 h post-transfection samples were examined for repair frequencies or aceton/ethanol fixed and propidium iodide stained to evaluate cell cycle and apoptosis. Bars represent means ± SEM, n=3 biologically independent experiments in triplicates, *p=0.0152, **p=0.0022 (absolute mean value of homologous repair frequency was 0.3%). Two sided Wilcoxon matched-pairs test in a and e.

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Extended Data Fig. 3 Analysis of mutant Vpu proteins and evaluation of known cellular Vpu targets.

Analysis of mutant Vpu proteins and evaluation of known cellular Vpu targets. a, Sequence of WITO Vpu with mutated positions indicated. b, Western blot depicting expression of different WITO Vpu mutants from (a) and CH106 wt and its KKDQ mutant with the ER retention sequence. Experiment repeated 2 times with similar outcome. c, Homologous repair in WTK1(HR/3′) cells transfected with different mutants of WITO Vpu or CH106 and the CH106 KKDQ mutant with ER retention sequence. 48 h post transfection cells were analyzed by FACS to evaluate DSB repair frequencies. Bars represent means ± SEM, n=3 or 4 biologically independent experiments in triplicates, *p=0.0273, **p=0.0078, **p=0.0039 (absolute mean value: 0.7 %). Statistical evaluation shown for the comparison between wt WITO Vpu and the indicated Ala substitution mutants. d, BrdU assay to analyze DNA processing mediated by different WITO Vpu mutants. WTK1(HR/3′) WITO transfected cells were IR (2 Gy), pre-extracted, fixed and stained against BrdU. Data presents 4 independent experiments where each dot (n=14-18) presents mean value of 30-50 analyzed nuclei from one image view, ± SEM. *p=0.0166, **p=0.0012, **p=0.0085, ****p=0.0001. Black stars presenting comparison to the control, blue stars comparison to WITO Vpu. The lower panel shows representative BrdU and AU1 microscopic images. e, Homologous repair in WTK1(HR/3′) (β-TrCP). Cells were transfected with wt, vpr* or vpu* pHIV-1-NL4-3-env*-IRES-mCherry constructs together with expression plasmids for I-SceI and the DN form of β-TrCP. 48 h later cells were harvested for FACS analysis. Bars represent means ± SEM, n=3 biologically independent experiments in triplicates, **p=0.039 (absolute mean value: 0.1%). Lower panels show Western blot demonstrating overexpression of β-TrCP-DN. f, Immunoprecipitation (IP) of RAD52 1h after γ-irradiation (2Gy) of WTK1(HR/3′) cells transfected 24h before with NL4-3 Vpu expression plasmid or empty vector (Control). Graph presents mean values ± SD from two independent experiments. Note that RAD52 is difficult to detect by Western blotting unless it is concentrated for example by IP. Thus, most analyses had to rely on mRNA expression levels. g, Evaluation of RAD52 mRNA levels in SaOS cells treated as described in the legend Fig. 2i. Bars represent means ± SEM, n=4. Two sided Wilcoxon matched-pairs test in c, d, e, g.

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Extended Data Fig. 4 Role of 53BP1 and cellular nucleases in HIV-1-mediated homologous repair regulation.

Role of 53BP1 and cellular nucleases in HIV-1-mediated homologous repair regulation. a, Detection of DSBs by 53BP1 foci. WTK1(HR/3′) cells transfected with wt or vpu* pHIV-1-NL4-3-env*-IRES-mCherry constructs were γ-irradiated (2Gy) or left untreated (0Gy), fixed and stained for 53BP1 at the indicated time points. Immunolabeled foci were scored by quantification in 150 nuclei each, n=3 biologically independent experiments. Maximum scores were set to 100% (absolute foci numbers for 53BP1: 7), bars represent means ±SD. The left panel shows representative microscopic images of 53BP1 foci. b, Role of DNase2, ERCC1 and FEN1 nucleases in HIV-1 mediated DNA repair. WTK1(HR/3′) cells were transfected with pHIV-1-NL4-3-env*-IRES-mCherry, I-SceI-HA and shRNA to silence DNase2, ERCC1 or FEN1. Bars represent means ± SEM, n=2 (DNase2, ERCC1) or n=4 (FEN1) biologically independent experiments in triplicates, *p=0.0313 (absolute mean values: Dnase2 2.2%, ERCC1 0.3% and FEN1 1.0%). Right three graphs presenting rtPCR analysis to show DNase2, ERCC1 and FEN1 knockdown efficiencies. Bars represent means ± SEM, n=4 biologically independent experiments, two sided unpaired t-test, ***p=0.0001. c, Homologous repair frequency in WTK1(HR/3′) cells 48 h after transfection with pHIV-1-NL4-3-env*-IRES-mCherry constructs containing intact or defective vpu genes, pCMV-HA-I-SceI and shRNA to silence SLX4. Bars represent means ± SEM, n=2 biologically independent experiments in triplicates, *p=0.0313 (absolute mean value: 0.1%). d, Western blot analysis of WTK1(HR/3′) cells 48h after transfection with pHIV-1-NL4-3-env*-IRES-mCherry and pCMV-HA-I-SceI plasmids. The left panel shows examples of primary data and the right panel mean values ± SD of 3-6 independent experiments after quantification of band intensities using Bio-Rad ChemiDoc. Two sided Wilcoxon matched-pairs test in b (left three panels) and c.

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Extended Data Fig. 5 Role of BLM and EXO1 in Vpu-mediated modulation of DNA end processing.

Role of BLM and EXO1 in Vpu-mediated modulation of DNA end processing. a, Representative images for immunofluorescence analysis of p-RPA and BrdU and b, knock-down efficiency of BLM and EXO1. c, p-RPA accumulation as a function of BLM. WTK1(HR/3′) cells were γ-irradiated (2Gy) 48h after transfection with wt or vpu* pHIV-1-NL4-3-env*-IRES-mCherry plus shRNA to silence BLM (BLMkd). 1h post-irradiation, samples were taken for p-RPA32 S4/8 and RPA Western Blot analysis. The right panel shows mean values (±SD) of p-RPA levels obtained in three independent experiments relative to control cells. d, p-RPA Western blot analysis in WTK1(HR/3′) cells transfected with wt or vpu* pHIV-1-NL4-3-env*-IRES-mCherry, together with shRNA against EXO1. 48 h after transfection cells were irradiated and taken for Western blotting. e, Expression of BLM and EXO1 in primary CD4+ T cells. CD4+ T cells isolated form two blood donors were either treated with IL-2 alone or stimulated with IL-2/PHA or IL-2 and CD3/CD28 magnetic beads or left untreated (U) for 3d when cells were harvested for Western blot analysis.

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Extended Data Fig. 6 Effect of Vpu on PML-NBs. Role of BLM and EXO1 in Vpu-medidated modulation of DNA end processing.

Effect of Vpu on PML-NBs. Role of BLM and EXO1 in Vpu-mediated modulation of DNA end processing. a, Immunofluorescence analysis of Sp100 foci formation in WTK1(HR/3′) cells transfected with 1µg NL4-3, WITO Vpu or empty control. Cells were fixed at indicated time points. Data presents 3 independent experiments where each dot (n=6) represents mean value of 30-50 analyzed cells from one microscopic view; bars represent means ±SEM, *p=0.0313. Right panel, representative microscopic images. b, c, Immunofluorescence analysis of PML-NB and Sp100 foci in WTK1(HR/3′) cells transfected with wt or vpu* pHIV-1-CH058-env*-IRES-BFP constructs. Data present three independent experiments where each dot represents mean value of 30-50 analyzed cells from one microscopic view; bars represent means ±SEM, *p=0.0313. Right panel, representative microscopic images. Two sided Wilcoxon matched-pairs test in a-c.

Extended Data Fig. 7 Localization of Vpu at the nuclear membrane and effects on RanBP2-RanGAP1 proximity and function.

Localization of Vpu at the nuclear membrane and effects on RanBP2-RanGAP1 proximity and function. a, Subcellular protein fractionation performed in CD4+ T cells transfected with pCG-WITO Vpu-AU1 expression plasmid or empty control. Experiment conducted 2 times with similar outcome. b, Confocal microscopic localization analysis of Vpu and RanBP2. SaOS cells were transfected with 1 µg SIVcpz MB897 Vpu-AU1. 24 h later cells were fixed, permeabilized and costained against AU1-tagged Vpu and RanBP2. Graph on left presents the probability of Vpu following RanBP2 calculated from the images such as displayed on the right, bars represent means ± SEM, Two sided unpaired t- test here and in c, n=6, ****p=0.0001. Size bar = 5 µm. c, Primary CD4+ T cells were transfected with 0.5 µg of a vector expressing MB897 Vpu-AU1. Cells were permeabilized, stained with anti-AU1 and RanBP2 Ab and analyzed by confocal microscopy 24h post-transfection. Graph on left presents the probability of Vpu following RanBP2 calculated from the images such as displayed on the right, bars represent means ±SEM, n=5, ****p=0.0001. Size bar =5 µm. d, RanBP2 and RanGAP1 PLA in primary CD4+ T cells. Cells were transfected with 0.5 µg WITO Vpu-AU1 expression plasmid or empty vector control. Left, evaluation of RanBP2-RanGAP1 PLA foci per cell from two different donors, n=32, bars represent means ± SEM, two sided Mann-Whitney test, ****p=0.0001. Right, representative microscopic images. e, PLA to evaluate RanGAP1 and SUMO1 interaction in WTK1(HR/3′) cells transfected with WITO Vpu-AU1 expression plasmid or empty control. Left panel presenting evaluation of RanGAP1-SUMO PLA foci per cell, n=4 biologically independent experiments; two sided Wilcoxon matched-pairs test, mean values ± SEM, right panel presenting microscopic images. f, PLA assay to evaluate EXO1 and SUMO2/3 interaction in WTK1(HR/3′) cells transfected with WITO Vpu-AU1 expression plasmid or empty control. Left panel presenting evaluation of EXO1-SUMO2/3 foci per cell, n=2 biologically independent experiments, two sided Wilcoxon matched-pairs test (control n= 27, WITO n=20), mean values ± SEM, ****p=0.0001. Right panel presenting microscopic images.

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Extended Data Fig. 8 Role of PIAS1, PIAS4 and RanBP2 SUMO ligases in BLM SUMOylation.

Role of PIAS1, PIAS4 and RanBP2 SUMO ligases in BLM SUMOylation. a, Role of PIAS1, PIAS4 and RanBP2 SUMO ligases in BLM SUMOylation. WTK1(HR/3′) cells were transfected with 1 µg WITO Vpu-AU1 or control plus 3 µg of shRNA to target PIAS1, PIAS4 and RanBP2. 24h later PLA was performed. Left, evaluation of BLM-SUMO2/3 foci per cell, n=3 biologically independent experiments where each dot (8-12) represents a mean from 30-50 cells analyzed from one microscopic view; bars represent means ± SEM, two sided Wilcoxon matched-pairs test, *p=0.0117, **p=0.002, **p=0.0039, **p=0.0078. Right, Western blots presenting knockdown of PIAS1, PIAS4 and RanBP2. Lower panels, representative microscopic images. b, WTK1(HR/3′) cells were transfected with 1 µg WITO Vpu-AU1 or control and stained against SUMO1, RanBP2 and RanGAP1 24h later. Intensity profiles including the corresponding DAPI (blue) staining in the presence (red) or absence (black) of WITO vpu were quantified using ImageJ. Displayed are the means (full lines) of 10µm profiles of 25 individually quantified and aligned cells including SEM (dotted line). Exemplary confocal images are shown next to the quantifications and the profile indicated by a white line.

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Extended Data Fig. 9 Effect of Vpu and RAD52 or BLM on unintegrated viral cDNA, integration and superinfection in primary CD4+ T cells.

Effect of Vpu and RAD52 or BLM on unintegrated viral cDNA, integration and superinfection in primary CD4+ T cells. a-c, rtPCR analysis of 1-LTR, 2-LTR circles and integration in CD4+ T cells infected with wt or vpu* pHIV-1-NL4-3 in the presence or absence of a RAD52 inhibitor (5 µM 6-Hydroxy-DL-DOPA) 48 h post infection. Bars represent means ± SEM, n=3 biologically independent experiments in triplicates, (a) *p=0.0156, **p=0.0039, (b) **p=0.0078, (c) *p=0.0273, **p=0.0039. d, Propidium iodide staining of acetone/ethanol fixed cells and p24 stain of primary CD4+ T cells treated as in Fig. 7 (a–c). n=3, bars represent means ± SEM of 6 (apoptosis) or 4 measurements, **p=0.0022. e, Western blot analysis of p24 protein expression in CD4+ T cells infected with wt or vpu* pHIV-1-NL4-3. Experiment conducted two times with similar outcome. f-k, rtPCR analysis of 1-LTR, 2-LTR circles and integration in CD4+ T cells transduced with wt, vpu* or vpr* defective pHIV-1-NL4-3-env* constructs (f-h) or infected with replication-competent pHIV-1-NL4-3 in the presence of the protease inhibitor saquinavir (i-k). Bars represent means ± SEM from 6 (f,g), 5 (h), 3(i), 2(j, k) donors are shown. Analysis done 48 h post-infection/transduction. l, Western blot demonstrating BLM knockdown efficiency in Jurkat cells. Experiment conducted 2 times with similar outcome. m-o, rtPCR analysis of 1-LTR and 2-LTR circles and integration in BLM silenced Jurkat cells infected with wt or vpu* VSV-G pseudotyped pHIV-1-NL4-3. Bars represent means ± SEM, n=3 biologically independent experiments in triplicates, (m) *p=0.0156, **p=0.0039; (n) *p=0.0313, **p=0.0039, (o) **p=0.0039. Analysis done 48 h post-infection. p, Supernatants from m-o were taken to analyze infectious yields determined by β-galactosidase assay in TZM-bl cells. Bars represent means ± SEM, n=2 biologically independent experiments in triplicates, *p=0.0313. Two sided Wilcoxon matched-pairs test in a-d, m-p.

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Extended Data Fig. 10 Effect of Vpu on HIV-1 superinfection in primary CD4+ T cells and BLM deficient cells.

Effect of Vpu on HIV-1 superinfection in primary CD4+ T cells and BLM deficient cells. a, Schematic presentation of the experimental design to measure and calculate HIV-1 superinfection. b, Primary flow cytometry data of CD4+ T cells treated as described in panel b; n=6. Calculated percentages of superinfected cells are shown between the dot plot panels. c, Primary flow cytometry data of BLM+/- cells treated as described in the legend to Fig. 6r.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Supplementary Table 1.

Reporting Summary

Supplementary Video 1

Video presenting colocalization between WITO Vpu and RanBP2 in transfected SaOS cells.

Supplementary Video 2

Video presenting colocalization between SIV Vpu and RanBP2 in transfected CD4+ T cells.

Supplementary Video 3

Video presenting colocalization between SIV Vpu and RanBP2 in transfected SaOS cells.

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Volcic, M., Sparrer, K.M.J., Koepke, L. et al. Vpu modulates DNA repair to suppress innate sensing and hyper-integration of HIV-1. Nat Microbiol 5, 1247–1261 (2020). https://doi.org/10.1038/s41564-020-0753-6

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