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.
The induction of DNA damage responses (DDRs) and innate immune activation are inevitable consequences of human immunodeficiency virus type 1 (HIV-1) infection. Reverse transcription (RT) converts the single-stranded viral RNA genome into linear double-stranded DNA. This process is associated with the formation of RNA:DNA hybrid intermediates and generation of circular forms of HIV-1 complementary DNA containing one or two copies of the long terminal repeat region (1-LTR and 2-LTR, respectively) that do not support productive infection1. However, accumulation of these viral nucleic acids may trigger antiviral immune responses2. To avoid this, HIV-1 uses its capsid core and cellular cofactors to shield its single-stranded RNA genome and RT intermediates from recognition by cytosolic sensors3,4. However, this ‘cloaking’ is imperfect and accumulating non-integrated HIV-1 DNA triggers immune activation and interferon production, unless it is degraded by cellular nucleases such as TREX1 (ref. 5).
HIV-1 also directly modulates DNA repair. The viral accessory protein R (Vpr) is a particularly well-known modulator of the DDR machinery. Vpr is contained in virions and induces proteasomal degradation of the DNA repair enzymes HLTF, UNG2 and MUS81 immediately after viral entry to prevent the restriction of viral cDNA RT products6,7,8. Following RT and nuclear import, a double-strand break (DSB) is introduced into the host cell genome for integration of the linear viral cDNA9,10. Completion of proviral integration requires non-homologous end joining (NHEJ)—that is, DNA-PKcs, Ku, XRCC4 and Nibrin—and other DDR factors, such as ATM11. Thus, DDRs play key roles in viral replication and HIV-1 needs to prevent accumulation of its nucleic acids to minimize immune activation.
Numerous studies have examined how HIV-1 avoids immune sensing and affects DNA repair during the early steps of viral replication. In contrast, it is poorly understood how HIV-1 modulates DNA repair in productively infected cells. Here, using fluorescence-based assays, we identified the HIV-1 accessory factor viral protein U (Vpu) as a key modulator of DNA repair. Vpu is a small, single-pass trans-membrane protein that is only present during the late stages of HIV-1 infection. It prevents superinfection by inducing CD4 degradation12,13 and promotes virus release by antagonizing the restriction factor tetherin14,15. We found that Vpu interacts with RanBP2–RanGAP1*SUMO1–Ubc9 SUMO E3-ligase complexes at the nuclear pore to manipulate functions of promyelocytic leukaemia protein nuclear bodies (PML-NBs) and DDR components such as RAD52 or Bloom syndrome protein (BLM). This allows Vpu to suppress superinfection by eliminating newly incoming viral cDNA species through two cooperative mechanisms: (1) inhibition of RAD52-dependent homologous repair and thereby circularization of non-integrated linear viral DNA to maintain susceptibility to nucleolytic attack and (2) enhancement of DNA processing by BLM to promote degradation of linear viral DNA suppressing innate immune activation and hyper-integration. We also demonstrate that BLM restricts proviral integration of HIV-1. In summary, our results show that Vpu modulates DNA repair to suppress the accumulation and sensing of nuclear viral DNA species and suggest a role for BLM in viral latency.
Proviral HIV-1 expression inhibits homologous repair
HIV-1 is known to modulate DSB repair but the underlying mechanisms have remained elusive16. We applied enhanced green fluorescent protein (EGFP)-based reporters for specific DSB repair pathways, namely NHEJ, microhomology-mediated end joining (MMEJ) and two types of homologous repair: homologous recombination (HR) and single-strand annealing (SSA)17,18. For the assessment of extrachromosomal repair, Jurkat T cells were co-transfected with an HIV-1 NL4-3 IRES-mCherry proviral construct (HIV-1 refers to NL4-3 unless specified otherwise), DSB repair substrates and an I-SceI expression vector for targeted cleavage (Fig. 1a–d, left panels). Scoring of red and green fluorescent cells allowed DSB repair to be monitored in HIV-1-positive cells (Extended Data Fig. 1a). HIV-1 had no effect on NHEJ (Fig. 1a) and/or MMEJ (Fig. 1b) following the transfection of proviral HIV-1 DNA, which bypasses the earliest steps of viral replication from entry to integration. In contrast, HR and SSA were strongly reduced (Fig. 1c,d). Addition of the pan-caspase inhibitor Z-VAD-FMK reduced the sub-G1 fraction—that is, the percentage of apoptotic cells (Extended Data Fig. 1b)—but did not alter the HIV-1-mediated repair patterns (Fig. 1b–d). Thus, no effects were under- (MMEJ) or overestimated (HR and SSA) due to HIV-1-induced apoptosis. Further analyses also excluded indirect effects through cell-cycle changes (Extended Data Fig. 1c). HIV-1-dependent inhibition of homologous repair (HR and SSA) was confirmed in the human lymphoblastoid cell line WTK1 (Extended Data Fig. 1d–f).
To examine the HIV-1-mediated effects on DSB repair in the chromatin environment, we used the WTK1(HR/3′) cell line19, which carries the chromosomally integrated HR-EGFP/3′EGFP substrate (Fig. 1e). Transfection with the HIV-1 IRES-mCherry construct decreased homologous repair by approximately 70% (Fig. 1e) without affecting I-SceI expression (Fig. 1f). Genomic PCR analysis of sorted EGFP+ WTK1(HR/3′) cells (Fig. 1g) revealed decreases of 40% for HR (Fig. 1h) and of 76% for SSA (Fig. 1i). Analysis of the repair construct integrity excluded simple destruction by nucleases (Fig. 1i). HIV-dependent suppression of homologous repair was confirmed in WTK1(Δ/3′) cells with chromosomally integrated ΔEGFP/3′EGFP substrate, which allowed distinction between HR and SSA in hygromycin B selection medium (Extended Data Fig. 1g)18. Together, these results show that proviral HIV-1 gene expression suppresses HR and SSA both extra- and intra-chromosomally.
RAD52 is critical for the HIV-1-dependent suppression of homologous repair
Silencing of ATM, ATR, Nibrin, CtIP and Ku70 involved in DDR and end processing neither diminished homologous repair nor abrogated the HIV-1-mediated decline in WTK1(HR/3′) cells (Extended Data Fig. 1h-l). Depletion of ATM and ATR even increased the basal homologous repair frequencies, consistent with de-repression of error-prone pathways like SSA20. In contrast, silencing of the DNA damage sensor PARP1 diminished homologous repair in control cells and reduced the inhibitory effect of HIV-1 (Fig. 1j). Dominant negative interference with NF-κB, which is modulated by HIV-1 (ref. 21) and activates DSB repair downstream of PARP1 (ref. 22), slightly reduced the HIV-1-mediated suppression of homologous repair (Fig. 1k). A similar reduction was observed following BRCA1 knockdown (Fig. 1l) but not following dominant negative interference with RAD51 (Fig. 1m). Strikingly, silencing or pharmacological inhibition of the SSA factor RAD52 abolished the inhibitory effect of HIV-1 entirely (Fig. 1n,o). Thus, although PARP1 and BRCA1 are also involved, RAD52 plays a dominant role in the HIV-1-dependent suppression of homologous repair.
HIV-1 Vpu inhibits homologous repair and modulates RAD52 function
The HIV-1 accessory proteins Vpr, Vpu and Nef are known to affect DNA repair and/or NF-κB signalling7,21,23,24. We thus examined their involvement in the suppression of homologous repair by HIV-1. Intriguingly, the absence of Vpu abrogated the inhibitory effect and even resulted in 1.4-fold enhanced homologous repair in lymphoid cells transfected with HIV-1, whereas the absence of Vpr or Nef had little-to-no effect (Fig. 2a). The Vpu proteins from HIV-1 NL4-3, the transmitted founder HIV-1 strain WITO, the chronic HIV-1 strain CH106 and the chimpanzee virus SIVcpz EK505 all suppressed homologous repair by about 50% (Fig. 2b). Vpu inhibited homologous repair already at low expression levels (Extended Data Fig. 2a) and was detected at similar levels in cells infected with HIV-1, transfected with proviral constructs or transfected with Vpu expression vectors (Extended Data Fig. 2b). Vpu did not induce apoptosis or cell-cycle changes (Extended Data Fig. 2c), suggesting direct effects on the DNA repair machinery.
In agreement with the results obtained for WTK1 cells (Fig. 2a), wild-type (wt) HIV-1 reduced homologous repair in primary CD4+ T cells by approximately 50% and a lack of Vpu disrupted this effect (Fig. 2c). Vpu-dependent suppression of homologous repair was confirmed for HIV-1 CH058 and CH198 containing defective env genes (Fig. 2d) and was not due to different infection rates (Extended Data Fig. 2d). The inhibitory effect of Vpu on DSB repair was confirmed in HIV-1-infected WTK1 cells (Fig. 2e). Homologous repair was inhibited by Vpu in the context of both env-defective (Fig. 2d) and wt HIV-1 (Extended Data Fig. 2e, left). HIV-1 integration was reported to affect cell survival25. However, the evaluation of apoptosis and the cell-cycle distribution revealed no changes between CD4+ T cells harbouring wt or vpu-defective HIV-1 (Extended Data Fig. 2e).
To obtain insights into the mechanism(s) underlying Vpu-mediated inhibition of homologous repair, we analysed mutant forms of the HIV-1 CH058, CH293, CH077 and STCO infectious molecular clones that are defective in NF-κB inhibition or at counteracting tetherin26,27,28. A lack of these Vpu functions did not impair the ability of HIV-1 to inhibit homologous repair in primary CD4+ T cells (Fig. 2f) or WTK1(HR/3′) cells (Fig. 2g). To map critical domains, we examined alanine-substitution mutants of WITO Vpu containing changes in potential trafficking domains29 or the β-transducin repeat-containing protein (β-TrCP) binding motif30 (Extended Data Fig. 3a) that is critical for the Vpu-mediated degradation of CD4 (ref. 31). All mutant Vpu proteins were expressed (Extended Data Fig. 3b) and the QEE61–63AAA mutation affecting an EXXXLV motif in HIV-1 Vpu disrupted its ability to inhibit homologous repair and end processing (Extended Data Figs. 3c,d). In comparison, changes in the YxxΦ trafficking and β-TrCP binding motifs did not disrupt these Vpu functions. Mutations in the DSGxxS and EXXXLV motifs in Vpu significantly reduced the number of BrdU foci measured as an indicator of DNA-end processing (Extended Data Fig. 3d). The addition of a carboxy (C)-terminal endoplasmic reticulum-retention signal to Vpu disrupted its effect on homologous repair (Extended Data Fig. 3c, compare CH106 KKDQ with CH106), which provides support for proper subcellular location of Vpu being critical. Consistent with the mutational analyses, coexpression of a dominant negative mutant of β-TrCP did not affect suppression of homologous repair by HIV-1 (Extended Data Fig. 3e). Together, these results show that the ability of Vpu to inhibit homologous repair is conserved among primary HIV-1 strains and genetically separable from other Vpu activities.
We next examined whether Vpu targets RAD52, which was critical for the suppression of homologous repair by HIV-1 (Fig. 1n,o). RAD52 silencing indeed lowered the repair frequency and abolished the effect of Vpu (Fig. 2h). Vpu had little effect on the levels of RAD52 protein or messenger RNA (Extended Data Figs. 3f,g) but prevented an increase in discrete nuclear RAD52 foci after irradiation of SaOS cells (Fig. 2i). The latter effect was only observed in the absence of irradiation (Fig. 2i), suggesting that Vpu prevents de novo formation of active RAD52 repair centres in response to DNA damage but does not disrupt pre-existing RAD52 foci.
Vpu enhances BLM/EXO1-dependent DNA-end processing
Nucleolytic DNA-end processing generates ssDNA and plays a key role in homologous repair (Fig. 3a)32. Immunofluorescence microscopy revealed that HIV-1 does not affect the formation or clearance kinetics of radiation-induced p53-binding protein 1 (53BP1) foci representing indicators of DSBs (Extended Data Fig. 4a). However, HIV-1 enhanced the formation of foci containing phosphorylated replication protein A (p-RPA) in a Vpu-dependent manner early (1 h) after radiation exposure (Fig. 3b). Phosphorylated RPA is generated by phosphorylation of the RPA32 subunit following binding to ssDNA and represents a marker for ssDNA formation33,34 (Fig. 3a). Thus, HIV-1 Vpu enhances ssDNA formation at DSBs and consequently promotes nucleolytic processing of DNA ends.
To clarify whether the Vpu-dependent effects on DNA-end processing and homologous repair are linked, we examined the involvement of various DNA repair nucleases and components of end-processing complexes using short hairpin RNA (shRNA) interference35. Similar to the MRE11–RAD50–Nibrin (MRN) nuclease complex components Nibrin and CtIP, initiating end resection (Extended Data Figs. 1j,k), FEN1, ERCC1, SLX4 and DNase2 did not alter the effect of HIV-1 on homologous repair (Extended Data Fig. 3b). Knockdown of the helicase BLM, coordinating end resection by exonuclease 1 (EXO1), DNase2 (Fig. 3c,d)35, and the structure-specific endonuclease subunit SLX4 targeted by Vpr23 (Extended Data Fig. 4c) also did not abrogate HIV-1 mediated suppression of homologous repair. In addition, HIV-1 did not affect the expression of various proteins involved in nucleolytic processing (Extended Data Fig. 4d).
We investigated the impact of BLM and EXO1 on HIV-1-induced p-RPA accumulation to further examine how Vpu promotes DNA-end processing. EXO1 is a nuclease involved in long-stretch DNA-end processing stimulated by interaction with BLM helicase36. Detection of p-RPA and BrdU under non-denaturing conditions as independent indicators of ssDNA34 revealed that a lack of Vpu or silencing of BLM or EXO1 expression eliminated HIV-1-mediated end processing (Fig. 3e and Extended Data Fig. 5a,b). Proximity ligation assays (PLA)37 demonstrated that wt HIV-1 as well as WITO Vpu increased the number of BLM–EXO1 PLA foci in irradiated cells (Fig. 3f), suggesting that Vpu recruits BLM to stimulate EXO1. HIV-1 moderately increased the levels of p-RPA but not RPA32 in a Vpu-, BLM- and EXO1-dependent manner (Extended Data Figs. 5c,d). Notably, BLM and EXO1 were induced in the main target cells of HIV-1 replication—that is, activated CD4+ T cells (Extended Data Fig. 5e). Collectively, these results show that Vpu promotes the processing of DNA ends by BLM and EXO1 independently of its effects on homologous repair.
Vpu manipulates PML-NBs and modulates SUMOylation
The function of BLM is governed by SUMOylation, which regulates its intra-nuclear trafficking between DNA damage-induced foci and PML-NBs38,39. Analysis by PLA revealed that Vpu reduces BLM–SUMO2/3 signals by approximately 50% (Fig. 4a) and immunoprecipitation confirmed a reduction in BLM SUMOylation (Fig. 4b). Similarly, Vpu reduced RAD52–SUMO PLA signals by approximately 70% (Fig. 4c). PML-NBs are hot spots for SUMOylation and harbour many DNA repair proteins, including BLM and RAD52 (ref. 40). Silencing of PML by about 60% reduced homologous repair and abolished further reduction by Vpu in cells transfected with HIV-1 (Fig. 4d). This is in agreement with results showing that PML is important for efficient HR41 and suggests that Vpu might affect PML function to modulate HR. We found that Vpu increased the number of PML- or Sp100-stained PML-NBs by approximately twofold by 24 h post transfection, followed by the disappearance of a fraction of PML-NBs (Fig. 4e and Extended Data Fig. 6a). This kinetic is reminiscent of PML-NB dispersal and disruption after genotoxic stress40. A marked reduction in PML-NBs at later time points was confirmed with wt HIV-1 CH058 (Extended Data Fig. 6b,c). In addition, Vpu reduced the PML–SUMO2/3 PLA signals by approximately 50% (Fig. 4f). HIV-1 NL4-3 in WTK1 cells (Fig. 4g) and CH058 in primary CD4+ T cells (Fig. 4h) reduced the PML–SUMO levels in a Vpu-dependent manner. Finally, HIV-1 impaired colocalization between BLM and PML-NBs (Fig. 4i), and Vpu was required and sufficient for this effect. Together, these data provide support for Vpu manipulating PML-NBs and mitigating SUMOylation and/or SUMO interactions of BLM and RAD52 to promote end processing and suppress homologous repair.
Vpu modulates the RanBP2–RanGAP1*SUMO1–UBC9 SUMO E3-ligase complex
Our findings were surprising because Vpu is known to localize in perinuclear compartments, including the trans-Golgi network, but not in the nucleus42. However, subcellular protein fractionation revealed the presence of Vpu in both, the membrane and nuclear fractions (Extended Data Fig. 7a), which is in agreement with the reported interaction of Vpu with RanBP2 in mass spectrometry analysis43. This nucleoporin (also known as Nup358) forms filaments at the cytoplasmic surface of the nuclear pores that assemble SUMO E3-ligase complexes modulating the SUMOylation and function of PML-NBs44. In support of an interaction, RanBP2 co-immunoprecipitated Vpu (Fig. 5a). Analysis of HIV-1-infected or -transfected SaOS cells (Fig. 5b, Extended Data Fig. 7b and Supplementary Video 1) and infected primary CD4+ T cells (Fig. 5c) confirmed the presence of Vpu at the nuclear envelope. Strikingly, Vpu and RanBP2 alternated at the nuclear rim in a non-random pattern (Fig. 5b,c). Analysis by PLA verified that Vpu and endogenous RanBP2 were in close proximity at the nuclear pore (Fig. 5d). Notably, SIVcpz MB897 Vpu showed stronger colocalization with RanBP2 than HIV-1 Vpu in CD4+ T and SaOS cells (Extended Data Fig. 7b,c and Supplementary Videos 2,3), conceivably because SIVcpz Vpu proteins lack the functional constraints associated with the anti-tetherin activity of HIV-1 Vpu at cytoplasmic membranes45.
RanBP2 filaments form complexes with RanGAP1 and UBC9 (ref. 46) that operate as E3 SUMO ligase targeting PML-NBs44. We thus analysed whether Vpu interferes with the RanBP2–RanGAP1*SUMO1–UBC9 complex to manipulate the SUMOylation and function of RAD52 and BLM in the nucleus. Vpu reduced the signal for RanBP2–RanGAP1 colocalization in WTK1(HR/3′) (Fig. 5e) and CD4+ T cells (Extended Data Fig. 7d) by approximately 70% without changing the levels of RanBP2, RanGAP1 and UBC9 expression (Fig. 5f). Vpu did not abrogate co-precipitation of RanBP2 and RanGAP1 (Fig. 5g,h) but it reduced the RanGAP1–SUMO PLA signals at the nuclear membrane (Extended Data Fig. 7e) as well as the nuclear EXO1–SUMO PLA signals (Extended Data Fig. 7f). Notably, Vpu also diminished the number of RanBP2–BLM PLA foci (Fig. 5i). Moreover, SUMOylation of BLM was dependent on both RanBP2 and the E3 SUMO-protein ligase PIAS4 but not on PIAS1 (Extended Data Fig. 8a). In addition, Vpu reduced the levels of SUMO1 in the nucleus but had no effect on the levels of RanBP2 and RanGAP1 at the nuclear membrane (Extended Data Fig. 8b). Together, these data suggest that Vpu interacts with RanBP2 to modulate the spatial organization of RanBP2–RanGAP1*SUMO1–UBC9 complexes rather than their integrity, thereby reducing their SUMOylation activity and consequently affecting the functions of PML, RAD52, BLM and EXO1.
Vpu prevents accumulation, sensing and hyper-integration of nuclear viral DNA
Our data showed that HIV-1 uses Vpu to suppress homologous repair in a RAD52-dependent manner and enhances BLM-mediated DNA-end processing. To elucidate the biological significance of these effects, we analysed the impact of Vpu on nuclear viral cDNA species. Quantitative PCR of HIV-1-infected primary CD4+ T cells revealed that a lack of Vpu was associated with a two- to fivefold increase in the 1-LTR and 2-LTR cDNA forms as well as proviral copies (Fig. 6a–c and Extended Data Fig. 9a–c). Silencing or pharmacological inhibition of RAD52 reduced the 1-LTR circle numbers in the absence of Vpu (Fig. 6a and Extended Data Fig. 9a) but had no effect on the 2-LTR circles (Fig. 6b and Extended Data Fig. 9b). This observation is consistent with the role of homologous repair in 1-LTR and NHEJ in 2-LTR formation47,48. Efficient integration was restored by RAD52 knockdown but not inhibition (Fig. 6c and Extended Data Fig. 9c). Notably, the RAD52 inhibitor 6-hydroxy-DL-DOPA prevents homologous repair but not ssDNA binding49. RAD52 was also reported to mediate the integration of recombinant adeno-associated virus50 but to suppress proviral integration by competition with the integrase for ssDNA51. This might explain why RAD52 knockdown and chemical inhibition affected integration differently. Control experiments showed that neither silencing of RAD52 nor HIV-1 infection markedly affected apoptosis or the cell cycle (Extended Data Fig. 9d). Vpu did not affect viral p24 and p55 Gag expression, although it promoted virion release due to tetherin antagonism (Extended Data Fig. 9e). Vpu-dependent reduction of 1-LTR and 2-LTR circular forms as well as proviral integration were confirmed using the transmitted founder CH058 HIV-1 strain (Fig. 6e–g). Importantly, the effects of Vpu on nuclear viral DNA species were not observed with env-defective HIV-1 or in the presence of the protease inhibitor saquinavir (Extended Data Fig. 9f–k). They thus required secondary rounds of HIV-1 infection.
To examine the role of BLM in the Vpu-dependent modulation of nuclear viral DNA forms, we transduced fibroblasts derived from a patient with Bloom syndrome and the corresponding BLM-reconstituted cells with HIV-1. Both 1-LTR and 2-LTR circles, proviral integration and infectious HIV-1 yield were strongly increased in the absence of BLM (Fig. 6h–k). Similarly, BLM knockdown increased the levels of nuclear viral DNA, proviral integration and infectious HIV-1 production in Jurkat T cells (Extended Data Fig. 9l–p). HIV-1 induced IFNβ mRNA expression in the absence of BLM but not in its presence (Fig. 6l). Together, the results provide support for BLM reducing HIV-1-induced innate immune activation and proviral integration by promoting the degradation of linear viral DNA in the nucleus.
Our results suggested that Vpu might suppress superinfection of HIV-1 independently of CD4 downmodulation by promoting BLM-dependent degradation of unintegrated linear viral DNA. To examine this, we transduced CD4+ T cells with wt or vpu-defective HIV-1-NL4-3-env* IRES-BFP constructs and exposed them to vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped env-defective HIV-1 IRES-GFP constructs allowing viral entry independently of CD4 (Extended Data Fig. 10a). An absence of Vpu enhanced superinfection by 1.5- and twofold when the cells were first infected by CH058 and subsequently challenged with NL4-3 or CH058, respectively (Fig. 6m,n and Extended Data Fig. 10b,c). Vpu suppressed superinfection independently of its ability to suppress NF-κB activation (Fig. 6n). Superinfection was even 7.3-fold increased when CD4+ T cells were initially infected with HIV-1 NL4-3 and subsequently exposed to CH058 (Fig. 6o).
An absence of Vpu increased T-cell activation in superinfected cells, as indicated by the upregulated expression of the activation markers CD69 and CD25 in the double-positive cell population (Fig. 6p,q). Superinfection was increased in the absence of BLM (Fig. 6r and Extended Data Fig. 10c). Interestingly, Vpu still suppressed superinfection in BLM-deficient fibroblasts. A possible reason for this is that Vpu might also affect other RanBP2-dependent processes such as docking of the HIV-1 pre-integration complex to the nuclear pore and nuclear entry52,53,54. A lack of BLM was associated with significantly increased expression of IFNβ in superinfected cells (Fig. 6s). Thus, BLM eliminates linear viral cDNA species and suppresses innate sensing and proviral integration of HIV-1.
Previous studies demonstrated that the HIV-1 accessory protein Vpu inhibits superinfection by downmodulating CD4 (ref. 13) and suppresses innate immune activation by inhibiting activation of NF-κB21,27,55. Here we show that Vpu also prevents superinfection and immune activation by modulating DNA repair. Specifically, Vpu inhibits homologous repair while stimulating nucleolytic processing of DNA ends to suppress the accumulation of nuclear viral cDNA species (summarized in Fig. 6t). To prevent circularization of viral cDNA and permit nucleolytic attack, Vpu manipulates the homologous repair factor RAD52. To stimulate processing of viral DNA ends, it activates BLM. Vpu achieves this by interacting with RanBP2 at the nuclear pore to suppress the SUMOylation activity of RanBP2–RanGAP1*SUMO1–UBC9 complexes and consequently PML-NB-mediated SUMOylation of BLM. Thus, Vpu prevents superinfection and immune sensing by synergistic mechanisms including CD4 degradation, suppression of NF-κB activation, inhibition of RAD52 and activation of BLM for the degradation of excess viral nuclear cDNA and prevention of proviral hyper-integration (summarized in Supplementary Fig. 1).
It may seem surprising that Vpu performs activities reducing all forms of nuclear HIV-1 cDNAs. However, it must be considered that Vpu is present only during the late stages of the viral replication cycle, when HIV-1 has already achieved integration and established productive infection56. In fact, reducing effects on viral DNA species were only observed under conditions that allow multiple rounds of HIV-1 infection and are in line with the emerging role of Vpu as a multi-functional inhibitor of immune activation and viral superinfection. Inhibition of superinfection by reducing the levels of CD4 on the cell surface seems important given that HIV-1 utilizes three of its gene products (Vpu, Nef and Env) to achieve this57. Nonetheless, CD4 downmodulation does not entirely prevent superinfection13. Thus, increased DNA-end processing by Vpu may represent a back-up mechanism preventing superinfection via degradation of linear viral DNA products and consequently proviral hyper-integration. Strikingly, reconstitution of BLM expression in cells from a patient with a truncating BLM mutation resulted in a reduction in the levels of proviral integration by approximately 30-fold (Fig. 6j). Thus, by preventing integration of new proviruses that might become latent, BLM may also play a role in the replenishment of latent viral reservoirs.
Another prominent function of Vpu is the inhibition of the expression of antiviral host genes by preventing nuclear translocation of p65 (ref. 21) and counteracting tetherin, which traps virions and acts as an immune sensor inducing NF-κB-dependent proinflammatory responses14,55. Prevention of superinfection will also suppress innate sensing because less viral material enters the cell. Increased degradation of nuclear viral DNA will further suppress the innate sensing of HIV-1 infection. The exact determinants of Vpu-mediated modulation of DNA repair need further investigation. However, our results show that this Vpu function is conserved between HIV-1 and SIVcpz, and genetically separable from the effects on NF-κB and tetherin.
Vpu is not localized in the nucleus and does not interact directly with homologous repair proteins. However, previous data indicate that substantial Vpu fractions are localized in the perinuclear region58 and might bind to RanBP2 (ref. 43), a component of the nuclear pore complex and part of a multi-subunit SUMO E3-ligase complex46. RanBP2 has been identified as a HIV-1 dependency factor required for the nuclear import of viral DNA43,59. In addition, depletion of RanBP2 compromises the SUMOylation and function of PML-NBs44. We found that RanBP2 co-immunoprecipitates Vpu (Fig. 5a,h) and show that Vpu and RanBP2 are located at the nuclear membrane (Fig. 5b,c and Extended Data Fig. 7b,c). Intriguingly, Vpu reduces the number of RanBP2–RanGAP1 foci (Fig. 5e and Extended Data Fig. 7d) and manipulates PML-NBs (Fig. 5e–i and Extended Data Fig. 6a–c). These nuclear organelles are major sites of SUMOylation and colocalize with DSB repair proteins, including RAD52 and BLM40,60. Thus, Vpu might suppress SUMOylation of these DNA repair proteins by altering the SUMO ligase activity of the RanBP2–RanGAP1*SUMO1–UBC9 complex and downstream PML-NB function. Vpu achieves this without disrupting the RanBP2–RanGAP1 interaction or altering their expression levels (Fig. 5g,h). Instead, Vpu seems to localize between RanBP2 molecules (Fig. 5b,c), presumably altering their spacing and consequently functionality. Manipulation of PML-NBs and SUMOylation to promote viral replication or immune evasion has already been reported for simian virus 40, adeno-, herpes simplex and influenza viruses40,61,62. Notably, proviral integration in close proximity to PML-NBs promotes HIV-1 latency and disruption of these organelles reactivates viral gene expression63. Thus, it will be interesting to further examine whether Vpu modulates HIV-1 latency by affecting the integrity and function of PML-NBs.
Our data show that Vpu downregulates SUMOylation of BLM (Fig. 4a,b) and promotes complex formation of BLM with the nuclease EXO1 (Fig. 3f). Intriguingly, BLM and EXO1 were shown to generate radiation-induced ssDNA fragments that leak into the cytosol of breast cancer cells, where they can be fully digested by TREX1 (ref. 64). We show that HIV-1 targets the same factors to prevent the accumulation of viral cDNA via the manipulation of SUMOylation, which controls DNA-end resection65. This mechanism may contribute to the downregulation of the number of RAD52 foci by Vpu because SUMOylation of BLM seems to be required for the efficient recruitment of RAD52 to DNA damage sites39.
The Vpu-mediated activation of BLM is reminiscent of Vpr-induced activation of SLX4, another SUMOylated nuclease co-factor associating with PML-NB and involved in Holliday junction cleavage during HR23,66. Both BLM and SLX4 suppress antiviral IFN expression23 (Fig. 6s). Thus, Vpu and Vpr may cooperate in activating nuclease complexes to suppress innate immune sensing via the destruction of excess viral DNA. In contrast to Vpu, however, Vpr is incorporated into viral particles and virion-associated Vpr is sufficient to mediate the degradation of DNA repair enzymes7. Thus, Vpr might predominantly prevent the accumulation of viral cDNA species during the initial establishment of HIV-1 infection. In contrast, Vpu acts during the late stage of infection and its effects on the DNA repair machinery seem mostly relevant for superinfection events. Notably, evidence suggests that mechanical forces acting on the nucleus during T-cell migration in vivo also induce changes, including the induction of DNA repair and sensing responses67. It will be of interest to further analyse how Vpu and Vpr cooperate to modulate DDRs and to facilitate HIV-1 escape from innate immune sensing.
In conclusion, our data indicate that HIV-1 utilizes Vpu to manipulate RanBP2–RanGAP1*SUMO1–UBC9 Sumo E3-ligase complexes and consequently PML-NBs and SUMOylation of DNA repair factors for the removal of immune-activating and/or dead-end viral DNA products in the nucleus. Vpu-activated BLM may synergize with TREX1 (ref. 5), located in the cytoplasm, to prevent the accumulation of viral DNA species that may serve as pathogen-associated molecular patterns to trigger innate immune activation and interferon responses. Our discovery of Vpu as a modulator of DNA repair pathways thus identifies a higher-level control mechanism exerted by HIV-1 within the network of virus–host circuits affecting DDRs and the antiviral immune defence. Critical players in this control mechanism may represent promising targets for the development of drugs preventing inflammation and premature ageing in HIV-1-infected individuals.
The pHIV-1-NL4-3-IRES-mCherry plasmids carrying STOP codons in the env, vpu and nef gene were cloned by replacing the IRES-eGFP cassette in the respective GFP reporter viruses13 with an IRES-mCherry cassette. The IRES-mCherry was PCR amplified using primers containing the unique single cutter MluI and XmaI. The vpr-defective pHIV-1-NL4-3-IRES-mCherry construct was created by site-directed mutagenesis as described previously68 for the pHIV-1-NL4-3-IRES-mCherry–env* construct. Proviral HIV-1 CH058 constructs were cloned in the pBR322 vector using the unique restriction sites MluI and NotI flanking the proviral sequence. For the BFP reporter constructs of CH058, the overlapping part of nef and LTR was duplicated and new unique PmeI and SacII restriction sites were introduced via overlap-extension PCR. The outer primers overlap with the unique restriction site StuI in the env gene of CH058 and the MluI site after the 3′ LTR. After introducing the overlap-extension-PCR product in the provirus, the new single cutters PmeI and SacII were used to introduce the reporter cassette. The IRES-BFP cassette was PCR amplified using primers containing PmeI and SacII restriction sites. To generate the env-deficient CH058 construct, nucleotides 122–1371 of the env gene were deleted using the Q5 site-directed mutagenesis kit (NEB) as recommended by the manufacturer. This kit was also used to introduce point mutations or premature stops in the second and third codon of the vpu gene. A NL4-3 proviral clone without overlapping env and vpu genes was generated via overlap-extension PCR as previously described45. During this process, a unique SacII restriction site was introduced upstream of vpu and a unique NcoI site downstream of vpu. The vpu of HIV-1 subtype B WITO was PCR amplified using primers (Supplementary Table 1) containing SacII and NcoI sites and an AU1 tag at the C terminus, and cloned into this NL4-3 proviral backbone.
Jurkat (American Type Culture Collection, ATCC), WTK1 (ref. 69) and WTK1(HR/3ʹ)18, K562(Δ/3)18 cells were cultivated in RPMI-1640 medium with 10% fetal calf serum (FCS), 2 mM l-glutamine, streptomycin and penicillin. The human osteogenic sarcoma cell line SaOS (ATCC) was cultivated in McCoys medium with 10% FCS, 2 mM l-glutamine, streptomycin and penicillin. Human embryonic kidney 293T cells (HEK293T; ATCC) and HeLa HIV-1 reporter TZM-bl (NIH) cells were maintained in DMEM medium with 10% FCS, 2 mM l-glutamine, streptomycin and penicillin. Fibroblast GMO8505 cells from a patient with Bloom syndrome70 were cultivated in α-MEM medium with 10 % FCS, 2 mM l-glutamine and 350 µg ml−1 G418 (ref. 70). CD4+ T cells were isolated from healthy donors using a combination of lymphocyte separation medium (Biocoll separating solution, Biochrom) and negative isolation (RosetteSep, Stemcell Technologies). Cells were stimulated with IL-2 (10 ng ml−1) and anti-CD3/CD28 beads (Life Technologies). The cells were cultured in RPMI-1640 medium with 20% FCS. All cell lines were routinely tested for mycoplasma contamination.
Cell-cycle and apoptosis analysis
Cell-cycle and apoptosis results were obtained by FACS analysis on cells fixed with ethanol/acetone and stained with propidium iodide as previously described71 processed under the conditions of the DSB repair assay.
Transfections to analyse DSB repair
Double-stranded-break repair was analysed using the EGFP-based test system as described previously18. Jurkat cells, 4 × 106 in 400 µl DMEM medium without phenol red, were transfected via electroporation (Bio-Rad Laboratories) with 10 µg pCMV-HA–I-SceI, 10 µg pBS/wt-EGFP, 5 µg pHIV-1-NL4-3-env*-IRES-mCherry or pBS as a control and 10 µg repair substrate EJ5SceGFP17, EJ-EGFP/3ʹEGFP, HR-EGFP/5ʹEGFP or 5ʹEGFP/HR-EGFP. WTK1(HR/3ʹ) cells, 4 × 106 in 400 µl DMEM medium without phenol red, were electroporated with a DNA mixture consisting of 10 µg pCMV-HA–I-SceI or 10 µg pBS/wt-EGFP, 10 µg pHIV-1-NL4-3-env*-IRES-mCherry or its vpr, vpu and nef STOP variants (vpr*, vpu* and nef*, respectively) or 1 µg expression plasmids for different vpu alleles, namely pCG-NL4-3 Vpu–AU1, pCG-CH106 Vpu, pCG-WITO Vpu and pCG-EK505 Vpu. For the analysis of the effects of different Vpu mutants on DSB repair, WTK1(HR/3ʹ) cells were transfected with 1 µg WITO Vpu, where amino acids 28–81 were substituted to alanine in triplicates, plus 10 µg pCMV-HA–I-SceI and 19 µg pBS. In another experiment, WTK1(HR/3ʹ) cells were transfected with 4 µg wt, vpu* or vpu NF-кB or BST mutants of the proviral transmitted founder viruses CH293, CH077 or STCO together with 1 µg pCMV-HA–I-SceI. Primary CD4+ T cells were transfected with a 3 μg DNA mixture of pCMV-HA–I-SceI, pBS, repair plasmid HR-EGFP/3ʹEGFP and the viral construct pHIV-1-NL4-3-env*-IRES-mCherry wt or its vpr* and vpu* variants, pCG-NL4-3 Vpu–AU1 expression plasmid; pHIV-1 M subtype B CH058, pHIV-1 M subtype B CH058 vpu*, pXL-TOPO HIV-1 M subtype C CH198, pXL-TOPO HIV-1 M subtype C CH198 vpu*; pHIV-1 M subtype B CH058 env+ wt, vpu* or vpr*; or pHIV-1 M subtype B CH058 env*, where the Vpu function to inhibit NF-кB or tetherin (BST) were lost. The mix contained all plasmids in a ratio of 1:1:1:1 using the Amaxa T-cell nucleofection kit, program U15.
Transfections to analyse DSB repair after gene silencing
To evaluate the role of specific DNA repair proteins in HIV-1-mediated DSB repair, a general DNA mix of 10 µg pCMV-HA–I-SceI or pBS/wt-EGFP and 10 µg pHIV-1-NL4-3-env*-IRES-mCherry or its vpu* variant was supplemented with 40 µg of distinct shRNA or dominant negative protein expression plasmid. The following shRNA and dominant negative protein expression plasmids were used: pRS-PARP1; pRS-EXO1 and pRS-SLX4 mixes of four shRNAs each; mixes of pRS-BRCA1–4 and 6; pRS-CtIP-6 and -8; pRS-NBN-4 and -5; pRS-FEN1-0, -7 and -8; pRS-ERCC1–5 and -6; pRS-PIAS1–5, -6 and -7; pRS-PIAS4–7, -8 and -9; pRS-RanBP2 mix of all four, respectively (all purchased from Origene); pSuper-midDNase2-1 and -3 (ref. 72); pcDNA3.0-IкBα-SR22; pSuper-Rad52 (ref. 73); pcDNA3.1-Rad51sm (ref. 74); pSuper-BLMmi (ref. 37); pCG-β-TrCP1 C-HA-IRES-DsRed2 (ref. 75); pSuper-PML-a and PML-c mixture76; pSHAG-ATM and pSHAG- ATR77; and pSuper-Ku70 (ref. 71).
Evaluation of DSB repair frequency
Following cultivation for 48 h, 5–20 × 104 living cells were examined to identify EGFP+ and EGFP− cells using flow cytometry (FACS Calibur or FACS Canto; BD). Transfected samples were individually corrected using the transfection efficiency of each sample, where pBS was substituted by the wt-EGFP-expression plasmid to correct for differences in transfection, transcription, translation, proliferation and lethality. The transfection efficiencies ranged from 50 to 80%. The mean ± s.e.m. values of ≥6 measurements were calculated and set to 100% for each control.
In situ PLA and immunofluorescence microscopy
WTK1(HR/3ʹ) cells were transfected with 1 μg pCG-WITO-Vpu–AU1 or the corresponding controls. SaOS cells engaged for immunofluorescence analysis were transfected with 5 μg pCG-NL4-3 Vpu–AU1 expression plasmid or an empty control using an Amaxa nucleofection kit V, program D24. The cells were used for the PLA or immunofluorescence microscopy 24 h later. The in situ PLA was performed as described by Hampp and colleagues37. The following antibodies were used: AU1 goat (Go; Novus Biologicals, NB600-452), RanBP2 rabbit (Rb; Abcam, ab64276), RanGAP1 mouse (Ms; Santa Cruz, sc-28322), RanGAP1 Ms (Abcam), PML Ms (Abcam, ab96051), PML (Bethyl, A301-167), BLM (Bethyl, A300-120), BLM Rb (Abcam, 2179), RAD52 (Novus Biologicals, NBP2-58116), SUMO2/3 (Novus Biologicals, NBP2-34384, NBP1-77163) and EXO1 (Abnova, H00009156-B01P). Imaging was performed using a Zeiss LSM 710 confocal microscope. Quantification of foci was carried out using the ImageJ software.
In the Vpu localization studies, CD4+ T cells were transfected with 0.5 μg pCG-WITO-Vpu–AU1 or pCR-XL TOPO-SIVcpz Ptt MB897.2 (Amaxa U15 program), whereas SaOS cells were transfected with 1 μg pCG-WITO-Vpu–AU1 (Amaxa D24 program). Vpu localization was also analysed in SaOS cells infected with HIV-1 constructs, where the vpu gene was AU1 tagged and separated from env as described previously45.
Immunofluorescence experiments were performed 24 h after transfection or infection. Briefly, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. After blocking with BSA, Vpu and RanBP2 were stained with antibodies to AU1 (Novus Biologicals), RanBP2 (Abcam), p-RPA32 S4/8 (Bethyl, A300-245A), RAD52 (Novus Biologicals), BLM (Bethyl), PML (Bethyl, Abcam), SUMO1 and SUMO2 (Novus Biologicals), RanGAP1 (Abcam), BrdU (Abcam), sp100 (Abcam) and 53BP1 (Novus Biologicals). Secondary antibodies conjugated to Alexa Fluor 488 and 647 were used for detection. A confocal microscope (LSM 710, Zeiss) with the corresponding software (LSM 710 Release version 5.5SP1, Zeiss; ImageJ) were used for analysis.
Virus production and infectivity assay
To generate virus stocks, HEK293T cells were transfected with 5 μg pHIV-1-NL4-3-IRES-mCherry wt or vpu* mutant plasmids thereof using a calcium phosphate transfection method13. For the evaluation of 1-LTR, 2-LTR circles and integration, HIV-1 NL4-3 without reporter was used. In the case of HIV-1 pseudo-type production, 1 μg pHIT-G-VSV-G was added to the sample mixture. HEK293T mock samples were transfected with only the transfection reagents. The supernatants were harvested 40 h post transfection. The viral infectivity was determined by infecting TZM-bl reporter cells; 6,000 cells were seeded per well on a 96-well plate and infected with the cell-culture supernatants. After 3 d, the supernatants were removed and a β-galactosidase assay was performed following the manufacturer’s instructions (Gal-Screen, Applied Bioscience). The β-galactosidase activity was quantified as relative light units per second (RLU s−1) using an Orion Microplate Luminometer.
Western blotting and antibodies
Cellular pellets were lysed in lysis buffer (50 mM Tris pH 7,4, 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, 25 mM NaF, 25 mM β-glycerophosphate, 0.1 mM NaV and proteinase inhibitor, Roche). To examine the phosphorylation status of proteins, phosphatase inhibitor was added into the lysis buffer (PhosSTOP phosphatase inhibitor cocktail tablets, Roche). The viral supernatants were centrifuged through a 20% sucrose cushion at 20,800g for 90 min at 4 °C and lysed in Western blot lysis buffer. The protein concentrations were determined using a BCA protein assay kit (Thermo Scientific). The samples were mixed with Protein sample loading buffer (Li-COR) with 10% β-mercaptoethanol, heated at 95 °C for 5 min and 60–80 μg protein was loaded onto 8–15% SDS–PAGE gels. The electrophoresed protein samples were blotted onto Hybond-C extra, Hybond-P (GE Healthcare) or Immobilon-FL PVDF (Merck Millipore) membranes.
The following antibodies were applied: actin (Abcam, ab8226), artemis Rb, ATM 2C1 Ms (Abcam), ATR Ab-2 Rb (Calbiochem), AU1 Ms (Convance), AU1 Go/Rb (Novus Biologicals), BLM C16 Go (Santa Cruz), BLM Rb (Bethyl), BLM Go (Bethyl), BLM Rb (Abcam), BRCA1 Ab-1 clone MS110 Ms (Calbiochem/Millipore), CtIP T-16 Go (Santa Cruz), DNase2 Rb (Imgenex), ERRC1 8F1 Ms (BD), EXO1 Rb (GeneTex), EXO1 Ms (Abnova), FEN1 Ms (BD), GAPDH rat (BioLegend), HA F-7 Ms, HA HAc5 Ms (Abcam), IкBα C-21 Rb (Santa Cruz), Ku70 S5C11 Ms (Abcam), Mre11 Rb (Novus Biologicals), Nbs1 1D7 Ms (Novus Biologicals), PARP1 Rb (Cell Signaling), PCNA Ms (Abcam), PIAS1 (Cell Signaling), PIAS4 (Cell Signaling), Rad51 H-92 Rb (Santa Cruz), Rad52 F-7 Ms (Santa Cruz), RAD52 (Novus Biologicals), RanBP2 Rb (Abcam), RanGAP1 Rb (Bethyl), RanGAP1 Ms (Abcam), RCC1 (Santa Cruz), RPA32 (Bethyl), p-RPA32 S4/S8 Rb (Bethyl), PML PG-M3 Ms (Santa Cruz), PML Rb (Bethyl), PML C7 Ms (Abcam), anti PML–SUMO Rb78, SLX4 Rb (Bethyl), SUMO1 (Ms) and SUMO2 (Ms and Rb; Novus Biologicals), tubulin DM1A Ms (Abcam) and Ubc9 Ms (Santa Cruz). Secondary mouse, rabbit and goat antibodies were purchased from Rockland or Thermo Fischer. The western blot signals were visualized using Super signal west pico chemiluminescent substrate, Super signal west dura extended duration substrate (Thermo Scientific) with the use of a ChemiDoc (Bio-Rad) or Odyssey (Li-COR Biosciences) scanner. The values of the band intensities for the proteins of interest were normalized to the values obtained for the corresponding loading controls.
Subcellular protein fractionation and immunoprecipitation
CD4+ T cells were transfected with the pCG-WITO-Vpu–AU1 plasmid, incubated for 24 h and then protein fractionation was performed according to the manufacturer’s instructions (Subcellular protein fractionation kit, Thermo Scientific).
The cells used for immunoprecipitation were transfected with 1 μg pCG-WITO-Vpu–AU1 or the corresponding controls. After 24 h, the samples were lysed with IP lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP40 and protease inhibitor) or RIPA buffer to demonstrate covalent SUMOylation (150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS, 50 mM Tris pH 8.0 and 25 mM NEM) for 30 min on ice and 10 min, respectively. The lysed samples were centrifuged and incubated for 3 h with Pierce protein A/G magnetic beads (88802) that were pre-incubated overnight with antibody (5 μg primary antibody per 10 μl of beads per sample).
PCR and quantitative real-time PCR (rtPCR)
WTK1(HR/3ʹ) cells were transfected with 10 µg pCMV-HA–I-SceI, 10 µg pNL4-3-IRES-mCherry-HIV-1-delta-env and 10 µg pBS, and EGFP+ and EGFP− cells were sorted by FACS after 48 h. Genomic DNA was isolated using a Blood and cell culture DNA mini kit (Qiagen) following the instructions of the manufacturer. A Taq PCR core kit (Qiagen) was used to perform PCR analysis of distinct repair pathways using the following primers: PCR1.1 (5ʹ-CCCGCAACCTCCCCTTCTAC-3ʹ) and Sce1.2 (5ʹ-ACCTTGAT-GCCGTTCTG-3ʹ) to detect construct integrity as a control; PCR1.1 and PCR1.2 (5ʹ-GAACCCGCTC-GTCTGGCTAAG-3ʹ) to detect HR; and PCR1.1 and PCR2.2 (5ʹ-CTGTCTTTAACAAATTG-GACTAATCG3ʹ) to detect SSA. Analysis of 1-LTR, 2-LTR and integrated provirus was done as described previously59,79. CD4+ T cells were first transfected with 2 µg shRNA to knockdown RAD52 or an empty control. Following cultivation for 24 h, the cells were infected with wt or vpu HIV-1 NL4-3 constructs normalized to the p24 content. In the case of RAD52 inhibitor, CD4+ T cells were pre-treated with 5 µM 6-hydroxy-DL-DOPA for 4 h before spinoculation. For the detection of 1-LTR, the HIV-1 gag (LA1)- and envelope (LA15)-specific primers were used. As shown by Jacque and Stevenson79, the PCR conditions used almost exclusively generate small 1-LTR products and large 2-LTR amplicons are inefficiently generated. The primers for the detection of 2-LTRs bind in the R-U3 region and are thereby specific for 2-LTR circles only. Quantification of the viral circles and integrants was normalized to the RNaseP housekeeping gene. The ΔΔCT value was calculated as described previously80. The specificity of the amplifications of the 1-LTR and 2-LTR viral cDNA was verified by DNA sequence analysis. Moreover, the rtPCR cycling conditions described by Munir et al.81 had the same outcome for the evaluation of 1-LTR, thereby proving the specificity of our assay.
To determine the mRNA levels of IFNβ and RAD52, mRNA was isolated and cDNA was synthesized using an RNase plus kit (Qiagen) and PrimeScript (TAKARA) as per the manufacturer’s instructions. Quantitative PCR was performed using a Taqman qPCR mix from BioBudget and quantification was normalized on the RNaseP housekeeping gene. All primers and probes were purchased from Applied Biosystems, Thermo Fischer.
Statistical analysis and quantification
All experiments were independently repeated at least twice with similar results. Where statistics were calculated (DSB repair assay, cell cycle and apoptosis, rtPCR, superinfection, immunofluorescence microscopy, PLA and pattern analysis), at least three replicates were measured in each experiment. Calculations were performed using the Prism 7.05 software (GraphPad). Graphical data presentations show the mean values and s.e.m.; statistical significance is indicated with asterisks (n ⩾ 4), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Significances for pattern analysis were determined using Mann–Whitney tests. The mean values and s.d. are shown for the western blots; no statistics were calculated for these data, unless otherwise specified.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
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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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).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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. Source data
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. Source data
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. Source data
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. Source data
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. Source data
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. Source data
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. Source data
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 Fig. 1 and Supplementary Table 1.
Video presenting colocalization between WITO Vpu and RanBP2 in transfected SaOS cells.
Video presenting colocalization between SIV Vpu and RanBP2 in transfected CD4+ T cells.
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