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• W2766673110 abstract "Article30 October 2017Open Access Source DataTransparent process DNA damage induced by topoisomerase inhibitors activates SAMHD1 and blocks HIV-1 infection of macrophages Petra Mlcochova Corresponding Author Petra Mlcochova [email protected] orcid.org/0000-0001-6908-9363 Division of Infection and Immunity, UCL, London, UK Search for more papers by this author Sarah J Caswell Sarah J Caswell Macromolecular Structure Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Ian A Taylor Ian A Taylor Macromolecular Structure Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Greg J Towers Greg J Towers Division of Infection and Immunity, UCL, London, UK Search for more papers by this author Ravindra K Gupta Ravindra K Gupta orcid.org/0000-0001-9751-1808 Division of Infection and Immunity, UCL, London, UK Africa Health Research Institute, Durban, KwaZulu Natal, South Africa Search for more papers by this author Petra Mlcochova Corresponding Author Petra Mlcochova [email protected] orcid.org/0000-0001-6908-9363 Division of Infection and Immunity, UCL, London, UK Search for more papers by this author Sarah J Caswell Sarah J Caswell Macromolecular Structure Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Ian A Taylor Ian A Taylor Macromolecular Structure Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Greg J Towers Greg J Towers Division of Infection and Immunity, UCL, London, UK Search for more papers by this author Ravindra K Gupta Ravindra K Gupta orcid.org/0000-0001-9751-1808 Division of Infection and Immunity, UCL, London, UK Africa Health Research Institute, Durban, KwaZulu Natal, South Africa Search for more papers by this author Author Information Petra Mlcochova *,1, Sarah J Caswell2, Ian A Taylor2, Greg J Towers1 and Ravindra K Gupta1,3 1Division of Infection and Immunity, UCL, London, UK 2Macromolecular Structure Laboratory, The Francis Crick Institute, London, UK 3Africa Health Research Institute, Durban, KwaZulu Natal, South Africa *Corresponding author. Tel: +44 20 7679 2000; E-mail: [email protected] The EMBO Journal (2018)37:50-62https://doi.org/10.15252/embj.201796880 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract We report that DNA damage induced by topoisomerase inhibitors, including etoposide (ETO), results in a potent block to HIV-1 infection in human monocyte-derived macrophages (MDM). SAMHD1 suppresses viral reverse transcription (RT) through depletion of cellular dNTPs but is naturally switched off by phosphorylation in a subpopulation of MDM found in a G1-like state. We report that SAMHD1 was activated by dephosphorylation following ETO treatment, along with loss of expression of MCM2 and CDK1, and reduction in dNTP levels. Suppression of infection occurred after completion of viral DNA synthesis, at the step of 2LTR circle and provirus formation. The ETO-induced block was completely rescued by depletion of SAMHD1 in MDM. Concordantly, infection by HIV-2 and SIVsm encoding the SAMHD1 antagonist Vpx was insensitive to ETO treatment. The mechanism of DNA damage-induced blockade of HIV-1 infection involved activation of p53, p21, decrease in CDK1 expression, and SAMHD1 dephosphorylation. Therefore, topoisomerase inhibitors regulate SAMHD1 and HIV permissivity at a post-RT step, revealing a mechanism by which the HIV-1 reservoir may be limited by chemotherapeutic drugs. Synopsis Etoposide/camptothecin-mediated DNA damage regulates SAMHD1 and HIV permissivity in human macrophages. DNA damage causes activation of p53 and p21, leading to transition of macrophages from G1-like to G0 state and decrease of CDK1 expression. In the absence of CDK1, SAMHD1 is activated by dephosphorylation at T592, resulting in restriction of HIV-1 infection. DNA damage-mediated suppression of HIV-1 infection occurs after completion of viral DNA synthesis, at the step of 2LTR circle and provirus formation. Introduction Retroviruses must reverse transcribe their RNA into DNA and integrate nascent viral DNA into the host genome in order to replicate (Skalka & Katz, 2005; Lusic & Siliciano, 2017). Increasing evidence suggests that macrophage infection contributes to the reservoir of infected cells that persist and prevent cure of HIV/AIDS (Alexaki et al, 2008; Siliciano & Greene, 2011; Watters et al, 2013; Honeycutt et al, 2016). Integration may be recognized as a form of DNA damage, and the host DNA damage response (DDR) (Daniel et al, 1999; Jackson & Bartek, 2009) is critical for “gap repair” during the integration process. Indeed, there is increasing evidence that key DDR proteins are involved in retroviral infection, specifically during integration (Daniel et al, 2003; Ariumi et al, 2005; DeHart et al, 2005; Lau et al, 2005). Furthermore, proteins able to restrict HIV infection, including APOBEC3G and SAMHD1, have been linked to DNA damage responses (Leonard et al, 2013; Roberts et al, 2013; Clifford et al, 2014; Kretschmer et al, 2015). SAMHD1 is a deoxynucleotide triphosphohydrolase (Goldstone et al, 2011), and the mechanism of HIV restriction is thought to be via depletion of dNTPs to levels that are insufficient for completion of retroviral reverse transcription (RT) (Goldstone et al, 2011; Lahouassa et al, 2012; Schmidt et al, 2015). The activity of SAMHD1 is thought to be regulated by CDK1/2-mediated phosphorylation at amino acid T592 (Cribier et al, 2013; White et al, 2013; Antonucci et al, 2016; Mlcochova et al, 2017). Here, we link DNA damage in primary myeloid cells and cellular SAMHD1 activation, in the absence of a type I interferon response. We show that DNA damage induced by topoisomerase inhibitors activates p53 and p21, leading to SAMHD1 T592 dephosphorylation/activation. Activated SAMHD1 mediates a block to HIV infection, which occurs after the synthesis of full-length HIV DNA. Importantly, the etoposide (ETO)-induced inhibition of HIV-1 can be fully abrogated by SAMHD1 depletion. Results DNA damage induces a post-reverse transcription block to HIV-1 in macrophages It has been reported that HIV can induce DNA damage during integration into host DNA (Daniel et al, 2004). Moreover, certain reports suggest that integration can be enhanced by DNA damage induction (Ebina et al, 2012; Koyama et al, 2013). The cellular response to DNA damage also typically involves cell cycle arrest and activation of DNA damage repair (DDR) (Branzei & Foiani, 2008). We reported recently that HDAC inhibitors, which are known to induce cell cycle arrest and/or apoptosis, inhibit HIV-1 infection in monocyte-derived macrophages (MDM) through activation of SAMHD1 (Mlcochova et al, 2017). Here, we investigate the impact of inducing DNA damage on HIV-1 infection in MDM. We employed topoisomerase inhibitors, which block the unwinding of dsDNA leading to inhibition of fundamental biological processes including DNA replication, transcription, DNA repair and chromatin remodelling, by stabilizing the DNA-single or -double breaks (Hsiang et al, 1989; Gorczyca et al, 1993). To test this, we treated MDM with 5 μM ETO for 18 h to confirm that ETO induced DNA damage, measured by staining for nuclear γH2AX or 53BP1 (Fig 1A) in uninfected cells. Further, we pretreated MDM with etoposide (ETO) or camptothecin (CTH), inhibitors of topoisomerase I and II, respectively. 18 h after treatment with titrations of inhibitors, we infected cells with VSV-G pseudotyped HIV-1 GFP and measured infection 48 h later by enumerating GFP-positive cells. Both topoisomerase inhibitors blocked HIV-1 infection in a dose-dependent manner (Fig 1B). We then used the most effective, non-cytotoxic, concentration of ETO (5 μM) (Fig 1C) to measure its effect on MDM infection by different viruses (Fig 1D). HIV-1 wild-type and capsid mutants N74D and P90A (known to use alternative host cofactors for nuclear translocation) were equally sensitive to topoisomerase inhibitors. However, HIV-2 and SIVsm, which encode Vpx, and degrade SAMHD1, were insensitive to drug treatment. Infection by DNA viruses, adenovirus (AdV) and herpes simplex virus (HSV), and RNA virus Semliki Forest virus (SFV), was also insensitive to topoisomerase inhibition. Figure 1. Etoposide/Camptothecin-induced DNA damage inhibits HIV-1 infection A. Monocyte-derived macrophages (MDM) were treated with 5 μM etoposide (ETO) for 18 h. Cells were stained for γH2AX and 53BP1 nuclear foci, acquired and analysed using the automated cell-imaging system Hermes WiScan and ImageJ. On average, 104 cells were acquired and analysed (n = 3, mean ± s.e.m.; *P-value ≤ 0.05; paired t-test). Scale bars, 10 μm. B. MDM were treated with increasing concentrations of etoposide (ETO) and camptothecin (CTH) 18 h before infection. Cells were infected with VSV-G pseudotyped HIV-1 GFP in the presence of ETO/CTH, and 104 cells in each experiment were recorded and analysed for GFP expression 48 h post-infection using an automated cell-imaging system Hermes WiScan and ImageJ (n = 3, mean ± s.e.m.). C. MDM were treated with increasing concentrations of ETO and CTH for 66 h. Cells were stained to distinguish between live and dead cells using LIVE/DEAD fixable Dead cell stain protocol. Percentages of live/dead cells were determined using automated cell-imaging system Hermes WiScan and ImageJ. Addition of 20% ethanol for 10 min into cells treated with ETO was used as positive control (n = 3, mean ± s.e.m.). D. MDM were treated with 5 μM ETO for 18 h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP viruses wild-type (wt) HIV-1 capsid mutants (N74D or P90A), HIV-2, SIV sooty mangabey (SIVsm E543), replication competent adenovirus type 5 AdV (AdV), Semliki Forest virus (SFV) and HSV-1. The percentages of infected cells were determined using an automated cell-imaging system Hermes WiScan and ImageJ and normalized to untreated control ˜100% (n = 3, mean ± s.e.m.; **P-value ≤ 0.01; ***P-value ≤ 0.001; (ns) non-significant, paired t-test). Cells from a representative donor were used for immunoblotting. E. MDM were treated with 5 μM ETO for 18 h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP. 104 cells were recorded and analysed for GFP expression 48 h post-infection using an automated cell-imaging system Hermes WiScan and ImageJ (n = 4, mean ± s.e.m.; ***P-value ≤ 0.001, paired t-test). F. MDM were treated with 5 μM ETO for 18 h and infected in the presence of ETO with full-length replication competent macrophage tropic HIV-1 virus BaL. Cells were stained for intracellular p24, and the percentage of infected cells was quantified 48 h post-infection by FACS (n = 3, mean ± s.e.m.; ***P-value ≤ 0.001, paired t-test). G–I. MDM were treated with 5 μM ETO for 18 h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post-infection for qPCR (n = 3, mean ± s.e.m.; *P-value ≤ 0.05; ***P-value ≤ 0.001; (ns) non-significant, paired t-test). (G) Late viral RT products. AZT: MDM treated with 20 μM AZT, a reverse-transcriptase inhibitor, were used as control. (H) 2LTR circles. (I) Integrated copies of viral DNA, Alu-Gag qPCR. J. MDM were treated with 5 μM ETO for 18 h and infected with HIV-1 BaL (3, 6, 12 ffu/cell). Cells were stained for intracellular p24, and the percentage of infected cells was quantified 48 h post-infection by FACS (n = 3, mean ± s.e.m.; **P-value ≤ 0.01; ***P-value ≤ 0.001, paired t-test). K. MDM were treated with 5 μM ETO for 18 h, infected with HIV-1 BaL (3, 6, 12 ffu/cell) and DNA isolated 18 h post-infection for qPCR (n = 3, mean ± s.e.m.; (ns) non-significant, paired t-test). L. MDM were treated with 5 μM ETO for 18 h, infected with HIV-1 BaL and DNA isolated 4, 6 and 18 h post-infection for qPCR (n = 3, mean ± s.e.m.; (ns) non-significant, paired t-test). Source data are available online for this figure. Source Data for Figure 1 [embj201796880-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint We next investigated where in the HIV-1 life cycle ETO acts. We treated MDM with 5 μM ETO for 18 h and infected cells with VSV-G pseudotyped HIV-1 GFP (known to use an endocytic entry route) or wild-type macrophage and CCR5 tropic HIV-1 isolate BaL and determined infection (Fig 1E and F). Both viruses were equally sensitive to the ETO-mediated inhibition of viral infection, suggesting independence of drug sensitivity from viral route of entry. MDM infected with HIV-1 BaL were used for DNA isolation and qPCR to determine efficiency of reverse transcription (Fig 1G), viral 2LTR circle formation (a measure of nuclear entry) (Fig 1H; De Iaco et al, 2013) and viral integration by Alu-gag PCR (Fig 1I). Surprisingly, reverse transcription was not affected by ETO treatment (Fig 1G), but we observed a decrease in 2LTR circles (Fig 1H) and viral integration (Fig 1I). We also confirmed that ETO did not affect RT products at increasing MOI (determined as ffu/target cell), even though infection was significantly decreased (Fig 1J and K). Importantly, ETO did not have any effect on RT products over a time-course (Fig 1L). In addition, although dNTP levels are already very low in MDM, we detected a decrease in dTTP and dCTP after ETO treatment (Fig EV1). Click here to expand this figure. Figure EV1. Quantification of dNTP levels in MDM treated with etoposide (ETO)Analysis of cellular dNTP levels in unstimulated and ETO-treated MDM. dNTPs were extracted for quantification from two donors. The bar graph shows the amounts (fmoles per 106 cells) of the indicated dNTP from each donor, with and without ETO treatment. BD: below level of detection. Download figure Download PowerPoint DNA damage-induced HIV-1 block is SAMHD1 dependent in macrophages We observed that lentiviruses encoding Vpx (HIV-2 and SIVsm), a SAMHD1 antagonist (Hrecka et al, 2011; Laguette et al, 2011), were insensitive to ETO in MDM suggesting that SAMHD1 might be responsible for the effect of ETO/CTH on HIV-1 infection (Fig 1D). To test this, we treated MDM with 5 μM ETO, infected cells with HIV-1 and assayed phosphorylation of SAMHD1 at T592 by immunoblot (Fig 2A–C). We found that SAMHD1 is phosphorylated in untreated MDM allowing efficient HIV infection, confirming previous data (Mlcochova et al, 2017). Addition of ETO led to loss of SAMHD1 phosphorylation and reduced HIV-1 infectivity. As expected, the inhibitory effect of ETO on HIV infection was lost after siRNA-mediated SAMHD1 depletion (SAMHD1 KD) (Fig 2A) or treatment of MDM with SIVmac virus-like particles containing Vpx/Vpr (SIV VLP; Figs 2B and EV2), which was confirmed by a dose titration of HIV-1 virus on SAMHD1 KD (Fig EV3A) and SIV VLP-treated cells (Fig EV3B) and by infecting control and SAMHD1 KD cells at MOI achieving equal percentage of infected cells (Fig EV3C and D). Importantly, the rescue was independent of the SIV Vpr protein, as evidenced by wild-type activity of SIV VLP deleted for Vpr (Figs 2C and EV2) but completely dependent on presence of Vpx. To investigate which step of the virus life cycle was inhibited by ETO, and consequently rescued by SAMHD1 depletion, we infected MDM with HIV-1 BaL with and without co-infection with SIV VLP and isolated DNA 18 h post-infection to measure late RT products (Fig 2D), 2LTR circles (Fig 2E) and integration products (Fig 2F) under conditions of FCS culture where SAMHD1 is phosphorylated in the absence of ETO. Infection was measured in parallel samples 48 h post-viral challenge (Fig 2G). Critically, the post-RT block seen on ETO treatment was abrogated by SAMHD1 depletion. We conclude that HIV inhibition following ETO treatment is mediated through SAMHD1 and impacts both nuclear import and viral integration, but not late viral DNA synthesis. Figure 2. SAMHD1 inhibits HIV-1 at a post-RT step following DNA damage A. MDM were transfected with control or pool of SAMHD1 (KD) siRNA and 3 days later treated with 5 μM ETO and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP 18 h later. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell-imaging system Hermes WiScan and ImageJ 48 h post-infection (n = 3, mean ± s.e.m.; **P-value ≤ 0.01; (ns) non-significant, paired t-test). B. MDM were treated with 5 μM ETO for 18 h and co-infected in the presence of ETO with VSV-G HIV-1 GFP and SIVmac virus-like particles containing Vpx/Vpr (SIV VLP). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the Hermes WiScan and ImageJ 48 h post-infection (n = 3, mean ± s.e.m.; **P-value ≤ 0.01; (ns) non-significant, paired t-test). C. MDM were treated with 5 μM ETO for 18 h and co-infected in the presence of ETO with VSV-G HIV-1 GFP and SIVmac virus-like particles containing Vpx/Vpr (SIV VLP) or deleted for Vpx (SIV VLP del Vpx) or deleted for Vpr (SIV VLP del Vpr). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell-imaging system Hermes WiScan and ImageJ 48 h post-infection (n = 4, mean ± s.e.m.; **P-value ≤ 0.01; (ns) non-significant, paired t-test). D–F. MDM were treated with 5 μM ETO for 18 h and co-infected in the presence of ETO with HIV-1 BaL and SIVmac virus-like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post-infection for qPCR quantification of (F) late RT products; (G) 2LTR circles; (H) integrated viral DNA (n = 3, mean ± s.e.m.; *P-value ≤ 0.05; **P-value ≤ 0.01; (ns) non-significant, paired t-test). (D) Late viral RT products. AZT: MDM treated with 20 μM AZT, a reverse-transcriptase inhibitor, were used as control. (E) 2LTR circles. (F) Integrated copies of viral DNA, Alu-Gag qPCR. G. The percentage of infected cells was quantified by Hermes WiScan and ImageJ 48 h post-infection (n = 3, mean ± s.e.m.; **P-value ≤ 0.01; (ns) non-significant, paired t-test). Source data are available online for this figure. Source Data for Figure 2 [embj201796880-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Schematic representation of SIVmac VLP used in the studySchematic representation of the packaging plasmids derived from SIVmac251. SIVmac virus-like (SIV VLP) particles were prepared as described in Materials and Methods. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. SAMHD1 inhibits HIV-1 following DNA damage MDM were transfected with control or pool of SAMHD1 (KD) siRNA and 3 days later treated with 5 μM ETO and infected in the presence of ETO with HIV-1 BaL (3, 5, 12 ffu/cell). Cells were stained for intracellular p24, and the percentage of infected cells was quantified 48 h post-infection by FACS (n = 3, mean ± s.e.m.). MDM were treated with 5 μM ETO for 18 h and co-infected in the presence of ETO with HIV-1 BaL (3, 5, 12 ffu/cell) and SIVmac virus-like particles containing Vpx/Vpr (SIV VLP). Cells were stained for intracellular p24, and the percentage of infected cells was quantified 48 h post-infection by FACS (n = 3, mean ± s.e.m.). MDM were transfected with control or pool of SAMHD1 (KD) siRNA and 3 days later treated with 5 μM ETO for 18 h and infected in the presence of ETO with VSV-G HIV-1 GFP. The percentage of infected cells was quantified by the automated cell-imaging system Hermes WiScan and ImageJ 48 h post-infection (n = 3, mean ± s.e.m.; **P-value ≤ 0.01; (ns) non-significant, paired t-test). Cells from a representative donor were used for immunoblotting. Download figure Download PowerPoint Surprisingly, we noted that HIV-1 2LTR circles were increased by nearly 20-fold by co-infection with SIV VLP (Fig 2E) in the absence of ETO. However, this increase in 2LTR circles was not mirrored by an increase in integrated proviral DNA (Fig 2E and F). SIV bearing Vpx mutant Q76A rescues HIV-1 infection following DNA damage To further probe the mechanism of ETO on HIV-1 infection, we treated MDM with 5 μM ETO for 18 h, co-infected cells with HIV-1 and SIV VLP (bearing Vpx WT/Vpr WT) or SIV VLP Q76A (bearing Vpx Q76A mutant/Vpr WT) and measured infection 48 h later. The Vpx mutant Q76A maintains the ability to interact with SAMHD1, but in previous studies did not rescue HIV-1 infection from SAMHD1, possibly because it cannot recruit DCAF1 to degrade SAMHD1 (Srivastava et al, 2008; Hrecka et al, 2011; Laguette et al, 2011; Reinhard et al, 2014). We used MDM cultured in human serum (low dNTP levels, dephosphorylated SAMHD1) and in FCS (high dNTP levels, phosphorylated SAMHD1) (Mlcochova et al, 2017) and co-infected cells with HIV-1 and SIV VLP or SIV VLP Q76A (Fig 3A and B). SIV VLP but not SIV VLP Q76A increased infection in MDM (Fig 3A and B). By contrast, ETO-mediated inhibition of HIV-1 infection had been rescued by both SIV VLP and SIV VLP Q76A (Fig 3A and B). ETO caused dephosphorylation of SAMHD1 and blocked HIV-1 infection in MDM. However, this block to infection was abrogated by SIV VLP Q76A, even though SAMHD1 was not degraded. As expected, dNTP levels following treatment with SIV VLP Q76A infected cells were not increased (Fig EV4). Critically, SAMHD1 was dephosphorylated (active) in the presence of ETO and SIV VLP Q76A (Fig 3C) suggesting inhibition of the active form of SAMHD1 by the Vpx mutant without manipulation of activation by T592 phosphorylation or degradation. Figure 3. Vpx Q76A rescues DNA damage-induced block to HIV-1 infection A, B. MDM were treated with 5 μM ETO for 18 h and co-infected in the presence of ETO with VSV-G HIV-1 GFP and SIVmac virus-like particles containing Vpx wild-type (WT)/Vpr or Vpx Q76A mutant/Vpr (SIV VLP Q76A) (n = 3, mean ± s.e.m.; *P-value ≤ 0.05; **P-value ≤ 0.01; (ns) non-significant, paired t-test). (A) MDM were differentiated and cultured in human serum instead of FCS. (B) MDM were differentiated and cultured in FCS. A standard culture condition used in all experiments. See Materials and Methods. C–F. MDM were treated with 5 μM ETO for 18 h and co-infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post-infection for qPCR quantification of (D) late RT products; (E) 2LTR circles; (F) integrated viral DNA (n = 3, mean ± s.e.m.; *P-value ≤ 0.05; **P-value ≤ 0.01; (ns) non-significant, paired t-test). (C) The percentage of infected cells was quantified by the automated cell-imaging system Hermes WiScan and ImageJ 48 h post-infection. Cells from a representative donor were used for immunoblotting. (D) Late viral RT products. AZT: MDM treated with 20 μM AZT, a reverse-transcriptase inhibitor, were used as control. (E) 2LTR circles. (F) Integrated copies of viral DNA, Alu-Gag qPCR. Source data are available online for this figure. Source Data for Figure 3 [embj201796880-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Quantification of dNTP levels in MDM following treatment with SIV VLP Q76AAnalysis of cellular dNTP levels in MDM and MDM expressing SIV Vpx(Q76A). dNTPs were extracted for quantification from two donors. The bar graph shows the amounts (fmoles per 106 cells) of the indicated dNTP from the MDM of each donor, before and after the introduction of SIV Vpx(Q76A) in SIV VLPs. Download figure Download PowerPoint To confirm which step of the virus life cycle was inhibited by ETO and rescued by SIV VLP Q76A, we infected MDM with HIV-1 BaL with and without co-infection with SIV VLP Q76A and measured infection (Fig 3C), late RT products (Fig 3D), 2LTR circles (Fig 3E) and integration products (Fig 3F). We detected a ~3- to 5-fold decrease in RT, integration of viral products and infection in the presence of Vpx Q76A. This was independent of ETO treatment (Fig 3C–F). Importantly, there was no reduction in reverse transcription but substantial restriction of integrated provirus formation after ETO treatment, which was rescued by co-infection with SIV VLP Q76A (Fig 3A–F). Interestingly, we noted that HIV-1 2LTR circles were not increased by co-infection with SIV VLP Q76A (Fig 3E) in the absence of ETO. These data suggest that following DNA damage, SAMHD1 can be antagonized without being degraded or phosphorylated at T592. ETO-induced DNA damage does not trigger type I IFN responses in MDM Recent evidence suggests that DNA damage activates the type I interferon system to anti-microbial responses (Brzostek-Racine et al, 2011; Hartlova et al, 2015). As SAMHD1 was shown to mediate spontaneous expression and release of IFN when mutated (Crow & Manel, 2015) or deleted (Behrendt et al, 2013), we investigated the possibility that DNA damage induction could activate a type I IFN response and block HIV-1 infection in MDM. To test this, we treated MDM with 5 μM ETO for 18 h to induce DNA damage. As a positive control for innate immune triggering, we treated MDM with cGAMP, the product of the activated DNA sensor cGAS (Sun et al, 2013; Wu et al, 2013) or IFN-β for 18 h. We then isolated RNA from the cells and tested for specific interferon-stimulated gene (ISG) transcripts (Fig 4A). None of the ISG transcripts tested was strongly elevated after ETO exposure of MDM. Moreover, an immunofluorescence/translocation assay detected 5–20% of IRF3-positive nuclei in MDM after treatment with cGAMP and LPS (positive controls), consistent with activation of innate immune responses. However, there was no IRF3 translocation to the nucleus after 2, 6 or 18 h of ETO treatment (Fig 4B and C), suggesting that a type I IFN response is not strongly activated after ETO-induced DNA damage in MDM. Figure 4. ETO-induced DNA damage does not activate type I IFN response in MDM MDM were treated with 5 μM ETO for 18 h, 3 μg/ml cGAMP and 10 ng/ml IFN-β for 18 h. RNA was isolated and qPCR performed for selected genes using TaqMan assays. Expression levels of target genes were normalized to GAPDH (n = 3, mean ± s.e.m.; *P-value ≤ 0.05; (ns) non-significant, paired t-test). MDM were treated with 5 μM ETO or cGAMP for 18 h or 100 ng/ml LPS for 2 h. Cells were stained and analysed for IRF3 translocation (green) into the nucleus (blue). Scale bars: 10 μm. Quantification of nuclei positive for IRF3 staining (n = 3, mean ± s.e.m.). Download figure Download PowerPoint DNA damage promotes a G0 state and regulates SAMHD1 phosphorylation via p53 and p21 In the absence of general type I IFN responses after ETO treatment, we hypothesized that the restriction of HIV-1 is mediated entirely by dephosphorylation and activation of SAMHD1. Recent evidence demonstrated that cell cycle status regulates phosphorylation of SAMHD1 (Cribier et al, 2013; Badia et al, 2016; Mlcochova et al, 2017). G0 state macrophages encode active, dephosphorylated SAMHD1, but SAMHD1 is phosphorylated and deactivated by CDK1 in a subpopulation of macrophages in a G1-like state (Mlcochova et al, 2017). To test whether ETO altered cell cycle status and therefore activated SAMHD1 through the same pathway, we treated MDM with ETO and CTH and measured the proportion of cells in G0 or G1 by detection of MCM2, a marker expressed throughout the cell cycle but not in G0. In fact, the proportion of MDM expressing MCM2 and thus being in a G1-like state was significantly reduced after ETO/CTH treatment, indicative of cells returning to G0, a state non-permissive to HIV-1 infection (Fig 5A). We mapped the pathway leading to SAMHD1 dephosphorylation and HIV restriction using immunoblotting (Fig 5B–D). ETO induced DNA damage, as measured by an increase in γH2AX, and resulted in increased expression of p53 and p53 phosphorylation at Ser15 (Fig 5B–D). We also observed increased expression of p21 but not p27 protein. Moreover, absence of PARP cleavage (Fig 5B–D) suggested lack of apoptosis, in addition supported by the lack of cell death measured using cell viability/cell survival analysis (Fig 1C). Loss of HIV-1 permissivity following ETO treatment also correlated with loss of CDK1 and SAMHD1 activation by dephosphorylation. No increase in expression or phosphorylation of CDK2 was detected (Fig 5B and C). The same results were obtained with the topoisomerase I inhibitor CTH (Fig 5D). These data suggest that a DNA damage-induced block to HIV infection in human macrophages is mediated through cell cycle arrest activated by a p53/p21/CDK1 pathway culminating in activation of SAMHD1 by dephosphorylation (Fig 6). Figure 5. ETO regulates SAMHD1 phosphorylation through the p53, p21 pathway MDM were treated with increasing concentrations of ETO and CTH. Cells were stained for MCM2 expression, acquired and analysed using the automated cell-imaging system Hermes WiScan and ImageJ. On average, 104 cells were acquired (n = 3, mean ± s.e.m.). MDM were treated with 5 μM ETO, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage-associated proteins. Quantification of specific proteins band intensities from immunoblot in panel (B) using a CCD camera. Intensities of protein bands were normalized to intensity of actin protein band. MDM were treated with 5 μM ETO or 0.01 μM CTH for 18 h, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage-associated protei" @default.
• W2766673110 created "2017-11-10" @default.
• W2766673110 creator A5007176093 @default.
• W2766673110 creator A5023254532 @default.
• W2766673110 creator A5036869978 @default.
• W2766673110 creator A5055046444 @default.
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• W2766673110 date "2017-10-30" @default.
• W2766673110 modified "2023-10-15" @default.
• W2766673110 title "<scp>DNA</scp> damage induced by topoisomerase inhibitors activates <scp>SAMHD</scp> 1 and blocks <scp>HIV</scp> ‐1 infection of macrophages" @default.
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