Disrupting HIV ‐1 capsid formation causes cGAS sensing of viral DNA
2020; Springer Nature; Volume: 39; Issue: 20 Linguagem: Inglês
10.15252/embj.2019103958
ISSN1460-2075
AutoresRebecca P. Sumner, Lauren Harrison, Emma Touizer, Thomas P. Peacock, Matthew Spencer, Lorena Zuliani‐Alvarez, Greg J. Towers,
Tópico(s)Immune Cell Function and Interaction
ResumoArticle27 August 2020Open Access Source Data Disrupting HIV-1 capsid formation causes cGAS sensing of viral DNA Rebecca P Sumner Corresponding Author Rebecca P Sumner [email protected] orcid.org/0000-0003-0735-8649 Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Lauren Harrison Lauren Harrison Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Emma Touizer Emma Touizer orcid.org/0000-0002-7164-8555 Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Thomas P Peacock Thomas P Peacock Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Matthew Spencer Matthew Spencer Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Lorena Zuliani-Alvarez Lorena Zuliani-Alvarez Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Greg J Towers Greg J Towers Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Rebecca P Sumner Corresponding Author Rebecca P Sumner [email protected] orcid.org/0000-0003-0735-8649 Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Lauren Harrison Lauren Harrison Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Emma Touizer Emma Touizer orcid.org/0000-0002-7164-8555 Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Thomas P Peacock Thomas P Peacock Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Matthew Spencer Matthew Spencer Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Lorena Zuliani-Alvarez Lorena Zuliani-Alvarez Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Greg J Towers Greg J Towers Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Author Information Rebecca P Sumner *,1, Lauren Harrison1, Emma Touizer1, Thomas P Peacock1,†, Matthew Spencer1, Lorena Zuliani-Alvarez1 and Greg J Towers1 1Division of Infection and Immunity, University College London, London, UK †Present address: Department of Medicine, Imperial College London, London, UK *Corresponding author. Tel: +44 20 3108 2422; E-mail: [email protected] The EMBO Journal (2020)39:e103958https://doi.org/10.15252/embj.2019103958 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 Detection of viral DNA by cyclic GMP-AMP synthase (cGAS) is a first line of defence leading to the production of type I interferon (IFN). As HIV-1 replication is not a strong inducer of IFN, we hypothesised that an intact capsid physically cloaks viral DNA from cGAS. To test this, we generated defective viral particles by treatment with HIV-1 protease inhibitors or by genetic manipulation of gag. These viruses had defective Gag cleavage, reduced infectivity and diminished capacity to saturate TRIM5α. Importantly, unlike wild-type HIV-1, infection with cleavage defective HIV-1 triggered an IFN response in THP-1 cells that was dependent on viral DNA and cGAS. An IFN response was also observed in primary human macrophages infected with cleavage defective viruses. Infection in the presence of the capsid destabilising small molecule PF-74 also induced a cGAS-dependent IFN response. These data demonstrate a protective role for capsid and suggest that antiviral activity of capsid- and protease-targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo. Synopsis Viruses must avoid or block activation of an interferon response in order to replicate. HIV-1 uses its capsid to physically protect viral reverse transcripts from detection by the DNA sensor cGAS, thus disruption of capsid integrity/stability enhances innate immune sensing that may be harnessed therapeutically. Treatment of HIV-1 with protease inhibitors or mutation of protease cleavage sites in gag leads to defective Gag cleavage, reduced particle infectivity and diminished capacity to saturate restriction factor TRIM5α. Gag cleavage defective viruses, unlike wild-type HIV-1, activate a potent interferon response in THP-1 cells and primary macrophages. Innate immune activation is dependent on viral reverse transcription and the cellular DNA sensor cGAS. Treatment of HIV-1 with capsid destabiliser PF-74 also leads to a potent cGAS-dependent interferon response. The HIV-1 capsid physically masks viral DNA from innate immune detection. Introduction The innate immune system provides the first line of defence against invading pathogens such as viruses. Cells are armed with pattern recognition receptors (PRRs) that recognise pathogen-associated molecular patterns (PAMPs), such as viral nucleic acids, and lead to the activation of a potent antiviral response in the form of secreted interferons (IFNs), proinflammatory cytokines and chemokines, the expression of which is driven by the activation of key transcription factors such as IFN regulatory factor 3 (IRF3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (Chow et al, 2015). For HIV-1, a number of cytosolic PRRs have been demonstrated to contribute to the detection of the virus in infected cells including DNA sensors cyclic GMP-AMP synthase (cGAS) (Gao et al, 2013; Lahaye et al, 2013; Rasaiyaah et al, 2013), IFI16 (Jakobsen et al, 2013; Jonsson et al, 2017), PQBP1 (Yoh et al, 2015), RNA sensors DDX3 (Gringhuis et al, 2017) and also MDA5, although only in the circumstance where the genome lacked 2′-O-methylation by 2′-O-methyltransferase FTSJ3 (Ringeard et al, 2019). The nuclear protein NONO has also been implicated in the detection of HIV cDNA (Lahaye et al, 2018). The best studied HIV sensor is cGAS, which upon binding double-stranded DNA, such as HIV-1 reverse transcription (RT) products, produces second messenger 2′3′-cGAMP (Ablasser et al, 2013; Sun et al, 2013; Wu et al, 2013) that binds and induces phosphorylation of ER-resident adaptor protein STING and its translocation to perinuclear regions (Tanaka & Chen, 2012). Phosphorylation of STING provides a platform for the recruitment of TBK1 and IRF3 leading to IRF3 phosphorylation and its subsequent translocation to the nucleus to drive expression of IFN and IFN-stimulated genes (ISGs) (Liu et al, 2015; Shang et al, 2019). Activation of STING by 2′3′-cGAMP also activates IKK and the transcription of NF-κB-dependent genes (Ishikawa & Barber, 2008). Of course, detection of infection by sensing is not universal and viruses are expected to hide their PAMPs and typically have mechanisms to antagonise specific sensors and downstream restriction factors. Work from our laboratory, and others, has demonstrated that primary monocyte-derived macrophages (MDMs) (Tsang et al, 2009; Rasaiyaah et al, 2013) and THP-1 cells (Cingoz & Goff, 2019) can be infected by wild-type (WT) HIV-1 without significant innate immune induction. However, MDM senses HIV-1 if, for example, mutations are made in the capsid protein to prevent the recruitment of cellular cofactors such as CPSF6 and cyclophilin A (Rasaiyaah et al, 2013), after depletion of the cellular exonuclease TREX1 (Yan et al, 2010; Rasaiyaah et al, 2013) and HIV can be sensed by a process requiring NONO if restriction by SAMHD1 is overcome (Lahaye et al, 2018). Sensing of HIV was found to be dependent on viral reverse transcription (RT) and the cellular DNA sensing machinery cGAS and STING. In addition to having a role in recruitment of cofactors for nuclear entry, a variety of evidence suggests that the viral capsid has a role in protecting the process of viral DNA synthesis, preventing degradation of RT products by cellular nucleases such as TREX1 and from detection by DNA sensors (Burdick et al, 2017; Francis & Melikyan, 2018). Here, we have tested the hypothesis that an intact viral capsid is crucial for evasion of innate immune sensing by disrupting the process of viral particle maturation, either biochemically, using protease inhibitors (PIs), or genetically, by mutating the cleavage site between the capsid protein and spacer peptide 1. The resulting viral particles had defective Gag cleavage, reduced infectivity and, unlike wild-type HIV-1, activated an IFN-dependent innate immune response in THP-1 cells and primary human macrophages. This response in THP-1 cells was mostly dependent on viral DNA synthesis and the cellular sensors cGAS and STING. Defective viruses were less able to saturate restriction by TRIM5α indicating a reduced ability to bind this restriction factor, likely due to aberrant particle formation. Finally, we show that the viral capsid-binding small molecule inhibitor PF-74, which has been proposed to accelerate capsid opening (Marquez et al, 2018), also induces HIV-1 to activate an innate response in THP-1 cells, which is dependent on cGAS. Together these data support the hypothesis that the viral capsid plays a physical role in protecting viral DNA from the cGAS/STING sensing machinery in macrophages and that disruption of Gag cleavage and particle maturation leads to aberrant viral capsid formation and activation of an IFN response that may be important in vivo during PI treatment of HIV-1. Results Protease inhibitor treatment of HIV-1 leads to innate immune induction in macrophages To test the hypothesis that intact viral capsids protect HIV-1 DNA from detection by DNA sensors, we sought to activate sensing using defective viral particles with disrupted capsid maturation. The protease inhibitor (PI) class of anti-retrovirals blocks the enzymatic activity of the viral protease, preventing Gag cleavage and proper particle formation, as observed by electron microscopy (Schatzl et al, 1991; Muller et al, 2009). By producing VSV-G-pseudotyped HIV-1ΔEnv.GFP (LAI strain (Peden et al, 1991) with the Nef coding region replaced by GFP, herein called HIV-1 GFP) in the presence of increasing doses of the PI lopinavir (LPV, up to 100 nM), we were able to generate viral particles with partially defective Gag cleavage, as assessed by immunoblot of extracted viral particles detecting HIV-1 CA protein (Fig 1A). At the highest dose of LPV (100 nM), increased amounts of intermediate cleavage products corresponding to capsid and spacer peptide 1 (CA-SP1), matrix and CA (MA-CA), MA, CA, SP1 and nucleocapsid (MA-NC) were particularly evident along with increased amounts of full length uncleaved Gag (Figs 1A and EV1A). Uncleaved CA-SP1 was also evident at 30 nM LPV. As expected, defects in Gag cleavage were accompanied by a reduction in HIV-1 GFP infectivity in both phorbol myristyl acetate (PMA)-treated THP-1 (Fig 1B) and U87 cells (Fig 1C). For the highest dose of LPV, this corresponded to a 24- and 48-fold defect in infectivity in each cell type, respectively. Viral titres were calculated according to the number of genomes, assessed by qPCR (see Methods), to account for small differences in viral production between conditions. These differences were no more than twofold from untreated virus. Figure 1. PI treatment induces HIV-1 to trigger an ISG response in macrophages A. Immunoblot of HIV-1 GFP virus particles (2 × 1011 genomes) produced with lopinavir (LPV, 0-100 nM) detecting p24. B, C. Titration of LPV-treated HIV-1 GFP viruses on PMA-treated THP-1 shSAMHD1 (B) or U87 (C) cells. Mean ± SD, n = 3. D–F. ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). G. CXCL-10 protein in cell supernatants from (D-F) (ELISA). H. ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.2 U RT/ml 30 nM LPV-treated HIV-1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib. A control was stimulated with 1 ng/ml IFNβ. I. RT products from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.3 U RT/ml LPV-treated HIV-1 GFP viruses. J. Immunoblot of HIV-1 R9 BaL virus particles (2 × 1011 genomes) produced with LPV (0–100 nM) detecting p24. K, L. ISG qRT-PCR (K) and infection data (L) from primary monocyte-derived macrophages (MDMs) infected for 24 h with LPV-treated HIV-1 R9 BaL viruses (0.2 U RT/ml). Data are collated from two donors (represented by circles and squares), n = 3. Horizontal line represents the median. Data information: Data are mean ± SD, n = 3, representative of 2 repeats (H, I), or 3 repeats (D–G). Statistical analyses were performed using the Student's t-test, with Welch's correction where appropriate and comparing to the 0 nM LPV condition. *P < 0.05, **P < 0.01, ***P < 0.001. See also Fig EV1. For experiments in which the virus dose used was normalised by RT activity, the number of genome copies was also measured by qPCR of virus. This gave dose equivalents of within twofold to threefold of RT equivalents. Source data are available online for this figure. Source Data for Figure 1 [embj2019103958-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. PI treatment induces HIV-1 to trigger an ISG response in macrophages A. Schematic of intermediate Gag cleavage products. MA: matrix, CA: capsid, SP1: spacer peptide 1, NC: nucleocapsid, SP2: spacer peptide 2. B. Infection data from Fig 1D–G. PMA-treated THP-1 shSAMHD1 cells transduced for 48 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml or 0.5 U RT/ml). C. Immunoblot of HIV-1 GFP virus particles (2 × 1011 genomes) produced with darunavir (DRV, 0–50 nM) detecting p24. D. Titration of DRV-treated HIV-1 GFP viruses on U87 cells. Mean ± SD, n = 3. E. Infection data from (F, G). F. IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells transduced for 24 h with DRV-treated HIV-1 GFP (1 × 1010 genomes/ml). G. CXCL-10 protein in supernatant from (F) (ELISA). H. ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.2 U RT/ml 30 nM LPV-treated HIV-1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib. A control was stimulated with 1 ng/ml IFNβ. I, J. Titration of LPV-treated HIV-1 R9 BaL viruses on primary MDM. Collated data (mean ± SD, n = 3) represented as percentage titre normalised to R9 BaL produced in the absence of LPV (0 nM) are in (I) and data from individual donors are in (J). K. CXCL-10 protein in supernatant of primary MDM 48 h post-transduction with WT HIV-1 GFP or DRV-treated (12.5 nM) HIV-1 GFP (6 × 107 genomes/ml or 3 × 108 genomes/ml) in the presence of DMSO vehicle or 2 μM ruxolitinib. L. Infection data from (K). Data information: Data are mean ± SD, n = 3, representative of 2 repeats (E–H, K, L) or 3 repeats (B). Statistical analyses were performed using the Student's t-test, with Welch's correction where appropriate and comparing to the 0 nM DRV condition. *P < 0.05, **P < 0.01. Source data are available online for this figure. Download figure Download PowerPoint To test the visibility of PI-inhibited viruses to innate sensing responses, we generated a THP-1 cell line that was stably depleted for the HIV restriction factor SAMHD1 (Appendix Fig S1A). Monocytic THP-1 cells can be differentiated to a macrophage-like transcriptome by treatment with PMA, to yield an adherent cell line that is highly competent for innate immune sensing, including DNA sensing. Differentiation of THP-1 normally leads to SAMHD1 activation by dephosphorylation and potent restriction of HIV-1 infection (Cribier et al, 2013). SAMHD1 depletion effectively relieved this restriction and allowed HIV-1 GFP infection (Appendix Fig S1B). SAMHD1-depleted THP-1 cells (herein referred to as THP-1 shSAMHD1 cells) remained fully competent for innate immune sensing and produced interferon-stimulated genes (ISGs) and inflammatory chemokines including CXCL-10, IFIT-2 (also known as ISG54) and CXCL-2 in response to a range of stimuli, including transfection of herring testis DNA (HT-DNA), exposure to 2′3′-cGAMP and infection by Sendai virus (Appendix Fig S1C–E). Infection of PMA-treated THP-1 shSAMHD1 cells with HIV-1 GFP that had been produced in the presence of increasing doses of LPV led to a virus and LPV dose-dependent increase in the expression of ISGs CXCL-10, IFIT-2 and MxA at the mRNA level (Fig 1D–F), and CXCL-10 protein secretion (Fig 1G). In agreement with previous reports (Cingoz & Goff, 2019), HIV-1 GFP produced in the absence of LPV induced very little, or no ISG expression in THP-1 cells at the doses tested, consistent with the hypothesis that HIV-1 shields its PAMPs from cellular PRRs (see Fig 1D–G, 0 nM drug dose). Virus dose in these experiments was normalised according to RT activity, as measured by SG-PERT (see Methods), which differed no more than fivefold in the LPV-treated versus LPV-untreated virus. Determination of genome by qRT-PCR gave similar dose values. Infection levels in differentiated THP-1 cells were approximately equivalent between the various LPV doses tested (Fig EV1B) because HIV-1 GFP infection of THP-1 is maximal at about 70% GFP positivity (Pizzato et al, 2015). Similar results were obtained with the PI darunavir (DRV); treatment of HIV-1 GFP with increasing doses of DRV (up to 50 nM) led to defects in Gag cleavage (Fig EV1C), decreased infectivity (Fig EV1D and E) and at 12.5 and 25 nM DRV-treated virus activated an ISG response in PMA-treated THP-1 shSAMHD1 cells (Fig EV1F and G). To test whether LPV-treated HIV-1-induced ISG expression depended on IFN production or direct activation of ISGs, infections were repeated in the presence of the JAK1/2 inhibitor ruxolitinib (Quintas-Cardama et al, 2010). Activation of STAT transcription factor downstream of IFN receptor engagement requires phosphorylation by JAKs, and hence, ruxolitinib inhibits IFN signalling (Fig 1H). Induction of MxA (Fig 1H) and CXCL-10 (Fig EV1H) expression by LPV-treated HIV-1 GFP was severely reduced in the presence of ruxolitinib, indicating that induction of ISG expression in these experiments requires an infection-driven type I IFN response. Treatment of cells with type I IFN provided a positive control for ruxolitinib activity (Figs 1H and EV1H). Importantly, measurement of viral DNA production in infected PMA-treated THP-1 shSAMHD1 cells, demonstrated that LPV did not increase DNA levels, ruling out increased DNA levels as an explanation for increased sensing (Fig 1I). We conclude that PI-inhibited HIV-1 fails to protect viral DNA from innate immune sensors by effective encapsidation. To test whether PI inhibition of HIV-1 caused similar innate immune activation in primary human cell infection, we turned to HIV-1 R9 (BaL-Env) infection of primary human macrophages. Production of R9 (BaL-Env) in HEK293T cells in the presence of 10–100 nM LPV induced the expected defects in Gag cleavage (Fig 1J) and infectivity (Fig EV1I and J) as observed with VSV-G-pseudotyped HIV-1 GFP (Fig 1A–C). Furthermore, virus produced in the presence of 30 and 100 nM LPV induced the expression of CXCL-10 on infection of primary MDM, whereas virus grown in the absence of LPV, or at low LPV concentrations (10 nM), induced very little CXCL-10 expression (Fig 1K). Increasing concentrations of LPV during HIV-1 production led to a decrease in MDM infection, read out by p24 positivity, in these experiments (Fig 1L). Similarly, DRV-treated HIV-1 GFP induced more CXCL-10 secretion in primary MDM than untreated HIV-1 GFP (0 nM DRV) and this was dependent on type I IFN production, as evidenced by the lack of CXCL-10 production in the presence of ruxolitinib (Fig EV1K). Infection levels were not changed by ruxolitinib treatment (Fig EV1L). Together, these data suggest that infection by PI-treated HIV-1 induces an IFN-dependent innate immune response in PMA-treated THP-1 cells and primary human MDM that is not observed after infection with untreated virus. HIV-1 bearing Gag cleavage mutations also induces innate immune activation Producing virus in the presence of PI suppresses Gag cleavage at multiple sites. Previous work suggested that inhibition of the CA-SP1 cleavage site was particularly toxic to infectivity and defective particles were irregular with partial polyhedral structures (Muller et al, 2009; Mattei et al, 2018). Concordantly, our data show a defect in cleavage at the CA-SP1 site in the presence of LPV (Fig 1A and J) or DRV (Fig EV1C). Importantly, the presence of even small proportions of CA-SP1 cleavage mutant exerted trans-dominant negative effects on HIV-1 particle maturation (Muller et al, 2009). To test whether a CA-SP1 cleavage defect can cause HIV-1 to trigger innate sensing, we prepared chimeric VSV-G pseudotyped HIV-1 GFP viruses by transfecting 293T cells with varying ratios of WT HIV-1 GFP and HIV-1 GFP with CA-SP1 Gag mutant L363I M367I (Wiegers et al, 1998; Checkley et al, 2010). Increasing the proportion of the ∆CA-SP1 mutant increased the presence of uncleaved CA-SP1 detected by immunoblot (Fig 2A). Defective cleavage was accompanied by a modest decrease in infectivity on U87 cells (Fig 2B). Figure 2. HIV-1 with Gag protease cleavage mutation induces ISGs in macrophages A. Immunoblot of HIV-1 GFP virus particles (2 × 1011 genomes) with varying proportions of ΔCA-SP1 protease cleavage mutation detecting p24. B. Titration of HIV-1 GFP ΔCA-SP1 viruses on U87 cells. Mean ± SD, n = 3. C, D. ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). E. CXCL-10 protein in supernatants from (C, D) (ELISA). F. RT products from THP-1 cells transduced for 24 h with 6 × 109 genomes/ml (approx. 0.5 U RT/ml) HIV-1 GFP ΔCA-SP1 viruses. G. IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.016 – 0.2 U RT/ml). Data are shown as individual measurements, representative of 2 repeats. H. IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced with HIV-1 GFP containing either 0% (WT) or 75% ΔCA-SP1 mutant, or stimulated with 4 μg/ml cGAMP as a control, in the presence of DMSO vehicle or 2 μM ruxolitinib. I. ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75% ΔCA-SP1 mutant (3 × 108 genomes/ml or 1.5 × 109 genomes/ml, equivalent to 0.1 U RT/ml and 0.5 U RT/ml). J. ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75% ΔCA-SP1 mutant (1.5 × 109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib. K. CXCL-10 protein in supernatants from (J) (ELISA). Data information: Data are mean ± SD, n = 3, representative of 2 repeats (F, I-K) or 3 repeats (C-E, H). Statistical analyses were performed using the Student's t-test, with Welch's correction where appropriate and comparing to the 0% ΔCA-SP1 virus (C–E, I) or the DMSO control (H, J, K). *P < 0.05, **P < 0.01, ***P < 0.001. See also Fig EV2. Source data are available online for this figure. Source Data for Figure 2 [embj2019103958-sup-0004-SDataFig2A.pdf] Download figure Download PowerPoint As with HIV-1 GFP produced in the presence of PIs, infection of PMA-treated THP-1 shSAMHD1 cells with the HIV-1 GFP ∆CA-SP1 mutants led to a ∆CA-SP1 dose-dependent increase in the expression of CXCL-10 (Fig 2C) and MxA mRNA (Fig 2D), and CXCL-10 at the protein level (Fig 2E). Induction was not explained by differences in the amount of viral DNA in infected cells, and similar levels of viral DNA (Fig 2F) and infection (Fig EV2A) were observed at the viral doses tested. Virus dose in these experiments was normalised according to RT activity, which differed no more than fivefold between viruses. Importantly, differences in RT activity, measured by SG-PERT, were mirrored by measurements of genome copy, measured by qPCR. This is consistent with variation in viral production rather than inhibition of RT activity by the ∆CA-SP1 mutation. Cleavage defective viruses, and not wild-type virus, also induced dose-dependent luciferase expression from an undifferentiated THP-1 cell line that had been modified to express Gaussia luciferase under the control of the IFIT-1 (also known as ISG56) promoter, herein called IFIT1-luc (Mankan et al, 2014) (Fig 2G). IFIT1-luc is both IRF-3- and IFN-sensitive (Mankan et al, 2014). HIV-1 bearing ∆CA-SP1 mutant also induced a type I IFN response, evidenced by suppression of IFIT1-luc by ruxolitinib (Fig 2H). In the IFIT1-luc cells, ∆CA-SP1 mutation did not impact infection levels (Fig EV2A–C) and neither did ruxolitinib treatment (Fig EV2C). We propose that during single round infection of THP-1 cells, the virus has already integrated by the time IFN is produced, and this is why ruxolitinib does not rescue infection and thus the percentage of GFP-positive cells. To corroborate these findings in primary cells, we infected MDM with HIV-1 GFP ∆CA-SP1 (75% mutant) and found enhanced CXCL-10, IFIT-2 and MxA expression compared with WT HIV-1 GFP (Figs 2I and EV2D). Furthermore, HIV-1 GFP ∆CA-SP1 induced an IFN response in these cells, as treatment with ruxolitinib significantly reduced IFIT-2 expression (Fig 2J) and CXCL-10 secretion (Fig 2K) induced by HIV-1 GFP ∆CA-SP1. Interestingly in primary MDM, treatment of cells with ruxolitinib did enhance infection levels of HIV-1 GFP ∆CA-SP1, but not WT HIV-1 GFP. This is consistent with the notion that HIV-1 GFP ∆CA-SP1 induces a IFN-dependent antiviral response in these cells that is, in this case, fast enough to inhibit single round infection (Fig EV2E and F). Click here to expand this figure. Figure EV2. HIV-1 with Gag protease cleavage mutation induces ISGs in macrophages Infection levels of cells from Fig 2C–E. PMA-treated THP-1 shSAMHD1 cells transduced for 48 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml or 0.5 U RT/ml). Infection data from Fig 2G. THP-1-IFIT-1 cells transduced for 48 h with HIV-1 GFP ΔCA-SP1 viruses (0.016–0.2 U RT/ml). Infection data from Fig 2H. THP-1-IFIT-1 cells transduced for 48 h with HIV-1 GFP containing either 0% (WT) or 75% ΔCA-SP1 mutant in the presence of DMSO vehicle or 2 μM ruxolitinib. Infection data from Fig 2I. Primary MDM transduced for 48 h with WT HIV-1 GFP or 75% ΔCA-SP1 mutant (3 × 108 genomes/ml or 1.5 × 109 genomes/ml). Infection data from Fig 2J and K. Primary MDM transduced for 48 h with WT HIV-1 GFP or 75% ΔCA-SP1 mutant (1.5 × 109 genomes/ml) in the presence of DMSO vehicle or 2 μM ruxolitinib. Repeat of (E) in a second donor. Data information: Data are individual measurements (B) or mean ± SD, n = 3 (A, C–F), representative of 2 repeats. Statistical analyses were performed using Student's t-test, with Welch's correction where appropriate and comparing to the DMSO control. **P < 0.01, ***P < 0.001. Download figure Download PowerPoint We also performed similar experiments measuring replication of HIV-1 in MDM over several days, inhibiting replication with various concentrations of LPV. In this case, neither blockade of IFN receptor with antibody, or inhibition of JAK/STAT signalling with ruxolitinib, significantly rescued infection over two independently performed experiments (Appendix Fig S2A–D). We hypothesise that prevention of IFN activity does not rescue viral replication because the replication inevitably remains suppressed by effective protease inhibition. However, in vivo, we might expect that IFN produced in this way would contribute to innate and adaptive immune suppression of infection. Together these data support our hypothesis that disruption of Gag maturation yields viral particles that fail to shield PAMP from innate sensors. Maximal innate immune activation by maturation defective viruses is dependent on viral DNA synthesis To determine whether viral DNA synthesis is required for HIV-1 bearing ∆CA-SP1 to trigger sensing, we infected THP-1 IFIT1-luc cells with HIV-1 75% ∆CA-SP1 in the presence of reverse transcriptase inhibitor nevirapine and assessed sensing by measuring IFIT1-luc expression and CXCL10 secretion. As expected, infectivity was severely diminished by 5 μM nevirapine (Fig EV3A and B) and both luciferase (Fig 3A) and CXCL-10 (Fig 3B) secretion was completely inhibited suggesting that viral DNA synthesis is required to activate sensing. Concordantly, expression of ISGs IFIT-2 (Fig 3C) and MxA (Fig 3D) induced by HIV-1 75% ∆CA-SP1 was also abolished in the presence of nevirapine. A small, but statistically significant, reduction in luciferase (Fig 3A) and CXCL-10 (Fig 3B) secretion was observed in the presence of the integrase inhibitor raltegravir, although this was not observed in every experiment (Fig 3C and D). We conclude that viral DNA is the active PAMP and this notion was also supported by the observation that mutation D185E in the RT active site (HIV-1 ∆CA-SP1 RT D185E) also reduced activation of IFIT-1 luc expression (Fig 3E) and CXCL10 secretion (Fig 3F) on infection of the THP-1 IFIT-1 reporter cells. Mutation D116N of the viral integrase (HIV-1 ∆CA-SP1 INT D116N) im
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