ABSTRACT
Brain-resident microglia and myeloid cells (perivascular macrophages) are important HIV reservoirs in vivo, especially in the central nervous system (CNS). Despite antiretroviral therapy (ART), low-level persistent HIV replication in these reservoirs remains detectable, which contributes to neuroinflammation and neurological disorders in HIV-infected patients. New approaches complementary to ART to repress residual HIV replication in CNS reservoirs are needed. Our group has recently identified a BRD4-selective small molecule modulator (ZL0580) that induces the epigenetic suppression of HIV. Here, we examined the effects of this compound on HIV in human myeloid cells. We found that ZL0580 induces potent and durable suppression of both induced and basal HIV transcription in microglial cells (HC69) and monocytic cell lines (U1 and OM10.1). Pretreatment of microglia with ZL0580 renders them more refractory to latent HIV reactivation, indicating an epigenetic reprogramming effect of ZL0580 on HIV long terminal repeat (LTR) in microglia. We also demonstrate that ZL0580 induces repressive effect on HIV in human primary monocyte-derived macrophages (MDMs) by promoting HIV suppression during ART treatment. Mechanistically, ZL0580 inhibits Tat transactivation in microglia by disrupting binding of Tat to CDK9, a process key to HIV transcription elongation. High-resolution micrococcal nuclease mapping showed that ZL0580 induces a repressive chromatin structure at the HIV LTR. Taken together, our data suggest that ZL0580 represents a potential approach that could be used in combination with ART to suppress residual HIV replication and/or latent HIV reactivation in CNS reservoirs, thereby reducing HIV-associated neuroinflammation.
IMPORTANCE Brain-resident microglia and perivascular macrophages are important HIV reservoirs in the CNS. Persistent viral replication and latent HIV reactivation in the CNS, even under ART, are believed to occur, causing neuroinflammation and neurological disorders in HIV-infected patients. It is critical to identify new approaches that can control residual HIV replication and/or latent HIV reactivation in these reservoirs. We here report that the BRD4-selective small molecule modulator, ZL0580, induces potent and durable suppression of HIV in human microglial and monocytic cell lines. Using an in vitro HIV-infected, ART-treated MDM model, we show that ZL0580 also induces suppressive effect on HIV in human primary macrophages. The significance of our research is that it suggests a potential new approach that has utility in combination with ART to suppress residual HIV replication and/or HIV reactivation in CNS reservoirs, thereby reducing neuroinflammation and neurological disorders in HIV-infected individuals.
INTRODUCTION
Currently close to 37 million people are infected with HIV worldwide. HIV integrates its provirus into the host cell genome and establishes latent infection, which has posed a major obstacle for HIV eradication or cure (1). The current standard treatment for HIV infection is antiretroviral therapy (ART) which consists of a cocktail of antiviral drugs targeting different steps of the HIV replication cycle (2). ART is highly effective in controlling active viral replication and peripheral viremia, but it does not eradicate HIV infection. Of note, basal low levels of viremia during proper ART regimen (3, 4), as well as rapid viral rebound during ART treatment interruption (5, 6), were reported in HIV-infected individuals. These factors could increase the risks for the expansion of ART-resistant strains and persistent inflammation (7). Therefore, new approaches that are complementary to ART and target host mechanisms to further suppress HIV transcription are needed.
The central nervous system (CNS) is considered a sanctuary site for HIV persistence (8). It has been shown that even under ART when HIV is fully suppressed in peripheral blood, a low level of persistent HIV replication remains detectable in the CNS, which correlates with HIV-associated neurocognitive disorders (HAND) in infected individuals (9). In the CNS, brain-resident microglia and perivascular macrophages cells are major HIV target cells and represent important HIV reservoirs (10, 11). Microglia are the resident macrophages of the spinal cord and the brain and are originally derived from the primitive myeloid precursors in the yolk sac (12, 13). Activation of microglia is essential to control CNS infections; however, microglial activation following HIV infection could result in chronic inflammation and neurodegeneration. There is correlation between hyperactivated microglia and HIV-associated dementia, as well as the less severe conditions known as HAND (14, 15). Therefore, efficient control of residual HIV replication in CNS reservoirs is considered critical for reducing neuroinflammation and neurological complications in HIV-infected individuals. In addition, compared to CD4 T cells (16, 17), microglia and macrophages are generally more resistant to cytopathic effect and are less sensitive to some ART drugs for inhibition (18), which could further increase the pool of HIV reservoirs in the CNS, as well as maximize the potential emergence of drug-resistant strains. Complementary approaches are needed to further control residual HIV replication and latent HIV reactivation in myeloid cells, especially in the CNS.
HIV proviral transcription is controlled by host epigenetic and transcriptional machinery (19). As an epigenetic reader, the bromodomain and extraterminal domain (BET) family protein BRD4 plays an important role in regulating HIV transcription (20–23) and has been investigated as a potential therapeutic target (24, 25). It was reported that BRD4 can compete with HIV Tat for cellular p-TEFb/CDK9, resulting in the suppression of HIV transcription elongation (20, 21, 23). Modulation of BET/BRD4 by a pan-BET inhibitor, JQ1, was shown to relieve such competition and therefore reactivate HIV transcription (20, 21). Accumulating evidence indicates that BRD4 is functionally versatile (26), and its activity on HIV transcription is associated with the partner proteins it interacts with (27). Based on structure-guided drug design, our recent work characterized a novel BRD4-selective small molecule modulator, ZL0580, that is distinct from JQ1 and induces epigenetic suppression of HIV in human T cells and peripheral blood mononuclear cells (PBMCs) (28).
In this study, we examine whether ZL0580 could suppress transcription and latent reactivation of HIV in CNS reservoirs, including microglia and other myeloid cell populations. By using an immortalized human microglial cell line that harbors integrated HIV provirus (29), we show that ZL0580 induced potent and durable suppression of both induced and basal HIV transcription in human microglial cells and in monocytic cell lines (U1 and OM10.1). We also demonstrate that a single treatment of microglial cells with ZL0580 rendered them more resistant to the ensuing latent HIV reactivation. Mechanistically, ZL0580 suppresses HIV transcription in microglial cells by inhibiting Tat transactivation, as well as by inducing a more repressive chromatin structures at the HIV long terminal repeat (LTR). Combination treatment of HIV-infected human primary monocyte-derived macrophages (MDMs) with ART and ZL0580 demonstrated that ZL0580 also induces a repressive effect on HIV in human macrophages by promoting rapid HIV suppression during ART.
RESULTS
ZL0580 suppresses HIV expression in microglia and monocytic cell lines.Our recent study has identified ZL0580 as a novel BRD4-selective small molecule modulator that suppresses HIV in human T cells (28). Here, we examined the effects of ZL0580 on HIV in human myeloid cells, which are considered important HIV reservoirs in vivo and play critical roles in the HIV pathogenesis in CNS. To test this, we took advantage of multiple HIV-infected myeloid cell lines (contain integrated HIV provirus), including microglial cells (29) and U1 and OM10.1 cells. The microglial cell line (HC69) is the immortalized human microglia superinfected with HIV carrying a green fluorescence protein (GFP) reporter, facilitating the study of HIV latency and transcriptional regulation (29). HIV activation (viral and GFP expression) in HC69 cells can be induced upon treatment with human tumor necrosis factor alpha (TNF-α) (29, 30). We first tested the effect of ZL0580 on HIV in the HC69 microglial cells. Cells were treated with dimethyl sulfoxide (DMSO) alone (negative control [NC]), TNF-α, TNF-α plus ZL0580 (TNF-α/ZL0580), or TNF-α plus JQ1 (TNF-α/JQ1) as a control for 24 h. The level of HIV activation was assessed by flow cytometry based on GFP expression. We found that ZL0580 significantly suppressed TNF-α-induced HIV activation in microglia (Fig. 1A and B). To determine whether the suppressive effect of ZL0580 on HIV occurs at the transcriptional level, we quantified expression of mRNAs (early multispliced HIV RNA and GFP RNA) at 24 h posttreatment and showed that ZL0580 substantially reduced the expression of both RNAs (Fig. 1C and D). Next, we examined dose-response effect of ZL0580 on HIV suppression in HC69 cells. Cells were treated with DMSO alone (NC) or with TNF-α in the absence or presence of different concentrations of ZL0580 (0 to 16 μM) for 24 h. The level of HIV expression was measured by flow cytometry based on GFP expression (%GFP+ cells). We showed that ZL0580 suppressed TNF-α-induced HIV activation in microglial cells in dose-dependent manner (Fig. 1E). Potential toxic effects of ZL0580 on HC69 cells were assessed by treating them with a wide range of concentrations of ZL0580 (0 to 128 μM) for various lengths of time (1 and 3 days), followed by Live/Dead aqua blue staining and flow cytometric analysis for cell viability. We observed that ZL0580 did not cause significant cell death at concentrations below 128 μM in resting cells (day 3 posttreatment [p.t.]) (Fig. 1F) and at concentrations below 64 μM in activated cells (day 3 p.t.) (Fig. 1G), indicating that the observed HIV-suppressive effect by ZL0580 (8 μM) in microglia was not simply due to cell toxicity. To validate and expand our findings on HIV-infected microglial cells, we also used U1 and OM10.1 cells, which are promonocytic cell lines that respectively carry two copies and a single copy of integrated HIV provirus, expressing minimal constitutive HIV-1 production under basal conditions (31, 32). For U1, the cells were treated with DMSO alone (NC), phorbol myristate acetate (PMA), or PMA plus ZL0580 (PMA/ZL0580); for OM 10.1, the cells were treated with DMSO alone (NC), TNF-α, or TNF-α plus ZL0580 (TNF-α/ZL0580). Consistently, we found that ZL0580 resulted in significant reduction in the expression of HIV mRNA (GAG) in both cell lines at 24 h posttreatment, compared to treatment with TNF-α or PMA alone (Fig. 1H and I), without causing overt cell toxicity (Fig. 1J).
ZL0580 suppresses HIV expression in multiple myeloid cell lines. (A and B) Microglial cells (HC69) were not treated (NC) or stimulated with TNF-α (300 pg/ml) to activate HIV in the absence or presence of ZL0580 (8 μM) or JQ1 (8 μM) (as a control) for 24 h. (A) Representative FACS plots for HIV (%GFP+) under different conditions. (B) Quantification of %GFP+ cells from two independent experiments. (C and D) Comparison of HIV transcription in microglia after different treatment conditions (24 h). HIV early multispliced (MS) RNA (C) and GFP RNA (D) were quantified by qPCR. The data are shown as the fold change compared to NC. (E) ZL0580 suppression of HIV in microglial cells is dose dependent. HC69 cells were untreated (NC) or stimulated with TNF-α (300 pg/ml) to activate HIV in the absence or presence of different concentrations of ZL0580 (1, 4, 8, and 16 μM) for 24 h. HIV activation was measured by flow cytometry (%GFP+). Quantification of GFP+ cells from two independent experiments. (F and G) Toxic effect of ZL0580 in microglia during resting (F) and activated (G) conditions. HC69 cells were untreated (NC) or treated with different concentrations of ZL0580 (1, 4, 8, 16, 32, 64, and 128 μM) for 24 h. At day 1 and day 3 after treatment, the cell viability was measured by flow cytometry based on Aqua Blue staining. HC69 cells were stimulated with TNF-α (300 pg/ml) in the absence or presence of different concentrations of ZL0580 (1, 4, 8, 16, 32, 64, and 128 μM) for 24 h. On day 1 and day 3 after treatment, the cell viability was measured by flow cytometry based on Aqua Blue staining. (H and I) Effect of ZL0580 treatment on HIV transcription in U1 (H) and OM 10.1 (I) cells. U1 were untreated (NC) or stimulated with PMA (0.05 μg) to activate HIV in the absence or presence of ZL0580 (8 μM) for 24 h. OM 10.1 cells were untreated (NC) or stimulated with TNF-α (20 ng) to activate HIV in the absence or presence of ZL0580 (8 μM) for 24 h. HIV transcription was quantified by qPCR. The data are shown as the fold change compared to NC. (J) Percentage of viable cells (OM10.1 and U1) under different conditions. Statistically significant differences between groups are indicated by asterisks (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
The HIV suppressive effect of ZL0580 in microglial cells is durable.Next, we sought to determine whether a single dose of ZL0580 has a durable suppressive effect on HIV in microglial cells. We treated the cells with DMSO alone (NC), TNF-α, or TNF-α plus ZL0580 (TNF-α/ZL0580) and quantified the mRNA (early multispliced mRNA and GFP mRNA) at different time points posttreatment (days 1, 3, 14, 21, and 28), respectively. We found that, compared to cells treated with TNF-α alone, ZL0580 treatment (TNF-α/ZL0580) induced significant suppression of HIV transcription through day 21 after single treatment, which waned somewhere between days 21 and 28 (Fig. 2A and B). In addition, we examined whether repeated doses of ZL0580 treatments prolong its suppressive effect. Microglia were treated with DMSO alone (NC), TNF-α, or TNF-α plus ZL0580 (TNF-α/ZL0580) at day 0, and then ZL0580 treatment was repeated on days 3 and 7 after the initial treatment (three doses of ZL0580). Interestingly, repeated ZL0580 treatments markedly prolonged its suppressive effect on HIV through 41 days after the initial treatment (Fig. 2C and D). Based on the data (fold reduction on day 41) (Fig. 2C and D), we speculate that the suppressive effect of ZL0580 would remain beyond this time point. To ensure that the suppressive effect was not due to cell toxicity, cell viability prior to experiment termination (day 41) was monitored and found to be comparable between the two treatment conditions (Fig. 2E). These data indicate that ZL0580 induces durable HIV suppression in microglial cells.
The HIV suppressive effect of ZL0580 is durable in microglia. (A and B) HC69 cells were stimulated with TNF-α (300 pg/ml) to activate HIV in the absence or presence of ZL0580 (8 μM) for 24 h, and HIV transcription was quantified by qPCR at different time points posttreatment. The expression of HIV mRNA (early multispliced [A] and GFP mRNA [B]) under different conditions was quantified by qPCR. Data were normalized to untreated (NC) control of each day. (C to E) HC69 cells were stimulated with TNF-α (300 pg/ml) to activate HIV in the absence or presence of ZL0580 (8 μM) for 24 h. ZL0580 treatment was repeated at days 3 and 7 after the initial treatment. The expression of HIV mRNA (early multispliced [C] and GFP mRNA [D]) under different conditions was quantified by qPCR. Data were normalized to the untreated (NC) control for each day. (E) The percentage of viable cells at day 41 posttreatment was compared between TNF-α and TNF-α/ZL0580 treatment. Statistically significant differences between groups are indicated by asterisks (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). n.s., not significant.
ZL0580 induces suppression of basal HIV expression in microglial cells in resting condition.In addition to transcriptionally active HIV, as described above, we also assessed the effect of the compound on basal HIV transcription in microglial cells under resting conditions. Microglial cells were treated with DMSO alone (NC) or ZL0580 (8 μM), followed by quantification of HIV and GFP RNAs at different time points after treatment (days 1, 3, and 14, respectively). Similarly, treatment of resting microglial cells with a single dose of ZL0580 resulted in suppression of HIV transcription compared to the untreated cells (Fig. 3A). No significance difference in cell viability was observed between NC and ZL0580 treatments on all days examined (Fig. 3B). These data together demonstrate that ZL0580 can also suppress basal HIV transcription in microglial cells.
ZL0580 suppresses basal HIV expression in microglial cells (HC69) in the resting condition. HC69 cells were untreated (NC) or treated with ZL0580 (8 μM) for 24 h. (A) Comparison of HIV early MS mRNA quantified by qPCR under different conditions. The data are shown as the fold change compared to NC. (B) Percentage of viable cells at different time points posttreatment determined using a trypan blue assay. (C) HC69 cells were untreated (NC) or treated with ZL0580 (8 μM), JQ1 (8 μM), or 8 μM ZL0580 and JQ1 for 24 h. HIV activation was measured by flow cytometry (%GFP+). The quantification of GFP+ cells from two independent experiments is shown. Statistically significant differences between groups are indicated by asterisks (*, P ≤ 0.01; **, P ≤ 0.001; ***, P ≤ 0.0001; n.s., not significant).
Since our data showed that JQ1 and ZL0580 induce opposing effects on HIV transcription in J-Lat cells (28) and microglia, we next examined whether ZL0580 could antagonize the enhancing effect of JQ1 on HIV transcription in microglia. Cells were respectively treated with DMSO (NC), ZL0580, JQ1, or ZL0580+JQ1 (all 8 μM) for 24 h, followed by assessment of HIV activation by flow cytometry based on GFP expression. Interestingly, ZL0580 could largely antagonize HIV activation induced by JQ1 in microglia (Fig. 3C and D).
Pretreatment of microglial cells with a single dose of ZL0580 induces durable resistance to latent HIV reactivation.Next, we determined whether ZL0580 could promote the blockage of latent HIV reactivation upon subsequent stimulation of microglia with TNF-α. Microglial cells were either treated with DMSO alone (NC) or exposed to ZL0580 (8 μM) for 24 h, followed by cell washing and continuous culture (the medium was replaced once every 3 days). Cells under both NC and ZL0580-pretreated conditions were reactivated with TNF-α at different time points after ZL0580 treatment (days 7, 14, and 21, respectively). HIV activation was assessed by quantifying HIV early multispliced mRNA (Fig. 4A) and GFP mRNA (Fig. 4B). We found that even under stimulation with this strong reactivator (TNF-α), ZL0580 rendered cells more resistant to latent HIV reactivation in microglial cells (Fig. 4A and B). Similarly, this suppressive effect was not due to cell toxicity (Fig. 4C). These findings show the durability of the compound in suppressing HIV transcription after a single dose of treatment, as well as its potency in rendering cells more resistant to the ensuing latent HIV reactivation.
Pretreatment with a single dose of ZL0580 induces durable resistance to latent HIV reactivation in microglia. HC69 cells were untreated (NC) or treated with ZL0580 (8 μM) for 24 h, followed by continuous culture. On days 7, 14, and 21 after the initial treatment, the cells were stimulated with TNF-α (300 pg/ml) for 24 h to activate latent HIV. HIV transcription was quantified by qPCR. The expression of HIV RNA (early multispliced [A] and GFP RNA [B]) was compared between different treatment conditions. (C) Cell viability was quantified at different time points and compared between different treatments. The data are shown as the fold change compared to NC/TNF-α. Statistically significant differences between groups are indicated by asterisks (**, P ≤ 0.01; ***, P ≤ 0.001; n.s., not significant).
ZL0580 inhibits Tat binding to CDK9.Binding of HIV Tat protein to cellular kinase CDK9 (the catalytic factor component of p-TEFb complex) is a critical step in Tat transactivation and is important for efficient HIV transcription (33). This process has been shown to be suppressed by cellular BRD4 protein due to the competition with Tat for CDK9 (21, 23). To understand potential mechanisms underlying ZL0580-induced HIV suppression in myeloid cells (microglial cells), we examined the protein-protein interaction between TAT and CDK9 using coimmunoprecipitation (Co-IP) in these cells following ZL0580 treatment or no treatment. Similar to the analyses described above, microglial cells were not treated (NC) or were treated with TNF-α alone or TNF-α/ZL0580 for 24 h. Intriguingly, Tat Co-IP analysis showed that ZL0580 markedly reduced the binding of Tat to CDK9 in microglial cells under TNF-α stimulatory condition (Fig. 5A). This was not simply due to differential input Tat levels, since they were comparable among different treatments (Fig. 5C), an observation consistent with our recent report in J-Lat cells (28). As an important control, the total/input CDK9 protein was comparable between the two conditions (Fig. 5A). A highly consistent pattern was observed in microglial cells under the resting condition, where cells were not stimulated but directly treated with ZL0580 compared to DMSO treatment (Fig. 5B). Furthermore, CDK9-Tat binding was explored in U1 monocytic cells, and we observed similar patterns during activated (Fig. 5D) and resting (Fig. 5E) conditions. These data together indicate that ZL0580 suppresses HIV transcription by inhibiting Tat binding to CDK9 in myeloid cells under both stimulated and resting conditions. We also assessed expression of major cellular factors involved in HIV transcription initiation and elongation (RNAP-II, BRD4, and NF-κB). Consistent with the Tat-CDK9 binding result, we observed that ZL0580 treatment reduced RNAP-II activation (phosphorylated RNA-polymerase II) (Fig. 5C), supporting the idea that ZL0580 induces inhibition of HIV transcription elongation. Of note, unlike RNAP-II, ZL0580 treatment did not appear to affect the expression of other cellular factors BRD4 and NF-κB (Fig. 5C).
ZL0580 inhibits TAT binding to CDK9 in microglia during activated and resting conditions. (A) HC69 cells were stimulated with TNF-α (300 pg/ml) to activate HIV in the absence or presence of ZL0580 (8 μM) for 24 h. Co-IP analysis results for the binding of TAT to CDK9 was performed. The cellular expression of CDK9 protein (input) results are shown. (B) HC69 cells were treated with ZL0580 (8 μM) or left untreated (NC) for 24 h. Co-IP analysis for binding of TAT to CDK9 was performed. Cellular expression of CDK9 protein (input) results are shown. (C) Western blot measurement of cellular proteins involved in HIV transcription. HC69 cells were treated as indicated in panel A, and the total cellular proteins were extracted for Western blot analysis at 24 h posttreatment. (D) U1 cells were stimulated with PMA (0.05 μg) to activate HIV expression in the presence of ZL0580 (8 μM) for 24 h, and cellular proteins were analyzed at 24 h posttreatment. Co-IP was conducted to measure the binding of TAT to CDK9. (E) U1 cells were treated with ZL0580 (10 μM) or left untreated (NC). Cellular proteins were analyzed at 24 h posttreatment Co-IP was conducted to measure the binding of TAT to CDK9.
ZL0580 inhibits Tat recruitment to the HIV 5′ LTR promoter in microglial cells.To directly examine the impact of ZL0580 on Tat transactivation, we next assessed the recruitment of TAT to HIV 5′-LTR promoter in microglial cells following ZL0580 treatment under activated condition. First, we treated cells with DMSO alone (NC), TNF-α, or TNF-α/ZL0580 for 24 h, and then we examined the binding of TAT to the HIV promoter (a small region overlapping with HIV transcription start site) (21) using chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR). We found that ZL0580 treatment led to a significant reduction in TAT binding to the HIV promoter compared to cells treated with TNF-α alone (Fig. 6). In addition, under resting conditions where cells were only treated with ZL0580 (8 μM or not treated [NC]), we observed a similar reduction pattern induced by ZL0580 compared to NC (data not shown). Together, these data suggest that ZL0580 inhibits Tat transactivation in microglial cells.
ZL0580 reduces the recruitment of TAT to the HIV 5′ LTR promoter in microglia. ChIP analysis for recruitment of Tat to HIV LTR in HC69 cells under the activated condition. Cells were untreated (NC) or treated with TNF-α (300 pg/ml) to activate HIV in the presence of or absence of ZL0580 (8 μM) for 24 h. ChIP analysis was performed with Tat-specific antibody or control nonspecific IgG. Data were normalized to nonspecific IgG and are expressed as the fold enrichment. Statistically significant differences between groups are indicated (**, P ≤ 0.01).
ZL0580 induces more repressive chromatin structures in HIV LTR.Organization of nucleosome and chromatin structure (e.g., DNA accessibility) at the HIV LTR correlates with HIV proviral transcription (34). Our recent study showed that ZL0580 can induce epigenetic reprogramming, leading to a more repressive chromatin structure at the HIV LTR in J-Lat cells (28). In the present study, we demonstrated that pretreatment of microglial cells with ZL0580 rendered them more resistant to HIV reactivation (Fig. 4A and B), indicating potential epigenetic reprogramming effect of ZL0580 in microglial cells as well. To test this, we used high-resolution micrococcal nuclease (MNase) nucleosomal mapping to assess the HIV LTR chromatin profile, as reported previously (28, 34). Microglial cells were either untreated (NC) or treated with ZL0580 (8 μM) for 24 h, followed by activation with TNF-α for 24 h. After the chromatins were cross-linked, they were divided into undigested and MNase-digested portions. DNA from digested and undigested samples were probed with 20 separate sets of overlapping primers (Table 1) to amplify different regions along the HIV LTR (Fig. 7A). MNase can cleave nucleosome-free and linker DNA connecting two nucleosomes, while DNA within nucleosomes will be more protected from MNase digestion. This allows the assessment of nucleosomal occupancy and DNA accessibility upon calculating the ratio for the amount of PCR products in the digested DNA to that of the undigested control for each primer pair. Intriguingly, we found that treatment of microglia with ZL0580 induced more nucleosomal DNA protection in majority of amplicon regions compared to untreated cells (NC/TNF-α) (Fig. 7B). To determine whether ZL0580 induces durable effect on HIV LTR nucleosomal structure, the same MNase nucleosome mapping was conducted in microglia at later time point after ZL0580 treatment. Cells were treated with mock (NC) or ZL0580 for 24 h, followed by washing to remove residual drugs. On day 6 after initial treatment, the cells were activated with TNF-α for 24 h. We observed that the LTR nucleosomal structure remains repressed, albeit to a lesser extent, in ZL0580-treated cells compared to mock (NC)-treated cells (Fig. 7C). These data are consistent with the observed durable effect of ZL0580 on HIV transcription in microglia. Taken together, these data suggest that ZL0580 remodels HIV LTR chromatins to induce more repressive nucleosomal structure.
Primer sequences for high-resolution MNase mapping
High-resolution MNase nucleosomal mapping for HIV LTR in microglia cells. (A) Diagram illustrating PCR amplicons at HIV LTR region covering 40 to 902 nucleotides corresponding to Nucl-0, DHS1, Nucl-1, DHS2, and Nucl-2. The sizes of the PCR products are 100 ± 10 bp and ∼30 bp apart from each other. (B) Profile of changes in chromatin structure in the HIV LTR in microglia posttreatment. HC69 cells were left untreated (NC) or treated with ZL0580 (8 μM) for overnight. At day 1 (B) and day 6 (C) posttreatment, the cells under both conditions were stimulated with TNF-α (300 pg/ml) for 24 h. The chromatin profile was determined by calculating the ratio (y axis) for the amount of PCR product in the MNase digested to that of undigested control DNA samples for each primer pair. The x axis shows the corresponding PCR amplicon and represents base pair units, with “0” as the start of LTR Nuc-0. Statistically significant differences between groups are indicated (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
ZL0580 induces suppressive effect on HIV in human primary monocyte-derived macrophages.After demonstrating the suppressive activity of ZL0580 on HIV in multiple human myeloid cell lines, we next explored the effect of ZL0580 on HIV in human primary macrophages. In addition, it has been reported that low level of viral replication remains during ART treatment (3, 4), and importantly, HIV rebound can occur quickly after ART interruption (5, 6). Hence, we examined the effect of ZL0580 on HIV in macrophages in the presence of ART treatment. Human monocyte-derived macrophages (MDMs) were prepared from monocytes isolated from two donor PBMCs (donor 1 and donor 2). We established an MDM HIV infection model using two different R5 strains (JR-FL and US-1) to understand the breadth of viruses that are potentially affected by ZL0580. After viral inoculation, productive HIV infection in MDMs was monitored in culture supernatants on day 4 and day 7 by PCR quantification of viral copies. After the establishment of HIV infection in MDMs (typically on day 7 postinfection), cells were either not treated (NC), treated with ART alone (400 nM lamivudine, 400 nM raltegravir, and 200 nM efavirenz), or treated with ART plus ZL0580 (2.5 μM; ART/ZL0580). The medium was replaced every 3 days, and the same drugs were used for continuous treatment. HIV production in supernatants was measured using ultrasensitive nested PCR (28, 35). Using this model, our goal was to examine whether ZL0580 could promote rapid HIV suppression during ART and prevent or delay HIV rebound in fully suppressed MDMs after treatment interruption. Therefore, when HIV was fully suppressed by ART or ART/ZL0580, treatments were then stopped to monitor HIV rebound. Interestingly, in the US-1 infections, ART alone could effectively suppress HIV, eventually leading to full suppression in both donor MDMs; of note, ZL0580 (ART/ZL0580) could promote HIV suppression during ART (prior to treatment cessation) (Fig. 8A). It appears that ZL0580 could also prevent HIV rebound in the donor 1 MDMs after treatment cessation compared to ART alone (Fig. 8A). In JR-FL infection, however, ART failed to effectively suppress HIV in both donor MDMs, indicating that the effects of ART on HIV in macrophages may be dependent on the viral strains used. Despite the inefficiency of ART in suppressing JR-FL HIV, a consistent observation was that ZL0580 (ART/ZL0580) could also promote HIV suppression during ART in JR-FL-infected MDMs (both donors) (Fig. 8A). In this case, treatment was not stopped since HIV was not fully suppressed by either ART or ART/ZL0580 treatment. Lastly, cell viability was monitored at the last time point (day 34; prior to experiment termination) and found to be comparable among the three treatment conditions (Fig. 8B). To longitudinally monitor cell viability, a parallel experiment was performed wherein MDMs were repeatedly treated as described above (NC, ART, and ART/ZL0580), followed by the collection of cells on days 18 and 29 (representing middle and late time points after treatments) for cell viability analysis. Similarly, the MDM viability was comparable among the treatment conditions at both time points (Fig. 8C). Collectively, these data provide evidence that ZL0580 also induces a repressive effect on HIV in human macrophages and could promote HIV suppression in combination with ART.
ZL0580 induces a suppressive effect on HIV in human MDMs by promoting HIV suppression during ART treatment. (A) Monocytes isolated from normal human PBMCs (n = 2; donors 1 and 2) were differentiated into macrophages (MDMs) and then infected with two different R5 HIV strains (US-1 or JR-FL). HIV replication in MDMs was measured on days 4 and 7 after viral inoculation in culture supernatants to confirm successful infection. On day 7 postinfection, the cells were either not treated (NC), treated with ART alone, or treated with ART plus ZL0580 (2.5 μM). The culture medium was replaced with fresh medium containing the same amounts of drugs every 3 days. For the US-1 infection (top two panels), treatment was stopped when the HIV was fully suppressed in the ART-alone group. Viral production in supernatants was continuously monitored up to day 34 as the last time point prior to experimental termination. For the JR-FL infection (bottom two panels), since our data showed that ART failed to efficiently suppress HIV, treatments were not stopped and maintained to the end of the experiments (day 34). In the experiment, HIV production in culture supernatants was measured every 3 days using the two-step nested reverse transcription-qPCR. Viral production kinetics from day 0 to day 34 for each donor are shown. (B) The percentage of viable MDM cells at the last time point (day 34) postinfection (JR-FL) was measured and compared between different conditions. (C) Longitudinal analysis of MDM cell viability following different treatments. MDMs derived from the same two donor PBMCs (not infected) were repeatedly mock treated (NC), treated with ART alone, or treated with ART plus ZL0580 (2.5 μM) once every 3 days to the end of experiment (day 34). On days 18 and 29 after the first treatments, the cell viability was measured by flow cytometry based on Aqua Blue staining (% viable cells) and compared between different treatments. n.s., not significant..
DISCUSSION
Brain-resident microglia and myeloid cell populations, such as monocytes and perivascular macrophages, play an important role in the establishment, persistence, and pathogenesis of HIV, especially in the CNS (36). Our findings demonstrate that modulating host BRD4 with a small-molecule modulator, ZL0580, can potently and durably suppress HIV transcription in several latently infected myeloid cell lines and block events of viral reactivation in microglia. One characterization of this state of suppression is a significant reduction in viral mRNA in an already low level of basal transcription and the durability of the suppression after a single dose of the treatment in microglia. Our data also demonstrate that ZL0580 induces a suppressive effect on HIV in human primary macrophages and promotes HIV suppression in combination with ART.
One of the proposed strategies for HIV eradication is the shock-and-kill approach (37). This strategy aims to reactivate the latent viral reservoir during the ART regimen using latency-reversing agents (LRA) such as protein kinase C agonists and HDAC inhibitors (37). The virally productive cells can then be lysed due to cytopathic effects or be targeted by HIV-specific cytolytic T lymphocytes, whereas the infection of new cells will be inhibited by continuous ART treatment (37). Several in vitro and ex vivo studies showed that the use of LRA is a potential promising approach to curing HIV infections; however, data from several clinical trials have not yet shown enough evidence in reducing HIV reservoirs (37). A potential concern for the application of the LRA strategy in the CNS is that reactivating of latently infected cells can possibly result in the production of neurotoxic viral proteins, such as Tat and the gp120, and these proteins have been implicated in CNS inflammation and neurodegeneration (8). In addition, reactivation of latently infected cells may also result in reinfection of new cells in CNS, where ART penetration is suboptimal (38). Recent data showed that a low level of ongoing viral replication continued to replenish HIV reservoirs even under ART (4). Therefore, the development of approaches that can repress residual HIV transcription and prevent or delay latent HIV reactivation (block and lock) are gaining increasing interest. In support of this, several recent studies showed that inhibition of viral Tat with didehydro-cortistatin A (dCA) (39), repressing the NF-κB pathway with telomerase-derived peptide (GV1001) (40), or targeting other host factors (41, 42) can repress basal HIV transcription and reactivation. BRD4 plays an important role in HIV transcriptional regulation (21–23). Our recent study identified a novel small molecule (ZL0580) that can modulate BRD4 to suppress HIV in human T cells (28). In the present study, we extended this finding and demonstrated that ZL0580 can induce potent and durable HIV suppression in human myeloid cells, including microglia and macrophages. Our findings collectively support the proof of concept that targeting host mechanisms to suppress and/or silence HIV by small molecule is feasible in multiple cell types of HIV reservoirs.
Microglia are the major target and reservoir for HIV in the CNS with a long lifespan (43, 44). Compared to CD4 T cells, microglia are more resistant to cytopathic effect and apoptosis (36). In the CNS, ART drugs have low efficiency, and this is partially due to their low penetration across the blood-brain barrier (45, 46). Several studies reported the existence of HIV DNA and RNA in the brain tissues of an aviremic patient on suppressive ART, indicating ongoing HIV replication in the CNS. Furthermore, a recent study showed that the efficacies of several ART drugs were reduced in human microglia compared to PBMCs (18). Hence, beyond ART, new approaches are needed to further repress HIV activity in the microglia. Here, we showed that ZL0580 induces the suppression of HIV transcription in both activated and resting microglial cells (Fig. 3), supporting that residual levels of HIV transcription could be controlled by modulating BRD4 in microglia. Mechanistically, BRD4 is an epigenetic reader and interacts with a range of host epigenetic/transcriptional regulators to modulate target gene expression. Our data indicate that ZL0580 manipulates multiple functions of BRD4 to suppress HIV transcription in microglia, including inhibition of Tat transactivation (Fig. 5 and 6) and repression of the LTR structure (Fig. 7). Based on our recent study (28) and previous literature (20, 21, 23), we speculate that mechanisms by which BRD4 modulates HIV transcription could be associated with cell types and cellular status. In a transformed cell line (e.g., HC69) and activated cells where HIV Tat level is considered relatively high, BRD4 can function to compete with Tat for cellular active CDK9, leading to inhibition of Tat transactivation and transcription elongation (Fig. 5 and 6). In latent conditions when the HIV Tat level is considered low, a potential mechanism for BRD4 to inhibit HIV is engaging chromatin remodeling proteins (e.g., SWI/SNF) (31) to induce a repressive HIV LTR nucleosomal structure. Indeed, our data showed that treatment of HC69 cells with ZL0580 induces a more repressive LTR structure that is refractory to subsequent TNF-α reactivation compared to mock treatment (Fig. 7). This mechanism could help explain why ZL0580 pretreatment renders microglia more resistant to LRA-stimulated HIV reactivation (possibly due to “repressed” LTR). Nevertheless, additional studies are warranted to further understand mechanisms by which ZL0580 modulates BRD4 to induce repressive LTR structure in myeloid cells, for example, through engaging BRD4-interacting histone modifiers and remodeling proteins (22, 34).
HIV blips may contribute in the replenishment of HIV reservoirs even under optimal ART therapy (47, 48). Cerebrospinal fluid blips are commonly seen in patients with HAND who are on suppressive ART and show undetectable viremia (49, 50). It has been shown that patients who experience no blips revealed faster decay in the viral reservoir compared to patients with blips (51, 52). Hence, durable repression of HIV activity in CNS reservoirs is important. Our data showed that single ZL0580 treatment induces durable HIV suppression through day 21 in microglia, which waned somewhere between day 21 and day 28 posttreatment (Fig. 2). This could be due to a gradual loss of activity for ZL0580, or it could be related to the fact that newly generated HC69 cells in the culture (cell line continuously proliferate in culture) may be suboptimally exposed to ZL0580. Of importance, the durability of HIV suppression in microglia could be further extended by repeated treatments (Fig. 2C and D). Based on the data (fold reduction in Fig. 2C and D), we speculate that the suppressive effect of ZL0580 would remain beyond day 41. Therefore, all of these data further support the utility of ZL0580 in durably suppressing HIV in CNS reservoirs. Future studies are warranted to test the activity of this compound on HIV reservoirs, including microglia in CNS, in vivo in animal models.
Due to limitations in the availability of human primary microglia, we employed MDMs as a primary myeloid cell model of HIV infection. Two different HIV strains, US-1 (53–56) and JR-FL (57), were used to infect MDMs to explore potential effects of breath of viruses. In the MDM model, a consistent finding we observed is that ZL0580 could promote HIV suppression during ART treatment in both US-1 and JR-FL infections (Fig. 8A), indicating that ZL0580 may have utility in combination with ART to suppress residual HIV replication. A limitation of these data is that significant variation was also noted between the two HIV infections in terms of their responsiveness to ART treatment in MDMs: while ART could efficiently suppress US-1 HIV in MDMs, it failed to do so for JR-FL HIV (Fig. 8A), suggesting that the breath of viruses plays an important role in this model and that our proposed approach should be further tested with an expanded panel of viruses in myeloid cells in the future. Related to this limitation, since ART failed to induce full HIV suppression in JR-FL-infected MDMs (Fig. 8A), we were unable to precisely determine in the present study whether ZL0580 could also prevent or delay HIV rebound after treatment cessation in JR-FL-infected MDMs (Fig. 8A), which could be further explored in the future by using additional donor MDMs and by optimizing the JR-FL virus concentration for infection. In addition, since different ART regimens have been used for HIV treatment, it would also be important to understand if ART regimens influence the effects of ZL0580 on HIV. In this study, we have not yet tested this point, but we speculate that the ART regimen would matter. For example, if protease inhibitor, instead of integrase inhibitor, is included in the ART regimen, we expect that more proviruses would be established in cells, which would require a higher concentration or more doses of ZL0580 to achieve sustained HIV suppression. Future studies should also test impact of different ART regimens on the effectiveness of ZL0580 in suppressing HIV in myeloid cells and other HIV reservoirs.
Potential off-target effects should be carefully considered for therapeutic approaches that target host mechanism (e.g., BRD4) to suppress HIV, although host-targeting approaches provide some critical advantages (e.g., sustained suppression and limited drug resistance). Thus far, our data support that the HIV LTR is a major target by ZL0580/BRD4, although potential effects of ZL0580 on non-HIV gene targets are not fully excluded. In our recent study (28), we examined the effects of ZL0580 on phenotypes (e.g., receptor, coreceptor, and activation markers) and the gene expression profile of human T cells (17 selected genes critical for T-cell functions). We noted that ZL0580 does not appear to induce a broad impact on T cells (28). However, while these focused analyses are helpful, they are not unbiased approaches. In the future, the use of genomic approaches, such as RNA-Seq and CHIP-Seq, will be critical for more comprehensively characterizing whether and to what extent modulation of BRD4 by ZL0580 induces global or substantial off-target effects in HIV target cells.
In summary, our data demonstrate that the BRD4-selective small molecule ZL0580 induces epigenetic suppression of HIV in human microglial cells and primary macrophages. We showed that in the MDM model (ART and ZL0580), ZL0580 could promote HIV suppression during ART, indicating its potential utility in combination with ART drugs to suppress residual HIV replication in CNS reservoirs and thereby to minimize HIV-associated neurological disorders. Future studies are needed to examine the activity of ZL0580 to penetrate blood-brain barrier, as well as the HIV-suppressive efficacy and toxicity of this compound in vivo in HIV-infected animal models.
MATERIALS AND METHODS
Cell lines.The microglial cell line (HC69) was a generous gift from Jonathan Karn (Case Western Reserve University) and was maintained in DMEM-F12 medium (ATCC) supplemented with penicillin G (100 U/ml), streptomycin (100 μg/ml), 1% (vol/vol) fetal bovine serum (FBS), 1 μM dexamethasone (Sigma D4902), and 1× N2 supplement (Gibco-Invitrogen). The U1 and OM10.1 cells (obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID) were maintained in RPMI 1640 medium (Gibco) supplemented with penicillin G (100 U/ml), streptomycin (100 μg/ml), l-glutamine (2 mM, 0.3 mg/ml), and 10% (vol/vol) FBS. Cells were cultured at 37°C in a humidified 5% CO2 incubator.
Small molecule BRD4 modulator ZL0580.The design, synthesis, and chemical structure of ZL0580 was described in detail in our recent study (28).
Cell treatments and flow cytometric analysis of HIV expression.Quantification of GFP-expressing cells in microglia (HC69) was carried out by fluorescence-activated cell sorting (FACS) analysis using a BD Accuri C6 flow cytometer and LSR-Fortessa, and the data were analyzed by using FlowJo software (Tree Star). Unless otherwise stated, HC69 cells were treated with 300 pg/ml TNF-α (R&D Systems, 210-TA) with or without 8 μM ZL0580 or treated with DMSO (NC) for 24 h. Cells were harvested, washed, and resuspended in 300 μl of phosphate-buffered saline (PBS) and then analyzed for cell viability stained for viability using trypan blue staining or Aqua Blue Live/Dead (Invitrogen), followed by flow cytometric analyses for GFP expression and cell viability (Aqua Blue). For latent HIV reactivation experiment, cells were treated with DMSO (NC) or 8 μM ZL0580 for 24 h, followed by cell washing and continuous culture. Cells were reactivated at the indicated time posttreatment with 300 pg/ml TNF-α for overnight. For OM10.1, the cells were treated with DMSO (NC) or 20 ng TNF-α with or without 10 μM ZL0580. For U1, the cells were also treated with DMSO (NC) or 0.05 μg/ml PMA with or without 10 μM ZL0580.
Real-time PCR quantification of cell-associated HIV RNAs.HIV mRNA quantifications (Gag, early multispliced, and GFP) in myeloid cell lines were performed. RNA was extracted by using Quick-RNA MicroPrep kit (Zymo) according to the manufacturer’s instructions. Extracted RNA was used to synthesize cDNA using iScript reverse transcription supermix (Bio-Rad). The gene of interest was quantified by qPCR using iTaq Universal SYBR green supermix (Bio-Rad) and the CFX Connect Real-Time PCR detection system (Bio-Rad). PCRs and conditions were follows: 20 μl of total PCR containing 10 μM primers, 90 ng of cDNA, 10 μl of iTaq universal SYBR green supermix (2×) (Bio-Rad), and molecular-grade water was subjected to PCR cycling conditions (95°C for 3 min, 45 cycles of 95°C for 5 s and 60°C for 30 s). The primer sequences used in the PCRs are presented in Table 2. Gene expression was normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and calculated using the 2–ΔΔCT method.
Primer sequences for quantitative PCR quantification of HIV DNA and RNA
Western blot analysis.Briefly, 5 × 106 cells per treatment condition were cultured and treated as indicated. The cells were harvested and washed twice with PBS. Whole-cell extracts were prepared in 1× RPA buffer (Sigma, catalog no. 20-188) containing 1× protease inhibitor cocktail (Sigma, p8340), and 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), followed by a brief sonication. The total protein concentration was quantified using a protein assay kit (Pierce; Thermo Fisher Scientific) according to the manufacturer’s instructions. Equal amounts of proteins from different treatment conditions (20 to 30 μg of total proteins) were subjected to SDS-PAGE gel separation, followed by protein transfer into Immuno-Blot polyvinylidene difluoride membranes. The membranes were blocked (5% skim milk powder in TBST) for 1 h, followed by incubation with protein-specific antibodies overnight at 4°C. The following primary antibodies were used: Tat (catalog no. 160189; AIDS Reagent Program), p-RNA Pol II-CTD (Ser2; MA5-23510; Thermo Fisher), BRD4 (catalog no. 730007; Thermo Fisher), NF-κB (33-9900; Thermo Fisher), and GAPDH (catalog no. 2118; Cell Signaling). The membranes were washed three times with TBST, followed by 1 h of incubation with secondary antibodies (anti-rabbit or anti-mouse IgG-HRP; Thermo Fisher) based on the nature of the primary antibody. Western blot analysis was carried out using Super Signal West Pico chemiluminescent substrate (Thermo Fisher) reagent. Markers were used to identify the target protein band. As a loading control, the expression of GAPDH was also measured.
Coimmunoprecipitation.Briefly, 15 × 10 6 cells per treatment condition were cultured in six-well plate and treated as indicated. Cells were harvested, washed with PBS, and lysed in 500 μl of NP-40 lysis buffer that contains 1× protease inhibitor cocktail (Sigma, p8340) and 1 mM PMSF (Cell Signaling, catalog no. 8553), followed by rotation at 4°C for 1 h. Cell lysates were centrifuged at 12,000 rpm for 30 min at 4°C, and the supernatants were collected for protein quantification using a protein assay kit (Pierce; Thermo Fisher Scientific) according to the manufacturer’s instructions. Then, 3 μg of anti-Tat (MA1-71509; Thermo Fisher) was incubated with an equal amount of protein from each treatment condition, with rotation overnight at 4°C. The immune complexes were precipitated with 50 μl of streptavidin-magnetic beads for 1 h at 4°C. Beads were washed six times using cold NP-40 cell lysis buffer and then subjected to SDS-PAGE. The membranes were blocked and immunoblotted with anti-CDK9 antibody (MA5-14912; Thermo Fisher), followed by washing and incubation with secondary antibody, as described under “Western blot analysis” above.
Chromatin immunoprecipitation and qPCR.A ChIP-IT Express kit (Active Motif) was used according to the manufacturer’s instructions. Briefly, 15 × 106 cells were transferred to six-well plates and treated as described in the figure legends. At 24 h posttreatment, the cells were washed and fixed in 1 ml of PBS containing 37% formaldehyde (Sigma, F8775) and then incubated on a shaking platform at room temperature for 10 min to allow DNA and protein cross-linkage. The reaction was ended by the addition of 110 μl of 10× glycine on a shaking platform at room temperature for 5 min. Cell mixtures were then centrifuged at 2,500 rpm for 10 min at 4°C. Supernatants were removed, and 1 ml of ice-cold lysis buffer was added to the pellet for 30 min on ice. Nuclei were pelleted with centrifugation at 5,000 rpm for 10 min at 4°C. Nuclear pellets were resuspended in 400 μl of shearing buffer containing 2 μl of protease inhibitor cocktail (PIC) and 2 μl of PMSF on ice for 10 min and then subjected to sonication. The mixtures were centrifuged at 15,000 rpm for 10 min at 4°C. The supernatant, which is the sheared chromatin, was used to set up ChIP reactions by adding the magnetic beads and 3 μg of anti-Tat (Thermo Fisher) or control mouse IgG (Cell Signaling) for overnight rotation at 4°C. Mixtures were washed multiple times with ChIP buffers, followed by chromatin reverse cross-linkage. The DNA was eluted and purified by phenol-chloroform extraction and then used for qPCR analyses with the primers listed in Table 2. PCRs and conditions were set as described above under “Real-time PCR quantification.” The results were analyzed by the fold enrichment method according to the following formula: % enrichment = 2 − (CT IP – CT mock).
High-resolution MNase nucleosomal mapping.Microglial cells were either treated with ZL0580 (8 μM) or left untreated (NC) overnight. Cells under both conditions were then restimulated with TNF-α (300 pg/ml) for 24 h. Cellular cross-linkage was then monitored as described for the ChIP assay. Cells were washed with 1 ml of buffer B (0.25% Triton X-100, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES [pH 7.6]) and then with 1 ml of buffer C (150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES [pH 7.6]). The cells were washed in cold PBS, and ∼15 × 106 cross-linked cells were suspended in 1 ml of buffer A (300 mM sucrose, 2 mM Mg acetate, 3 mM CaCl2, 10 mM Tris [pH 8.0], 0.1% Triton X-100, 0.5 mM dithiothreitol [DTT]), followed by incubation for 5 min on ice, followed by 20× Dounce grinding (tight pestle, Kontes, 2-ml grinder). Nuclei were pelleted by centrifuging at 4°C and 750 × g for 5 min, followed by two washes with 1 ml of buffer D (25% glycerol, 5 mM Mg acetate, 50 mM Tris [pH 8.0], 0.1 mM EDTA, 5 mM DTT) at 15 × 106 nuclei/ml. Samples were centrifuged at 4°C and 750 × g for 5 min to pellet chromatins, and the pellets were suspended in 1 ml of buffer MN (60 mM KCl, 15 mM NaCl, 15 mM Tris [pH 7.4], 0.5 mM DTT, 0.25 mM sucrose, 1.0 mM CaCl2) at 1.5 × 107 nuclei/ml. Then, portions (150 μl) of chromatins (2.25 × 106 nuclei) were treated with 0, 0.5, 5, 20, 50, or 500 U/ml MNase (USB) for 30 min at 37°C, followed by the addition of EDTA (12.5 mM) and SDS (0.5%) to stop the enzymatic reactions, followed in turn by a proteinase K digestion step at 37°C for 4 h. The DNA was eluted and purified by phenol-chloroform extraction, and then 5 ng/μl DNA was used for real-time qPCR analysis with the primers shown in Table 1. The ΔCT method was used to calculate the fold change, and the ratio of the amount of digested DNA to the undigested DNA for each primer was then calculated.
In vitro HIV infection of human primary MDMs and nested PCR for quantification of viral RNA in cell supernatants.PBMCs of healthy donors were obtained from the blood bank at the University of Texas Medical Branch (UTMB). The use of deidentified PBMCs of health donors was approved by Institutional Review Board (IRB) of the UTMB. Total PBMCs were cultured as adherent monolayers at a density of 5 × 106 cells/well in six-well plates in RPMI 1640 medium (Gibco) (with no FBS) for 2 h. Adherent cells were differentiated into macrophages for 6 days in differentiation medium consisting of RPMI 1640 medium (Gibco) supplemented with penicillin G (100 U/ml), streptomycin (100 μg/ml), l-glutamine (2 mM, 0.3 mg/ml), 10% (vol/vol) FBS, and 100 ng/ml granulocyte-macrophage colony-stimulating factor (Gibco, PHC2015) for 6 days. At day 3 of cell culture, half of the culture medium was replaced with fresh differentiation medium. At day 6, the cells were infected with HIV (US-1 or JR-FL) using spinoculation (1,200 × g for 1 h), followed by overnight incubation at 37°C in a humidified 5% CO2 incubator. The cells were then dissociated nonenzymatically (Sigma, C5914), and equal numbers of cells were distributed into 48-well plates and maintained RPMI 1640 medium (Gibco) supplemented with penicillin G (100 U/ml), streptomycin (100 μg/ml), l-glutamine (2 mM, 0.3 mg/ml), and 10% (vol/vol) FBS. At day 7 postinfection, the cells were either not treated (NC), treated with ART alone (400 nM lamivudine, 400 nM raltegravir, and 200 nM efavirenz), or treated with ART plus ZL0580 (2.5 μM). Fresh medium containing the same treatments was changed every 3 days, and HIV production in the supernatants was measured by ultrasensitive nested PCR (as explained below) prior to medium change. Treatments were discontinued when ART fully suppressed HIV production, and HIV production in cell culture was monitored after treatment discontinuation. The establishment of ultrasensitive nested PCR for quantification of HIV production in culture supernatants was determined according to our recent study (28). Briefly, viral RNA from culture supernatants was extracted using a QIAamp viral RNA kit (Qiagen) according to the manufacturer’s protocol, followed by two-step nested PCR approach. After RNA extraction, cDNA was synthesized from the extracted viral RNA and then subjected to first-round PCR amplification (16 cycles) using Gag-out-F and Gag-out-R (Table 2). The products of the PCR were serially diluted and then subjected to a second-round nested qPCR (40 cycles) with the Gag-F/Gag-R primer set (Table 2). PCRs, conditions, and analysis were performed as described above under “Real-time PCR quantification.” The quantification of HIV copies was performed using the established standard curve.
Statistical analysis.Where indicated, experiments were analyzed by one-way analysis of variance, followed by the Student t test, in Prism 6.0 (GraphPad, San Diego, CA). Significance is indicated in the figures by asterisks (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001).
ACKNOWLEDGMENTS
We thank Jonathan Karn (Case Western Reserve University) for kindly providing the microglial cells (HC69). The U1 cell line (catalog no. 165), the OM10.1 cell line (catalog no. 1319), anti-Tat Ab (catalog no. 160189), and JR-FL HIV (catalog no. 395) were obtained from the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH.
This study was funded by National Institutes of Health grants (R56AI145666 and R21AI147903; H.H.) and a UTMB Institute for Human Infection and Immunity pilot grant (H.H.). E.A. was supported by a predoctoral training fellowship from the Saudi Arabian Cultural Mission, Jazan University.
FOOTNOTES
- Received 4 November 2019.
- Accepted 8 March 2020.
- Accepted manuscript posted online 18 March 2020.
- Copyright © 2020 American Society for Microbiology.
