Small-molecule screening using a human primary cell model of HIV latency identifies compounds that reverse latency without cellular activation (original) (raw)
Transduction of primary human CD4+ T cells with Bcl-2. To allow long-term in vitro survival, we transduced primary CD4+ T cells with Bcl-2 using a lentiviral vector, EB-FLV, in which Bcl-2 expression is driven by the constitutively active promoter of elongation factor 1 α (EF1α) (Figure 1, A and B). Freshly isolated primary human CD4+ T cells were costimulated with anti-CD3 and anti-CD28 and then transduced with the Bcl-2–expressing lentiviral vector. Transduced cells were maintained in the absence of TCR stimulants and exogenous cytokines for more than 3 weeks. Viable cells were then isolated using Ficoll-Hypaque density gradient centrifugation. As shown in Supplemental Figure 1A (supplemental material available online with this article; doi:10.1172/JCI39199DS1), more than 80% of the activated cells are lost during the first 3 weeks of cytokine withdrawal. However, the remaining viable cells have greatly improved survival (see below).
Generation of the Bcl-2–transduced primary CD4+ T cells. (A) The structure of the lentiviral vector carrying the human Bcl-2 gene (EB-FLV). The U3 region of 3′ LTR is deleted (3′ΔLTR) for self inactivation. The expression of human Bcl-2 is driven by EF1α promoter. Ψ, packaging signal; RRE, rev responsive element; cPPT, central polypurine tract; pEF1α, EF1α promoter. (B) Strategy to generate Bcl-2–transduced primary CD4+ T cells. Primary CD4+ T cells from normal donors were activated and transduced with the Bcl-2–expressing lentiviral vector. Viable cells were isolated after 3 to 4 weeks of culture in the absence of TCR stimulants or cytokines. (C) Intracellular staining for Bcl-2 with FITC-conjugated anti–Bcl-2 antibody in freshly isolated CD4+ T cells and Bcl-2–transduced cells. The Bcl-2–transduced cells were maintained in culture without cytokines for 4 weeks following transduction. Freshly isolated CD4+ T cells stained with FITC-conjugated isotype control antibodies served as a negative control (purple).
Activated primary T cells die quickly in culture without trophic cytokines (29). Therefore, only Bcl-2–transduced cells survive under the above culture conditions. Bcl-2 was overexpressed in the vast majority of viable cells present after 4 weeks of culture (Figure 1C). In control experiments, cells transduced with the control lentiviral vector that did not express Bcl-2 died within 2 weeks. To further confirm the longevity of the Bcl-2–transduced cells and the stability of Bcl-2 expression, we quantified viability and Bcl-2 expression weekly in cultures of isolated Bcl-2–transduced cells maintained without supplemental cytokines. We found less than 20% cell loss (Supplemental Figure 1B) and stable Bcl-2 expression (Supplemental Figure 1C) over a 4-week follow-up period.
Bcl-2–transduced cells reach a quiescent state similar to that of freshly isolated primary resting CD4+ T cells. Because resting memory CD4+ T cells are a major reservoir for latent HIV-1 in vivo, an in vitro latency model should consist of infected cells in a similarly quiescent state. After 4 weeks in culture without TCR stimulation or cytokines, surviving Bcl-2–transduced cells were characterized with regard to properties unique to primary resting T cells. These include small cell size, absence of cell proliferation and cytokine production, and lack of activation markers.
Resting Bcl-2–transduced cells exhibited small cell size (Figure 2A) and scanty cytoplasm (Supplemental Figure 2A). Using DNA/RNA staining (39–41), we found that the majority of resting Bcl-2–transduced cells were in the G0/G1a phase of the cell cycle, as is the case with freshly isolated primary resting CD4+ T cells (Figure 2B). Both cell populations readily entered the cell cycle following activation with anti-CD3 and anti-CD28 (Figure 2B). Surface expression of classic T cell activation markers such as CD25, CD69, and HLA-DR was very low on resting Bcl-2–transduced CD4+ T cells (Figure 2C). Less than 5% of these cells expressed CD25, and expression of HLA-DR was similarly low. Approximately 15%–20% of these cells had low but detectable levels of CD69. However, following 3 days of activation by anti-CD3 and anti-CD28, more than 90% of cells expressed high levels of CD25 and CD69 and more than 25% of cells expressed HLA-DR (Figure 2C). The resting status of the Bcl-2–transduced CD4+ T cells was further confirmed by measurement of cytokine mRNA levels using real-time RT-PCR (Figure 2D). Both the IL-2 and IFN-γ transcripts were present only at very low levels in the resting Bcl-2–transduced cells, while transcript levels were increased 8-fold and 90-fold, respectively, following activation by anti-CD3 and anti-CD28.
Characterization of resting Bcl-2–transduced cells. (A) Flow cytometric measurement of cell size. The forward scatter profile of Bcl-2–transduced cells coincided with that of freshly isolated resting CD4+ T cells. (B) Cell-cycle analysis in resting and activated freshly isolated CD4+ T cells, Bcl-2–transduced cells, and latently infected Bcl-2–transduced cells. Data are plotted with DNA staining (Hoechst 33342) on the y axis versus RNA staining (pyronin Y) on the x axis. Cells were either left in a resting state or activated with anti-CD3 and anti-CD28 antibodies for 2 days. The percentage of cells in each quadrant is indicated. (C) The expression of the activation markers CD25, CD69, and HLA-DR on resting and activated Bcl-2–transduced cells. The percentage of cells in each quadrant is indicated. (D) Levels of IL-2 and IFN-γ mRNA in resting and activated freshly isolated and Bcl-2–transduced CD4+ T cells. The levels of IL-2 and IFN-γ mRNA were quantified using real-time RT-PCR and normalized to the ubiquitin mRNA levels. The fold change was relative to that observed in the freshly isolated resting CD4+ T cells. (E) Levels of nuclear NF-κB p65 in resting and activated freshly isolated and Bcl-2–transduced CD4+ T cells. The nuclear NF-κB p65 was quantified by an ELISA-based assay and normalized to the total protein concentration of each nuclear extract. Results shown are relative OD450 values. Data in D and E are mean ± SD of triplicate samples from 1 of 2 independent experiments, all of which produced similar results.
The host transcription factor NF-κB is critical for HIV-1 replication (14–16, 42). Lack of NF-κB activation in resting CD4+ T cells is a major factor in HIV-1 latency (43). We therefore examined the activation state of NF-κB in resting Bcl-2–transduced cells by ELISA measurement of the nuclear levels of NF-κB p65. As Bcl-2–transduced cells returned to a quiescent state, nuclear levels of NF-κB fell to below those seen in freshly isolated resting CD4+ T cells (Figure 2E). Upon stimulation with anti-CD3 and anti-CD28, nuclear NF-κB was readily induced to high levels equivalent to those of activated primary CD4+ T cells (Figure 2E).
An additional characteristic of resting CD4+ T cells is their resistance to productive HIV-1 infection. Using a recombinant HIV-1 vector capable of expressing GFP, we showed that resting Bcl-2–transduced CD4+ T cells were resistant to HIV-1 infection regardless of whether the viruses were pseudotyped with an X4 HIV-1 envelope (Figure 3A) or VSV-G (Supplemental Figure 2B). However, T cell activation overcame this resistance (Figure 3A and Supplemental Figure 2B). Taken together, the above data demonstrate that the Bcl-2–transduced cells reach a resting state similar to that of primary resting CD4+ T cells while retaining the capability to be fully activated.
Resting Bcl-2–transduced cells exhibit resistance to HIV-1 infection and phenotypes of TEM. (A) Susceptibility of resting and activated freshly isolated and Bcl-2–transduced CD4+ cells to HIV-1 infection. Cells were infected with reporter virus pNL4-3-Δ6-drEGFP pseudotyped with HIV-1–X4 envelope. The number in each plot indicates the percentage of infected (GFP-positive) cells. (B) The expression of CD45RA, CD45RO, CCR7, and CD62L on resting Bcl-2–transduced cells. The percentage of cells in each quadrant is indicated.
Because latent HIV-1 primarily resides in memory CD4+ T cells (44), we further characterized Bcl-2–transduced cells for expression of memory T cell markers. Memory T cells are CD45RO positive. Central memory T cells (TCM) constitutively express CCR7 and CD62L, while effector memory T cells (TEM) show heterogeneous expression of CD62L but not CCR7 (45). A large portion of Bcl-2–transduced cells (~85%) expressed CD45RO (Figure 3B). Interestingly, approximately 30% of cells expressed both CD45RA and CD45RO. Only a few cells (~6.5%) expressed CCR7. Therefore, we concluded that resting Bcl-2–transduced cells are more like TEM than TCM. Of note, a recent study reported that CD4+ TCM and transition memory T cells (TTM) are 2 major cellular reservoirs for HIV-1 in vivo (46). Determining whether the latently infected cells generated in our model exhibit properties similar to those of TTM cells requires further investigation
Establishment of HIV-1 latency in Bcl-2–transduced resting CD4+ T cells. Latently infected cells are rare in vivo (47) because most infected cells die from viral cytopathic effects or host cytolytic responses before they can revert to a resting state in which HIV-1 gene expression is shut off. Preliminary results confirmed the previously reported cytopathic effects of HIV-1 gene products (48, 49). To increase the efficiency with which HIV-1 latency was established, we mutated the gag, vif, vpr, vpu, and nef genes of the reference strain NL4-3. Point mutations were introduced to produce premature stop codons in each gene except for nef, which was truncated (Figure 4A). Our primary goal was to study the reactivation of latent HIV-1, and thus the rev and tat genes were left unchanged because their products are crucial for the expression of viral genes. To keep splicing sites intact and mimic the processing of viral transcripts, the overall genome structure of HIV-1 was preserved. Destabilized EGFP was introduced to track the viral infection. Compared with NL4-3, this modified viral vector, pNL4-3-Δ6-drEGFP, gave dramatically improved viability of infected Bcl-2–transduced cells. As shown below, this approach made it possible to obtain large numbers of latently infected primary resting CD4+ T cells that could be used to study upregulation of HIV-1 gene expression. The ultimate yield of latently infected cells was much lower (approximately 5- to 10-fold) when Bcl-2–transduced cells were infected with an HIV-1 construct in which all the reading frames except env were open. For this reason, subsequent experiments were performed with the NL4-3Δ6-drEGFP virus.
Establishment of in vitro HIV-1 latency in resting Bcl-2–transduced CD4+ T cells. (A) Genome structure of the reporter virus NL4-3-Δ6-drEGFP. It contains a truncated nef and premature stop codons in the ORFs of gag, vif, vpr, and vpu that alter the indicated amino acids shown in the standard single-letter code. A portion of env was replaced with destabilized EGFP, and the signal peptide of env was mutated to allow the destabilized EGFP to remain in the cytoplasm. The red letters indicate the mutated amino acids in the signal peptide. (B) Strategy for generating latently infected Bcl-2–transduced cells. (C) Detection of latently infected cells in the sorted GFP-negative population. The sorted GFP-negative cells were activated with anti-CD3 and anti-CD28 or PMA for 2 days and then analyzed by flow cytometry to quantify the number of GFP-positive cells. FL2-H, red fluorescence channel. (D) Latently infected cells contain integrated viral genomes. Latently infected Bcl-2–transduced cells were left untreated (upper panel) or were pretreated with either medium alone (middle panel) or 1 μM raltegravir (lower panel) for 1 day and then activated with anti-CD3 and anti-CD28 monoclonal antibodies for 2 days. Cells were analyzed using flow cytometry. (E) Cell-cycle status of latently infected cells was determined using Hoechst 33342/pyronin Y staining for DNA/RNA. The controls for the resting and activated cells are the same as in Figure 2B. The percentage of cells in each quadrant is indicated.
To establish HIV-1 latency, Bcl-2–transduced cells were activated and then infected with pseudotyped NL4-3-Δ6-drEGFP. Because the env gene is disrupted by insertion of EGFP, an X4 envelope was provided in trans to achieve single-round infection. The infection rate, based on the percentage of GFP+ cells 3 days after infection, was 5% to 10%. Following more than 4 weeks of culture in the absence of cytokines and other activating stimuli, approximately 20%–30% of cells that initially expressed GFP became GFP negative. We then isolated the GFP-negative cells by sorting. To determine whether latency had been established, the GFP-negative cells were activated and analyzed for GFP expression by flow cytometry. Of the sorted GFP-negative cells, 1% to 3% could be induced to express GFP by activating stimuli such as anti-CD3 plus anti-CD28 or PMA (Figure 4, B and C). In control infected cultures that were not stimulated, the fraction of GFP-positive cells remained less than 0.2%. Taken together, these results indicate that latent infection was established in this system. We could further increase the frequency of latently infected cells by enriching the GFP-positive cells via sorting following the initial infection (Supplemental Figure 3A). The frequency of latently infected cells using this strategy could reach 15%–50% (Supplemental Figure 3, A and B). Although this enrichment increased the frequency of latently infected cells, it did not increase their absolute number. Our preliminary data showed that populations containing 1%–3% latently infected cells generated without enrichment were adequate for high-throughput screening. We therefore performed subsequent experiments without enrichment (Figure 4B).
An important feature of the stable reservoir for HIV-1 in vivo is that the viral genome is integrated into a host-cell chromosome (27, 47). To prove that the latently infected cells generated in this system contain an integrated form of the viral genome, we activated the cells in the presence of an HIV-1 integrase inhibitor, raltegravir. The concentration used was sufficient to block 99% of integration events (50). Raltegravir did not block the reactivation of latent HIV-1 (Figure 4D). This result confirms that this in vitro system is a model for postintegration latency.
Even after prolonged culture in the absence of activating stimuli, a small fraction of GFP-negative cells still expressed low levels of activation markers such as CD25, CD69, and HLA-DR. To exclude the possibility that latent HIV-1 might reside in the cells with some extent of activation, we removed these CD25+, CD69+, or HLA-DR+ cells by negative depletion. A very similar frequency of cells with inducible GFP expression was found in the CD25–CD69–HLA-DR– cells, excluding the possibility that latent HIV-1 resided in the cells with low levels of activation. We also evaluated the cell-cycle status of latently infected cells using DNA/RNA staining. As shown in Figure 4E, the majority of latently infected cells were in G0/G1a phase. Taken together, these observations demonstrate the establishment of HIV-1 latency in vitro in resting Bcl-2–transduced primary CD4+ T cells.
To investigate whether there was any residual integrated provirus that did not respond to T cell activation signals in this system, we sorted the GFP– cells following activation of latently infected cells by anti-CD3 and anti-CD28 for 3 days. The frequency of cells with proviral DNA among the GFP– cells determined using real-time PCR was similar to the percentage of GFP+ cells following activation. This suggests that approximately 50% of integrated proviruses were unresponsive to T cell activation (refer to Supplemental Material for details). Whether these viruses can be reactivated by subsequent rounds of activation requires future exploration.
The signaling pathways leading to the reactivation of latent HIV-1 remain intact in this in vitro model. To validate that the signaling pathways known to induce reactivation of latent HIV-1 remain intact in this in vitro model, we tested known HIV-1 activators, including TCR agonists, mitogens, cytokines, and small molecules alone or in combination. Representative flow cytometry plots are shown in Supplemental Figure 4, A and B. The results are summarized in Figure 5, A and B. PMA, prostratin, and 12-deoxyphorbol 13-phenylacetate (DPP), known activators of PKC, all strongly activated latent HIV-1. Ionomycin, an activator of nuclear factor of activated T cells (NFAT), reactivated a smaller fraction of latent HIV-1. Interestingly, valproic acid (VA), a known histone deacetylase (HDAC) inhibitor and HIV-1 activator (13), barely activated latent virus, even at concentrations as high as 5 mM. However, trichostatin A (TsA), a more potent HDAC inhibitor, activated a greater fraction of the cells at a concentration of 200 nM. Hexamethylene bisacetamide (HMBA), which activates latent HIV-1 in some cell line systems through regulation of active and inactive forms of positive transcription elongation factor b (pTEFb) (51, 52), only caused slight activation of latent HIV-1 at a concentration of 5 mM in our assay.
Response of latently infected Bcl-2–transduced CD4+ T cells to small molecules and cytokines known to reactivate latent HIV-1. Latently infected, Bcl-2–transduced cells were treated with known small-molecule activators (A) or cytokines (B). Bars represent the percentage of GFP-positive cells normalized to the response to anti-CD3 plus anti-CD28 antibodies. Data are mean ± SD of triplicate samples from 1 of 2 independent experiments. The concentrations of small molecules and cytokines used for the experiments are as follows: phytohemagglutinin (PHA) (1 μg/ml), PMA (10 ng/ml), ionomycin (1 μM), prostratin (1 μM), DPP (1 μM), TsA (200 nM), VA (5 mM), HMBA (5 mM), IL-2 (100 U/ml), IL-1b (5 ng/ml), IL-4 (3 ng/ml), IL-6 (5 ng/ml), TNF-α (10 ng/ml), IL-7 (10 ng/ml), and IL-12 (10 ng/ml). For a positive control, cells were activated with 2.5 μg/ml anti-CD3 and 1 μg/ml anti-CD28.
We also tested some cytokines that are known to activate latent HIV-1 (12, 53). Cytokines were used at concentrations previously reported to induce activation of latent HIV-1 in various primary cell systems. IL-7 alone strongly induced reactivation of latent HIV-1 as previously reported (12, 32), while TNF-α, a well-known activator of latent HIV-1 in transformed T cell lines (16, 21), activated only a small fraction of latent virus (Figure 5B). Consistent with this observation, previous studies using the resting CD4+ T cells isolated from patients on HAART also revealed the low activity of TNF- α in reactivation of latent HIV-1 (53). However, a combination of IL-2, IL-6, and TNF-α dramatically increased the reactivation of latent HIV-1 in our system. This response is also similar to that reported in resting CD4+ T cells isolated from patients on HAART (53).
Overall, the responses of the latently infected cells generated in this system to known activators were very similar to those previously reported in other primary cell systems or in resting CD4+ T cells isolated from patients on HAART (10, 12, 53, 54). These results strongly suggest that this in vitro HIV-1 latency model can mimic the latently infected CD4+ T cells present in vivo.
Screening of small-molecule libraries for compounds that reactivate latent HIV-1. Using the system described above, we screened a library of 2000 drugs and natural products and another set of more than 2400 compounds from the Johns Hopkins Drug Library (JHDL) (55). Representative results are shown in Figure 6A. In total, 9 and 8 compounds that could activate latent HIV-1 were discovered in the former and latter libraries, respectively. For further study, we selected 1 of these compounds, 5-hydroxynaphthalene-1,4-dione (5HN), because it was detected in both libraries and displayed the highest capacity to reactivate latent HIV-1. 5HN is a natural quinone (Figure 6B) found in the leaves, roots, and bark of the black walnut tree. 5HN was comparable to the combination of anti-CD3 and anti-CD28 antibodies in its ability to reactivate latent HIV-1 (Figure 6A and Supplemental Figure 5A).
Screening of small-molecule libraries identifies 5HN as a candidate activator. (A) Summary of screening results from JHDL. The results were expressed as the percentage of GFP-positive cells after normalization to the response to anti-CD3 plus anti-CD28. For simplicity, only 500 drugs including the hits PMA and 5HN are shown. (B) Chemical structure of 5HN. (C) Effects of 5HN, PMA, and anti-CD3 plus anti-CD28 on the size of latently infected resting CD4+ T cells. Cell size was measured by flow cytometry using the forward scatter. (D) Effect of 5HN on the transcription of HIV-1. Latently infected Bcl-2–transduced cells were left unstimulated or were stimulated with the indicated concentrations of 5HN or anti-CD3 plus anti-CD28 antibodies. The levels of viral mRNA were quantified using real-time RT-PCR and were normalized to the β-actin mRNA levels. The fold change is shown relative to that observed in the unstimulated samples. Data are mean ± SD of triplicate samples from 1 of 2 independent experiments, all of which produced similar results.
To confirm the effect of 5HN on latent HIV-1, we used real-time RT-PCR to examine levels of HIV-1 transcripts before and after stimulation with 5HN. Viral transcription was induced up to 8.8-fold in a dose-dependent manner by 5HN (Figure 6C). The ED50 of 5HN in reactivating latent HIV-1 was 0.5 μM (Supplemental Figure 5B), while the LD50 measurements for freshly isolated and Bcl-2–transduced primary CD4+ T cells were 9.9 μM and 7.7 μM, respectively (Supplemental Figure 5C). These results indicate the narrow therapeutic range of 5HN.
We also examined the effect of 5HN in a transformed cell line model of HIV-1 latency, the J-Lat system (22). We measured the expression of viral marker GFP and viral transcripts. 5HN also strongly activated latent HIV-1 in J-Lat cells (Supplemental Figure 6, A and B). Interestingly, although the potency of 5HN to reactivate latent HIV-1 was comparable to that of PMA in our primary cell model, 5HN was less effective than PMA in J-Lat cells. These results highlight the importance of using primary cells to screen for compounds that target latent HIV-1.
5HN does not cause global T cell activation. Agents that induce latent HIV-1 by causing global T cell activation are likely to be too toxic for clinical use. Therefore, we determined whether 5HN activates T cells. Unlike cells stimulated with PMA or anti-CD3 and anti-CD28 antibodies, the 5HN-treated latently infected cells retained the same small size as untreated cells as determined by flow cytometric analysis (Figure 6D). We further showed that 5HN does not cause upregulation of classic T cell activation markers such as CD69, CD25, or HLA-DR on freshly isolated resting CD4+ T cells (Figure 7A). Since the toxicity induced by T cell–activating agents is likely to be due to cytokine release, we determined whether 5HN induced production of T cell cytokines. IL-2 expression was not induced by 5HN, while IFN-γ transcripts were upregulated by approximately 10-fold. However, this level of IFN-γ activation is much less than that induced by costimulation with anti-CD3 and anti-CD28, which caused a greater than 1000-fold upregulation (Figure 7B). Importantly, at concentrations that induce reactivation of latent HIV-1, 5HN did not render uninfected resting CD4+ T cells susceptible to HIV-1 infection (Figure 7C). Finally, 5HN did not stimulate the proliferation of CD4+ T cells. This was evidenced by measurement of DNA/RNA staining (Figure 7D) or the dilution of CFSE in the 5HN-treated cells (Supplemental Figure 7). The above results demonstrate that 5HN did not cause global T cell activation at concentrations that activated latent HIV-1.
5HN does not activate CD4+ T cells. (A) Effects of 5HN on the expression of activation markers in primary resting CD4+ T cells. Freshly isolated CD4+ T cells were treated with the indicated concentrations of 5HN or with anti-CD3 plus anti-CD28 antibodies for 3 days. Expression of activation markers was quantified by flow cytometry. The values indicated the percentage of cells expressing individual markers. Data are mean ± SD of triplicate samples from 1 of 2 independent experiments. (B) Effects of 5HN on transcription of IL-2 and IFN-γ genes. Resting Bcl-2–transduced CD4+ T cells were left unstimulated or were stimulated with 5HN or anti-CD3/anti-CD28. Levels of IL-2 and IFN-γ transcripts in total cellular RNA were quantified by real-time RT-PCR and normalized to β-actin mRNA levels. The fold change relative to unstimulated samples is shown. Data are mean ± SD of triplicate samples from 1 of 2 independent experiments. (C) Effect of 5HN on the susceptibility of freshly isolated resting CD4+ T cells to HIV-1 infection. Cells were incubated with medium alone, 5HN, or anti-CD3/anti-CD28 antibodies for 3 days and were then infected with reporter virus NL4-3-ΔE-GFP. The number in each plot indicates the percentage of GFP-positive cells quantified by flow cytometry. (D) The effects of 5HN on the proliferation of latently infected cells. Cell proliferation was determined using Hoechst 33342/pyronin Y staining for DNA/RNA. The controls for the resting and activated cells are the same as in Figure 2B. The percentage of cells in each quadrant is indicated.
ROS are involved in the reactivation of latent HIV-1 by 5HN. 5HN is a quinone that can be reduced to a semiquinone radical by enzymes such as NADPH oxidoreductase. Under aerobic conditions, the semiquinone radical then generates superoxide anion (O2–) and hydrogen peroxide (H2O2), which induce oxidative stress (56, 57). Because ROS can indirectly activate the host transcription factor NF-κB that can in turn activate latent HIV-1 (58, 59), we hypothesized that induction of oxidative stress by 5HN may play a role in the reactivation of latent virus. To test this hypothesis, we first assessed the effects of 5HN on the production of ROS in freshly isolated primary resting CD4+ T cells using dihydrorhodamine 123 (DHR123). DHR123, a nonfluorogenic dye, is oxidized intracellularly by ROS into the fluorescent dye rhodamine 123 (60). Production of ROS in primary CD4+ T cells was detected within 2 hours of addition of concentrations of 5HN known to reactivate latent HIV-1 (Figure 8A). Because ROS can indirectly activate NF-κB, we next assessed the activation of NF-κB by measuring the nuclear levels of NF-κB using an ELISA-based assay. 5HN activated NF-κB in a dose-dependent manner (Figure 8B). To ensure that 5HN activates NF-κB in primary CD4+ T cells, we also quantified the transcripts of IκBα, an NF-κB–responsive gene, and showed that 5HN stimulates the expression of IκBα (Figure 8C). Antioxidants like N-acetylcysteine (NAC) or pyrrolidine dithiocarbamate (PDTC) suppress the effects of ROS on the activation of NF-κB (59). Consistent with this observation, preincubation of the latently infected cells with NAC or PDTC dramatically suppressed the stimulatory effect of 5HN on the expression of latent HIV-1 (Figure 8D). This result supports the role of ROS in the activation of latent HIV-1 by 5HN.
5HN activates latent HIV-1 via ROS and NF-κB. (A) ROS generation in primary resting CD4+ T cells. Cells were incubated with DHR123 prior to 5HN treatment. DHR123 conversion, an indicator of intracellular ROS, was evaluated by flow cytometry. (B) 5HN activates NF-κB in primary CD4+ T cells. Cells were left untreated, treated with 5HN, or stimulated with anti-CD3/anti-CD28 antibodies. The nuclear NF-κB p65 was quantified and normalized to the total protein concentration of each sample. Results are relative OD450 values. (C) Effects of 5HN on IκBα transcription. Freshly isolated CD4+ T cells were left unstimulated or were stimulated with 5HN for different periods of time. IκBα transcripts in total cellular RNA were quantified by real-time RT-PCR and normalized to β-actin mRNA levels. The fold change is relative to unstimulated samples. (D) Antioxidants NAC and PDTC block 5HN reactivation of latent HIV-1. Latently infected cells were treated with NAC or PDTC 1 hour prior to 5HN treatment. The percentage of the GFP-positive cells was measured using flow cytometry and normalized to that of cells receiving 5HN without prior treatment. (E) NF-κB is involved in the reactivation of latent HIV-1 by 5HN. Bcl-2–transduced cells latently infected with NL4-3-Δ6-drEGFP or mκ2-NL4-3-Δ6-drEGFP were treated with 5HN. GFP-positive cells were measured using flow cytometry and normalized to the maximal percentage of GFP-positive cells in each population following treatment with PMA plus ionomycin. Data in B–E are mean ± SD of triplicate samples from 1 of 2 independent experiments, all of which produced similar results.
To further confirm that NF-κB is required for the activation of latent HIV-1 by 5HN, we mutated the 2 tandem NF-κB–binding sites in the enhancer region of 3′ LTR of pNL4-3-Δ6-drEGFP (mκ2-LTR- NL4-3-Δ6-drEGFP). Following reverse transcription and integration into the genome of infected cells, the 5′ LTR of integrated reporter virus is derived from the 3′ LTR that contains mutated NF-κB–binding sites (61). The response to 5HN in the cells latently infected with mκ2-LTR NL4-3-Δ6-drEGFP was much lower than that in cells infected with wild-type NL4-3-Δ6-drEGFP (Figure 8E), indicating the essential role of NF-κB in the signaling pathways downstream of 5HN. Interestingly, at higher concentrations of 5HN (5 μM), there was partial activation of the mκ2-LTR NL4-3-Δ6-drEGFP reporter virus, suggesting the possibility of NF-κB–independent pathways for the reactivation of latent HIV-1 by 5HN.
PKCθ is a master regulator in TCR signaling pathways (62). Activation of PKC leads to activation of key transcription factors in the immune response, including NF-κB and activator protein 1 (AP-1). Because 5HN activates latent HIV-1 via NF-κB, we determined whether the effects of 5HN depend on PKC. Using the pan PKC inhibitor Gö6983, we found that inhibition of PKC completely suppressed PMA-induced activation of latent HIV-1 and reduced by approximately 50% the response to anti-CD3 and anti-CD28 costimulation. However, it almost had no effect on the activation of latent HIV-1 by 5HN. This suggests that 5HN acts downstream of PKC in the NF-κB signaling pathway leading to the activation of latent HIV-1 (Figure 9A). NFAT, another essential transcription factor for T cell activation, is also known to promote HIV-1 gene expression (63). To exclude the possibility that NFAT plays a role downstream of 5HN, we showed that cyclosporin A (CsA), an inhibitor of the NFAT signaling pathway, does not suppress the effect of 5HN on the reactivation of latent HIV-1, while it inhibits the effect of ionomycin (Figure 9B).
5HN activates latent HIV-1 independent of PKC and NFAT. (A) Effect of a PKC inhibitor on the reactivation of latent HIV-1 by 5HN. Latently infected Bcl-2–transduced cells were incubated with PKC inhibitor Gö6983 1 hour prior to the treatment with each activator. Cells were collected and analyzed for GFP-positive cells after 2 days of incubation. The results were normalized to the effect of anti-CD3 plus anti-CD28 costimulation. Data are mean ± SD of triplicate samples from 1 of 2 independent experiments, all of which produced similar results. (B) Effect of CsA on the reactivation of latent HIV-1 by 5HN. Latently infected Bcl-2–transduced cells were incubated with CsA 1 hour prior to the treatment with each activator. Cells were collected and analyzed for GFP-positive cells after 2 days of incubation. The results were normalized to the effect of PMA treatment. Data are mean ± SD of triplicate samples from 1 of 3 independent experiments.
5HN is also known to inhibit Pin1, a peptidyl-prolyl isomerase that plays an important role in transcription regulation (64, 65). To determine whether inhibition of Pin1 is involved in reactivation of latent HIV-1, we knocked down Pin1 expression in the latently infected Bcl-2–transduced cells using Pin1-specific RNAi. Downregulation of Pin1 did not activate latent HIV-1 (data now shown). We also demonstrated that fredericamycin A, a Pin1 inhibitor (66), failed to induce latent HIV-1 in our in vitro primary cell model (data not shown). Together, these results suggest that inhibition of Pin1 by 5HN is not responsible for reactivation of latent HIV-1.