HIV-1 reservoirs prevent eradication of HIV-1 from infected individuals. They arise at a very early clinical phase and also occur in patients that initiated early antiretroviral therapy (ART) [1,2]. Virus reservoirs are long-lived cells with persisting HIV-1 genomes which support chronic viraemia even in optimally treated patients with undetectable plasma virus levels. Latently infected reservoirs contain silenced virus genomes that can be reactivated by extracellular stimuli or stochastic events. Resting memory CD4+ T cells are currently the best characterized virus reservoirs, but other cells of the immune system like proliferating T cells , macrophages, monocytes or haematopoietic stem cells [4,5] can also form HIV-1 reservoirs. In addition to the immune system, the virus can also invade the brain within a few weeks after primary infection, presumably via the blood–brain barrier [6,7] and can persist in this organ lifelong [8,9]. HIV-1 infection can lead to HIV-associated neurocognitive disorders (HANDs), with a prevalence of up to 50% of HIV-1-infected individuals, irrespective of HAART . Restricted immunological surveillance and longevity of brain cells favour establishment of virus reservoirs in the brain. Ex-vivo studies of brain tissues from HIV-1-infected individuals, as well as studies with cultured human brain cells, demonstrate HIV-1 infection of various brain cell types [8,11–14]. Productive infection was shown for brain macrophages, which include perivascular and non-resident macrophages, as well as resident microglia. In addition, macroglial cells like astrocytes and neural progenitor cells were shown to contain proviral DNA, although these cells rarely displayed HIV-1 production markers. Up to 19% of the astrocyte population in neurocognitively impaired individuals contained HIV-1 DNA , amounting to a substantial population of HIV-1-positive cells, considering the abundance of human astrocytes (total number estimated to be >17 × 109 cells  and Pelvig et al.). Early infection of astrocytes was demonstrated in macaques exposed to simian immunodeficiency virus , and numerous cell culture studies confirmed persistence of HIV-1 in astrocytes and neural progenitor cell populations [13,18,19]. Infected astrocytes exposed to pro-inflammatory cytokines were capable of releasing infectious virus into the culture supernatant  and of transmitting virus to T cells and monocytes . Together, these findings suggest a key role for astrocytes and their precursors as reservoirs with latent HIV-1 in the brain.
Currently, the ‘shock-and-kill’ strategy is under intensive discussion for eliminating virus reservoirs. This strategy proposes killing of reservoir cells by compound-induced virus reactivation, combined with HAART intensification, to prevent de-novo infection [21,22]. Potential effects of ‘shock-and-kill’ strategies on HIV-1 reservoirs in the brain are still unknown. This issue is extremely critical for several reasons , including potential neurotoxicity of HIV-1 proteins [24,25]; poor penetration of antiretroviral drugs into the brain  and loss of irreplaceable cells in the brain .
Studies with CD4+ T-cell models of HIV-1 latency [27–29] have identified several pharmacological agents for activation of latent HIV-1 [28,30], as well as complex mechanisms involved in suppressing HIV-1 expression during latency in CD4+ T cells . Whereas a model for latent HIV-1 infection in microglial cells has been established , a model for easy monitoring of HIV-1 latency in macroglial cells of neuro-ectodermal lineage is still lacking.
Here, we report the establishment of a model for transcriptional HIV-1 latency in neuro-ectodermal brain cells, using a human neural stem cell (HNSC) line that can be cultured as self-renewing progenitor populations, as well as differentiated astrocyte-enriched cell populations [19,33]. We show that selected latency reversing agents (LRAs) can increase virus expression in the HNSC model and provide proof of concept for inhibition of HIV-1 reactivation in macroglial brain reservoirs by treatment with small molecules. Thus, we propose incorporation of the HNSC model for the development of HIV-1 cure strategies.
Materials and methods
HNSC.100 is a clonal cell line established from human fetal neural stem cells [34,35]. HNSC cells were cultivated on poly-L-lysine-coated plastic ware and incubated at 37°C and 5% CO2. Basal medium for proliferating HNSC.100 consisted of Dulbeccos Modified Eagle Medium (DMEM) F-12, 1% bovine serum albumin (BSA), 1× N2 supplement, 5 mmol/l HEPES, 0.5% fetal calf serum (FCS) (Gibco, Darmstadt, Germany). 1% PenStrep (Sigma–Aldrich, Taufkirchen, Germany), 20 ng/ml basic fibroblast growth factor 2 (FGF-2) and 20 ng/ml epidermal growth factor (EGF) (TebuBio, Offenbach, Germany). For differentiation, HNSC.100 cells were incubated for at least 2 weeks in the basal medium without growth factors. All other cells are described in the supplemental digital content (SDC) 1 (http://links.lww.com/QAD/A694).
Vesicular stomatitis virus (VSV)-G protein (VSV-G)-pseudotyped lentiviral particles were produced in human embryonic kidney (HEK) 293T cells co-transfected with pSG3.1Δenv or pNL4-3-deltaE-EGFP and pMD2.G, using Extreme Gene HP (Roche, Mannheim, Germany) according to the manufacturer's protocol (for plasmid details, see SDC 1). Virus-containing supernatants were harvested 72 h after transfection and concentrated with Amicon Ultra 100 K centrifugal filters (Millipore, Darmstadt, Germany). Virus preparations were diluted with HNSC basal medium, and viral titres were determined by HIV-1 p24 antigen capture assay according to the manufacturer's protocol (ABL Inc., Rockville, Maryland, USA).
HNSC.100 cell lines were exposed to VSV-G-pseudotyped SG3.1Δenv virus particles at 2.2 pg p24/cell for 48 h, or to NL4-3-deltaE-EGFP virus particles at 0.4 pg p24/cell were treated as described for 6 h. Subsequently, the cells were washed twice with medium to remove inoculum, and incubation was continued in fresh medium. Cells were harvested and stored at −20°C for isolation of nucleic acids or fixed with 2% paraformaldehyde (PFA) overnight at 4°C for flow cytometry analysis.
HNSCLatGFP1.2 cells were transfected with pMD2.G or a standard yellow fluorescent protein (YFP)-expressing plasmid, using Extreme Gene HP (Roche) as transfection reagent. Twenty-four-hour post-transfection, the medium was changed, and the cells were treated with compounds for 24 h before the medium was changed again. After additional 24 h, supernatants were collected and the cells were harvested for flow cytometry analysis. The p24 levels in the supernatants were determined by HIV-1 p24 antigen capture assay according to the manufacturer's protocol (ABL Inc.). To detect infectious virus, 20 μl of HNSC supernatants were used to inoculate 1 × 104 LC5-RIC cells. After 48 h, the red fluorescence signal was measured with a Tecan infinite M200 (Tecan, Männedorf, Switzerland) (for details see )
Flow cytometry analysis
Flow cytometry analysis was performed with a FACS CANTO II and the FACSDiva software (BD Bioscience, Heidelberg, Germany) using PFA-fixed cells.
Quantitative polymerase chain reaction (qPCR)
Levels of HIV-1 DNA or transcripts were quantified by quantitative polymerase chain reaction (qPCR) with the Roche LightCycler 1.5 System, using LightCycler FastStart DNA Master SYBR Green I-Kit and standard LightCycler software (Roche Diagnostics, Mannheim, Germany). RNA levels were quantified by relative qPCR with cDNA generated by reverse transcription with specific primers for HIV-1. HIV-1 DNA loads were determined by absolute qPCR quantification, using the TH4-7-5 cell line as external standard (for details and primer sequences, see SDC 1).
Immunofluorescent stainings and confocal microscopy
HNSCLatGFP1.2 cell lines were seeded on poly-L-lysine-coated glass cover slips. For detection of intracellular localization of p65, cells were left untreated or treated with nuclear factor-kappaB (NF-κB) inhibitors for 2 h before 10 ng/ml tumour necrosis factor (TNF)-alpha was added for 30 min. For GFP expression analysis, cells were left untreated, treated with 10 ng/ml TNF-alpha or 5 μmol/l suberoylanilide hydroxamic acid (SAHA) for 30 h. After treatments, cells were washed and fixed with 2% PFA for 4°C overnight before immunofluorescent stainings were performed as described in SDC 1. Localization of p65 or GFP expression was then analysed by fluorescence microscopy (Nikon TiE equipped with Perkin Elmer UltraView Vox System) using the Volocity 6.2.1-software (Perkin Elmer, Rodgau, Germany) (for details, see SDC 1).
Treatments with test compounds
TNF-alpha, Withaferin A (WTA), valproic acid (VPA), sodium butyrate (NaB), Prostratin A (Pro), 5-azacytidine (5-aza), Trichostatin A (TSA), phorbol 12-myristate 13-acetate (PMA), ammonium pyrrolidinedithiocarbamate (PDTC) and BMS-345541 were purchased from Sigma–Aldrich. SAHA/Vorinostat and Flavopiridol were obtained from the NIH AIDS Reagents Program (Cat. Nr.: 12130 and 9925). Bortezomib/Velcade (Biomol, Hamburg, Germany), R-Roscovitine (Adipogen AG, San Diego, California, USA), Fascaplysin (Santa Cruz Biotechnology, Heidelberg, Germany), PHA767491 hydrochloride (Tocris Bioscience, Wiesbaden, Germany) and all other substances were solved in DMSO and stored at −20°C. TNF-alpha was solved in PBS. LRAs were used at concentrations reported to reverse latency in HIV-1 latency immune cell models (compiled in Table S1, SDC2, http://links.lww.com/QAD/A694). Inhibitory compounds were used at concentrations reported to be effective in other studies (see Tables S2, SDC2, http://links.lww.com/QAD/A694 and S£, SDC2, http://links.lww.com/QAD/A694). Cell viabilities in treated proliferating and differentiated HNSCLatGFP1.2 populations were determined by MTT assay as described in Kremb et al. and were generally at least 80% (see Fig. S1). Toxicities of compounds during J-Lat 8.4 treatment were determined by CellTox Green Cytotoxicity Assay (Promega, Mannheim, Germany) and were 20% or less (Fig. S2).
Chromatin immunoprecipitation assays
HNSCLatGFP1.2 and J-Lat 8.4 cells were treated with 10 ng/ml TNF-alpha or were left untreated for 30 min before chromatin was prepared from 1 × 107 cells. Chromatin immunoprecipitation (ChIP) assays were performed with the Imprint Chromatin Immunoprecipitation Kit (Sigma–Aldrich) according to the manufacturer's protocol (for details, see SDC 1).
Statistical analysis was performed with Prism software (Graph Pad Software, San Diego, California, USA) using the one-way analysis of variance (ANOVA) test for pair-wise comparisons of selected columns referring to values of differently treated/differentiated cells to values of untreated/proliferating cells or values of untreated, reactivated samples to treated, reactivated cells, respectively. Statistical significance was also expressed (*P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001; NS = not significant).
Establishment of human neural stem cell progenitor populations with transcriptionally silenced HIV-1
To generate an experimental model for HIV-1 latency, proliferating HNSC.100 populations were infected with VSV-G-pseudotyped envelope (Env)-defective HIV-1. Monitoring quantities of HIV-1 nucleic acids for over 50 days in HNSC populations infected with SG3.1ΔEnv (HNSCLat) showed that HIV-1 RNA levels decreased to undetectable levels within 13 days, whereas HIV-1 DNA levels remained stable from day 20 onward (Fig. 1a, b). Similarly, HNSC.100 populations infected with the green fluorescent protein (GFP)-reporter virus NL4-3-deltaE-EGFP (HNSCLatGFP1.2) showed declining proportions of GFP-positive cells, whereas the copy numbers of HIV-1 DNA again remained stable (Fig. 1c, d). These results confirm generation of HNSC.100 populations with persisting, quiescent HIV-1 proviruses.
Effects of latency reversing agents on provirus quiescence in human neural stem cell progenitor populations
To investigate reactivation of HIV-1, HNSCLat and HNSCLatGFP1.2 were exposed to a panel of potential LRAs at concentration reported to reactivate HIV-1 in latently infected T cells (Table S1, SDC2). Initial experiments showed that exposure to TNF-alpha or SAHA clearly increased GFP expression in HNSCLatGFP1.2 cultures (Fig. 2a, b). In contrast, treatment with other compounds, including several histone deacetylase inhibitors (NaB, VPA and TSA), protein kinase C agonists (Pro, PMA) and the DNA methyltransferase inhibitor 5-aza led to no or only very low HIV-1 activation in both HNSCLatGFP1.2 and HNSCLat populations (Fig. 2b, c).
Next, we investigated possible synergistic effects of the compounds. Co-treatment of HNSCLatGFP1.2 with TNF-alpha and SAHA resulted in about 50% GFP-positive cells, compared to approximately 10% obtained by treatment with the single compounds (Fig. 2d). This indicated that at least 50% of the HNSCLatGFP1.2 population contained inducible HIV-1 proviral DNA. Synergistic effects were also observed for co-treatment with TNF-alpha and VPA, although VPA treatment alone did not activate HIV-1 expression. Co-treatment with TNF-alpha and SAHA also markedly increased levels of Gag-p24 in the culture supernatant of HNSCLatGFP1.2 cells, confirming production of virus-like particles (Fig. 2e). Release of infectious virus particles by HNSCLatGFP1.2 cells transfected with plasmids directing expression of VSV-G envelope proteins was also measurable upon co-treatment (Fig. 2f).
These results demonstrate differential effects of known LRAs on latent HIV-1 in HNSC populations.
HIV-1 latency in differentiated human neural stem cells
To investigate HIV-1 latency in differentiated HNSC populations, HNSCLatGFP1.2 were cultured without growth factors for at least 2 weeks. Differentiation was verified by up-regulated expression of the astrocyte marker GFAP (Fig. S3, SDC3) and by termination of cell proliferation beginning at day 13 after growth factor withdrawal (Fig. 3a). qPCR analysis confirmed that, after growth factor withdrawal (Fig. 3b) HIV-1 DNA levels remained constant over time (Fig. 3b). Treatment of differentiated HNSCLatGFP1.2 with the same panel of LRAs and under the same conditions used to test activation of HIV-1 latency in proliferating HNSCLatGFP1.2 populations revealed elevated GFP expression only after TNF-alpha treatment (Fig. 3c, d). In agreement, HIV-1 transcript levels in differentiated HNSCLat were strongly increased by TNF-alpha treatment, whereas SAHA had only moderate effects on transcript levels, and VPA was ineffective to induce HIV-1 transcription (Fig. 3e). Combined treatment of HNSCLatGFP1.2 with TNF-alpha and SAHA yielded similar GFP induction levels as single treatment with TNF-alpha (Fig. 3f), indicating that TNF-alpha and SAHA do not synergistically activate HIV-1 in differentiated HNSC populations. These results show that HNSC populations maintain latent HIV-1 proviruses after differentiation, and are less responsive to HIV-1 reactivation by SAHA than progenitor populations.
Involvement of the NF-κB pathway in tumor necrosis factor-alpha-mediated HIV-1 induction in latently infected human neural stem cell populations
Activation of the NF-κB pathway with binding of p65/RelA to NF-κB-binding sites in the HIV-1 ong terminal repeat (LTR) is a key feature of HIV-1 transcription initiation in latently infected T cells [31,37,38]. Since TNF-alpha is an inducer of the NF-κB pathway, we elucidated a possible involvement of NF-κB in HIV-1 activation in HNSCLatGFP1.2 by investigating nuclear translocation and binding of p65 to the HIV-1 in TNF-alpha-treated cells. Immunofluorescence analysis revealed that TNF-alpha treatment strongly increased nuclear localization of p65, compared to untreated cells (Fig. 4a). Next, we compared levels of p65 associated with HIV-1 LTR sequences in TNF-alpha-treated and untreated HNSCLat and HNSCLatGFP1.2 populations by ChIP assays. TNF-alpha treatment clearly increased the association of p65 to the HIV-1 LTR compared to untreated cells (Fig. 4b, c). These results confirmed that TNF-alpha treatment of latently infected HNSC populations activated the NF-κB signalling pathway and increased association of p65 with the HIV-1 LTR.
Small molecule inhibitors of NF-κB signalling and Cdk9 diminish activation of latent HIV-1
Since TNF-alpha-mediated reactivation of HIV-1 in HNSC populations depends on NF-κB signalling, we investigated whether NF-κB inhibitors can prevent TNF-induced HIV-1 activation. To this end, HNSCLatGFP1.2 cells were treated with NF-κB inhibitors (BMS-345541; Bortezomib, PDTC and Withaferin A) for 2–4 h (for concentrations, see Table S2, SDC2) before NF-κB was activated by TNF-alpha. Immunofluorescent stainings showed that TNF-alpha-exposed cells treated with NF-κB inhibitors had lower levels of nuclear p65 than cells exposed to TNF-alpha without inhibitor treatment (Fig. 5a). NF-κB inhibitors did not affect p65 localization in cells which were not exposed to TNF-alpha. Next, we examined the effect of NF-κB inhibition on TNF-alpha-mediated activation of latent HIV-1 in HNSCLatGFP1.2 populations. All inhibitors reduced the proportions of GFP-positive cells in both progenitor and differentiated HNSCLatGFP1.2 populations compared to populations exposed to TNF-alpha alone (Fig. 5b). In progenitors, bortezomib, WTA, PDTC or BMS-345541 reduced HIV-1 activation levels to less than 40%. In differentiated HNSCLatGFP1.2 cells, treatment with BMS-345541 most effectively suppressed HIV-1 activation. For further proof of concept, we investigated whether these inhibitors could also prevent reactivation of HIV-1 in a Jurkat T-cell model for post-integration latency (J-Lat 8.4). Indeed, all NF-κB inhibitors potently reduced TNF-alpha mediated reactivation in J-Lat 8.4 cells (Fig. 5c).
Suberoylanilide hydroxamic acid reactivated HIV-1 in HNSC progenitor populations much more potently than other HDAC inhibitors. Recent studies in proliferating T cells link HIV-1 activation by SAHA with the activation of the positive transcription elongation factor b (p-TEFb), which is a complex of the cyclin-dependent kinase Cdk9 and cyclin T1 or T2 [39,40]. Thus, we investigated whether exemplary Cdk inhibitors can reduce HIV-1 reactivation by SAHA in proliferating HNSCs. Therefore, proliferating HNSCLatGFP1.2 were either pre-treated with Cdk9 inhibitors (i.e. Flavopiridol, Roscovitine and PHA-767491) [41–43] or with the highly selective Cdk4 inhibitor Fascaplysin for 2–3 h (for concentrations see Table S3, SDC2) before HIV-1 expression was activated with SAHA. Pre-treatment with Roscovitine, Flavopiridol and PHA-767491 substantially diminished HIV-1 reactivation by SAHA, with Flavopiridol showing the strongest inhibition (Fig. 5d). In contrast, treatment with Fascaplysin did not reduce HIV-1 reactivation by SAHA. Cdk9 inhibitor treatments also reduced HIV-1 reactivation by SAHA in the J-Lat model, whereas Fascaplysin was also ineffective (Fig. 5e). These results demonstrate that small molecules that interfere with NF-κB signalling or Cdk9 activity can potently inhibit TNF-alpha or SAHA-induced activation of latent HIV-1 in proliferating and terminally differentiated HNSCLatGFP1.2, as well as in latently infected T cells.
Cells of the neuro-ectodermal lineage, particularly astrocytes and their precursors, are key candidates of HIV-1 reservoirs in the brain. Here, we report a novel model for HIV-1 proviral latency that consists of human neural progenitor cell populations (HNSC.100) carrying stable envelope-defective HIV-1 proviral genomes. The HNSC model has several properties that support its use in investigating HIV-1 latency in the brain. It mirrors the reactivation potential of an entire cell population, bypassing heterogeneity of latency responses in clones of individually selected cells [29,44]. Furthermore, the HNSC model allows investigation of HIV-1 latency in dividing progenitor as well as in non-dividing, differentiated brain cell populations. This is important since the astrocyte population in the brain consists of cells at different developmental stages and with different mitotic potentials [45–47].
Macroglial HIV-1 reservoirs show both differences and similarities to T-cell reservoirs in their sensitivities towards individual LRAs. Whereas several LRAs with high reactivation potential in T-cell reservoirs (Table S1, SDC2) did not reactivate HIV-1 in HNSCs, the pro-inflammatory cytokine TNF-alpha potently induced latent HIV-1 also in HNSCs. TNF-alpha is increased in different body compartments of HIV-1-infected individuals, including the brain [48,49], and is a hallmark of the chronic neuroinflammation accompanying HIV-1 infection of the brain [7,50]. Activation of latent HIV-1 by TNF-alpha in progenitor and terminally differentiated HNSC, as well as in T cells, strengthens its role as a ‘pan’ inducer of transcriptionally latent HIV-1 in different virus reservoirs.
Suberoylanilide hydroxamic acid is a primary LRA candidate for clinical flush-out of HIV-1 reservoirs and has been shown to activate virus expression in HIV-1-infected individuals  and in animal models . Studies in rodents also support access of systemically administered SAHA to the brain . SAHA potently reactivated HIV-1 in proliferating HNSCs, but showed reduced capacity in terminally differentiated HNSCs. This suggests differential responses of subsets of latently infected brain reservoir populations to SAHA treatment. Selective latency reversing activity of SAHA may also occur in T-cell populations. Thus, SAHA-reversed HIV-1 latency in only a minor fraction of resting CD4+ T cells derived from patients , but showed efficient latency reversing activity in various T-cell lines . SAHA's weak reactivation potential in non-proliferating cells may be caused by low expression or activity of HIV-1 key transcription factors in these cells. Thus, resting CD4+ T cells express only low levels of cyclin T1, which is a component of active p-TEFb complexes  and is essential for HIV-1 transcription. Alternatively, non-dividing cells may increase expression of HIV-1 transcription inhibitors like the bromodomain-containing protein 4 (BRD-4) or the negative elongation factor NELF, which are also linked to p-TEFb activity [57,58]. In proliferating HNSCs, co-treatment with SAHA and TNF-alpha led to substantially higher HIV-1 reactivation levels than treatment with single compounds. This synergism indicates that multiple cellular pathways contribute to and cooperate in reactivation of HIV-1 in proliferating macroglial reservoirs.
Small molecules that target the NF-κB pathway or Cdk9 inhibited HIV-1 reactivation in latently infected HNSCs and in the J-Lat 8.4 model of latently infected T cells. Thus, our study supports the existence of common cellular effectors/pathways for the control of HIV-1 expression and latency in the lymphoid system and the brain. Drugs targeting these pathways could prevent HIV-1 reactivation from multiple virus reservoirs and thus could be part of functional cure strategies. These primarily aim at maximum suppression of viraemia rather than elimination of cellular reservoirs, and may therefore be safer for organs with limited cell replacement like the brain. Several of the drugs used here are already clinically relevant: The Cdk inhibitors Roscovitine and Flavopiridol are in clinical development for treatment of haematologic malignancies . The proteasomal inhibitor Bortezomib (Velcade/PS-341) is approved for the treatment of multiple myelomas and mantle cell lymphoma . Furthermore, Roscovitine, Flavopiridol and Bortezomib inhibit HIV-1 replication in various experimental models [60,61]. However, the suitability of these drugs for clinical applications in HIV-1-infected individuals needs to be established in future studies.
We conclude that reactivation of HIV-1 latency in macroglial reservoirs displays both similarities and differences to T-cell reservoirs. Similarities include common cellular pathways for regulation of HIV-1 transcription. These provide targets for simultaneous manipulation of HIV-1 latency in multiple reservoirs. Our study supports blocking activation of HIV-1 proviruses as a strategy for functional HIV-1 cure and provides a powerful model for comparison of latency responses in the brain and the immune system.
The authors thank Ulrike Protzer for continuous support and encouragement. Furthermore, we thank Johanna Goetz for expert technical assistance. We especially thank Alberto Martinez-Serrano for providing the HNSC.100 cell line and the NIH AIDS Reagents Program for providing the plasmids pSG3.1Δenv and pNL4-3-deltaE-EGFP, the J-Lat 8.4 cell line and the compounds SAHA and Flavopiridol.
Author contributions: Conceived and designed the experiments: M.S., B.T., M.M., M.H., R.B.W.; performed experiments: M.S., B.T., M.M., C.Z.; analysed and interpreted the data: M.S., B.T., M.M., M.H.; conceived, drafted and finalized the manuscript: M.S., R.B.W.
Conflicts of interest
The authors have no conflict of interest.
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