Gene Therapy (2014) 21, 490–495 © 2014 Macmillan Publishers Limited All rights reserved 0969-7128/14 www.nature.com/gt
ORIGINAL ARTICLE
Specific reactivation of latent HIV-1 with designer zinc-finger transcription factors targeting the HIV-1 5′-LTR promoter P Wang1, X Qu1, X Wang1, X Zhu1, H Zeng1, H Chen2 and H Zhu1 HIV-1 latency remains the primary obstacle to the eradication of this virus. The current latency-reversing agents cannot effectively and specifically eliminate latent HIV-1 reservoirs. Therefore, better approaches are urgently needed. In this study, we describe a novel strategy to reactivate latent HIV-1 using zinc-finger transcription factors composed of designer zinc-finger proteins and the transcriptional activation domain VP64. For the first time, we demonstrate that ZF-VP64 with HIV-1 long terminal repeat (LTR) promoter-specific affinity could significantly reactivate HIV-1 expression from latently infected cells without altering cell proliferation or cell cycle progression. We also provide evidence that the reactivation of HIV-1 by ZF-VP64 occurs through specific binding to the 5′-LTR promoter. Our results demonstrate the potential of this novel approach for anti-HIV-1 latency therapy. Gene Therapy (2014) 21, 490–495; doi:10.1038/gt.2014.21; published online 13 March 2014
INTRODUCTION Despite being potent and life prolonging, the current combination antiretroviral therapy is not curative and does not eradicate HIV-1 infection. In long-lived latent reservoirs, the integrated HIV-1 provirus remains transcriptionally silent but replication competent, and the interruption of treatment inevitably results in a rapid rebound of viremia.1–5 Therefore, these latent reservoirs remain the primary obstacle to eradicating HIV-1 infection. Although the mechanisms contributing to the maintenance of HIV-1 latency are not completely understood, the majority involve the suppression of transcription.6 One strategy for eliminating latent HIV-1 reservoirs, termed ‘shock and kill’, which involves reactivating these reservoirs by inducing the transcription of the quiescent provirus followed by the elimination of reactivated virus-producing cells by viral cytopathic effect or host immune response, is currently the most widely discussed approach.1,7 Many novel latency-reversing agents have been identified by mechanism-directed approaches or large-scale screening. Some of these drugs have already entered clinical testing in humans; however, the results have been mixed or unsatisfactory.5,6 Potential limitations to the use of these drugs to reactivate latent HIV-1 include toxicity, a lack of target specificity and the development of acquired drug resistance. Most existing drugs cannot be targeted to HIV-1-infected cells or to the proviral genome; thus, innovative approaches involving HIV-1 specificity are urgently needed to address these issues. Artificial transcription factors are proteins designed to bind to the promoter region of a gene of interest and modulate target gene expression. This method has been applied to regulate endogenous gene expression in a number of instances.8–13 An artificial transcription factor is typically composed of a DNA-binding domain to bind to the promoter of the target gene, an effector domain (ED) to up- or downregulate the target gene and a nuclear localization signal to deliver the artificial transcription factors into the nuclei.14 Because of their DNA-binding selectivity and modular organization, the Cys2His2-type zinc-finger
proteins (ZFPs) are the most frequently used DNA-binding domains in ATFs.14,15 In zinc-finger transcription factors (ZF-TFs), DNA-binding domains can be linked to a variety of EDs with different functions, such as activator domains (VP16,16 VP6417 or p6518), repressor domains (KRAB19 or SID20) or DNA-modifying domains (methyltransferase,21 integrase22 or nuclease23). The control of gene expression with ZF-TFs can be applied to a broad range of drug discovery and therapeutic applications.24 Several reports have described the fusion of the KRAB repressor domain to ZFPs to repress the transcription and replication of HIV-1.25–28 However, to our knowledge, there are no applications of ZF-TFs with activator domains to reactivate latent HIV-1. Herein, we describe a novel approach using ZF-TFs to specifically recognize the HIV-1 long terminal repeat (LTR) promoter region and activate HIV-1 expression. RESULTS Construction of ZF-VP64 targeting the HIV-1 LTR promoter The HIV-1 LTR is highly conserved across all HIV-1 genomes; therefore, it represents an attractive and ideal anti-HIV-1 target. To design zinc-finger motifs specifically targeting the HIV-1 LTR, the DNA sequence of HIV-1 strain NL4-3 was submitted to SigmaAldrich (Shanghai, China), and four ZFP expression plasmids targeting the HIV-1 LTR were designed and assembled. To investigate whether these ZFPs could be used to engineer ZF-TFs to specifically reactivate HIV-1 from latency, the transcriptional activator VP64, a tetrameric repeat of the VP16’s minimal activation domain,17 was inserted downstream of the zinc-finger motifs. Thus, we generated four ZF-TFs: ZF1-VP64, ZF2-VP64, ZF3VP64 and ZF4-VP64. Each protein contained one zinc-finger DNAbinding module with one triple-FLAG tag and a nuclear localization signal at its N-terminus and one VP64 domain at its C-terminus (Figure 1a and Supplementary Figure S1). All the LTR target sites of the ZF-VP64 were near the transcription start site (+1): the target sites of ZF1-VP64 and ZF2-VP64 were downstream
1 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China and 2Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Boston, MA, USA. Correspondence: Dr H Zhu, State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China. E-mail: [email protected] Received 9 September 2013; revised 9 January 2014; accepted 24 January 2014; published online 13 March 2014
Specific reactivation of latent HIV by ZF-TFs P Wang et al
491 HIV-1 LTR transcription with Tat. HIV-1 LTR-luc reporter vector was cotransfected with ZF3-VP64 or ZF4-VP64 in the presence or absence of a Tat expression vector. The results showed that Tat alone induced an approximately 20-fold increase in luciferase reporter expression; when Tat was coexpressed with ZF3-VP64 or ZF4-VP64, the induction level increased to as high as 35–50-fold (Figure 2b). These data indicate that ZF-VP64 and Tat function synergistically to promote HIV-1 LTR transcription.
Figure 1. Basic structure and target sites of ZF-VP64. (a) Schematic representation of ZF-VP64-mediated HIV-1 expression activation. A ZF-VP64 is composed of a zinc-finger DNA-binding domain, a VP64 activator domain, a triple FLAG tag and an NLS. The red box in the 5′ LTR of the HIV-1 genome indicates the ZF-VP64 target sites. (b) Binding sites of ZF-VP64 to the HIV-1 LTR. The DNA sequence shown is from the 5′-LTR of HIV-1 (HXB2 reference strain, GenBank accession no. K03455). The nucleotides are numbered relative to the transcription start site (+1). The sites targeted by ZF-VP64 are underlined. Binding sites for NF-κB and Sp1 are also highlighted.
of the transcription start site, and the target sites of ZF3-VP64 and ZF4-VP64 were upstream of the transcription start site (Figure 1b). Potential of ZF-VP64 to activate the HIV-1 LTR in transient luciferase reporter assays To determine whether the designed ZF-VP64 constructs were able to activate HIV-1 expression from the 5′-LTR promoter, we cotransfected HEK-293T cells with the ZF-VP64 expression vectors and a reporter vector comprising the luciferase gene driven by the HIV-1 LTR promoter (LTR-luc). The luciferase levels were measured 48 h post transfection. As a negative control, the plasmid backbone lacking the zinc-finger domain (the ZF empty vector) was also tested. In this transient luciferase reporter assay, all four ZF-VP64 vectors successfully mediated the transcriptional activation of the HIV-1 LTR. Specifically, ZF3-VP64 produced a fourfold induction in reporter expression, and ZF4-VP64 generated a 2.5-fold induction. However, an induction of less than twofold was produced by ZF1-VP64 and ZF2-VP64. As expected, no significant activation was observed for the ZF empty vector (Figure 2a). Moreover, the potential of ZF-VP64 to activate HIV-1 LTR was dose-dependent and the minimal amount of DNA necessary for induction was about 50 ng per well of 24-well plates (Supplementary Figure S2). Based on these data, we chose ZF3-VP64 and ZF4-VP64 to use in further experiments. Transcription initiated from the HIV-1 LTR promoter is controlled predominantly at the level of elongation. By binding to the transactivation response element (TAR), the viral transactivator Tat can stimulate viral gene expression by recruiting transcription elongation complexes to the promoter.29–31 Therefore, we next asked whether a ZF-VP64 vector could synergistically promote © 2014 Macmillan Publishers Limited
ZF-VP64 reactivates HIV-1 in different latently infected cells The regulation of gene expression in a transient assay may differ from that observed with a genomic target because the reporter plasmid is not packaged into the chromatin in the same manner as the chromosomal gene.28 Therefore, we performed a more relevant assay to test the ability of ZF-VP64 to induce HIV-1 expression in latently infected cells. We first used C11 cells, a clonal, latently infected Jurkat T cell line established in our laboratory. These cells contain a single provirus integrated into an intron of RNPS1 and a green fluorescent protein (GFP) gene under the control of the HIV-1 LTR.32 The C11 cells remained untransfected (mock) or were nucleofected with the ZF empty vector or a ZF3-VP64 or ZF4-VP64 expression vector. The percentage of GFP-positive cells (indicating the transcriptional activity of the HIV-1 promoter) was measured by flow cytometry. At 48 h post transfection with ZF3-VP64 or ZF4-VP64, the percentage of GFP-positive cells was found to be as high as 32.65% or 27.12%, respectively, which was approximately 10-fold that of the untransfected cells (3.15%). Furthermore, the induction of GFP expression persisted for at least 4 days (Figures 3a and b). Similar results were obtained from another cell line latently infected with HIV-1 (J-Lat clone A10.6 cells), albeit with a weaker induction potential for ZF4-VP64 (Figure 3c). In both cell lines, the expression of HIV-1 by the control cells transfected with the ZF empty vector was higher than expected, perhaps due to a partial activation of these cells by nucleofection.33 These results suggested that ZF-VP64 could effectively reactivate HIV-1 expression from latently infected cells and could be further investigated as a novel antiretroviral agent. ZF-VP64 does not affect cell proliferation or cell cycle progression We next examined whether ZF-VP64 stimulated cell proliferation or affected cell cycle progression. The CCK-8 assay kit was used to evaluate cell proliferation 72 h after Jurkat T cells or HEK-293T cells were transfected with the ZF empty vector or with the ZF3-VP64 or ZF4-VP64 expression vector. The results showed that ZF-VP64 had no significant effect on cell proliferation (Figure 4a and Supplementary Figure S3a). The potentialof ZF-VP64 to induce cell cycle progression was further investigated by staining these cells with propidium iodide and analyzing the DNA content using flow cytometry. Using this method, the progression of cells through the different cell cycle phases (G0/G1, S and G2/M) can be assessed. Compared with the mock-treated cells, the transfection of the ZF empty vector, ZF3-VP64 or ZF4-VP64 did not affect cell cycle progression (Figure 4b and Supplementary Figure S3b). ZF3-VP64 specifically binds to the 5′-LTR promoter to reactivate HIV-1 We next investigated whether ZF-VP64 reactivated HIV-1 through a direct interaction with the 5′-LTR; ZF3-VP64 was selected for this assay. First, we deleted the ZF3-VP64 target site from the HIV-1 LTR-luc reporter vector (Figure 5a). Then, we transfected HEK-293T cells with the ZF3-VP64 expression vector and the wild-type HIV-1 LTR-luc reporter vector (LTR-luc) or the HIV-1 LTR-luc reporter vector lacking the ZF3-VP64 target site (LTR(ΔZF3)-luc). ZF3-VP64 induced an approximately fourfold increase in the stimulation of the wild-type LTR-luc reporter relative to the ZF empty vector Gene Therapy (2014) 490 – 495
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Figure 2. ZF-VP64-mediated activation of the HIV-1 LTR in transient transfection assays. (a) Luciferase assays for HIV-1 5′-LTR activity in transiently transfected cells comparing the activation potentials of various designer ZF-VP64 transcription factors. HEK-293T cells were cotransfected with pcDNA3.0 (mock), the ZF empty vector or a ZF-VP64 plasmid and the LTR-luc reporter plasmid for 48 h, and the relative luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). (b) Luciferase assays for HIV-1 5′-LTR activity in the presence or absence of Tat and the ZF3-VP64 or ZF4-VP64 activator construct were performed in transiently transfected cells. The data represent the mean ± s.d. of three independent experiments.
Figure 3. ZF-VP64-mediated activation of the HIV-1 provirus in latently infected cells. (a) C11 cells were untransfected (mock) or nucleofected with 2 μg of the ZF empty vector or a ZF-VP64 plasmid. At 48 h post transfection, the percentage of GFP-positive cells was measured by flow cytometry. The results are presented as fluorescence histograms. (b) The time-dependent effects of ZF-VP64-mediated activation of the HIV-1 provirus in C11 cells or (c) J-Lat clone A10.6 cells. C11 cells or J-Lat clone A10.6 cells were untransfected (mock) or nucleofected with 2 μg of the ZF empty vector or a ZF-VP64 plasmid for the indicated times. The GFP expression was monitored in gated live cells at 24, 48, 72 and 96 h by standard flow cytometric techniques. The data represent the mean ± s.d. of three independent experiments.
control, similar to the previous results, but failed to activate the LTR(ΔZF3)-luc reporter (Figure 5b), indicating that the induction of HIV-1 by ZF3-VP64 occurred through specific binding to the 5′-LTR promoter. Gene Therapy (2014) 490 – 495
To confirm that ZF3-VP64 bound to HIV-1 LTR in vivo, a chromatin immunoprecipitation (ChIP) assay was performed. C11 cells were nucleofected with ZF3-VP64 or the ZF empty vector and crosslinked with formaldehyde, and the chromatin © 2014 Macmillan Publishers Limited
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Figure 4. ZF-VP64 did not affect cell proliferation or cell cycle progression. (a) Jurkat T cells were nucleofected with pcDNA3.0 (mock), the ZF empty vector or a ZF-VP64 plasmid for 72 h, and cell viability was measured using a CCK-8 kit (Dojindo). The division of the OD450 between the treated and control groups indicated the percentage of cell viability. The data represent the mean ± s.d. of three independent experiments. (b) Jurkat T cells were nucleofected with pcDNA3.0 (mock), the ZF empty vector or a ZF-VP64 plasmid for 72 h, and cell cycle progression was analyzed by propidium iodide staining and flow cytometry. Similar results were observed in three independent experiments.
fragments were immunoprecipitated with an antibody that recognizes the FLAG tag at the N-terminus of ZF3-VP64 or with normal mouse IgG. DNA was isolated from the immunoprecipitates and was analyzed by PCR using primers specific for HIV-1 LTR. We observed that the anti-FLAG antibody was able to efficiently immunoprecipitate the HIV-1 LTR promoter region from the ZF3-VP64-transfected cells but not from the ZF empty vectortransfected cells. No immunoprecipitated DNA fragments were detected by PCR in the normal mouse IgG controls (Figure 5c). These findings further confirmed that the observed reactivation of HIV-1 was by a direct interaction between ZF3-VP64 and the 5′-LTR. DISCUSSION None of the current latency-reversing agents can safely and effectively eliminate the long-lived HIV-1 reservoirs; therefore, © 2014 Macmillan Publishers Limited
new anti-latency approaches need to be explored. ZF-TFs using ZFPs as DNA-binding domains are useful for validating genes as drug targets. In this study, we explored a novel strategy to reactivate latent HIV-1 using ZF-TFs composed of designer ZFPs and the transcriptional activation domain VP64. To our knowledge, this was the first demonstration that ZF-VP64 with HIV-1 LTR promoter-specific affinity could specifically reactivate HIV-1 expression from latently infected cells. The first step in creating productive ZF-TFs is selecting the DNA site to be targeted. A good target site should be located in a chromatin region that is accessible to DNA-binding proteins and close to known transcriptional regulators of the gene.34 In our previous work, the zinc-finger motifs used in ZF-VP64 were assembled into zinc-finger nucleases and were confirmed to specifically target DNA sequences in the HIV-1 5′-LTR and 3′-LTR,35 suggesting that these target sites are accessible to these DNA-binding proteins. Moreover, all the target sites of ZF-VP64 were located in the vicinity of the transcription start site. One of them, the target site of ZF3-VP64, was immediately adjacent to nuclear factor-κB and Sp1 cis-regulatory sites (Figure 1b). This location may be advantageous because activators positioned close together on the promoter often act synergistically to boost transcription,34 which may partially explain why ZF3-VP64 exhibited the highest activation potential. Furthermore, these target sites have also been proven to be well conserved across various HIV-1 subtypes.35 Considering all these factors, we were confident that the target sites of ZF-VP64 were well selected and could effectively promote HIV-1 expression driven by the LTR. Next, we tested the ability of these ZF-TFs in luciferase reportertransfected cells and in cells latently infected with HIV-1. The cotransfection of HEK-293T cells with plasmids expressing ZF-VP64 and the HIV-1 LTR-driven luciferase reporter resulted in diverse luciferase expression levels, indicating different activation potentials for ZF-VP64 constructs with different target sites. In consistency with our results, many previous studies have demonstrated that targeting DNA-binding proteins to different regions of a gene of interest may lead to different effects on gene regulation.25,26,36 ZF3-VP64, which was identified in the reporter assays as having the greatest potential, was confirmed to also be the most effective in cells latently infected with HIV-1. ZF3-VP64 effectively reactivated HIV-1 expression from ~ 30% of both the latently infected C11 cells and J-Lat clone A10.6 cells, a proportion greater than 10-fold that of the untreated cells. After demonstrating the effectiveness of ZF-VP64, we investigated its safety and specificity. First, we found no apparent effects of ZF-VP64 on cell proliferation or cell cycle progression in Jurkat T cells and HEK-293T cells. Then, to demonstrate that the regulation of HIV-1 expression was direct, we deleted the target site of ZF3-VP64; the LTR promotion disappeared immediately. Furthermore, the direct in vivo interaction between our designed transcription factor and the HIV-1 LTR promoter was verified in ChIP experiments. All these results provide strong evidence that ZF3-VP64 specifically targets the LTR. The targeting specificity of this strategy is its major advantage over already existing drugs and supports further investigation into this method as a novel antiretroviral approach. In conclusion, this study showed that a representative ZF3-VP64 could potently and specifically reactivate HIV-1 with no apparent toxicity. It is too early to suggest that this method can be used in anti-HIV-1 latency therapy because there are many unsolved problems, such as the means of transgene delivery and possible immunogenicity. However, these problems are also widely present in other gene therapies, which are constantly under development.25 The gene-based strategy that specifically reactivates latent HIV-1 like the one described here will be a useful basis for the development of HIV-1 gene therapy in the future. Gene Therapy (2014) 490 – 495
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Figure 5. ZF3-VP64 specifically bound to the LTR to reactivate latent HIV-1. (a) Sequencing confirmation of the deletion of the target site of ZF3-VP64 in the HIV-1 LTR-luc reporter vector. (b) HEK-293T cells were cotransfected with the ZF empty vector or ZF3-VP64 plasmid and the LTR-luc or LTR(ΔZF3)-luc reporter plasmid for 48 h, and the relative luciferase activity was measured. (c) Analysis of binding of ZF3-VP64 to HIV-1 LTR promoter by ChIP assay. C11 cells were nucleofected with ZF3-VP64 or the ZF empty vector for 48 h, and chromatin fragments were immunoprecipitated with an anti-FLAG antibody or normal mouse IgG. PCR primers specific for the HIV-1 LTR promoter were used to amplify the DNA isolated from the immunoprecipitated chromatin.
MATERIALS AND METHODS Cell culture C11 cells, a type of latently infected Jurkat T cell, were first created in our laboratory and are now used more widely.32,37,38 J-Lat clone A10.6 cells were kindly provided by the NIH AIDS Research and Reference Reagent Program (Dr Eric Verdin).39 The C11 and J-Lat clone A10.6 cells were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U ml − 1 penicillin and 100 μg ml − 1 streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C under 5% CO2. Human embryonic kidney 293 cells stably expressing the SV40 large T antigen (HEK-293T) were purchased from the American Type Culture Collection (Manassas, VA, USA) and were grown in Dulbecco’s modified Eagle’s medium (Gibco) with 10% fetal bovine serum , 100 U ml − 1 penicillin and 100 μg ml − 1 streptomycin at 37 °C under 5% CO2.
Generation of ZF-VP64 targeting the HIV-1 LTR Four ZFP expression plasmids targeting the HIV-1 LTR were designed and assembled by Sigma-Aldrich. The transcriptional activator VP64 was synthesized into the pUC57 vector by Sangon Biotech (Shanghai, China) with a 5′-BamHI restriction site and a 3′-AvaI restriction site. After a double digestion with BamHI and AvaI, the pUC57-VP64 constructs were ligated into the ZFP expression plasmids. These new constructs, in which VP64 was located downstream of the zinc-finger motifs, were named ZF-VP64. The target sites for these constructs in the HIV-1 LTR are shown in Figure 1, and the sequences are provided in Supplementary Figure S1.
Construction of reporter vectors and dual luciferase reporter assay To produce the HIV-1 LTR-luciferase reporter vector (LTR-luc), a full-length LTR fragment was PCR-amplified from pNL4-3-EGFP using the sense primer F-LTR: 5′-CGGGGTACCTGGAAGGGCTAATTCACTCCCAAAG-3′ and the antisense primer R-LTR: 5′-CCGCTCGAGTGCTAGAGATTTTCCACACTGACTA-3′. This PCR product was gel purified, digested with KpnI and XhoI, and then ligated into the KpnI–XhoI site of the pGL3-basic plasmid (Promega, Madison, WI, USA). Subsequently, an HIV-1 LTR-luc reporter vector lacking the ZF3-VP64 target site (LTR(ΔZF3)-luc) was constructed by overlapping PCR using the following primers: forward 5′-CTGGGGAGTGGTGCATAT AAGCAGCTGCTTTTTGC-3′ and reverse 5′-TGCTTATATGCACCACTCCCCA GTCCCGCCCAG-3′. All the constructed plasmids were confirmed by sequencing. Gene Therapy (2014) 490 – 495
To examine the effects of ZF-VP64 on the HIV-1 LTR, each ZF-VP64 construct, pcDNA3.0 (mock) or a ZF empty vector (700 ng) was cotransfected into HEK-293T cells with pRL-SV40 (10 ng) and LTR-luc or LTR(ΔZF3)-luc (100 ng) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cells were harvested 48 h post transfection, and the lysate was assayed for luciferase activity using the DualLuciferase Reporter Assay System (Promega). Three independent transfection experiments were performed, and each luciferase assay was performed in triplicate.
Nucleofection and flow cytometry assays To analyze the HIV-1 expression in latently infected cells after ZF-VP64 treatment, the cells were mock treated or were nucleofected with ZF-VP64 or the ZF empty vector using the Amaxa Cell Line Nucleofector Kit V (Lonza, Gaithersburg, MD, USA). The expression of GFP was used as a marker of HIV-1 activation in latently infected cells and was observed by fluorescence microscopy. At the indicated times after transfection, the cells were washed and resuspended in phosphate-buffered saline, and the percentage of GFP-positive cells was measured by flow cytometry to determine the level of HIV-1 expression. GFP expression was measured using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA), and the data were analyzed using CellQuest software (Macintosh, Sunnyvale, CA, USA).
Cell proliferation and cell cycle analysis The Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) was used to measure cell proliferation after the ZF-VP64 treatment. Briefly, Jurkat T cells or HEK-293T cells were transfected with pcDNA3.0 (mock), the ZF empty vector or ZF-VP64 constructs. At 72 h post transfection, 10 μl of CCK-8 solution was added to each well of the 96-well culture plates. After 4 h of incubation at 37 °C, the absorbance at 450 nm was measured using a microplate reader. To generate the cell cycle profiles, the transfected cells were fixed with 500 μl of 70% ethanol at − 20 °C for 2 h. Subsequently, the cells were washed twice with phosphate-buffered saline and then stained with propidium iodide (50 μg ml − 1 propidium iodide and 100 μg ml − 1 RNase A in phosphate-buffered saline) at 37 °C for 30 min. The cell cycle analysis was performed using a FACScan flow cytometer. All the experiments were performed independently at least three times in triplicate per experimental point. © 2014 Macmillan Publishers Limited
Specific reactivation of latent HIV by ZF-TFs P Wang et al
495 Chromatin immunoprecipitation ChIP assays were performed according to the manufacturer’s protocol (Millipore, Billerica, MA, USA) and a previously described procedure.37,38 Briefly, C11 cells were nucleofected with ZF3-VP64 or the ZF empty vector using the Amaxa Cell Line Nucleofector Kit V. At 48 h post transfection, the cells were crosslinked with 1% formaldehyde, and the lysates were sonicated to produce 500–1000 bp chromatin fragments. The supernatant, which contained the precleared chromatin, was then incubated at 0 °C overnight with 2 μg of anti-FLAG antibody M2 (Sigma-Aldrich) or normal mouse IgG (Millipore). After the samples were reverse cross-linked by an overnight incubation at 65 °C, the DNA was isolated and analyzed by PCR for 30 cycles (using the forward 5′-AGACTGCTGACATCGAGCTTTCT-3′ and reverse 5′-GTGGGTTCCCTAGTTAGCCAGAG-3′ primers), which produced a 192-bp PCR fragment containing the ZF3-VP64 target site.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We thank the NIH AIDS Research and Reference Reagent Program for providing the J-Lat clone A10.6 cells. We also thank Prof Jianqing Xu (Shanghai Medical College of Fudan University) for providing the pNL4-3-EGFP plasmid. This work was supported by the National Grand Program on Key Infectious Disease (2014ZX10001003) and National Natural Science Foundation of China (31271418 to H Zhu).
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16 Sadowski I, Ma J, Triezenberg S, Ptashne M. GAL4-VP16 is an unusually potent transcriptional activator. Nature 1988; 335: 563–564. 17 Segal DJ, Dreier B, Beerli RR, Barbas CF. III. Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences. Proc Natl Acad Sci USA 1999; 96: 2758–2763. 18 Ruben SM, Dillon PJ, Schreck R, Henkel T, Chen CH, Maher M et al. Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NFkappa B. Science 1991; 251: 1490–1493. 19 Margolin JF, Friedman JR, Meyer WK, Vissing H, Thiesen HJ, Rauscher FJ. III. Krüppel-associated boxes are potent transcriptional repression domains. Proc Natl Acad Sci USA 1994; 91: 4509–4513. 20 Magnenat L, Blancafort P, Barbas CF. III. In vivo selection of combinatorial libraries and designed affinity maturation of polydactyl zinc finger transcription factors for ICAM-1 provides new insights into gene regulation. J Mol Biol 2004; 341: 635–649. 21 Xu GL, Bestor TH. Cytosine methylation targetted to pre-determined sequences. Nat Genet 1997; 17: 376–378. 22 Bushman FD, Miller MD. Tethering human immunodeficiency virus type 1 preintegration complexes to target DNA promotes integration at nearby sites. J Virol 1997; 71: 458–464. 23 Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA 1996; 93: 1156–1160. 24 Jamieson AC, Miller JC, Pabo CO. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov 2003; 2: 361–368. 25 Reynolds L, Ullman C, Moore M, Isalan M, West MJ, Clapham P et al. Repression of the HIV-1 5′ LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors. Proc Natl Acad Sci USA 2003; 100: 1615–1620. 26 Segal DJ, Gonçalves J, Eberhardy S, Swan CH, Torbett BE, Li X et al. Attenuation of HIV-1 replication in primary human cells with a designed zinc finger transcription factor. J Biol Chem 2004; 279: 14509–14519. 27 Kim YS, Kim JM, Jung DL, Kang JE, Lee S, Kim JS et al. Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replicationof HIV-1. J Biol Chem 2005; 280: 21545–21552. 28 Eberhardy SR, Goncalves J, Coelho S, Segal DJ, Berkhout B, Barbas CF. III. Inhibition of human immunodeficiency virus type 1 replication with artificial transcription factors targeting the highly conserved primer-binding site. J Virol 2006; 80: 2873–2883. 29 Marciniak RA, Calnan BJ, Frankel AD, Sharp PA. HIV-1 Tat protein trans-activates transcription in vitro. Cell 1990; 63: 791–802. 30 Wei P, Garber ME, Fang S-M, Fischer WH, Jones KA. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 1998; 92: 451–462. 31 Muniz L, Egloff S, Ughy B, Jády BE, Kiss T. Controlling cellular P-TEFb activity by the HIV-1 transcriptional transactivator Tat. PLoS Pathog 2010; 6: e1001152. 32 Ding D, Qu X, Li L, Zhou X, Liu S, Lin S et al. Involvement of histone methyltransferase GLP in HIV-1 latency through catalysis of H3K9 dimethylation. Virology 2013; 440: 182–189. 33 Cohen N, Mouly E, Hamdi H, Maillot M-C, Pallardy M, Godot V et al. GILZ expression in human dendritic cells redirects their maturation and prevents antigen-specific T lymphocyte response. Blood 2006; 107: 2037–2044. 34 Gräslund T, Li X, Magnenat L, Popkov M, Barbas CF. Exploring strategies for the design of artificial transcription factors targeting sites proximal to known regulatory regions for the induction oF γ-globin expression and the treatment of sickle cell disease. J Biol Chem 2005; 280: 3707–3714. 35 Qu X, Wang P, Ding D, Li L, Wang H, Ma L et al. Zinc-finger-nucleases mediate specific and efficient excision of HIV-1 proviral DNA from infected and latently infected human T cells. Nucleic Acids Res 2013; 41: 7771–7782. 36 Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013; 152: 1173–1183. 37 Ying H, Zhang Y, Zhou X, Qu X, Wang P, Liu S et al. Selective histonedeacetylase inhibitor M344 intervenes in HIV-1 latency through increasing histone acetylation and activation of NF-kappaB. PLoS One 2012; 7: e48832. 38 Wang P, Qu X, Wang X, Liu L, Zhu X, Zeng H et al. As2O3 synergistically reactivate latent HIV-1 by induction of NF-κB. Antiviral Res 2013; 100: 688–697. 39 Jordan A, Bisgrove D, Verdin E. HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J 2003; 22: 1868–1877.
Supplementary Information accompanies this paper on Gene Therapy website (http://www.nature.com/gt)
© 2014 Macmillan Publishers Limited
Gene Therapy (2014) 490 – 495
ORIGINAL ARTICLE
Specific reactivation of latent HIV-1 with designer zinc-finger transcription factors targeting the HIV-1 5′-LTR promoter P Wang1, X Qu1, X Wang1, X Zhu1, H Zeng1, H Chen2 and H Zhu1 HIV-1 latency remains the primary obstacle to the eradication of this virus. The current latency-reversing agents cannot effectively and specifically eliminate latent HIV-1 reservoirs. Therefore, better approaches are urgently needed. In this study, we describe a novel strategy to reactivate latent HIV-1 using zinc-finger transcription factors composed of designer zinc-finger proteins and the transcriptional activation domain VP64. For the first time, we demonstrate that ZF-VP64 with HIV-1 long terminal repeat (LTR) promoter-specific affinity could significantly reactivate HIV-1 expression from latently infected cells without altering cell proliferation or cell cycle progression. We also provide evidence that the reactivation of HIV-1 by ZF-VP64 occurs through specific binding to the 5′-LTR promoter. Our results demonstrate the potential of this novel approach for anti-HIV-1 latency therapy. Gene Therapy (2014) 21, 490–495; doi:10.1038/gt.2014.21; published online 13 March 2014
INTRODUCTION Despite being potent and life prolonging, the current combination antiretroviral therapy is not curative and does not eradicate HIV-1 infection. In long-lived latent reservoirs, the integrated HIV-1 provirus remains transcriptionally silent but replication competent, and the interruption of treatment inevitably results in a rapid rebound of viremia.1–5 Therefore, these latent reservoirs remain the primary obstacle to eradicating HIV-1 infection. Although the mechanisms contributing to the maintenance of HIV-1 latency are not completely understood, the majority involve the suppression of transcription.6 One strategy for eliminating latent HIV-1 reservoirs, termed ‘shock and kill’, which involves reactivating these reservoirs by inducing the transcription of the quiescent provirus followed by the elimination of reactivated virus-producing cells by viral cytopathic effect or host immune response, is currently the most widely discussed approach.1,7 Many novel latency-reversing agents have been identified by mechanism-directed approaches or large-scale screening. Some of these drugs have already entered clinical testing in humans; however, the results have been mixed or unsatisfactory.5,6 Potential limitations to the use of these drugs to reactivate latent HIV-1 include toxicity, a lack of target specificity and the development of acquired drug resistance. Most existing drugs cannot be targeted to HIV-1-infected cells or to the proviral genome; thus, innovative approaches involving HIV-1 specificity are urgently needed to address these issues. Artificial transcription factors are proteins designed to bind to the promoter region of a gene of interest and modulate target gene expression. This method has been applied to regulate endogenous gene expression in a number of instances.8–13 An artificial transcription factor is typically composed of a DNA-binding domain to bind to the promoter of the target gene, an effector domain (ED) to up- or downregulate the target gene and a nuclear localization signal to deliver the artificial transcription factors into the nuclei.14 Because of their DNA-binding selectivity and modular organization, the Cys2His2-type zinc-finger
proteins (ZFPs) are the most frequently used DNA-binding domains in ATFs.14,15 In zinc-finger transcription factors (ZF-TFs), DNA-binding domains can be linked to a variety of EDs with different functions, such as activator domains (VP16,16 VP6417 or p6518), repressor domains (KRAB19 or SID20) or DNA-modifying domains (methyltransferase,21 integrase22 or nuclease23). The control of gene expression with ZF-TFs can be applied to a broad range of drug discovery and therapeutic applications.24 Several reports have described the fusion of the KRAB repressor domain to ZFPs to repress the transcription and replication of HIV-1.25–28 However, to our knowledge, there are no applications of ZF-TFs with activator domains to reactivate latent HIV-1. Herein, we describe a novel approach using ZF-TFs to specifically recognize the HIV-1 long terminal repeat (LTR) promoter region and activate HIV-1 expression. RESULTS Construction of ZF-VP64 targeting the HIV-1 LTR promoter The HIV-1 LTR is highly conserved across all HIV-1 genomes; therefore, it represents an attractive and ideal anti-HIV-1 target. To design zinc-finger motifs specifically targeting the HIV-1 LTR, the DNA sequence of HIV-1 strain NL4-3 was submitted to SigmaAldrich (Shanghai, China), and four ZFP expression plasmids targeting the HIV-1 LTR were designed and assembled. To investigate whether these ZFPs could be used to engineer ZF-TFs to specifically reactivate HIV-1 from latency, the transcriptional activator VP64, a tetrameric repeat of the VP16’s minimal activation domain,17 was inserted downstream of the zinc-finger motifs. Thus, we generated four ZF-TFs: ZF1-VP64, ZF2-VP64, ZF3VP64 and ZF4-VP64. Each protein contained one zinc-finger DNAbinding module with one triple-FLAG tag and a nuclear localization signal at its N-terminus and one VP64 domain at its C-terminus (Figure 1a and Supplementary Figure S1). All the LTR target sites of the ZF-VP64 were near the transcription start site (+1): the target sites of ZF1-VP64 and ZF2-VP64 were downstream
1 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China and 2Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Boston, MA, USA. Correspondence: Dr H Zhu, State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China. E-mail: [email protected] Received 9 September 2013; revised 9 January 2014; accepted 24 January 2014; published online 13 March 2014
Specific reactivation of latent HIV by ZF-TFs P Wang et al
491 HIV-1 LTR transcription with Tat. HIV-1 LTR-luc reporter vector was cotransfected with ZF3-VP64 or ZF4-VP64 in the presence or absence of a Tat expression vector. The results showed that Tat alone induced an approximately 20-fold increase in luciferase reporter expression; when Tat was coexpressed with ZF3-VP64 or ZF4-VP64, the induction level increased to as high as 35–50-fold (Figure 2b). These data indicate that ZF-VP64 and Tat function synergistically to promote HIV-1 LTR transcription.
Figure 1. Basic structure and target sites of ZF-VP64. (a) Schematic representation of ZF-VP64-mediated HIV-1 expression activation. A ZF-VP64 is composed of a zinc-finger DNA-binding domain, a VP64 activator domain, a triple FLAG tag and an NLS. The red box in the 5′ LTR of the HIV-1 genome indicates the ZF-VP64 target sites. (b) Binding sites of ZF-VP64 to the HIV-1 LTR. The DNA sequence shown is from the 5′-LTR of HIV-1 (HXB2 reference strain, GenBank accession no. K03455). The nucleotides are numbered relative to the transcription start site (+1). The sites targeted by ZF-VP64 are underlined. Binding sites for NF-κB and Sp1 are also highlighted.
of the transcription start site, and the target sites of ZF3-VP64 and ZF4-VP64 were upstream of the transcription start site (Figure 1b). Potential of ZF-VP64 to activate the HIV-1 LTR in transient luciferase reporter assays To determine whether the designed ZF-VP64 constructs were able to activate HIV-1 expression from the 5′-LTR promoter, we cotransfected HEK-293T cells with the ZF-VP64 expression vectors and a reporter vector comprising the luciferase gene driven by the HIV-1 LTR promoter (LTR-luc). The luciferase levels were measured 48 h post transfection. As a negative control, the plasmid backbone lacking the zinc-finger domain (the ZF empty vector) was also tested. In this transient luciferase reporter assay, all four ZF-VP64 vectors successfully mediated the transcriptional activation of the HIV-1 LTR. Specifically, ZF3-VP64 produced a fourfold induction in reporter expression, and ZF4-VP64 generated a 2.5-fold induction. However, an induction of less than twofold was produced by ZF1-VP64 and ZF2-VP64. As expected, no significant activation was observed for the ZF empty vector (Figure 2a). Moreover, the potential of ZF-VP64 to activate HIV-1 LTR was dose-dependent and the minimal amount of DNA necessary for induction was about 50 ng per well of 24-well plates (Supplementary Figure S2). Based on these data, we chose ZF3-VP64 and ZF4-VP64 to use in further experiments. Transcription initiated from the HIV-1 LTR promoter is controlled predominantly at the level of elongation. By binding to the transactivation response element (TAR), the viral transactivator Tat can stimulate viral gene expression by recruiting transcription elongation complexes to the promoter.29–31 Therefore, we next asked whether a ZF-VP64 vector could synergistically promote © 2014 Macmillan Publishers Limited
ZF-VP64 reactivates HIV-1 in different latently infected cells The regulation of gene expression in a transient assay may differ from that observed with a genomic target because the reporter plasmid is not packaged into the chromatin in the same manner as the chromosomal gene.28 Therefore, we performed a more relevant assay to test the ability of ZF-VP64 to induce HIV-1 expression in latently infected cells. We first used C11 cells, a clonal, latently infected Jurkat T cell line established in our laboratory. These cells contain a single provirus integrated into an intron of RNPS1 and a green fluorescent protein (GFP) gene under the control of the HIV-1 LTR.32 The C11 cells remained untransfected (mock) or were nucleofected with the ZF empty vector or a ZF3-VP64 or ZF4-VP64 expression vector. The percentage of GFP-positive cells (indicating the transcriptional activity of the HIV-1 promoter) was measured by flow cytometry. At 48 h post transfection with ZF3-VP64 or ZF4-VP64, the percentage of GFP-positive cells was found to be as high as 32.65% or 27.12%, respectively, which was approximately 10-fold that of the untransfected cells (3.15%). Furthermore, the induction of GFP expression persisted for at least 4 days (Figures 3a and b). Similar results were obtained from another cell line latently infected with HIV-1 (J-Lat clone A10.6 cells), albeit with a weaker induction potential for ZF4-VP64 (Figure 3c). In both cell lines, the expression of HIV-1 by the control cells transfected with the ZF empty vector was higher than expected, perhaps due to a partial activation of these cells by nucleofection.33 These results suggested that ZF-VP64 could effectively reactivate HIV-1 expression from latently infected cells and could be further investigated as a novel antiretroviral agent. ZF-VP64 does not affect cell proliferation or cell cycle progression We next examined whether ZF-VP64 stimulated cell proliferation or affected cell cycle progression. The CCK-8 assay kit was used to evaluate cell proliferation 72 h after Jurkat T cells or HEK-293T cells were transfected with the ZF empty vector or with the ZF3-VP64 or ZF4-VP64 expression vector. The results showed that ZF-VP64 had no significant effect on cell proliferation (Figure 4a and Supplementary Figure S3a). The potentialof ZF-VP64 to induce cell cycle progression was further investigated by staining these cells with propidium iodide and analyzing the DNA content using flow cytometry. Using this method, the progression of cells through the different cell cycle phases (G0/G1, S and G2/M) can be assessed. Compared with the mock-treated cells, the transfection of the ZF empty vector, ZF3-VP64 or ZF4-VP64 did not affect cell cycle progression (Figure 4b and Supplementary Figure S3b). ZF3-VP64 specifically binds to the 5′-LTR promoter to reactivate HIV-1 We next investigated whether ZF-VP64 reactivated HIV-1 through a direct interaction with the 5′-LTR; ZF3-VP64 was selected for this assay. First, we deleted the ZF3-VP64 target site from the HIV-1 LTR-luc reporter vector (Figure 5a). Then, we transfected HEK-293T cells with the ZF3-VP64 expression vector and the wild-type HIV-1 LTR-luc reporter vector (LTR-luc) or the HIV-1 LTR-luc reporter vector lacking the ZF3-VP64 target site (LTR(ΔZF3)-luc). ZF3-VP64 induced an approximately fourfold increase in the stimulation of the wild-type LTR-luc reporter relative to the ZF empty vector Gene Therapy (2014) 490 – 495
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Figure 2. ZF-VP64-mediated activation of the HIV-1 LTR in transient transfection assays. (a) Luciferase assays for HIV-1 5′-LTR activity in transiently transfected cells comparing the activation potentials of various designer ZF-VP64 transcription factors. HEK-293T cells were cotransfected with pcDNA3.0 (mock), the ZF empty vector or a ZF-VP64 plasmid and the LTR-luc reporter plasmid for 48 h, and the relative luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). (b) Luciferase assays for HIV-1 5′-LTR activity in the presence or absence of Tat and the ZF3-VP64 or ZF4-VP64 activator construct were performed in transiently transfected cells. The data represent the mean ± s.d. of three independent experiments.
Figure 3. ZF-VP64-mediated activation of the HIV-1 provirus in latently infected cells. (a) C11 cells were untransfected (mock) or nucleofected with 2 μg of the ZF empty vector or a ZF-VP64 plasmid. At 48 h post transfection, the percentage of GFP-positive cells was measured by flow cytometry. The results are presented as fluorescence histograms. (b) The time-dependent effects of ZF-VP64-mediated activation of the HIV-1 provirus in C11 cells or (c) J-Lat clone A10.6 cells. C11 cells or J-Lat clone A10.6 cells were untransfected (mock) or nucleofected with 2 μg of the ZF empty vector or a ZF-VP64 plasmid for the indicated times. The GFP expression was monitored in gated live cells at 24, 48, 72 and 96 h by standard flow cytometric techniques. The data represent the mean ± s.d. of three independent experiments.
control, similar to the previous results, but failed to activate the LTR(ΔZF3)-luc reporter (Figure 5b), indicating that the induction of HIV-1 by ZF3-VP64 occurred through specific binding to the 5′-LTR promoter. Gene Therapy (2014) 490 – 495
To confirm that ZF3-VP64 bound to HIV-1 LTR in vivo, a chromatin immunoprecipitation (ChIP) assay was performed. C11 cells were nucleofected with ZF3-VP64 or the ZF empty vector and crosslinked with formaldehyde, and the chromatin © 2014 Macmillan Publishers Limited
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Figure 4. ZF-VP64 did not affect cell proliferation or cell cycle progression. (a) Jurkat T cells were nucleofected with pcDNA3.0 (mock), the ZF empty vector or a ZF-VP64 plasmid for 72 h, and cell viability was measured using a CCK-8 kit (Dojindo). The division of the OD450 between the treated and control groups indicated the percentage of cell viability. The data represent the mean ± s.d. of three independent experiments. (b) Jurkat T cells were nucleofected with pcDNA3.0 (mock), the ZF empty vector or a ZF-VP64 plasmid for 72 h, and cell cycle progression was analyzed by propidium iodide staining and flow cytometry. Similar results were observed in three independent experiments.
fragments were immunoprecipitated with an antibody that recognizes the FLAG tag at the N-terminus of ZF3-VP64 or with normal mouse IgG. DNA was isolated from the immunoprecipitates and was analyzed by PCR using primers specific for HIV-1 LTR. We observed that the anti-FLAG antibody was able to efficiently immunoprecipitate the HIV-1 LTR promoter region from the ZF3-VP64-transfected cells but not from the ZF empty vectortransfected cells. No immunoprecipitated DNA fragments were detected by PCR in the normal mouse IgG controls (Figure 5c). These findings further confirmed that the observed reactivation of HIV-1 was by a direct interaction between ZF3-VP64 and the 5′-LTR. DISCUSSION None of the current latency-reversing agents can safely and effectively eliminate the long-lived HIV-1 reservoirs; therefore, © 2014 Macmillan Publishers Limited
new anti-latency approaches need to be explored. ZF-TFs using ZFPs as DNA-binding domains are useful for validating genes as drug targets. In this study, we explored a novel strategy to reactivate latent HIV-1 using ZF-TFs composed of designer ZFPs and the transcriptional activation domain VP64. To our knowledge, this was the first demonstration that ZF-VP64 with HIV-1 LTR promoter-specific affinity could specifically reactivate HIV-1 expression from latently infected cells. The first step in creating productive ZF-TFs is selecting the DNA site to be targeted. A good target site should be located in a chromatin region that is accessible to DNA-binding proteins and close to known transcriptional regulators of the gene.34 In our previous work, the zinc-finger motifs used in ZF-VP64 were assembled into zinc-finger nucleases and were confirmed to specifically target DNA sequences in the HIV-1 5′-LTR and 3′-LTR,35 suggesting that these target sites are accessible to these DNA-binding proteins. Moreover, all the target sites of ZF-VP64 were located in the vicinity of the transcription start site. One of them, the target site of ZF3-VP64, was immediately adjacent to nuclear factor-κB and Sp1 cis-regulatory sites (Figure 1b). This location may be advantageous because activators positioned close together on the promoter often act synergistically to boost transcription,34 which may partially explain why ZF3-VP64 exhibited the highest activation potential. Furthermore, these target sites have also been proven to be well conserved across various HIV-1 subtypes.35 Considering all these factors, we were confident that the target sites of ZF-VP64 were well selected and could effectively promote HIV-1 expression driven by the LTR. Next, we tested the ability of these ZF-TFs in luciferase reportertransfected cells and in cells latently infected with HIV-1. The cotransfection of HEK-293T cells with plasmids expressing ZF-VP64 and the HIV-1 LTR-driven luciferase reporter resulted in diverse luciferase expression levels, indicating different activation potentials for ZF-VP64 constructs with different target sites. In consistency with our results, many previous studies have demonstrated that targeting DNA-binding proteins to different regions of a gene of interest may lead to different effects on gene regulation.25,26,36 ZF3-VP64, which was identified in the reporter assays as having the greatest potential, was confirmed to also be the most effective in cells latently infected with HIV-1. ZF3-VP64 effectively reactivated HIV-1 expression from ~ 30% of both the latently infected C11 cells and J-Lat clone A10.6 cells, a proportion greater than 10-fold that of the untreated cells. After demonstrating the effectiveness of ZF-VP64, we investigated its safety and specificity. First, we found no apparent effects of ZF-VP64 on cell proliferation or cell cycle progression in Jurkat T cells and HEK-293T cells. Then, to demonstrate that the regulation of HIV-1 expression was direct, we deleted the target site of ZF3-VP64; the LTR promotion disappeared immediately. Furthermore, the direct in vivo interaction between our designed transcription factor and the HIV-1 LTR promoter was verified in ChIP experiments. All these results provide strong evidence that ZF3-VP64 specifically targets the LTR. The targeting specificity of this strategy is its major advantage over already existing drugs and supports further investigation into this method as a novel antiretroviral approach. In conclusion, this study showed that a representative ZF3-VP64 could potently and specifically reactivate HIV-1 with no apparent toxicity. It is too early to suggest that this method can be used in anti-HIV-1 latency therapy because there are many unsolved problems, such as the means of transgene delivery and possible immunogenicity. However, these problems are also widely present in other gene therapies, which are constantly under development.25 The gene-based strategy that specifically reactivates latent HIV-1 like the one described here will be a useful basis for the development of HIV-1 gene therapy in the future. Gene Therapy (2014) 490 – 495
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Figure 5. ZF3-VP64 specifically bound to the LTR to reactivate latent HIV-1. (a) Sequencing confirmation of the deletion of the target site of ZF3-VP64 in the HIV-1 LTR-luc reporter vector. (b) HEK-293T cells were cotransfected with the ZF empty vector or ZF3-VP64 plasmid and the LTR-luc or LTR(ΔZF3)-luc reporter plasmid for 48 h, and the relative luciferase activity was measured. (c) Analysis of binding of ZF3-VP64 to HIV-1 LTR promoter by ChIP assay. C11 cells were nucleofected with ZF3-VP64 or the ZF empty vector for 48 h, and chromatin fragments were immunoprecipitated with an anti-FLAG antibody or normal mouse IgG. PCR primers specific for the HIV-1 LTR promoter were used to amplify the DNA isolated from the immunoprecipitated chromatin.
MATERIALS AND METHODS Cell culture C11 cells, a type of latently infected Jurkat T cell, were first created in our laboratory and are now used more widely.32,37,38 J-Lat clone A10.6 cells were kindly provided by the NIH AIDS Research and Reference Reagent Program (Dr Eric Verdin).39 The C11 and J-Lat clone A10.6 cells were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U ml − 1 penicillin and 100 μg ml − 1 streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C under 5% CO2. Human embryonic kidney 293 cells stably expressing the SV40 large T antigen (HEK-293T) were purchased from the American Type Culture Collection (Manassas, VA, USA) and were grown in Dulbecco’s modified Eagle’s medium (Gibco) with 10% fetal bovine serum , 100 U ml − 1 penicillin and 100 μg ml − 1 streptomycin at 37 °C under 5% CO2.
Generation of ZF-VP64 targeting the HIV-1 LTR Four ZFP expression plasmids targeting the HIV-1 LTR were designed and assembled by Sigma-Aldrich. The transcriptional activator VP64 was synthesized into the pUC57 vector by Sangon Biotech (Shanghai, China) with a 5′-BamHI restriction site and a 3′-AvaI restriction site. After a double digestion with BamHI and AvaI, the pUC57-VP64 constructs were ligated into the ZFP expression plasmids. These new constructs, in which VP64 was located downstream of the zinc-finger motifs, were named ZF-VP64. The target sites for these constructs in the HIV-1 LTR are shown in Figure 1, and the sequences are provided in Supplementary Figure S1.
Construction of reporter vectors and dual luciferase reporter assay To produce the HIV-1 LTR-luciferase reporter vector (LTR-luc), a full-length LTR fragment was PCR-amplified from pNL4-3-EGFP using the sense primer F-LTR: 5′-CGGGGTACCTGGAAGGGCTAATTCACTCCCAAAG-3′ and the antisense primer R-LTR: 5′-CCGCTCGAGTGCTAGAGATTTTCCACACTGACTA-3′. This PCR product was gel purified, digested with KpnI and XhoI, and then ligated into the KpnI–XhoI site of the pGL3-basic plasmid (Promega, Madison, WI, USA). Subsequently, an HIV-1 LTR-luc reporter vector lacking the ZF3-VP64 target site (LTR(ΔZF3)-luc) was constructed by overlapping PCR using the following primers: forward 5′-CTGGGGAGTGGTGCATAT AAGCAGCTGCTTTTTGC-3′ and reverse 5′-TGCTTATATGCACCACTCCCCA GTCCCGCCCAG-3′. All the constructed plasmids were confirmed by sequencing. Gene Therapy (2014) 490 – 495
To examine the effects of ZF-VP64 on the HIV-1 LTR, each ZF-VP64 construct, pcDNA3.0 (mock) or a ZF empty vector (700 ng) was cotransfected into HEK-293T cells with pRL-SV40 (10 ng) and LTR-luc or LTR(ΔZF3)-luc (100 ng) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cells were harvested 48 h post transfection, and the lysate was assayed for luciferase activity using the DualLuciferase Reporter Assay System (Promega). Three independent transfection experiments were performed, and each luciferase assay was performed in triplicate.
Nucleofection and flow cytometry assays To analyze the HIV-1 expression in latently infected cells after ZF-VP64 treatment, the cells were mock treated or were nucleofected with ZF-VP64 or the ZF empty vector using the Amaxa Cell Line Nucleofector Kit V (Lonza, Gaithersburg, MD, USA). The expression of GFP was used as a marker of HIV-1 activation in latently infected cells and was observed by fluorescence microscopy. At the indicated times after transfection, the cells were washed and resuspended in phosphate-buffered saline, and the percentage of GFP-positive cells was measured by flow cytometry to determine the level of HIV-1 expression. GFP expression was measured using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA), and the data were analyzed using CellQuest software (Macintosh, Sunnyvale, CA, USA).
Cell proliferation and cell cycle analysis The Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) was used to measure cell proliferation after the ZF-VP64 treatment. Briefly, Jurkat T cells or HEK-293T cells were transfected with pcDNA3.0 (mock), the ZF empty vector or ZF-VP64 constructs. At 72 h post transfection, 10 μl of CCK-8 solution was added to each well of the 96-well culture plates. After 4 h of incubation at 37 °C, the absorbance at 450 nm was measured using a microplate reader. To generate the cell cycle profiles, the transfected cells were fixed with 500 μl of 70% ethanol at − 20 °C for 2 h. Subsequently, the cells were washed twice with phosphate-buffered saline and then stained with propidium iodide (50 μg ml − 1 propidium iodide and 100 μg ml − 1 RNase A in phosphate-buffered saline) at 37 °C for 30 min. The cell cycle analysis was performed using a FACScan flow cytometer. All the experiments were performed independently at least three times in triplicate per experimental point. © 2014 Macmillan Publishers Limited
Specific reactivation of latent HIV by ZF-TFs P Wang et al
495 Chromatin immunoprecipitation ChIP assays were performed according to the manufacturer’s protocol (Millipore, Billerica, MA, USA) and a previously described procedure.37,38 Briefly, C11 cells were nucleofected with ZF3-VP64 or the ZF empty vector using the Amaxa Cell Line Nucleofector Kit V. At 48 h post transfection, the cells were crosslinked with 1% formaldehyde, and the lysates were sonicated to produce 500–1000 bp chromatin fragments. The supernatant, which contained the precleared chromatin, was then incubated at 0 °C overnight with 2 μg of anti-FLAG antibody M2 (Sigma-Aldrich) or normal mouse IgG (Millipore). After the samples were reverse cross-linked by an overnight incubation at 65 °C, the DNA was isolated and analyzed by PCR for 30 cycles (using the forward 5′-AGACTGCTGACATCGAGCTTTCT-3′ and reverse 5′-GTGGGTTCCCTAGTTAGCCAGAG-3′ primers), which produced a 192-bp PCR fragment containing the ZF3-VP64 target site.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We thank the NIH AIDS Research and Reference Reagent Program for providing the J-Lat clone A10.6 cells. We also thank Prof Jianqing Xu (Shanghai Medical College of Fudan University) for providing the pNL4-3-EGFP plasmid. This work was supported by the National Grand Program on Key Infectious Disease (2014ZX10001003) and National Natural Science Foundation of China (31271418 to H Zhu).
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