Efficient inhibition of HIV-1 replication by an artificial polycistronic miRNA construct
- Tao Zhang†1,
- Tong Cheng†1,
- Lihua Wei1,
- Yijun Cai1,
- Anthony Et Yeo1,
- Jiahuai Han2,
- Y Adam Yuan3, 4,
- Jun Zhang1 and
- Ningshao Xia1Email author
© Zhang et al.; licensee BioMed Central Ltd. 2012
Received: 20 November 2011
Accepted: 1 June 2012
Published: 18 June 2012
RNA interference (RNAi) has been used as a promising approach to inhibit human immunodeficiency virus type 1 (HIV-1) replication for both in vitro and in vivo animal models. However, HIV-1 escape mutants after RNAi treatment have been reported. Expressing multiple small interfering RNAs (siRNAs) against conserved viral sequences can serve as a genetic barrier for viral escape, and optimization of the efficiency of this process was the aim of this study.
An artificial polycistronic transcript driven by a CMV promoter was designed to inhibit HIV-1 replication. The artificial polycistronic transcript contained two pre-miR-30a backbones and one pre-miR-155 backbone, which are linked by a sequence derived from antisense RNA sequence targeting the HIV-1 env gene. Our results demonstrated that this artificial polycistronic transcript simultaneously expresses three anti-HIV siRNAs and efficiently inhibits HIV-1 replication. In addition, the biosafety of MT-4 cells expressing this polycistronic miRNA transcript was evaluated, and no apparent impacts on cell proliferation rate, interferon response, and interruption of native miRNA processing were observed.
The strategy described here to generate an artificial polycistronic transcript to inhibit viral replication provided an opportunity to select and optimize many factors to yield highly efficient constructs expressing multiple siRNAs against viral infection.
RNA interference (RNAi) is a sequence-specific post-transcriptional gene-silencing mechanism, that was first discovered in Caenorhabditis elegans. RNAi can be triggered by small interfering RNAs (siRNAs) or endogenous microRNAs (miRNAs), which are processed by RNase III-like enzymes [2, 3]. Most miRNAs are processed from longer primary miRNA transcripts (pri-miRNA), which are transcribed from genome sequence by RNA polymerase II promoter [4, 5]. Subsequently, pri-miRNAs are processed into miRNA precursors (pre-miRNAs) that are approximately 60 nucleotides (nt) long by miRNA processing machinery consisting of the nuclear Drospha-DGCR8 complex [6, 7]. Next, pre-miRNAs are transported to the cytoplasm by Exportin-5 and further processed by Dicer to produce miRNA duplexes of approximately 22 nt in length [8, 9]. The miRNA duplexes are loaded into the RNA-induced silencing complex (RISC) to guide RISC-mediated gene regulation via mRNA cleavage or translational repression [10, 11]. Notably, several miRNA genes are encoded as clusters within the genome sequence and transcribed into pri-miRNAs simultaneously as clusters. Hence, multiple miRNAs can be transcribed and processed from a single transcription unit [13, 14].
Regarded as a potent post-transcription gene silencing tool, RNAi is now used as a standard laboratory tool to knock down gene expression at the cellular level, as well as at the organismal level [15, 16]. In addition, RNAi has been successfully used as a promising approach to inhibit the replication of different viruses, including human immunodeficiency virus type 1 (HIV-1) [17–22]. Many strategies have been proposed to inhibit HIV-1 replication in cell culture and animal models, including siRNA or short hairpin RNA (shRNA) vector-based or pri-miRNA vector-based approaches [23–25]. These vector-based approaches have demonstrated long-term inhibition of HIV replication. However, due to the restriction of the RNAi mechanism (sequence-specificity) and high mutation rate of HIV-1, escape mutants after RNAi treatment have been reported [26–28]. Therefore, a combination of multiple antiviral inhibitors to overcome escape has been proposed.
Currently, both multiple shRNAs in a combinatorial vector approach and multiple antiviral siRNAs embedded in a single polycistronic miRNA transcript approach have been used to reduce the chance of viral escape [29–32]. Of these approaches, polycistronic miRNA has been shown to be safer, since the expression of miRNA-like transcripts is low and regulated, therefore reducing the risk of toxicity .
Native miRNA clusters and tandem copes of miR155 have been employed as the basis for the design of a polycistronic transcript that simultaneously expresses multiple antiviral siRNAs [29, 31, 32]. The replacement of the mature miRNA sequence by the miRNA-like stem will produce mature siRNAs that specifically target viruses. Several studies have shown that the native flanking pri-miRNA sequences and key structural features of the native miRNAs were retained, as they were thought to be critical for efficient siRNA processing .
In this study, we designed an artificial polycistronic transcript containing two pre-miR-30a backbones and one pre-miR-155 backbone, which is driven by a cytomegalovirus (CMV) promoter. Moreover, we used antisense RNA sequence targeting the HIV-1 env gene as the linker to connect the pre-miRNA backbones. This study demonstrated that the flanking pri-miRNA sequence can be replaced and optimized with artificial sequence to construct the polycistronic transcript that expresses three anti-HIV siRNAs simultaneously and efficiently inhibits HIV-1 replication. This strategy provides a feasible method to replace the flanking pri-miRNA sequences with other antiviral elements to design more complicated and efficient inhibitors against pathogens that are prone to escape.
Screening of shRNA constructs inhibiting HIV-1 replication
Construction of single antiviral miRNA transcripts
To determine the inhibitory efficiency, the artificial miRNA expressing plasmids were cotransfected with pNL4-3. Artificial miR-LacZ against β-gal-expressing plasmids was used as negative control, whereas the original shRNA vectors were used as a positive control. Among the various artificial miRNA constructs screened, miR-A2, miR-B3, and miR-C1 had the most inhibition activity against HIV-1 replication (Figure 2B).
Construction of artificial polycistronic miRNA transcripts
In the constructs, e1, e2 or e3 was added to the downstream region of selected miR-A2, miR-C1 or miR-B3, respectively, for individual miR-A2-e1, miR-C1-e2, and miR-B3-e3 transcripts. Determination of the inhibitory activities of these basic structural elements was evaluated by the firefly luciferase reporter assay. Firefly luciferase expression was normalized to the Renilla luciferase expression from the co-transfected pRL plasmid. These three basic constructs were able to inhibit the expression of the reporter gene, although the inhibitory efficiency of miR-B3-e3 and miR-A2-e1 decreased by approximately 50% (Figure 3B).
To investigate whether linkers exerted anti-HIV-1 activity, plasmids expressing linkers only were co-transfected with pNL4-3. Our data demonstrated that linkers exhibited little antiviral activity (Additional file 1: Figure S1), which is consistent with the observation that antisense RNA shorter than 400 nucleotides is incapable of inhibiting HIV-1 replication. The individual artificial miRNA transcripts were then ligated to construct artificial polycistronic miRNA transcripts, which were named for the miRNA transcript followed by the linker name. For example, miR-AB stands for polycistronic miRNA transcript miR-A2-e1 connected by polycistronic miRNA transcript miR-B3-e3 successively, whereas miR-BA stands for miR-B3-e3 connected by miR-A2-e1 successively (Additional file 1: Figure S2).
Construction of MT-4 cells expressing polycistronic miRNA transcripts
Mature miRNA levels of miR-ACB in MT-4
Evaluation of off-target effects of miR-ACB
Potential prevention of HIV-1 escape by miR-ACB
The selection and construction of cell lines expressing multiple effective antiviral elements are critical to avoid escape mutations in HIV-1 by RNAi techniques. Expressing multiple shRNAs from separate promoters and long hairpin RNAs has been reported to achieve inhibition of viral replication [30, 38, 39]. However, the high expression level of RNA polymerase III promoters that are used to transcribe shRNAs and lhRNAs may increase toxicity due to saturation of the RNAi machinery [33, 40]. Another attractive approach is to express multiple antiviral siRNAs from a single polycistronic miRNA transcript that can be expressed from a single RNA polymerase II promoter to allow lower and regulated expression [29, 31]. The native miRNA clusters (mir-17-92 or mir-106b) were used as the backbone for insertion of multiple antiviral siRNAs. It was reported that the native flanking primary miRNA (pri-miRNA) sequences are maintained to keep the structural features of the native miRNAs, which are critical for efficient siRNA processing . In this study, we investigated a method to link multiple antiviral miRNA against HIV-1 with artificial flanking pri-miRNA sequence.
Firstly, we employed a conventional antiviral shRNA approach to select the antiviral small RNA sequences specifically targeting the two different HIV-1 targets, pol and vif. Secondly, the three selected small RNA sequences (pol22, pol25, and vif1 from 95 different sequences) were modified and ligated into the respectively pre-miR-30a or miR-155 backbones to generate three different artificial miRNA transcripts (miR-A2, miR-B3 or miR-C1). The individual miRNA transcripts were linked by arbitrary antisense RNA sequences targeted to HIV-1 env to construct an artificial single cistronic miRNA construct. Finally, the relative positions and the combinations of these artificial miRNA transcripts, together with the antisense RNA linker, were optimized based on a luciferase reporter system to acquire a tri-cistronic miRNA transcript (miR-ACB) with high expression levels of three individual antiviral siRNAs simultaneously. As a result, the MT-4 cells expressing the selected miR-ACB transcript exerted stronger inhibition of HIV-1 replication than any of the single antiviral miRNAs and had better suppressive activity against escaped virus replication than individual miRNA transcripts. Off-target effects of miRNA on cellular transcripts with partial sequence complementarity may induce negative effects on the treated cell. In this study, we did not observed any off-target effects of miR-ACB by evaluating cell proliferation rate, interferon response, and interruption of native mRNAs.
The strategy described here provides a method to construct miRNA polycistrons with artificial flanking pri-miRNA sequences. The results of this study showed that the inhibition efficiency of each miRNA embedded in the tri-cistron construction was lower than that for single miRNA. However, the opposite was observed for the native mir-17-92 backbone. These conflicting findings imply that the miRNA tri-cistron can be further optimized to yield more efficient constructs expressing multiple siRNAs against viral replication. The factors that might affect the optimization include the miRNA backbones, the combination, and the linkers. Future studies should investigate the incorporation of other antiviral elements, such as zinc-finger nucleases, single-chain antibodies, and ribozymes, into the linker sequence to produce more antiviral elements from a single miRNA polycistron to inhibit viral infection/escape.
The tri-cistronic transcript constructed with artificial flanking pri-miRNA sequences simultaneously expresses three anti-HIV siRNAs and efficiently inhibits HIV-1 replication without off-target effects. The strategy described here provides a feasible method to replace the flanking pri-miRNA sequences with other antiviral elements to design more complicated and efficient polycistronic miRNAs.
The shRNA expression plasmids were constructed according to the pSUPER instructions provided by the manufacturer (Oligoengine, WA, USA). Target sequences of shRNAs are shown in the Additional file 1: Table S1. The pcDNA3.1 vector was mutated to remove BglII site using QuikChange II Site-Directed Mutagenesis Kits (Agilent, CA, USA). The miRNA expression plasmids were constructed by inserting annealed oligonucleotides (Additional file 1: Table S2), encoding the miRNA target transcript, into the pcDNA3.1 vector at multiple cloning sites (BamHI/EcoRI). The basic elements were obtained by inserting the fragments (EcoRI/XhoI) of the linker (the 3′ end of each linker contains a BglII cloning sites.) into the cloning site (EcoRI/XhoI) of the miRNA-expressing vector. The miRNA cluster was obtained by inserting the fragment (BamHI/XhoI) of one basic structure into the cloning site (BglII/XhoI) of pcDNA3.1 expressing upstream basic structures of the cluster.
An H-2Kk expression cassette was cloned from pMACSKK.II (Miltenyi, Bergisch Gladbach, Germany) using primers (5′-TTTACTAGTCATGTTTGACAGCTTATCATCG-3′and 5′-TTTCTCGAGATACAAGGATCCATCTACC CTCCTTTTCCACC-3′). The cleaved fragment (SpeI/XhoI) was inserted into the cloning site (XbaI/XhoI) of pLL3.7 to obtain the pLLKk vector. pCDNA3.1 vectors expressing miRNAs or miRNA clusters were cleaved (BamHI/XhoI) and inserted into the same cleaved pLLKk. Fragments containing the RNAi target sequences were generated from pNL4-3 by adding XbaI at 5′ termination site and FseI at 3′ termination site. PCR products were cleaved (XbaI/FseI) and inserted into the cloning site (XbaI/FseI) of the pGL3-control vector (Promega, Madison, WI) to obtain luciferase reporters.
Human embryonic kidney 293FT adherent cells were purchase from Invitrogen and grown in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 μg/mL). MT-4 and TZM-bl cells were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Program and grown in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 μg/mL) (complete medium).
Infectious clone co-transfection experiments
500 ng miRNA expressing vectors (or 500 ng shRNA expressing vectors), 50 ng pRL Renilla Luciferase Control Reporter Vectors (Promega) and 100 ng pNL4-3 were co-transfected into 293FT cells in 24-wells plates at 80% confluency with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). At 48 h post-transfection, CA-p24 levels in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA) 48 h post-transfection. The cells were lysed with 120 μL Passive Lysis Buffer (Promega) and luciferase levels were analyzed from 10 μL lysate using the Dual Luciferase reporter assay (50 μL of substrate reagents; Promega) on a Centro LB 960 Microplate Luminometer (Berthold, Bad Wildbad, Germany).
Reporter co-transfection experiments
300 ng miRNA expressing vectors, 50 ng pRL, and 300 ng luciferase reporters were co-transfected into 293FT cells in 24-well plates with Lipofectamine 2000 (Invitrogen). Cells were lysed to measure luciferase activity 48 h post-transfection. Changes in the expression of firefly luciferase (target) were calculated relative to Renilla luciferase (internal control) and normalized to levels in cells transfected with the pcDNA3.1 control plasmid expressing miR-LacZ.
Lentiviral vector production and transduction
The pSUPER-Drosha is a shRNA expressing plasmid based on pSUPER vector to decrease the level of Drosha protein (the target sequence is AACGAGUAGGCUUCGUGACUU33). The 293FT cells were seeded in a 10 cm dish (5 × 106/dish). After 6 h, 12 μg pLLKk-miRNA vectors were co-transfected with 6 μg pVSVG, 6 μg pMDL, 6 μg pREV, and 6 μg pSUPER-Drosha vectors into 293FT cells using Lipofectamine 2000 reagent (Invitrogen). Culture medium was replaced 12 h post-transfection. On the third day of culture, cell culture supernatant containing lentiviral vectors was harvested and pooled. Cellular debris was removed by filtration through a 0.45 μm filter. Lentiviral stocks were titrated on 293 T cells. MT-4 cells (1 × 105) were transduced with lentivirus expressing miRNA at a multiplicity of infection (MOI) of 40. Ten days post-transduction, cells were sorted with live fluorescence-activated cell sorting (FACS), and green fluorescent protein (GFP)-positive cells were selected.
miRNA detection by Northern blotting
Total RNA was isolated using Trizol reagent from MT-4 cells or MT-4 cells expressing miRNAs. The miRNAs were detected using a miRNA Northern Blot Assay Kit (Signosis, Sunnyvale, CA), according to the manufacturer’s instructions. Biotinylated probes were used for detection: GTATGTAGGATCTGACTTA (miR-A), GGATTTACCACACCAGACA (miR-B) and GTAGACAGGATGAGGATTA (miR-C).
MTT cell viability assay
Cells were seeded at a density of 2000/well in 96-well plates and grown three days. MTT (Promega) was added at 15 μL/well and incubated at 37°C for 4 hours. Optical density was measured at 570 nm. All experiments were done in triplicate.
miRNA detection by quantitative PCR
Total RNA was isolated using Trizol reagent from MT-4 cells or MT-4 cells expressing miRNAs. The miRNAs were detected using a Ncode miRNA qRT-PCR Kit (Invitrogen), according to the manufacturer’s instructions. Sequences of primers are shown in the Additional file 1: Table S3.
HIV-1 challenge assays
The titer of HIV-1NL4-3 was determined by infecting TZM-bl cells and scored for β–galactosidase-positive cells . Briefly, TZM-bl cells were grown in 96-well plates at 1 × 104 cells per well. Cells were infected with 50 μL of 10-fold serially diluted virus. Two days post-infection, cultured cells were fixed and stained. Blue cells with β-galactosidase activity were counted under a light microscope. MT-4 cells or MT-4 cells expressing miRNA were infected by HIV-1 at MOI = 0.1. After 24 h of infection, cells were washed twice with RPMI 1640 medium and cultured in complete medium at 37°C. Viral spread was monitored by measuring CA-p24 production by ELISA.
The authors acknowledge funding support from the National Natural Science Foundation (Grant no. 30500092, 30600106, 30870514), the Project 863 (Grant no. 2006AA020905, 2006AA02A209), the Key Program in Infectious Diseases (Grant No. 2008ZX10004-015) and the Project 111 of the Ministry of Education (Grant no. B06016), People's Republic of China. Work performed at the National University of Singapore was supported by the AcRF Tier 2 grant from the Ministry of Education, Singapore (T208A3124).
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