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Specific inhibition of HIV-1 replication by short hairpin RNAs targeting human cyclin T1 without inducing apoptosis
FEBS Letters 579 (2005) 3100–3106
FEBS 29612
Specific inhibition of HIV-1 replication by short hairpin RNAs targeting human cyclin T1 without inducing apoptosis Zhaoyang Li, Yong Xiong, Yu Peng, JiÕan Pan, Yu Chen, Xiaoyun Wu, Snawar Hussain, Po Tien, Deyin Guo* The Modern Virology Research Centre and State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China Received 16 March 2005; revised 10 April 2005; accepted 17 April 2005 Available online 16 May 2005 Edited by Shou-Wei Ding
Abstract RNA interference (RNAi), a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), can be used not only as a research tool but also as a therapeutic strategy for viral infection. We demonstrated that intracellular expression of short hairpin RNA (shRNA) targeting human cyclin T1 (hCycT1), a cellular factor essential for transcription of messenger and genomic RNAs from the long terminal repeat promoter of provirus of human immunodeficiency virus type 1 (HIV-1), could effectively suppress the replication of HIV-1. We also showed that downregulation of hCycT1 did not cause apoptotic cell death, therefore, targeting cellular factor hCycT1 by shRNAs may provide an attractive approach for genetic therapy of HIV-1 infection in the future. 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Keywords: RNA interference; Human immunodeficiency virus type 1; Short hairpin RNA; Cyclin T1
1. Introduction Acquired immune deficiency syndrome (AIDS), caused by human immunodeficiency virus type 1 (HIV-1), has reached pandemic degree in many parts of the world with no quick solution in sight. Although the development and use of double or triple combinations of anti-retroviral drugs for the treatment of HIV-1 infection has led to dramatic improvements in the lives of many HIV-infected individuals, the therapy cannot effect a radical cure of the virus and may result in drugresistant viral variants. Therefore, it is imminent to develop new antiviral therapeutic or preventive approaches, and RNA interference (RNAi) holds considerable promise in this respect. RNAi is a process in which double-stranded RNA triggers the silencing of gene expression in a sequence-specific manner [1,2]. In mammalian cells, RNAi can be triggered by 21-nucleotide (nt) small interfering RNA (siRNA) duplexes [3,4]. The siRNA can be delivered as synthetic RNA or short hairpin * Corresponding author. Fax: +86 27 6875 2897. E-mail address: [email protected] (D. Guo).
Abbreviations: RNAi, RNA interference; siRNA, small interfering RNA; shRNA, short hairpin RNA; HIV-1, human immunodeficiency virus type 1; hCycT1, human cyclin T1; P-TEFb, positive transcription elongation factor b
RNA (shRNA) that is processed into siRNA by cellular enzyme Dicer [5]. The ability to stably express siRNA/shRNA using viral vectors renders the possibility of using RNAi as a form of gene therapy to specifically inhibit viral infection. Previously reports have shown that RNAi can be used to successfully downregulate the expression of a number of HIV-1 genes including gag, pol, tat, vif, nef and rev, and reduce HIV-1 infectivity [6–18]. However, the high mutation rate of HIV will lead to viral mutants that can escape from the inhibition of siRNAs targeting viral genes [19–21]. Therefore, RNAi-mediated knockdown of cellular factors implicated in supporting the HIV-1 life cycle offers an alternative approach to overcome this obstacle. In fact, cellular factors such as CD4, CXCR4, CCR5, NF-kB, P-TEFb, Cyclophilin A, DC-SIGN, SPT-5 and PARP-1 have been successfully downregulated, resulting in the inhibition of HIV-1 replication and infection [6,11,13,15,16,22–32]. However, whether downregulation of cellular genes can result in apoptotic cell death has not been reported. In this report, we selected human cyclin T1 (hCycT1) as the target for RNAi-mediated inhibition of HIV-1 replication. The hCycT1 is a subunit of positive transcription elongation factor b (P-TEFb) and plays a key role in the activation of HIV-1 transcription by direct interaction with the viral protein Tat which binds to HIV-1 transactivation response element of HIV-1 RNA [33–36]. We showed that cellular expression of shRNAs could specifically silence hCycT1 without causing apoptotic cell death and downregulation of hCycT1 efficiently inhibited HIV-1 replication. During the process of current work, Chiu et al. [24] showed that silencing hCycT1 with synthetic siRNA could suppress HIV replication, and our observation with shRNA is supporting their conclusions.
2. Materials and methods 2.1. shRNA design and plasmid constructs We used the siRNA design tools (http://www.ambion.com/techlib/ misc/siRNA_tools.html) to find the candidate target sites in the mRNAs and design the hairpin siRNA-encoding DNA oligonucleotides. Five targeting sites of hCycT1 were selected and their coordinates were according to the sequence of GenBank NM001240: CT-1, GAACTTTCTTATCGCCAGC (100–118); CT-2, CAGCCTTGTTT CTAGCAGC (257–275); CT-3, CACTGAAAGAATACCGCGC (1163–1181); CT-4, GCCAAGAGTACTAAATCCT (1174–1792); CT-5, GCCAATGGTCACAACACGA (1936–1955). After we showed the functionality of these shRNAs, we saw a report by Chiu et al., that a synthetic siRNA to hCycT1 could effectively inhibit HIV-1 replication [24], therefore, we also made a corresponding shRNA construct
0014-5793/$30.00 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2005.04.074
Z. Li et al. / FEBS Letters 579 (2005) 3100–3106 (named as CT-6) for comparison. Four other control siRNAs included Tat, LucF, LucR and Daxx that targeted HIV-1 Tat (TATGGCAGGAAGAAGCGGA), firefly luciferase (CATTATCCTCTAGAGGATG), Renilla luciferase (GTAGCGCGGTGTATTATAC) and Daxx (GGAGUUGGAUCUCUCAGAA), respectively [7,13,37]. All of the siRNA sequences were converted to shRNAs with the loop sequence of UUCAAGAGA and cloned as double-stranded DNA oligonucleotides between BamHI and HindIII sites of the shRNA expression vector pSilencer-H1 (3.0) (Ambion, USA). All constructs were verified by sequencing. Plasmid pNL4-3lucRE carrying the HIV-1 cDNA clone with a firefly luciferase gene inserted into the nef gene of pNL4-3 and plasmid pJRFL carrying env gene were obtained through the National Institutes of Health AIDS Reagent Repository [38,39]. 2.2. Cell culture and transient transfection HeLa and 293T cells were obtained from China Center for Type Culture Collection (CCTCC), Wuhan, China. Cells were cultured and maintained in DMEM (DulbeccoÕs minimal essential medium) containing 10% heat-inactivated fetal bovine serum (FBS) (Hyclone), 2 mM L -glutamine (Gibco-BRL) and 100 U/ml of penicillin and 100 lg/ml of streptomycin (Gibco-BRL) in a humidified incubator (37 C, 5% CO2). U373-MAGI-CCR5e cells (NIH, USA) was a subclone of U373MG cells expressing CD4 linked to neomycin resistance gene, the HIV-1-LTR-b-gal sequence linked to hygromycin resistance gene and a modified CCR5 gene linked to a puromycin resistance gene [40]. The cells were cultured and maintained in DMEM containing 10% FBS, 0.2 mg/ml G418, 0.1 mg/ml hygromycin B, and 1.0 lg/ml puromycin (Invitrogen). Twelve hours before transfection, cells were seeded onto plates (Falcon) in DMEM containing 10% FBS. Approximately 50–70% confluences were plated. Transfections of shRNAs expression plasmid and control plasmid were performed using Lipofectamine 2000 (Invitrogen). Cells were incubated in the transfection mixture for 6 h, and then added antibiotic-free DMEM to each well. In order to harvest the cells in different time, the transfected cells were trypsinized and washed, then stored 20 C for Western blot analysis. 2.3. Western blot The transfected cells were harvested and rinsed twice with phosphate-buffered saline, pH 7.4 (PBS; Hyclone). Cell extracts were prepared with lysis buffer (50 mM Tris–HCl, pH 7.4; 150 mM NaCl; 1 mM PMSF; 1 mM EDTA; 5 lg/ml Aprotinin; 5 lg/ml Leupeptin; 1% Triton-100; 1% sodium deoxycholate; 0.1% SDS) and cleared by centrifugation at 12 000 · g and 4 C. Total protein concentration in clear lysates was measured using the Bio-Rad protein assay kit (BioRad) with bovine serum albumin as a standard according to the manufacturerÕs instructions. Proteins of 20 lg in total cell lysates were subjected to 8% SDS–PAGE, and the resolved proteins were transferred onto a polyvinylidene difluoride membrane (PVDF; Invitrogen). After blocking with PBS containing 10% non-fat milk for 1 h at room temperature, membranes were incubated with mouse monoclonal antibody to hCycT1 (Novocastra) or b-actin (KPL) in PBS containing 0.5% Tween 20 overnight at 4 C. The blot was detected by peroxidase labeled goat anti-mouse secondary antibody (KPL) and chemiluminescent substrate (KPL) and exposed to X-ray film (FUJI superRX). 2.4. Propidium iodide staining Cells treated with shRNA were trypsinized and washed, then resuspended in PBS, fixed in 70% ethanol, and stored at 20 C overnight. In preparation for apoptosis analysis, the cells were pelleted, resuspended in 1 ml of PBS containing 0.2 mg/ml propidium iodide (PI) and 0.15 g/ml RNase A, and incubated at 37 C for 30 min in dark. Stained cells were analyzed on flow cytometry (FACSan, Beckman Coulter). The apoptotic index was calculated by scoring no less than 104 cells. All observations were reproduced at least twice by independent experiments. 2.5. TUNEL labeling assay Cells were grown on coverslips in 24-well plates and transfected with shRNA constructs. Later, the cells were fixed with 4% (w/v) paraformadehyde in PBS for 1 h at room temperature, permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. The cells were subsequently
3101 incubated with TUNEL reaction mixture (Roche) at 37 C for 30 min. The patterns were visualized and recorded using fluorescence microscopy with a digital camera (Nikon). 2.6. Luciferase activity assay Luciferase activity was quantified using a Bright-Glo Luciferase Assay System Kit (Promega) in accordance with the manufacturerÕs instructions. After transfection or infection, 100 ll substrate solution was added per well and incubated for 2 min, and chemoluminescence was measured in a luminometer (Tecan GENios). All of the values were expressed as a mean of three independent experiments. 2.7. Preparation of viral stocks and virus infection Fresh viral stocks were prepared by co-transfection of 293T cells with 5 lg of pNL4-3LucR-E- and 5 lg of pJRFL with Lipofectamine 2000 reagent in a 10 cm2 tissue culture dish. After 48 h, the cells were washed once with phosphate-buffered saline (PBS), and newly produced viral particles were harvested over 3 h in 0.5 ml of fresh medium and were then filtered in a 0.45 lm filter. The concentration of p24 antigen released into cultured media was measured by ELISA (RETRO-TEK). For virus infection, we plated U373-MAGICCR5e cells at 0.6 · 105 cells per well (24 well plate), and the cells was 30% confluent one day after plating. After 12 h, transfection was done as described above. On the second day, virus was diluted to the concentration of 0.05, 0.1, 0.2, 0.4, 0.8 ng/ll of p24 in culture medium and 150 ll of virus was added to each well (12-well plate). DEAE Dextran was added at a final concentration of 20 lg/ml. After 2 h in a 37 C, 5% CO2 incubator, 1–2 ml of fresh culture medium was added to each well. Virus samples were tested in duplicate. 2.8. X-gal staining After U373-MAGI-CCR5e cells were infected for 2 days, cells were washed twice with PBS and were then fixed with 1% formaldehyde, 0.2% glutaraldehyde in PBS for 5 min at room temperature. Then cells were washed with PBS to remove traces of formaldehyde and glutaraldehyde and were treated with staining solution (5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl2, and 0.2% Xgal in PBS) for 1–16 h at 37 C. When the blue-stained cells were visible, X-Gal solution was removed and cells were covered with PBS and photographed.
3. Results 3.1. Specific inhibition of hCycT1 protein expression by shRNAs targeting hCycT1 The protein hCycT1 is a subunit of the P-TEFb complex that interacts with the transactivation domain of Tat and is involved in the species-specific activation of HIV-1 transcription. We first tested whether intracellularly expressed shRNAs could downregulate hCycT1 expression. Six constructs were obtained which led to expression of 6 shRNAs targeting hCycT1 (named as shRNA CT1–6, respectively). The shRNA CT6 corresponded to a synthetic siRNA sequence that had been shown to be capable to inhibit hCycT1 expression [24] and thus was used for comparison of effectiveness of shRNAs. The shRNA LucR that targeted specifically to Renilla luciferase gene was used as negative control. HeLa cells were transfected with individual shRNA-expressing constructs, and expression of hCycT1 was subjected to Western blot analysis with specific antibody to hCycT1. As shown in Fig. 1A, the expression of hCycT1 in HeLa cells transfected with hCycT1 shRNA CT-4 (lane 4) and shRNA CT-5 (lane 5) was significantly downregulated compared with that of control shRNA LucR (lane 7) whereas shRNA CT-6 (lane 6) only slightly reduced hCycT1. In contrast,
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specifically downregulate hCycT1 expression. Similar phenomena were demonstrated in 293T and U373-MAGICCR5e cells (data not shown). As hCycT1 shRNA CT-4 was the most effective shRNA to suppress the expression of hCycT1 in all the cell lines mentioned above, we used hCycT1 shRNA-4 for further studies. We next tested whether the inhibition of hCycT1 by shRNAs was dose- and time-dependent. As shown in Fig. 1B, increase of the quantity of the hCycT1 shRNA-expressing plasmid from 0.2 to 1.6 lg per well lowered the expression level of hCycT1 accordingly. Moreover, hCycT1 expression was detected at different times post transfection and the results showed (Fig. 1C) that down regulation of hCycT1 were most significant 36 h post transfection. These results indicated that the inhibition of hCycT1 expression by shRNAs was both dose- and time-dependent.
Fig. 1. Specific down regulation of human CycT1 expression by shRNAs. (A) HeLa cells were transfected with 0.8 lg shRNAexpressing constructs or mock transfected in 24 well plates, and hCycT1 expression was subjected to Western blot analysis after 48 h. Lanes 1–6 represent hCycT1 shRNAs 1–6, respectively. M indicates mock-transfected control while lane 7 is negative control shRNA LucR. (B) and (C) HeLa cells were transfected with 0 to 1.6 lg (B) or 0.8 lg (C) CT-4 shRNA construct in 24 well plates for 48 h (B) or 0, 12, 24, 36, 48 h (C), and then subjected to western blot analysis. bActin expression was used as internal control for all the western blot experiments.
shRNA CT-1 (lane 1), CT-2 (lane 2) and CT-3 (lane 3) had no effect on the expression of hCycT1, indicating positional effects of targeting sites. Moreover, the level of b-actin was unaffected by either control or hCycT1 shRNAs. These results indicated that intracellularly generated shRNAs could
3.2. Knockdown of hCycT1 protein did not induce apoptosis The prerequisite for inhibition of virus replication by interference with cellular gene expression is that knocking down the cellular gene would not lead to host cell apoptosis. To test the effect of hCycT1 on cell growth, we transfected shRNA CT-4 and control shRNA into different cell lines including 293T, HeLa and U373-MAGI-CCR5e. The transfection efficiency was determined by co-transfection of a reporter plasmid, pEGFP-C1, to confirm that the transfection efficiency was similar. Forty-eight hours post-transfection, cells were harvested and sub-G1 peak were determined by flow cytometry through apoptosis staining. The result showed that there was no statistically significant difference in sub-G1 between cells treated with control shRNA and those treated with hCycT1 shRNA in monolayer cell culture (Fig. 2A). Furthermore, the knockdown of hCycT1 did not affect the cell cycle through comparing with the control and
Fig. 2. Knockdown of hCycT1 by hCycT1 shRNA-4 does not induce apoptosis. HeLa cells were transfected with 4 lg hCycT1 shRNA CT-4, Daxx shRNA or negative control LucR shRNA constructs in 6 well plates and 48 h later, the sub-G1 peak was examined using FACS by PI staining (A) or the nuclei of apoptotic cells was stained by TUNEL labeling assay with fluorescein (B).
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mock transfection. In order to confirm this result, we did TUNEL labeling assay through analyzing the DNA fragments of the treated cells. The result showed that no apoptotic cells were found whereas, as a control, knocking down the Daxx protein in HeLa cells can cause apoptosis (Fig. 2B). These data indicated that down regulation of hCycT1 did not induce significant apoptosis of transfected cells and hCycT1 could be used as a potential target for inhibiting HIV-1 replication. 3.3. Knockdown of hCycT1 protein inhibits HIV-1 replication in 293T cells We used luciferase-expressing HIV-1 clone (pNL4-3LucRE-) to test whether knockdown of hCycT1 by shRNA could inhibit HIV replication. We co-transfected shRNA and pNL4-3LucR-E- plasmid into 293T cells and measured the luciferase activity at 48 h post transfection. As shown in Fig. 3A, the shRNA CT-4 reduced the luciferase activity significantly when compared with the cells transfected with control Renilla luciferase shRNA LucR or mock (Fig. 3A). The inhibition efficiency of different hCycT1 shRNAs was in accordance with the down regulation of hCycT1 protein expression levels (Fig. 1A and Fig. 3A). The inhibition time course and efficiency of the hCycT1 shRNA CT-4 was similar to that of shRNAs LucF and Tat which targeted the firefly luciferase reporter and HIV-1 Tat gene, respectively, indicating the strong inhibition effect of shRNA CT-4 (Fig. 3B). In addition, the inhibition effect of shRNA is dose-dependent as shown in Fig. 3C. Taken together, the shRNA CT-4 to hCycT1 could inhibit HIV-1 gene expression and replication specifically and efficiently in 293T cells. 3.4. Effects of hCycT1 shRNA on early stages of HIV-1 infection We further tested the influence of the hCycT1 interference on the early stages of HIV-1 infection with single-round replication HIV-1 viral particles. We cotransfected pNL4-3lucRE- and pJRFL into 293T cells and harvested the HIV-1 particles. At 12 h post transfection with shRNAs, the U373MAGI-CCR5e cells, which express b-galactosidase ( b-gal) under the control of HIV-1 LTR, were infected by the viral particles. The luciferase activity was measured at 48 h post infection and shRNA LucF targeting the luciferase reporter was used as control (Fig. 4 A and B). The Fig. 4A showed that Tat and hCycT1 shRNAs dramatically decrease the luciferase activity, indicating their significant suppression of viral replication. With different doses of viral particles (Fig. 4C), luciferase activity remained low with Tat and hCycT1 shRNAs, suggesting that viral replication could be inhibited regardless of the dose of viral particles used. U373-MAGI-CCR5e cells contain a single integrated copy of HIV-1 LTR linked to the b-gal gene. We next examined whether shRNAs against hCycT1 could suppress the activation of integrated HIV-1 LTR promoter. Cells were treated according to the method mentioned above and were stained by X-gal. Fig. 4D showed that shRNA directed against hCycT1 resulted in very low levels of b-gal gene expression. These results suggested that the activation of integrated HIV-1 promoter was suppressed in cells transfected with shRNA directed against hCycT1.
Fig. 3. Specific inhibition of HIV-1 replication by hCycT1 shRNAs in 293T cells. (A) 293T cells were cotransfected with 0.2 lg pNL43lucRE and indicated 0.2 lg shRNAs (LucF, firefly luciferase shRNA; Tat, Tat shRNA; CT-1 to CT-6, hCycT1 shRNA 1–6, respectively and LucR, Rennila luciferase shRNA) or mock in 96 well plates and the luciferase activity that was driven by HIV-1 replication was examined 48 h later. (B) 293T cells were cotransfected with 0.2 lg pNL4-3lucRE and indicated dose of CT-4, Tat, LucF or LucR in 96 well plates, 48 h later, luciferase activity assay were performed. (C) 293T cells were cotransfected with 0.2 lg pNL4-3lucRE and 0.2 lg CT-4, Tat, LucF, LucR or Mock in 96 well plates, luciferase activity assay were performed in 0, 12, 24, 36, 48 h.
4. Discussion RNAi with shRNA has been successfully utilized in a number of recent studies in cultured mammalian cells to reduce the expression of specific cellular and viral genes. Here, intracellularly expressed shRNA based on plasmid vector were utilized in 293T, HeLa and U373-MAGI-CCR5e cells to assay whether it could inhibit the expression of the hCycT1 protein and, in turn, whether inhibition of hCycT1 expression could modulate
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Fig. 4. Suppression of HIV-1 infection by knockdown of hCycT1 protein. (A) U373-MAGI-CCR5e cells were transfected with 0.8 lg Tat, LucF, CT-4 and LucR shRNAs or mock transfected in 24 well plates and 12 h post transfection, the cells were infected by 64 ng (p24) single-round replication HIV-1 particles. The luciferase activity was analyzed after 48 h. (B) and (C) U373-MAGI-CCR5e cells were transfected with indicated dose (B) or 0.8 lg (C) of Tat, LucF, CT-4 and LucR shRNAs in 24 well plates and 12 h post transfection, the cells were infected by 64 ng (p24) (B) or 8, 16, 32, 48, 64 ng (p24) (C) single-round replication HIV-1 particles. The luciferase activity was analyzed after 48 h. (D) Inhibition of HIV-1 5 0 -LTR promoter activation in cells transfected with CT-4 shRNA in 24 well plates shown by X-gal staining. U373-MAGI-CCR5e cells were transfected with 0.8 lg CT-4 and LucR shRNAs or mock transfected in 24 well plates and 12 h post transfection, the cells were infected by 64 ng (p24) single-round replication HIV-1 particles. 48 h later, the cells were stained by X-gal and photographed.
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HIV-1 gene expression and replication. In this study, we have demonstrated that hCycT1 shRNA suppressed hCycT1 protein expression but did not induce significant apoptosis in cultured cells. We have also shown that knockdown of hCycT1 expression by RNAi inhibited HIV-1 replication specifically and effectively. Furthermore, we observed the correlation between the levels of downregulation of hCycT1 expression and the inhibition of HIV-1 replication (Fig. 1B, Fig. 3B, Fig. 4B), indicating that the replication level of HIV-1 may be dependent on the amount of hCycT1 in cultured cells. This result was in contrast with one previous observation that low level of hCycT1 expression does not limit HIV-1 Tat function in cultured cells [41]. Cyclins act as regulatory elements of transcription activation protein complexes (P-TEFb) together with CDKs that function as catalytic subunits to phosphorylate carboxyl-terminal domain (CTD) of Pol II. CDK/cyclin complexes not only regulate cell division by phosphorylating different substrates, but also control several cellular pathways such as signal transduction, differentiation, and apoptosis [42–44]. CDK9/cyclin T directs its activity in a cell cycle independent manner and was involved in transcription during the elongation steps [45]. This means that hCycT1 is important for cell survival. Surprisingly, knockdown of hCycT1 protein neither influenced the cell cycle nor induced apoptosis in cultured cell lines, and this was consistent with a previous report that knockdown of P-TEFb led to inhibition of HIV-1 replication but not resulted in cell death [24]. This may indicate that very low level of P-TEFb kinase activity is sufficient for cell viability or the cells can compensate for lower P-TEFb protein levels by converting kinase-inactive P-TEFb to active forms [24]. Our results are supporting these explanations and demonstrated that the essential genes for cell viability like hCycT1 could also serve as target for suppression of HIV-1 infection and replications. In previous reports about RNAi with HIV-1 replication and infection, chemically synthesized siRNAs were most commonly used [9,11,24], however, synthetic siRNAs do not persist for long period in cells and are not feasible to be delivered into the vast number of target cells like T lymphocytes and macrophages. Here, we showed that intracellular expression of shRNAs from a DNA vector could efficiently downregulate hCycT1 expression and specifically suppress HIV-1 replication. Thus, this result renders a possibility to deliver the anti-HIV shRNA-encoding genes by viral vectors into target cells and stably express the shRNAs. In this study, the shRNA CT-6 to hCycT1 was designed according to the sequence of a synthetic siRNA which was effective to silence hCycT1 [24], however, the shRNA CT-6 appeared not as efficient as the corresponding synthetic siRNA (Fig. 1A) although we did not have direct comparison. Instead, the shRNA CT-4 to hCycT1 was most effective among the 6 shRNAs to hCycT1 (Fig. 1A). The discrepancy suggests that the efficiency of DNA vector-derived shRNA and the synthetic siRNAs may have different requirements for sequence configuration and, thus, the sequence of synthetic siRNA might not be translated into shRNA without changing its efficiency. Taken together, this study demonstrated the hCycT1 could be a promising cellular target for suppression of HIV-1 replication by DNA-based shRNAs. Our future study will focus on the delivery of shRNA-encoding genes into primary cells, such as T lymphocytes, monocytes and macrophages by lentiviral vectors and examine the feasibility to use hCycT1 as target in genetic therapy of AIDS.
Z. Li et al. / FEBS Letters 579 (2005) 3100–3106 Acknowledgments: We thank Juanjie Tang, Ping Chen, Ke Zhang, Ying Wu, Xin Qiu, Weijun Zhu, Yong Yu and Xiaoxing Huang for stimulating discussion and help in experiments. The HIV-1 reagents was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pNL4-3.Luc.RE from Dr. Nathaniel Landau and U373-MAGI-CCR5E from Dr. Michael Emerman. This study was supported by China NSFC Grants (30270313 and 30440030). D.G.Õs lab is supported by the startup package and ‘‘Luojia’’ Professorship Program of Wuhan University.
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FEBS 29612
Specific inhibition of HIV-1 replication by short hairpin RNAs targeting human cyclin T1 without inducing apoptosis Zhaoyang Li, Yong Xiong, Yu Peng, JiÕan Pan, Yu Chen, Xiaoyun Wu, Snawar Hussain, Po Tien, Deyin Guo* The Modern Virology Research Centre and State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China Received 16 March 2005; revised 10 April 2005; accepted 17 April 2005 Available online 16 May 2005 Edited by Shou-Wei Ding
Abstract RNA interference (RNAi), a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), can be used not only as a research tool but also as a therapeutic strategy for viral infection. We demonstrated that intracellular expression of short hairpin RNA (shRNA) targeting human cyclin T1 (hCycT1), a cellular factor essential for transcription of messenger and genomic RNAs from the long terminal repeat promoter of provirus of human immunodeficiency virus type 1 (HIV-1), could effectively suppress the replication of HIV-1. We also showed that downregulation of hCycT1 did not cause apoptotic cell death, therefore, targeting cellular factor hCycT1 by shRNAs may provide an attractive approach for genetic therapy of HIV-1 infection in the future. 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Keywords: RNA interference; Human immunodeficiency virus type 1; Short hairpin RNA; Cyclin T1
1. Introduction Acquired immune deficiency syndrome (AIDS), caused by human immunodeficiency virus type 1 (HIV-1), has reached pandemic degree in many parts of the world with no quick solution in sight. Although the development and use of double or triple combinations of anti-retroviral drugs for the treatment of HIV-1 infection has led to dramatic improvements in the lives of many HIV-infected individuals, the therapy cannot effect a radical cure of the virus and may result in drugresistant viral variants. Therefore, it is imminent to develop new antiviral therapeutic or preventive approaches, and RNA interference (RNAi) holds considerable promise in this respect. RNAi is a process in which double-stranded RNA triggers the silencing of gene expression in a sequence-specific manner [1,2]. In mammalian cells, RNAi can be triggered by 21-nucleotide (nt) small interfering RNA (siRNA) duplexes [3,4]. The siRNA can be delivered as synthetic RNA or short hairpin * Corresponding author. Fax: +86 27 6875 2897. E-mail address: [email protected] (D. Guo).
Abbreviations: RNAi, RNA interference; siRNA, small interfering RNA; shRNA, short hairpin RNA; HIV-1, human immunodeficiency virus type 1; hCycT1, human cyclin T1; P-TEFb, positive transcription elongation factor b
RNA (shRNA) that is processed into siRNA by cellular enzyme Dicer [5]. The ability to stably express siRNA/shRNA using viral vectors renders the possibility of using RNAi as a form of gene therapy to specifically inhibit viral infection. Previously reports have shown that RNAi can be used to successfully downregulate the expression of a number of HIV-1 genes including gag, pol, tat, vif, nef and rev, and reduce HIV-1 infectivity [6–18]. However, the high mutation rate of HIV will lead to viral mutants that can escape from the inhibition of siRNAs targeting viral genes [19–21]. Therefore, RNAi-mediated knockdown of cellular factors implicated in supporting the HIV-1 life cycle offers an alternative approach to overcome this obstacle. In fact, cellular factors such as CD4, CXCR4, CCR5, NF-kB, P-TEFb, Cyclophilin A, DC-SIGN, SPT-5 and PARP-1 have been successfully downregulated, resulting in the inhibition of HIV-1 replication and infection [6,11,13,15,16,22–32]. However, whether downregulation of cellular genes can result in apoptotic cell death has not been reported. In this report, we selected human cyclin T1 (hCycT1) as the target for RNAi-mediated inhibition of HIV-1 replication. The hCycT1 is a subunit of positive transcription elongation factor b (P-TEFb) and plays a key role in the activation of HIV-1 transcription by direct interaction with the viral protein Tat which binds to HIV-1 transactivation response element of HIV-1 RNA [33–36]. We showed that cellular expression of shRNAs could specifically silence hCycT1 without causing apoptotic cell death and downregulation of hCycT1 efficiently inhibited HIV-1 replication. During the process of current work, Chiu et al. [24] showed that silencing hCycT1 with synthetic siRNA could suppress HIV replication, and our observation with shRNA is supporting their conclusions.
2. Materials and methods 2.1. shRNA design and plasmid constructs We used the siRNA design tools (http://www.ambion.com/techlib/ misc/siRNA_tools.html) to find the candidate target sites in the mRNAs and design the hairpin siRNA-encoding DNA oligonucleotides. Five targeting sites of hCycT1 were selected and their coordinates were according to the sequence of GenBank NM001240: CT-1, GAACTTTCTTATCGCCAGC (100–118); CT-2, CAGCCTTGTTT CTAGCAGC (257–275); CT-3, CACTGAAAGAATACCGCGC (1163–1181); CT-4, GCCAAGAGTACTAAATCCT (1174–1792); CT-5, GCCAATGGTCACAACACGA (1936–1955). After we showed the functionality of these shRNAs, we saw a report by Chiu et al., that a synthetic siRNA to hCycT1 could effectively inhibit HIV-1 replication [24], therefore, we also made a corresponding shRNA construct
0014-5793/$30.00 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2005.04.074
Z. Li et al. / FEBS Letters 579 (2005) 3100–3106 (named as CT-6) for comparison. Four other control siRNAs included Tat, LucF, LucR and Daxx that targeted HIV-1 Tat (TATGGCAGGAAGAAGCGGA), firefly luciferase (CATTATCCTCTAGAGGATG), Renilla luciferase (GTAGCGCGGTGTATTATAC) and Daxx (GGAGUUGGAUCUCUCAGAA), respectively [7,13,37]. All of the siRNA sequences were converted to shRNAs with the loop sequence of UUCAAGAGA and cloned as double-stranded DNA oligonucleotides between BamHI and HindIII sites of the shRNA expression vector pSilencer-H1 (3.0) (Ambion, USA). All constructs were verified by sequencing. Plasmid pNL4-3lucRE carrying the HIV-1 cDNA clone with a firefly luciferase gene inserted into the nef gene of pNL4-3 and plasmid pJRFL carrying env gene were obtained through the National Institutes of Health AIDS Reagent Repository [38,39]. 2.2. Cell culture and transient transfection HeLa and 293T cells were obtained from China Center for Type Culture Collection (CCTCC), Wuhan, China. Cells were cultured and maintained in DMEM (DulbeccoÕs minimal essential medium) containing 10% heat-inactivated fetal bovine serum (FBS) (Hyclone), 2 mM L -glutamine (Gibco-BRL) and 100 U/ml of penicillin and 100 lg/ml of streptomycin (Gibco-BRL) in a humidified incubator (37 C, 5% CO2). U373-MAGI-CCR5e cells (NIH, USA) was a subclone of U373MG cells expressing CD4 linked to neomycin resistance gene, the HIV-1-LTR-b-gal sequence linked to hygromycin resistance gene and a modified CCR5 gene linked to a puromycin resistance gene [40]. The cells were cultured and maintained in DMEM containing 10% FBS, 0.2 mg/ml G418, 0.1 mg/ml hygromycin B, and 1.0 lg/ml puromycin (Invitrogen). Twelve hours before transfection, cells were seeded onto plates (Falcon) in DMEM containing 10% FBS. Approximately 50–70% confluences were plated. Transfections of shRNAs expression plasmid and control plasmid were performed using Lipofectamine 2000 (Invitrogen). Cells were incubated in the transfection mixture for 6 h, and then added antibiotic-free DMEM to each well. In order to harvest the cells in different time, the transfected cells were trypsinized and washed, then stored 20 C for Western blot analysis. 2.3. Western blot The transfected cells were harvested and rinsed twice with phosphate-buffered saline, pH 7.4 (PBS; Hyclone). Cell extracts were prepared with lysis buffer (50 mM Tris–HCl, pH 7.4; 150 mM NaCl; 1 mM PMSF; 1 mM EDTA; 5 lg/ml Aprotinin; 5 lg/ml Leupeptin; 1% Triton-100; 1% sodium deoxycholate; 0.1% SDS) and cleared by centrifugation at 12 000 · g and 4 C. Total protein concentration in clear lysates was measured using the Bio-Rad protein assay kit (BioRad) with bovine serum albumin as a standard according to the manufacturerÕs instructions. Proteins of 20 lg in total cell lysates were subjected to 8% SDS–PAGE, and the resolved proteins were transferred onto a polyvinylidene difluoride membrane (PVDF; Invitrogen). After blocking with PBS containing 10% non-fat milk for 1 h at room temperature, membranes were incubated with mouse monoclonal antibody to hCycT1 (Novocastra) or b-actin (KPL) in PBS containing 0.5% Tween 20 overnight at 4 C. The blot was detected by peroxidase labeled goat anti-mouse secondary antibody (KPL) and chemiluminescent substrate (KPL) and exposed to X-ray film (FUJI superRX). 2.4. Propidium iodide staining Cells treated with shRNA were trypsinized and washed, then resuspended in PBS, fixed in 70% ethanol, and stored at 20 C overnight. In preparation for apoptosis analysis, the cells were pelleted, resuspended in 1 ml of PBS containing 0.2 mg/ml propidium iodide (PI) and 0.15 g/ml RNase A, and incubated at 37 C for 30 min in dark. Stained cells were analyzed on flow cytometry (FACSan, Beckman Coulter). The apoptotic index was calculated by scoring no less than 104 cells. All observations were reproduced at least twice by independent experiments. 2.5. TUNEL labeling assay Cells were grown on coverslips in 24-well plates and transfected with shRNA constructs. Later, the cells were fixed with 4% (w/v) paraformadehyde in PBS for 1 h at room temperature, permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. The cells were subsequently
3101 incubated with TUNEL reaction mixture (Roche) at 37 C for 30 min. The patterns were visualized and recorded using fluorescence microscopy with a digital camera (Nikon). 2.6. Luciferase activity assay Luciferase activity was quantified using a Bright-Glo Luciferase Assay System Kit (Promega) in accordance with the manufacturerÕs instructions. After transfection or infection, 100 ll substrate solution was added per well and incubated for 2 min, and chemoluminescence was measured in a luminometer (Tecan GENios). All of the values were expressed as a mean of three independent experiments. 2.7. Preparation of viral stocks and virus infection Fresh viral stocks were prepared by co-transfection of 293T cells with 5 lg of pNL4-3LucR-E- and 5 lg of pJRFL with Lipofectamine 2000 reagent in a 10 cm2 tissue culture dish. After 48 h, the cells were washed once with phosphate-buffered saline (PBS), and newly produced viral particles were harvested over 3 h in 0.5 ml of fresh medium and were then filtered in a 0.45 lm filter. The concentration of p24 antigen released into cultured media was measured by ELISA (RETRO-TEK). For virus infection, we plated U373-MAGICCR5e cells at 0.6 · 105 cells per well (24 well plate), and the cells was 30% confluent one day after plating. After 12 h, transfection was done as described above. On the second day, virus was diluted to the concentration of 0.05, 0.1, 0.2, 0.4, 0.8 ng/ll of p24 in culture medium and 150 ll of virus was added to each well (12-well plate). DEAE Dextran was added at a final concentration of 20 lg/ml. After 2 h in a 37 C, 5% CO2 incubator, 1–2 ml of fresh culture medium was added to each well. Virus samples were tested in duplicate. 2.8. X-gal staining After U373-MAGI-CCR5e cells were infected for 2 days, cells were washed twice with PBS and were then fixed with 1% formaldehyde, 0.2% glutaraldehyde in PBS for 5 min at room temperature. Then cells were washed with PBS to remove traces of formaldehyde and glutaraldehyde and were treated with staining solution (5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl2, and 0.2% Xgal in PBS) for 1–16 h at 37 C. When the blue-stained cells were visible, X-Gal solution was removed and cells were covered with PBS and photographed.
3. Results 3.1. Specific inhibition of hCycT1 protein expression by shRNAs targeting hCycT1 The protein hCycT1 is a subunit of the P-TEFb complex that interacts with the transactivation domain of Tat and is involved in the species-specific activation of HIV-1 transcription. We first tested whether intracellularly expressed shRNAs could downregulate hCycT1 expression. Six constructs were obtained which led to expression of 6 shRNAs targeting hCycT1 (named as shRNA CT1–6, respectively). The shRNA CT6 corresponded to a synthetic siRNA sequence that had been shown to be capable to inhibit hCycT1 expression [24] and thus was used for comparison of effectiveness of shRNAs. The shRNA LucR that targeted specifically to Renilla luciferase gene was used as negative control. HeLa cells were transfected with individual shRNA-expressing constructs, and expression of hCycT1 was subjected to Western blot analysis with specific antibody to hCycT1. As shown in Fig. 1A, the expression of hCycT1 in HeLa cells transfected with hCycT1 shRNA CT-4 (lane 4) and shRNA CT-5 (lane 5) was significantly downregulated compared with that of control shRNA LucR (lane 7) whereas shRNA CT-6 (lane 6) only slightly reduced hCycT1. In contrast,
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specifically downregulate hCycT1 expression. Similar phenomena were demonstrated in 293T and U373-MAGICCR5e cells (data not shown). As hCycT1 shRNA CT-4 was the most effective shRNA to suppress the expression of hCycT1 in all the cell lines mentioned above, we used hCycT1 shRNA-4 for further studies. We next tested whether the inhibition of hCycT1 by shRNAs was dose- and time-dependent. As shown in Fig. 1B, increase of the quantity of the hCycT1 shRNA-expressing plasmid from 0.2 to 1.6 lg per well lowered the expression level of hCycT1 accordingly. Moreover, hCycT1 expression was detected at different times post transfection and the results showed (Fig. 1C) that down regulation of hCycT1 were most significant 36 h post transfection. These results indicated that the inhibition of hCycT1 expression by shRNAs was both dose- and time-dependent.
Fig. 1. Specific down regulation of human CycT1 expression by shRNAs. (A) HeLa cells were transfected with 0.8 lg shRNAexpressing constructs or mock transfected in 24 well plates, and hCycT1 expression was subjected to Western blot analysis after 48 h. Lanes 1–6 represent hCycT1 shRNAs 1–6, respectively. M indicates mock-transfected control while lane 7 is negative control shRNA LucR. (B) and (C) HeLa cells were transfected with 0 to 1.6 lg (B) or 0.8 lg (C) CT-4 shRNA construct in 24 well plates for 48 h (B) or 0, 12, 24, 36, 48 h (C), and then subjected to western blot analysis. bActin expression was used as internal control for all the western blot experiments.
shRNA CT-1 (lane 1), CT-2 (lane 2) and CT-3 (lane 3) had no effect on the expression of hCycT1, indicating positional effects of targeting sites. Moreover, the level of b-actin was unaffected by either control or hCycT1 shRNAs. These results indicated that intracellularly generated shRNAs could
3.2. Knockdown of hCycT1 protein did not induce apoptosis The prerequisite for inhibition of virus replication by interference with cellular gene expression is that knocking down the cellular gene would not lead to host cell apoptosis. To test the effect of hCycT1 on cell growth, we transfected shRNA CT-4 and control shRNA into different cell lines including 293T, HeLa and U373-MAGI-CCR5e. The transfection efficiency was determined by co-transfection of a reporter plasmid, pEGFP-C1, to confirm that the transfection efficiency was similar. Forty-eight hours post-transfection, cells were harvested and sub-G1 peak were determined by flow cytometry through apoptosis staining. The result showed that there was no statistically significant difference in sub-G1 between cells treated with control shRNA and those treated with hCycT1 shRNA in monolayer cell culture (Fig. 2A). Furthermore, the knockdown of hCycT1 did not affect the cell cycle through comparing with the control and
Fig. 2. Knockdown of hCycT1 by hCycT1 shRNA-4 does not induce apoptosis. HeLa cells were transfected with 4 lg hCycT1 shRNA CT-4, Daxx shRNA or negative control LucR shRNA constructs in 6 well plates and 48 h later, the sub-G1 peak was examined using FACS by PI staining (A) or the nuclei of apoptotic cells was stained by TUNEL labeling assay with fluorescein (B).
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mock transfection. In order to confirm this result, we did TUNEL labeling assay through analyzing the DNA fragments of the treated cells. The result showed that no apoptotic cells were found whereas, as a control, knocking down the Daxx protein in HeLa cells can cause apoptosis (Fig. 2B). These data indicated that down regulation of hCycT1 did not induce significant apoptosis of transfected cells and hCycT1 could be used as a potential target for inhibiting HIV-1 replication. 3.3. Knockdown of hCycT1 protein inhibits HIV-1 replication in 293T cells We used luciferase-expressing HIV-1 clone (pNL4-3LucRE-) to test whether knockdown of hCycT1 by shRNA could inhibit HIV replication. We co-transfected shRNA and pNL4-3LucR-E- plasmid into 293T cells and measured the luciferase activity at 48 h post transfection. As shown in Fig. 3A, the shRNA CT-4 reduced the luciferase activity significantly when compared with the cells transfected with control Renilla luciferase shRNA LucR or mock (Fig. 3A). The inhibition efficiency of different hCycT1 shRNAs was in accordance with the down regulation of hCycT1 protein expression levels (Fig. 1A and Fig. 3A). The inhibition time course and efficiency of the hCycT1 shRNA CT-4 was similar to that of shRNAs LucF and Tat which targeted the firefly luciferase reporter and HIV-1 Tat gene, respectively, indicating the strong inhibition effect of shRNA CT-4 (Fig. 3B). In addition, the inhibition effect of shRNA is dose-dependent as shown in Fig. 3C. Taken together, the shRNA CT-4 to hCycT1 could inhibit HIV-1 gene expression and replication specifically and efficiently in 293T cells. 3.4. Effects of hCycT1 shRNA on early stages of HIV-1 infection We further tested the influence of the hCycT1 interference on the early stages of HIV-1 infection with single-round replication HIV-1 viral particles. We cotransfected pNL4-3lucRE- and pJRFL into 293T cells and harvested the HIV-1 particles. At 12 h post transfection with shRNAs, the U373MAGI-CCR5e cells, which express b-galactosidase ( b-gal) under the control of HIV-1 LTR, were infected by the viral particles. The luciferase activity was measured at 48 h post infection and shRNA LucF targeting the luciferase reporter was used as control (Fig. 4 A and B). The Fig. 4A showed that Tat and hCycT1 shRNAs dramatically decrease the luciferase activity, indicating their significant suppression of viral replication. With different doses of viral particles (Fig. 4C), luciferase activity remained low with Tat and hCycT1 shRNAs, suggesting that viral replication could be inhibited regardless of the dose of viral particles used. U373-MAGI-CCR5e cells contain a single integrated copy of HIV-1 LTR linked to the b-gal gene. We next examined whether shRNAs against hCycT1 could suppress the activation of integrated HIV-1 LTR promoter. Cells were treated according to the method mentioned above and were stained by X-gal. Fig. 4D showed that shRNA directed against hCycT1 resulted in very low levels of b-gal gene expression. These results suggested that the activation of integrated HIV-1 promoter was suppressed in cells transfected with shRNA directed against hCycT1.
Fig. 3. Specific inhibition of HIV-1 replication by hCycT1 shRNAs in 293T cells. (A) 293T cells were cotransfected with 0.2 lg pNL43lucRE and indicated 0.2 lg shRNAs (LucF, firefly luciferase shRNA; Tat, Tat shRNA; CT-1 to CT-6, hCycT1 shRNA 1–6, respectively and LucR, Rennila luciferase shRNA) or mock in 96 well plates and the luciferase activity that was driven by HIV-1 replication was examined 48 h later. (B) 293T cells were cotransfected with 0.2 lg pNL4-3lucRE and indicated dose of CT-4, Tat, LucF or LucR in 96 well plates, 48 h later, luciferase activity assay were performed. (C) 293T cells were cotransfected with 0.2 lg pNL4-3lucRE and 0.2 lg CT-4, Tat, LucF, LucR or Mock in 96 well plates, luciferase activity assay were performed in 0, 12, 24, 36, 48 h.
4. Discussion RNAi with shRNA has been successfully utilized in a number of recent studies in cultured mammalian cells to reduce the expression of specific cellular and viral genes. Here, intracellularly expressed shRNA based on plasmid vector were utilized in 293T, HeLa and U373-MAGI-CCR5e cells to assay whether it could inhibit the expression of the hCycT1 protein and, in turn, whether inhibition of hCycT1 expression could modulate
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Fig. 4. Suppression of HIV-1 infection by knockdown of hCycT1 protein. (A) U373-MAGI-CCR5e cells were transfected with 0.8 lg Tat, LucF, CT-4 and LucR shRNAs or mock transfected in 24 well plates and 12 h post transfection, the cells were infected by 64 ng (p24) single-round replication HIV-1 particles. The luciferase activity was analyzed after 48 h. (B) and (C) U373-MAGI-CCR5e cells were transfected with indicated dose (B) or 0.8 lg (C) of Tat, LucF, CT-4 and LucR shRNAs in 24 well plates and 12 h post transfection, the cells were infected by 64 ng (p24) (B) or 8, 16, 32, 48, 64 ng (p24) (C) single-round replication HIV-1 particles. The luciferase activity was analyzed after 48 h. (D) Inhibition of HIV-1 5 0 -LTR promoter activation in cells transfected with CT-4 shRNA in 24 well plates shown by X-gal staining. U373-MAGI-CCR5e cells were transfected with 0.8 lg CT-4 and LucR shRNAs or mock transfected in 24 well plates and 12 h post transfection, the cells were infected by 64 ng (p24) single-round replication HIV-1 particles. 48 h later, the cells were stained by X-gal and photographed.
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HIV-1 gene expression and replication. In this study, we have demonstrated that hCycT1 shRNA suppressed hCycT1 protein expression but did not induce significant apoptosis in cultured cells. We have also shown that knockdown of hCycT1 expression by RNAi inhibited HIV-1 replication specifically and effectively. Furthermore, we observed the correlation between the levels of downregulation of hCycT1 expression and the inhibition of HIV-1 replication (Fig. 1B, Fig. 3B, Fig. 4B), indicating that the replication level of HIV-1 may be dependent on the amount of hCycT1 in cultured cells. This result was in contrast with one previous observation that low level of hCycT1 expression does not limit HIV-1 Tat function in cultured cells [41]. Cyclins act as regulatory elements of transcription activation protein complexes (P-TEFb) together with CDKs that function as catalytic subunits to phosphorylate carboxyl-terminal domain (CTD) of Pol II. CDK/cyclin complexes not only regulate cell division by phosphorylating different substrates, but also control several cellular pathways such as signal transduction, differentiation, and apoptosis [42–44]. CDK9/cyclin T directs its activity in a cell cycle independent manner and was involved in transcription during the elongation steps [45]. This means that hCycT1 is important for cell survival. Surprisingly, knockdown of hCycT1 protein neither influenced the cell cycle nor induced apoptosis in cultured cell lines, and this was consistent with a previous report that knockdown of P-TEFb led to inhibition of HIV-1 replication but not resulted in cell death [24]. This may indicate that very low level of P-TEFb kinase activity is sufficient for cell viability or the cells can compensate for lower P-TEFb protein levels by converting kinase-inactive P-TEFb to active forms [24]. Our results are supporting these explanations and demonstrated that the essential genes for cell viability like hCycT1 could also serve as target for suppression of HIV-1 infection and replications. In previous reports about RNAi with HIV-1 replication and infection, chemically synthesized siRNAs were most commonly used [9,11,24], however, synthetic siRNAs do not persist for long period in cells and are not feasible to be delivered into the vast number of target cells like T lymphocytes and macrophages. Here, we showed that intracellular expression of shRNAs from a DNA vector could efficiently downregulate hCycT1 expression and specifically suppress HIV-1 replication. Thus, this result renders a possibility to deliver the anti-HIV shRNA-encoding genes by viral vectors into target cells and stably express the shRNAs. In this study, the shRNA CT-6 to hCycT1 was designed according to the sequence of a synthetic siRNA which was effective to silence hCycT1 [24], however, the shRNA CT-6 appeared not as efficient as the corresponding synthetic siRNA (Fig. 1A) although we did not have direct comparison. Instead, the shRNA CT-4 to hCycT1 was most effective among the 6 shRNAs to hCycT1 (Fig. 1A). The discrepancy suggests that the efficiency of DNA vector-derived shRNA and the synthetic siRNAs may have different requirements for sequence configuration and, thus, the sequence of synthetic siRNA might not be translated into shRNA without changing its efficiency. Taken together, this study demonstrated the hCycT1 could be a promising cellular target for suppression of HIV-1 replication by DNA-based shRNAs. Our future study will focus on the delivery of shRNA-encoding genes into primary cells, such as T lymphocytes, monocytes and macrophages by lentiviral vectors and examine the feasibility to use hCycT1 as target in genetic therapy of AIDS.
Z. Li et al. / FEBS Letters 579 (2005) 3100–3106 Acknowledgments: We thank Juanjie Tang, Ping Chen, Ke Zhang, Ying Wu, Xin Qiu, Weijun Zhu, Yong Yu and Xiaoxing Huang for stimulating discussion and help in experiments. The HIV-1 reagents was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pNL4-3.Luc.RE from Dr. Nathaniel Landau and U373-MAGI-CCR5E from Dr. Michael Emerman. This study was supported by China NSFC Grants (30270313 and 30440030). D.G.Õs lab is supported by the startup package and ‘‘Luojia’’ Professorship Program of Wuhan University.
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