Original Article Fluoroquinolones Inhibit HCV by Targeting Its Helicase

Antiviral Therapy 2012; 17:467–476 (doi: 10.3851/IMP1937) Original article Fluoroquinolones inhibit HCV by targeting its helicase Irfan A Khan1,2, Sammer Siddiqui1, Sadiq Rehmani1, Shahana U Kazmi2, Syed H Ali1,3* 1Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan 2Department of Microbiology, University of Karachi, Karachi, Pakistan 3Department of Microbiology, Dow University of Health Sciences, Karachi, Pakistan *Corresponding author e-mail: [email protected] Background: HCV has infected >170 million individuals of 12 different fluoroquinolones. Afterwards, Huh-7 and worldwide. Effective therapy against HCV is still lacking and Huh-8 cells were lysed and viral RNA was extracted. The there is a need to develop potent drugs against the virus. extracted RNA was reverse transcribed and quantified by In the present study, we have employed two culture models real-time quantitative PCR. Fluoroquinolones were also to test the activity of fluoroquinolone drugs against HCV: a tested on purified NS3 protein in a molecular-beacon- subgenomic replicon that is able to replicate independently based in vitro helicase assay. in the cell line Huh-8 and the Huh-7 cell culture model Results: To varying degrees, all of the tested fluoroqui- that employs cells transfected with synthetic HCV RNA to nolones effectively inhibited HCV replication in both produce the infectious HCV particles. Fluoroquinolones have Huh-7 and Huh-8 culture models. The inhibition of HCV also been shown to have inhibitory activity against certain NS3 helicase activity was also observed with all 12 of the viruses, possibly by targeting the viral helicase. To tease out fluoroquinolones. the mechanism of the antiviral activity of fluoroquinolones, Conclusions: Fluoroquinolones inhibit HCV replication their effect on HCV NS3 helicase protein was also tested. possibly by targeting the HCV NS3 helicase. These drugs Methods: Huh-7 cells producing the HCV virion as well hold promise for the treatment of HCV infection. as Huh-8 cells were grown in the presence or absence Introduction The burden of HCV infection is substantial world- Fluoroquinolones have been found effective against wide, with an overall prevalence of 2.2–3.0% [1]. The vaccinia virus and papovaviruses [6,7]. Such studies current recommended treatment for HCV combines prompted a series of efforts to synthesize modified fluo- pegylated interferon (IFN)-a2a and a broad spectrum roquinolones to enhance their antiviral activities [6]. antiviral drug, ribavirin. Such a regimen has been Various fluoroquinolones with broad-spectrum antivi- shown to be only moderately effective in combating ral activities were identified and tested in clinical trials HCV infection, with considerable variation across the with varying outcomes [6,8,9]. On the basis of these HCV genotypes [2,3]. Hence, more effective thera- and our own studies on fluoroquinolone-mediated inhi- peutic options need to be identified, which should bition of simian virus 40 [10], we hypothesized that preferably incorporate a short treatment duration and fluoroquinolones might possess an inhibitory activity low toxicity profile. against HCV. Quinolones form a major group of antibacterial Recently, the availability of efficient virus culture drugs, active against a broad spectrum of microorgan- systems for HCV [11,12] has made it possible to screen isms. These drugs target the bacterial enzymes DNA for antiviral drugs against this virus. To test antiviral gyrase and topoisomerase IV [4], specifically interact- activity of fluoroquinolones against HCV, two cell cul- ing with the DNA-binding unit of gyrase called Gyr A. ture systems were employed in the current study: first, Such an interaction leads to impaired bacterial DNA the human hepatoma (Huh)-7 cell line, transfected with replication and double-stranded DNA breaks [5]. The a chimeric HCV RNA, capable of producing complete versatility of the quinolone molecule has led to the dis- HCV virions and second, the Huh-8 cell line, harbour- covery of various quinolone-based bioactive agents that ing an autonomously replicating HCV subgenomic also exhibit antiviral activities [6]. replicon [11,13]. A fluorescence-based helicase assay ©2012 International Medical Press 1359-6535 (print) 2040-2058 (online) 467 AVT-11-OA-2098_Khan.indd 467 10/04/2012 11:36:25 IA Khan et al. [14] was also used to test the effect of fluoroquinolones centrifuge tube. To this mixture was added 500 µl on the activity of NS3 helicase. Here, we show that all HEPES buffer, pH 7.0 (50 mM HEPES, 280 mM NaCl of the 12 fluoroquinolones used in this study inhibited and 1.5 mM Na2HPO4) dropwise on a vortex mixer HCV production and HCV RNA replication, as well as for 30 s. After incubation for 30 min, the mixture was the activity of HCV NS3 helicase. added to Huh-7 cells plated in 60×15 mm cell culture dishes [15]. After 3–4 h, the medium was aspirated and Methods the cells were replenished with fresh complete medium [10,12]. After 48 h, the expression of transfected Plasmids and cell lines RNA in the cells was confirmed by immunostain- Full-length FL-J6/JFH genotype 2a/2a chimeric clone, ing, using a monoclonal antibody against HCV NS3 Huh-7 (gift from Dr Charles M Rice) and Huh-8 har- protein (AbCam, Cambridge, UK), and by detecting bouring an autonomous Con1/SG-Neo genotype 1b HCV RNA in a PCR, using primers against HCV NS3 subgenomic replicon with neomycin phosphotransfer- gene: forward (5′-CATGGCATGCATGTCGG-3′) and sase (NPTII) selectable marker were used. reverse (5′-CGATGTAAGGGAGGTGTGAGG-3′). The transfected cells were then allowed to grow for Cell culture 2–3 weeks until cell death became apparent. The Huh-7 cells were maintained in complete growth supernatant containing HCV virion was then collected medium (Dulbecco’s modified Eagle medium contain- and filtered through a 0.22 mm filter. The supernatant ing 10% heat-inactivated fetal bovine serum, 0.1 mM was then stored at 4°C, and used to infect fresh Huh-7 non-essential amino acids and 1% penicillin/strepto- cells. The success of infection was confirmed by immu- mycin solution). All reagents were purchased from Inv- nostaining and PCR, as described above. itrogen (Carlsbad, CA, USA). Huh-8 cells, carrying the self-replicating subgenomic replicon, were maintained Determination of antiviral activity of fluoroquinolones in the same growth medium, additionally containing Twelve fluoroquinolones (ofloxacin, 8-quinolinol, G418 (0.75 mg/ml; Sigma–Aldrich, St Louis, MO, 8-hydroxyquinoline, cinoxacin, enoxacin, enrofloxacin, USA) [11,13]. fleroxacin, flumiquine, lumifloxacin, norfloxacin, balo- floxacin and difloxacin) were obtained from Sigma– FL-J6/JFH in vitro transcription Aldrich [10]. The desired fluoroquinolones were added FL-J6/JFH plasmid was linearized with XbaI (New to Huh-7 or Huh-8 cells at concentrations of 0.01 mM, England BioLabs, Ipswich, MA, USA), purified using 0.1 mM, 1.0 mM or 10 mM. After 4 days (96 h), total gel filtration columns (Promega, Madison, WI, USA) RNA was isolated from both the cell lines and HCV and used for in vitro transcription of HCV genomic RNA levels were quantitated in triplicate in a quantita- RNA with T7 Express RiboMAX (Promega). RNA tive real-time PCR (qPCR), using primers against the transcription reaction mixture (20 ml) contained 2 ml HCV NS3 gene (described above). As an internal con- enzyme mix T7 Express (T7 RNA polymerase, recom- trol, the house-keeping gene b-globin was also quanti- binant RNasin ribonuclease inhibitor and recombinant tated in all samples. HCV RNA levels were corrected inorganic pyrophosphatase), 10 ml RiboMAX Express for the levels of b-globin RNA in all samples. T7 2X buffer, 110 units RQ1 RNase-Free DNase, 1 mg linearized DNA template and Nuclease-Free water up Time-point assay to determine effects of to 20 ml (Promega). Following a 0.5 h synthesis at 37°C, fluoroquinolones on cellular and viral RNA replication the synthesized RNA was DNase-treated with RQ1 Both Huh-7 and Huh-8 cells were split in six-well RNase-Free DNase (Promega). RNA concentration tissue culture plates (Corning Inc., Corning, NY, was measured by spectrophotometry and aliquots were USA), grown overnight and then treated with 1.0 mM stored at -80°C until use. or 10 mM fluoroquinolones. Total RNA was isolated from treated and untreated cells after 24, 48, 72 and HCV RNA transfection of Huh-7 cells 96 h. HCV RNA was quantitated in triplicate by For RNA transfection, cells were washed with amplifying the HCV NS3 gene in a qPCR. b-Globin phosphate- buffered saline (PBS), trypsinized, re- messenger RNA (mRNA) levels were measured as well suspended in complete growth medium and plated to assess the inhibitory effects of fluoroquinolones on overnight, aiming for 80–90% confluency. The fol- cellular RNA production. lowing day, cells were placed in fresh medium 2–3 h prior to transfection. Transfection was achieved using HCV RNA analysis and quantitation a CaCl2 transfection protocol [15]. FL-J6/JFH RNA Total RNA was isolated from Huh-8 or HCV-infected transcripts (2–3 mg) were mixed with 50 ml 2.5M Huh-7 cells using an AquaPure RNA Isolation kit CaCl2 and 450 ml sterile distilled water in a 1.5 ml (Bio-Rad Laboratories, Hercules, CA, USA). For 468 ©2012 International Medical Press AVT-11-OA-2098_Khan.indd 468 10/04/2012 11:36:25 Inhibition of HCV by fluoroquinolones complementary DNA (cDNA) synthesis, 2 mg RNA with 14,000× rpm for 15 min, dissolved in 1.5 ml of 20 mM 0.5 mg random primers was added in 15 ml sterile water Tris (pH 8), 50 mM NaCl, 1 mM EDTA, 0.1 mM DTT and heated at 70°C for 5 min. The reaction was placed (gel filtration buffer), and centrifuged at 14,000× rpm on ice and 2 ml 10 mM dNTPs mix, 2 ml 5×M-MLV RT for 15 min (fraction III).

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