HCV is the major causative agent of acute and chronic liver diseases [1 Dienstag JL, McHutchison JG. American Gastroenterological Association technical review on the management of hepatitis C Gastroenterology 2006; 130(1): 231-64. quiz 14-7]. Nearly 200 million people worldwide (3% of the population), including 4 to 5 million in the US, are chronically infected with HCV and 4 million new infections occur every year [2 Alter MJ. Epidemiology of hepatitis C virus infection World J Gastroenterol 2007; 13(17): 2436-41.,3 Soriano V, Madejon A, Vispo E, et al. Emerging drugs for hepatitis C Expert Opin Emerg Drugs 2008; 13(1): 1-19.]. In the developed world, HCV accounts for two-thirds of all cases of liver cancer and transplants [4 Shepard CW, Finelli L, Alter MJ. Global epidemiology of hepatitis C virus infection Lancet Infect Dis 2005; 5(9): 558-67.], and in the US, approximately 12,000 people are estimated to die from HCV each year [5 Armstrong GL, Wasley A, Simard EP, McQuillan GM, Kuhnert WL, Alter MJ. The prevalence of hepatitis C virus infection in the United States, 1999 through 2002 Ann Intern Med 2006; 144(10): 705-14.].

The introduction of IFNα and the nucleoside analog ribavirin (RBV) greatly improved the percentage of chronically HCV-infected patients able to reach a sustained antiviral response (SVR) [6 Tong MJ, Reddy KR, Lee WM, et al. Treatment of chronic hepatitis C with consensus interferon: a multicenter, randomized, controlled trial. Consensus Interferon Study Group Hepatology 1997; 26(3): 747-54.,7 Sy T, Jamal MM. Epidemiology of hepatitis C virus (HCV) infection Int J Med Sci 2006; 3(2): 41-6.]. Yet, the current standard PEGylated IFNα plus RBV (pIFNα/RBV) therapy has a low success rate of approximately 50% in patients with genotype 1 HCV [8 Simmonds P, Bukh J, Combet C, et al. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes Hepatology 2005; 42(4): 962-73.,9 Cross TJ, Antoniades CG, Harrison PM. Current and future management of chronic hepatitis C infection Postgrad Med J 2008; 84(990): 172-6.] and can be associated with significant side effects [10 Manns MP, Wedemeyer H, Cornberg M. Treating viral hepatitis C: efficacy, side effects, and complications Gut 2006; 55(9): 1350-9.]. Not only is genotype 1 the most prevalent HCV genotype in Europe, North and South America, China and Japan, it is also the most difficult to treat [11Global surveillance and control of hepatitis C. Report of a WHO Consultation organized in collaboration with the Viral Hepatitis Prevention Board, Antwerp, Belgium J Viral Hepat 1999; 6(1): 35-47.]. Thus, there is an urgent need for the development of additional anti-HCV agents with novel mechanisms of antiviral action in order to provide alternate treatments for the increasing number of patients who are unresponsive to the current pIFNα/RBV treatment [2 Alter MJ. Epidemiology of hepatitis C virus infection World J Gastroenterol 2007; 13(17): 2436-41.,12 Davis GL, Albright JE, Cook SF, Rosenberg DM. Projecting future complications of chronic hepatitis C in the United States Liver Transpl 2003; 9(4): 331-8.].

The availability of assays, which permit the identification of new anti-HCV agents, is limited. Two main assays were developed for the precise quantification of HCV replicon replication and for the discovery of antivirals: a real-time RT-PCR assay and a luciferase-based reporter assay [13 Lee JC, Chang CF, Chi YH, Hwang DR, Hsu JT. A reporter-based assay for identifying hepatitis C virus inhibitors based on subgenomic replicon cells J Virolog Methods 2004; 116(1): 27-33.,14 Krieger N, Lohmann V, Bartenschlager R. Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations J Virol 2001; 75(10): 4614-24.]. Although these systems have proven to be vital in understanding many aspects of HCV replication, their main disadvantage is cost, both RT-PCR and luciferase reagents are expensive. In this study, we developed a rapid, sensitive and reproducible flow cytometry-based assay, which permits the identification of new anti-HCV agents in live cells in a quantitative manner to complement the existing HCV replicon replication assays currently available.

We found that both Luc-Con1 and Luc-Con1-NS5A-YFP cell lines efficiently replicated the viral genome as demonstrated by high and sustained luciferase levels over a period of one month and behaved in nearly identical manners when titrated with Alisporivir (Fig. 1C, D). This further suggests that the YFP insertion into the NS5A gene does not significantly alter the replication of the replicon. Second, we used confocal microscopy to analyze the expression of the NS5A-YFP protein in cells harboring the Luc-Con1-NS5A-YFP replicon. The YFP fluorescence signal was found in the cytoplasm as bright dots in a reticular staining pattern that surrounds the nucleus that likely represents the endoplasmic reticulum (ER) compartment (Fig. 1E). This pattern is similar to the distribution of HCV nonstructural proteins in Huh-7 cells harboring subgenomic HCV replicons, as determined by immunofluorescence microscopy [20 Gosert R, Egger D, Lohmann V, et al. Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons J Virol 2003; 77(9): 5487-92.]. Taken together, the similarity of size, distribution, and morphology of the dot-like YFP structures identified here with those previously identified as HCV RNA replication complexes, strongly suggests that the NS5A-YFP fusion protein is incorporated into such complexes.

We then assessed whether the established Luc-Con1-NS5A-YFP replicon cell line permits the screening of antiviral agents. We selected a panel of anti-HCV compounds including direct-acting (DAA) and host-targeting antivirals (HTA). As DAA, we chose the recently FDA-approved protease inhibitor telaprevir [21 Zeuzem S, Andreone P, Pol S, et al. Telaprevir for retreatment of HCV infection N Eng J Med 2011; 364(25): 2417-8.,22 Jacobson IM, McHutchison JG, Dusheiko G, et al. Telaprevir for previously untreated chronic hepatitis C virus infection N Eng J Med 2011; 364(25): 2405-16.] and the potent investigational NS5A inhibitor, BMS-790052, which is currently in phase II studies [23 Gao M, Nettles RE, Belema M, et al. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect Nature 2010; 465(7294): 96-100.]. As HTA agents, we chose cyclophilin inhibitors such as cyclosporine A (CsA) [24 Watashi K, Hijikata M, Hosaka M, Yamaji M, Shimotohno K. Cyclosporin A suppresses replication of hepatitis C virus genome in cultured hepatocytes Hepatology 2003; 38(5): 1282-8.], two non-immunosuppressive CsA analogs: Alisporivir [25 Watashi K. Alisporivir, a cyclosporin derivative that selectively inhibits cyclophilin, for the treatment of HCV infection Curr Opin Investig Drugs 2010; 11(2): 213-4.] and SCY-635 [26 Hopkins S, Scorneaux B, Huang Z, et al. SCY-635, a novel nonimmunosuppressive analog of cyclosporine that exhibits potent inhibition of hepatitis C virus RNA replication in vitro Antimicrob Agents Chemother 2010; 54(2): 660-72.] as well as sanglifehrin A (SfA) and one SfA analog: BC544 [27 Gregory MA, Bobardt M, Obeid S, et al. Preclinical characterization of naturally occurring polyketide cyclophilin inhibitors from the sanglifehrin family Antimicrob Agents Chemother 2011; 55(5): 1975-81.]. Alisporivir and SCY-635 are currently being tested for safety and efficacy in phase III and II studies, respectively, whilst BC544 is in preclinical development.

Efficacy of the selected compounds was first examined by luciferase production in lysates of Luc-Con1-NS5A-YFP replicon cells 3 days post-drug exposure. The 50% effective concentrations (EC₅₀) for inhibition of Luc-Con1-NS5A-YFP replication in Huh-7.5.1 cells by telaprevir, BMS-790052, CsA, Alisporivir, SCY-635, SfA, BC544 and BC556 were 8.1 ± 1.4, 1.4 ± 0.2, 311 ± 23, 7.9 ± 0.4, 8.2 ± 1.1, 288 ± 33, 6.1 ± 1.2 and 5.4 ± 0.6 nM, respectively (Table 1). Except for CsA, a majority of the drugs exhibit low cytotoxicity (CC₅₀ >10 µM) (Table 1). After verifying high antiviral activities of the selected compounds using luciferase as the reporter gene, we examined their potency using NS5A-YFP as the reporter gene by flow cytometry. Stable Luc-Con1-NS5A-YFP replicon cells were exposed to control (DMSO) or selected drugs and analyzed by FACS for YFP content. Parental Huh-7.5.1 cells express minimal autofluorescence (0.3%), whereas Luc-Con1-NS5A-YFP cells exhibit a strong YFP signal (81.2%). All drugs when used at a concentration of 2μM and their antiviral activity assessed between a 24-96 hr period reduced the percentage of YFP+ cells, indicating that they efficiently block HCV RNA replication. Specifically, at the 72hr time point the percentage of YFP+ cells was reduced from 81.2% to 1.22% for telaprevir, 2.42% for BMS-790052, 3.94% for CsA, 5.3% for Alisporivir, 1.77% for SCY-635, 33.5% for SfA, and 9.36% for BC544 (Fig. 2A). There is thus a direct correlation for antiviral potency as measured by luciferase in cell lysates and YFP fluorescence in live cells.

Table 1.

Inhibitory and Cytotoxic Effects of Antivirals on Luc-Con1-NS5A-YFP Subgenomic Replicon Replication

Kinetic studies indicate that the percentage of YFP+ cells 2 days post-drug exposure is already reduced (Fig. 2B), but 3 days post-drug treatment, all compounds, except SfA, dramatically reduced the YFP fluorescence signal (Fig. 2B), demonstrating the high potency of the selected compounds. Results were unchanged 4 days post-drug treatment (Fig. 2B), suggesting that the inhibitory action of the selected antivirals reaches a maximal effect after 3 days. Titration studies using the cyclophilin inhibitor Alisporivir as an antiviral prototype demonstrate that the flow cytometry-based live cell assay represents a useful tool to quantitatively screen HCV inhibitors (Fig. 2C). We verified the FACS results (Fig. 2B) by confocal analysis. Exposure of Luc-Con1-NS5A-YFP cells to cyclophilin inhibitors such as Alisporivir and SCY-635 totally abolished the YFP fluorescence signal compared to the control (Fig. 2D). We also verified the block of viral replication by examining HCV protein levels in Luc-Con1-NS5A-YFP cells by Western blotting. NS5A-YFP levels were profoundly reduced 2 and 3 days post-drug treatment (Fig. 2E). Similar results were obtained for NS5B (Fig. 2E). Interestingly, an NS5B signal was still detectable three days post-SfA treatment (Fig. 2E), correlating well with residual YFP levels detected by flow cytometry in SfA-treated live cells (Fig. 2B). Similar levels of calnexin and cyclophilin A (CypA) indicate that similar levels of proteins were analyzed (Fig. 2E). Intracellular cyclophilin B (CypB) levels were dramatically reduced in Huh-7.5.1 cells treated with all cyclophilin inhibitors (CsA, Alisporivir, SCY-635, SfA or BC544), but not with non-cyclophilin inhibitors (telaprevir and BMS-790052) (Fig. 2E). These data are in accordance with previous findings including ours that cyclophilin inhibitors such as CsA and Alisporivir, by binding to the enzymatic hydrophobic pocket of CypB, trigger the release of CypB from the ER into the extracellular medium [29 Price ER, Jin M, Lim D, Pati S, Walsh CT, McKeon FD. Cyclophilin B trafficking through the secretory pathway is altered by binding of cyclosporin A Proc Natl Acad Sci USA 1994; 91(9): 3931-5.,30 Flisiak R, Horban A, Gallay P, et al. The cyclophilin inhibitor Debio-025 shows potent anti-hepatitis C effect in patients coinfected with hepatitis C and human immunodeficiency virus Hepatol 2008; 47(3): 817-26.].

In this study, we established a flow cytometry live cell-based assay that permits a rapid and sensitive screening of HCV inhibitors. Specifically, we created a stable cell line, which harbors a subgenomic replicon encoding an NS5A-YFP fusion protein. This system allows direct and sensitive measure of YFP fluorescence in live hepatoma cells, which support autonomous viral RNA replication and viral protein expression from the HCV replicon. Importantly, this flow cytometry system is not only fast, but it also exhibits an extremely low variability between assays. Furthermore, we demonstrated that this stable fluorescent system permits the precise quantification of HCV replication inhibition by mechanistically distinct classes of inhibitors including direct-acting (DAA) and host-targeting antiviral (HTA) agents. In conclusion, this flow cytometry live cell-based assay is well suited for multiple applications including the evaluation of HCV replication and anti-HCV drug screening.