HCV is the major causative agent of acute and chronic liver diseases [1]. 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 [2,3]. In the developed world, HCV accounts for two-thirds of all cases of liver cancer and transplants [4], and in the US, approximately 12,000 people are estimated to die from HCV each year [5].

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) [6,7]. 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 [8,9] and can be associated with significant side effects [10]. 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 [11]. 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 [2,12].

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 [13,14]. 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]. 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 [21,22] and the potent investigational NS5A inhibitor, BMS-790052, which is currently in phase II studies [23]. As HTA agents, we chose cyclophilin inhibitors such as cyclosporine A (CsA) [24], two non-immunosuppressive CsA analogs: Alisporivir [25] and SCY-635 [26] as well as sanglifehrin A (SfA) and one SfA analog: BC544 [27]. 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.

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 [29,30].

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.