Fast hepatitis C virus RNA elimination and NS5A redistribution by NS5A inhibitors studied by a multiplex assay approach

Abstract

While earlier therapeutic strategies for the treatment of hepatitis C virus (HCV) infection relied exclusively on interferon (IFN) and ribavirin (RBV), four direct-acting antiviral agents (DAAs) have now been approved, aiming for an interferon-free strategy with a short treatment duration and fewer side effects. To facilitate studies on the mechanism of action (MOA) and efficacy of DAAs, we established a multiplex assay approach, which employs flow cytometry, a Gaussia luciferase reporter system, Western blot analysis, reverse transcription-quantitative PCR (RT-qPCR), a limited dilution assay (50% tissue culture infectious dose [TCID50]), and an image profiling assay that follows the NS5A redistribution in response to drug treatment. We used this approach to compare the relative potency of various DAAs and the kinetics of their antiviral effects as a potential preclinical measure of their potential clinical utility. We evaluated the NS5A inhibitors ledipasvir (LDV) and daclatasvir (DCV), the NS3/4A inhibitor danoprevir (DNV), and the NS5B inhibitor sofosbuvir (SOF). In terms of kinetics, our data demonstrate that the NS5A inhibitor LDV, followed closely by DCV, has the fastest effect on suppression of viral proteins and RNA and on redistribution of NS5A. In terms of MOA, LDV has a more pronounced effect than DCV on the viral replication, assembly, and infectivity of released virus. Our approach can be used to facilitate the study of the biological processes involved in HCV replication and help identify optimal drug combinations.

FIG 1

HCV gt2a viral genome organization and experimental design of HCV infection and DAA treatment. (A) Schematic diagrams of the HCV gt2a virus expressing an NS5A-Ypet fusion protein as a marker for viral replication and the HCV gt2a virus carrying a Gaussia luciferase reporter gene at the p7-NS2 junction as a secretable marker for viral replication. (B) Huh-7.5.1 cells were seeded into wells followed by infection with either Jc1/Ypet virus or Jc1/Gluc2A virus for 48 h and then treated with compounds from 3 different classes of HCV DAAs: NS5A inhibitors (DCV and LDV), NS3/4A protease inhibitor (DNV), or NS5B polymerase inhibitor (SOF). IRES, internal ribosome entry site.

FIG 2

Assessment of HCV inhibition by DAAs from 3 classes using flow cytometry assay. Dose-response curves are shown for DCV (A), LDV (B), DNV (C), and SOF (D). Jc1/Ypet virus-infected Huh-7.5.1 cells were treated with DMSO (control) or different concentrations of the HCV inhibitors. Samples were analyzed for Ypet cell populations at the indicated time points as described in Materials and Methods. Data were from gated live cell population (dead cells were excluded). Each data point represents the average value from 2 individual experiments. Error bars represent standard errors of the mean (SEM).

FIG 3

Assessment of HCV inhibition by DAAs from 3 classes using a Gaussia luciferase reporter system. Dose-response curves are shown for DCV (A), LDV (B), DNV (C), and SOF (D). Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or different concentrations of the HCV inhibitors. Culture supernatants were analyzed for luciferase activity at the indicated time points as described in Materials and Methods. Data were normalized to data for DMSO controls. Each data point represents the average value from 3 individual experiments. Error bars represent SEM.

FIG 4

Assessment of HCV inhibition by DAAs from 3 classes using Western blot analysis. Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or the HCV inhibitors at concentrations of 100× EC₅₀ (DCV, 3.2 nM; LDV, 3 μM; DNV, 0.32 μM; SOF, 20 μM). Cell lysates were harvested at 8 hpt (C and D) or at 24 hpt (A and B) and blotted for NS5A (A and C) or core (B and D). Data were normalized to GAPDH and quantified as relative fold change with respect to DMSO. Each data point represents the average value from 3 individual experiments. Error bars represent SEM. Statistical analysis was performed by using Student's t test (*P < 0.05).

FIG 5

Schematic diagram of the image profiling assay. Cells were seeded in 96-well plates and infected with Jc1/Gluc2A virus followed by DAA treatment as described in Materials and Methods. (A) Nine images for every condition were obtained by an automated autofocus Zeiss LSM 510 Meta confocal microscope. Data were processed using the LSM Toolbox functions in the ImageJ program and converted to a format compatible with the CellProfiler software. CellProfiler was used to identify cells and define their cytoplasm and nuclei. Morphological changes in the cytoplasm were analyzed using image profiling methods. (B) Representative analyses based on changes of the Gabor texture are shown for DCV (left panel) and DNV (right panel). Dose-dependent NS5A texture changes were seen for DCV treatment but not for DNV treatment. Nuclei were stained with DRAQ5 (blue channel) and HCV NS5A with green fluorescent protein (GFP) (green channel).

FIG 6

Assessment of HCV inhibition by DAAs from 3 classes using the image profiling assay. Dose-response curves are shown for DCV (A), LDV (B), DNV (C), and SOF (D). Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or different concentrations of the HCV inhibitors. Cells were fixed and stained with DRAQ5 and 9E10 anti-NS5A antibodies as described in Materials and Methods. Images were acquired at ×40 magnification and analyzed for NS5A Gabor texture changes at the indicated time points. Data were normalized to data for DMSO controls. Each data point represents the average value from 2 individual experiments. Error bars represent SEM.

FIG 7

Assessment of HCV inhibition by DAAs from 3 classes using the RT-qPCR assay. Dose-response curves are shown for DCV (A), LDV (B), DNV (C), and SOF (D). Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or different concentrations of the HCV inhibitors. The total cellular RNA was extracted by TRIzol reagent and analyzed for HCV 5′ UTR RNA by RT-qPCR at the indicated time points as described in Materials and Methods. Cycle threshold (C_T) values were converted to copy numbers based on the standard curve of pure Jc1/Gluc2A genome-encoded plasmid. Data were normalized to data for DMSO controls. Each data point represents the average value from 2 individual experiments. Error bars represent SEM.

FIG 8

Assessment of HCV inhibition by DAAs from 3 classes using the infectivity assay. Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or 100× EC₅₀ of the HCV inhibitors. Extracellular virus was precipitated by PEG and analyzed for viral titer by a TCID₅₀ assay, and extracellular viral RNA was detected by RT-qPCR at 8 hpt as described in Materials and Methods. Data were normalized to data for DMSO controls. Each data point represents the average value from two individual experiments. Error bars represent SEM. The statistical analysis was performed by using Student's t test (*P < 0.05). (A) Extracellular RNA copy number per milliliter; (B) extracellular viral infectivity per milliliter.