AACF
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NIAID's Antimicrobial Acquisition and
Coordinating Facility AACF |
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Submission Info Screening Assays |
Hepatitis C Virus Assays
HCV RNA Replicon Protocol Introduction We use the cell line Huh7 ET (luc-ubi-neo/ET), which contains a new HCV RNA replicon with a stable luciferase (LUC) reporter. This particular construct has not been described in the scientific literature. It is similar to the cell line 5-2 (1), but contains additional modifications that make the cell line more robust and provide stable LUC expression for antiviral screening. This composition of the replicon is shown diagrammatically in Fig. 1.
The LUC reporter is used as an indirect measure of HCV replication. The activity of the LUC reporter is directly proportional to HCV RNA levels and positive control antiviral compounds behave comparably using either LUC or RNA endpoints. The use of the LUC endpoint is more economical than HCV RNA and can be used for high-throughput applications to screen libraries of compounds.
Primary HCV RNA replicon assayFirst we examine the effect of drugs added in triplicate at a single high-test concentration of 20 mM on HCV RNA-derived LUC activity and cytotoxicity. Human interferon alpha-2b is included in each run as a positive control compound. Subconfluent cultures of the ET line are plated out into 96-well plates that are dedicated for the analysis of cell numbers (cytotoxicity) or antiviral activity and the next day drugs are added to the appropriate wells. Cells are processed 72 hr later when the cells are still subconfluent. Compounds that reduced the LUC signal by 50% or more relative to the untreated cell controls move forward in the program. Compound cytotoxicity is assessed as the percent viable cells relative to the untreated cell controls. This data is also reported.
HCV RNA replicon confirmatory assayThe HCV RNA replicon comfirmatory assay is then used to examine the effects of compounds at five half-log concentrations each (Fig. 2). Human interferon alpha-2b is included in each run as a positive control compound. Subconfluent cultures of the ET line are plated out into 96-well plates that are dedicated for the analysis of cell numbers (cytotoxicity) or antiviral activity and the next day drugs are added to the appropriate wells. Cells are processed 72 hr later when the cells are still subconfluent. Compound EC50 and EC90 values (antiviral activity) are derived from HCV RNA levels assessed as either HCV RNA replicon-derived LUC activity or as HCV RNA using TaqMan RT-PCR. Compound IC50 and IC90 values (cytotoxicity) are calculated using CytoTox-1 (Promega), a colorimetric assay used as an indicator of cell numbers and cytotoxicity when the LUC assay system is employed, while ribosomal (rRNA) levels determined via TaqMan RT-PCR are used as an indication of cell numbers in the RNA-based assay. Compound selectivity indices SI50 and SI90 values are calculated from the spreadsheets (Fig. 3).
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