Screening of Hepatitis C Virus Inhibitors Using Genotype 1a HCV Replicon Cell Lines

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1 Screening of Hepatitis C Virus Inhibitors Using Genotype 1a HCV Replicon Cell Lines Margaret Robinson, 1 Yang Tian, 1 Nikos Pagratis, 1 and William E. Delaney, IV 1 UNIT Gilead Sciences, Foster City, California ABSTRACT Hepatitis C virus (HCV) replicons are the primary tools for identifying and evaluating anti-hcv compounds during drug discovery research. Genotype 1a and 1b are the most common genotypes in North America and Europe. These genotypes display significant genetic divergence ( 20% at the nucleotide level and 14% at the amino acid level), which can translate into marked differences in drug susceptibility. Thus, it is critical that potential therapeutics be assessed against both genotypes to ensure antiviral efficacy in the broad genotype 1 patient population. This unit describes assays for screening HCV inhibitors using replicons with a focus on genotype 1a. Specific protocols are provided for screening 1a replicons that encode the Renilla luciferase gene and for replicons that do not encode exogenous reporter genes, both in 96-well (manual) and in 384-well (highthroughput) formats. Curr. Protoc. Microbiol. 22: C 2011 by John Wiley & Sons, Inc. Keywords: hepatitis C virus (HCV) genotype 1a drug screening replicon INTRODUCTION HCV replicons are self-replicating RNA sequences derived from the HCV genome (Bartenschlager, 2005). The discovery of the HCV replicon by Lohman et al. (1999) was a major advance for drug discovery efforts as it provided the first opportunity to study HCV inhibitors in a cell-based system dependent on viral RNA replication. Replicons quickly became crucial for discovering and optimizing new HCV inhibitors. HCV is a genetically diverse virus that can be classified into six major genotypes and multiple subtypes (Kuiken and Simmonds, 2009). To date, three HCV genotypes have been demonstrated to replicate robustly in vitro as replicons: genotypes 1b, (Lohmann et al., 1999), 1a (Blight et al., 2000), and 2a (Kato et al., 2003). Marked differences in inhibitor potency have been observed between genotypes for many inhibitor classes, making it important to screen individual HCV genotypes and subtypes for all potential therapeutics. In this unit, protocols and guidance for screening HCV inhibitors using genotype 1a HCV replicons are provided. Basic Protocol 1 describes a facile way to screen inhibitors using replicons that do not express exogenous reporter genes by taking advantage of the endogenous NS3/4A protease activity of HCV. This protocol is intended to be run on 96- well plates, which are commonly used for manual antiviral (EC 50 ) assays. The Alternate Protocol is a modified version of Basic Protocol 1 that allows for an endpoint read (instead of kinetic), which shortens the assay read time and increases the assay throughput. The availability of newer genotype 1a replicons, which incorporate luciferase reporter genes, enables HCV replication to be conveniently assessed using robust, high signalto-noise ratio bioluminescence assays. Basic Protocol 2 provides a protocol optimized for assaying antiviral activity in genotype 1a replicons encoding Renilla luciferase, also in a 96-well format. Although the throughput provided by 96-well assays is sufficient Current Protocols in Microbiology , August 2011 Published online August 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: / mc1707s12 Copyright C 2011 John Wiley & Sons, Inc

2 for many antiviral applications, the throughput can be further enhanced by running these assays in a 384-well format. Basic Protocols 3 and 4 thus provide methods for testing replicons without or with, respectively, a Renilla luciferase reporter in fully automated high-throughput formats (384-well). BASIC PROTOCOL 1 DETERMINATION OF ANTIVIRAL ACTIVITY (EC 50 VALUES) IN GENOTYPE 1a HCV REPLICONS USING A KINETIC NS3 PROTEASE ASSAY The following protocol provides a method to screen the potency of HCV replicon inhibitors in vitro using genotype 1a (H77 strain) replicon cell lines in a 96-well format. The EC 50 value is defined as the compound concentration required to inhibit 50% of viral replication and in this protocol is determined using a previously reported NS3 protease kinetic assay (Yang and Delaney, 2006). Briefly, this assay uses a sensitive NS3/4A protease substrate to quantify the presence of active NS3/4A in replicon cell cultures. Cells robustly replicating the HCV replicon express quantities of active NS3/4A protease that can readily be detected in lysates prepared from small scale cultures (e.g., 96- or 384-well plates). Therefore, the NS3/4A protease activity within cell lysates can be used as an indirect measure of HCV replication levels (O Boyle et al., 2005; Yang and Delaney, 2006). The 96-well assay format is ideal for low-throughput experiments involving small sets of compounds. The NS3 protease assay is appropriate for any HCV replicon cell lines that do not express a reporter gene (this assay has also been successfully applied to genotype 1b and 2a replicon systems), although it may also be used in replicon cell lines that express exogenous reporters as well. The fluorescence resonance energy transfer quench (FRET-Q) pair of the europium chelate W1284 and the dye QSY7 conjugated to a peptide substrate allows efficient substrate cleavage by NS3/4A protease, which separates the fluorophore from the quencher, providing high detection sensitivity using common fluorometers (Yang and Delaney, 2006). An advantage of this particular FRET-Q pair is that it allows time-resolved (TR) fluorescence to be measured, which substantially reduces assay noise by separating the times of fluorophore excitation and quantification. Consequently, this TR-FRET-Q substrate allows detection of the NS3/4A activity in cell lysates using low and therefore cost-effective concentrations of the protease substrate. Screening of Hepatitis C Virus Inhibitors Using Replicon Cell Lines Materials Genotype 1a HCV replicon cell line (e.g., H77 replicon cell line, Apath; or any cell line with similar or higher levels of HCV replicon expression) Complete Dulbecco s modified Eagle medium (DMEM; see recipe) 1 Dulbecco s phosphate-buffered saline (DPBS) without calcium and magnesium (Cellgro, cat. no ) 0.5% (10 ) trypsin with EDTA 4Na (Invitrogen, cat. no ) Replicon cell assay medium (see recipe) Antiviral test compounds, suggested control compounds are the nucleoside, 2 C-methyl adenosine (2 CMA), or the NS3 protease inhibitor BILN-2061 (both available for purchase from Acme Biosciences) DMSO 5 cell lysis buffer (Promega, cat. no. E1531) 5 M NaCl solution (Sigma Aldrich, cat. no. S6546) TR-FRET-Q NS3 protease substrate (see recipe) 37 C, 5% CO 2 humidified cell culture incubator Cell culture flasks (e.g., Corning 175-cm 2, cat. no ) Aspirator 10-ml pipets Current Protocols in Microbiology

3 50-ml disposable tubes (BD Falcon, cat. no ) Microscope and hemacytometer 96-well plates for drug dilution (V-bottom plate; VWR International, cat. no ), media dilution (deep-well; VWR International, cat. no ), and fluorescence assays (white plate with flat bottom; Sigma-Aldrich, cat. no. CLS3610) Multi-channel pipettors (10-μl, 200-μl, 1000-μl; e.g., Rainin P10, P200, P1000) Microplate shaker (Thermo Scientific Barnstead/Lab-Line titer plate shaker or similar) Fluorescence plate reader (e.g., PerkinElmer Victor 3 fluorescence/luminescence reader or similar, e.g., Biotek Synergy, Molecular Devices Analyst) Prepare and maintain cells 1. Maintain 1a-H77 replicon cells in complete DMEM cell culture medium in a 37 C, 5% CO 2 humidified incubator. For general maintenance, passage cells two times a week to maintain cell density at <80% confluence. A large culture flask (175-cm 2 ) will grow to 70% to 80% confluence within 4 days when seeded at a density of cells/flask in 50 ml of medium. HCV subgenomic replicons typically encode the neomycin phosphotransferase gene, which is a selection marker conferring resistance to Geneticin. Geneticin is added into the complete cell culture medium to maintain a population of cells that contain replicons. The correct seeding density of replicon cells is important and may need optimization. If the seeding cell density is too low, cells grow slowly so it may take a long period of time to produce enough cells for experiments. It is also critical to prevent cells from becoming over-confluent. Cells reaching confluence will slow their growth, which has been shown to significantly reduce HCV replication (Pietschmann et al., 2001; Scholle et al., 2004). Under these conditions, the level of NS3 protease will drop significantly. It is further recommended that replicon cell lines be passaged no more than 20 to 25 times since replicon levels progressively decline with long-term passaging. 2. When cells are ready to be seeded for antiviral assays ( 80% confluent), detach them from tissue culture flasks as follows. Aspirate culture medium from the flask and wash cells once with 15 ml of room temperature DPBS. Add 5 ml of 1 trypsin (diluted in DPBS) and incubate flask 5 min in 37 C incubator to allow the trypsin to loosen the monolayer. Add 15 ml of replicon cell assay medium to stop the trypsin digestion. Separate cells with a 10-ml pipet by gently pipetting up and down a few times. Transfer cell suspension to a 50-ml polypropylene centrifuge tube. 3. Count cells under a microscope using a hemacytometer and calculate the total cell number (see APPENDIX 4A). 4. Add appropriate volume of replicon cell assay medium (excluding Geneticin) to achieve a final concentration of cells/ml. A confluent 175-cm 2 flask should typically yield sufficient cells to seed four to seven replicon assay plates. Geneticin is excluded from the assay medium because it is toxic to the cells during replicon inhibition. Seed cells in assay plates for EC 50 assay (96-well format) Cell seeding (day 1) 5. Dispense replicon cells in 100 μl of replicon cell assay medium into each well of columns 1 through 11 of a clear-bottom, white 96-well plate. Fill column 12 with 100 μl of complete cell culture medium only. Incubate plate 24 hr in a 37 C, 5% CO 2 humidified incubator Current Protocols in Microbiology

4 Four compounds can be tested per plate in this protocol seed one plate for one to four compounds, two plates for five to eight compounds, etc. Cells are plated 1 day before the addition of antiviral compounds to ensure optimal attachment and growth of cells during the 72-hr antiviral assay. However, it is possible to seed cells in the morning of day 1 and treat with compounds later in the same day; if this is done, the cells should be given a minimum of 3 hr to attach before proceeding. Column 12 is not seeded with cells as it will later serve as a background control for the assay readout. Preparation of antiviral compound dilutions (day 2) 6. Dissolve test compounds in DMSO at a concentration of 10 mm. A concentration of 10 mm is recommended for stock drug solutions; solubility or compound limitations may require lower stock concentrations to be prepared. DMSO is the most common solvent used in pharmaceutical research since it solubilizes most compounds; however, some compounds may be prepared in other solvents as solubility permits. 7. Dilute test compounds serially in a V-bottom 96-well plate (Fig A) as follows. Dispense 20 μl of DMSO into all wells in columns 2 through 11 using a multi-channel pipettor. Place 30 μl of the 10 mm test compounds in the wells of column 1. Serially dilute the compounds 1:3 across the plate (horizontally, going from left to right, using 8 channels of a multichannel pipettor for a full plate of compounds) by transferring and mixing 10 μl of compounds into the 20 μl of DMSO in the adjacent well (to the right). Do not dilute compounds into the final columns (11 and 12), as these last two columns contain the untreated control and noise/background control for the assay, respectively. A V-bottom plate is recommended, since it concentrates small volumes at the bottom of the wells. A 1:3 dilution scheme is recommended here as a starting point; however, higher or lower dilution factors could be used to cover broader concentration ranges, or provide greater precision over a smaller concentration range. The starting concentrations can be revised along with the dilution factor to best measure the potency of the compounds being tested. The starting concentration of test compounds (concentrations in the left-most column of Fig A) can be adjusted based on anticipated potency. 8. Dispense 500 μl of replicon cell assay medium (excluding Geneticin) to all wells of a 96-well deep-well plate using a multi-channel pipettor. Transfer 5 μl of DMSO/drug dilutions from the V-bottom serial dilution 96-well plate to the 96-well deep-well plate containing 500 μl medium per well (this step achieves a test compound dilution of 1:100; Fig B). A volume of 500 μl is the recommended volume to make enough medium/drug dilution for two duplicate cell assay plates. This volume can be increased or decreased depending on the number of replicates being tested. 9. Using a multi-channel pipettor, thoroughly mix the medium/drug dilutions in the 96-well, deep-well plate. Remove plates containing replicon cells from the cell culture incubator and dispense 100 μl of each medium/compound dilution (row) to two independent rows of a 96-well plate containing replicon cells, achieving a final concentration of 0.5% DMSO and a 1:200 dilution compared to the original drug dilution plate in a total volume of 200 μl (each cell plate contains four test compounds in duplicate; Fig C). Incubate plate for 72 hr in a 37 C, 5% CO 2 humidified incubator. Screening of Hepatitis C Virus Inhibitors Using Replicon Cell Lines When dispensing medium containing drug dilutions to cell culture plates, do not disturb cell monolayer on the bottom of the plate. To preserve cell monolayer, place multi-channel pipettor tips against the side of the well when dispensing medium. It is critical to maintain a final DMSO concentration of 0.5%. Concentrations of 1% significantly affect the growth of the replicon cells. Current Protocols in Microbiology

5 DMSO 96-well (V-bottom) compound plate serially dilute compounds three-fold in DMSO Column number dilute DMSO compound dilutions 100 into cell culture medium Medium 96-well (deep-well) compound plate transfer 100 l to assay plates HCV replicon cell assay plates with compounds Figure A 96-well plate map and schema for dilution protocol. (A) Drugs are initially diluted serially in 100% DMSO as described in Basic Protocol 1, step 7, then 30 μl of test compounds (dissolved in DMSO) are added to wells in column 1; 20 μl of DMSO is then added to all wells in columns 2 through 11. Compounds are diluted 1:3 from columns 1 to 10 using a multichannel pipettor (horizontally from left to right). Compounds are not added to column 11 (which later serves as an untreated control) or to column 12 (which later serves as a background control during assay readouts). (B) Drug dilutions are transferred into an intermediate plate wherein they are further diluted in replicon cell assay medium at a factor of 1:100. (C) 100 μl of drug dilutions are then transferred from the intermediate dilution plate into the final replicon cell assay plate (which contains replicon cells previously seeded in a volume of 100 μl replicon cell assay medium) achieving the indicated final drug concentrations in a final volume of 200 μl and 0.5% DMSO Current Protocols in Microbiology

6 Pipetting the same dilutions into two rows of replicon cells provides a more robust analysis by overcoming some cell-based variability (e.g., potential uneven seeding or cell growth between wells). A 3-day (72 hr) assay is the industry standard for calculating EC 50 values. Compound activity can be measured at earlier or later time points; however, potency may vary based on the length of drug exposure. NS3 protease assay (day 4) 10. Prepare fresh solutions for detecting NS3 protease activity in cell lysates as follows: a. Prepare cell lysate assay buffer by combining 5 cell culture lysis buffer, 5 M NaCl, and water to generate a final solution of 1 cell culture lysis buffer with 0.15 M NaCl. Mix well. The presence of 0.15 M NaCl in cell lysate assay buffer significantly enhances NS3-4A protease activity when using a TR-FRET-Q substrate. b. Prepare 1 μm TR-FRET-Q NS3 substrate by diluting the 250 μm substrate stock to 1 μm with cell lysate assay buffer prior to use. This substrate is a short peptide with europium chelate W1284 linked at one end and a fluorescence quencher QSY7 linked at the other end. This substrate is custom synthesized by Perkin Elmer. Refer to Yang and Delaney (2006) for details. 11. Remove cell culture medium from the replicon assay plates by inverting plates and decanting the liquid into a waste receptacle; then gently tap inverted plates onto paper towels to remove any remaining medium. Wash cells by adding 100 μl of1 DPBS to each well and repeating the plate inversion step. Washing cells with DPBS is critical when testing antiviral activity of protease inhibitors because any residual protease inhibitor in the well can inhibit the NS3 protease assay and affect EC 50 values. It is recommended to wash the cells two times with DPBS when testing activity of protease inhibitors. 12. Dispense 80 μl of cell lysis buffer into each well of the 96-well cell plate. Shake the plate for 15 min at room temperature on a microplate shaker to facilitate complete cell lysis. Cell lysates can be processed for NS3 protease activity immediately (proceed to next step). Alternatively, cell lysates can be frozen at this point (up to 1 month at 20 C) and thawed at a later time for protease quantification. This can be convenient if numerous plates are being processed simultaneously or there is not time to finish the assay in 1 day. 13. Add 10 μl of1μm TR-FRET-Q substrate into each well and mix by pipetting up and down a few times. Measure fluorescence 14. Monitor fluorescence increase over time using a fluorescence plate reader with the excitation wavelength set at 340 nm and emission wavelength set at 615 nm with a 200-μsec delay. Screening of Hepatitis C Virus Inhibitors Using Replicon Cell Lines Typical assays take between 20 and 30 min, but run times may vary depending on the type of plate reader used. Initial velocities should be calculated at each inhibitor concentration using software accompanying the fluorescent plate reader. The maximum slope is normally observed within the first couple of cycles of the NS3 assay, but it can also be reached later in the assay depending on the NS3 signal of the replicon cells. This protocol was optimized for reading on a PerkinElmer Victor 3 fluorometer, which uses Workout 1.5 software (DAZDAQ) the maximal slope algorithm is used to calculate initial slope Current Protocols in Microbiology

7 HCV replicon signal (% untreated control) Compound concentration (log 10 M) Figure Typical dose response resulting from an EC 50 assay. Antiviral data from an NS3 protease or Renilla luciferase assay are processed as described in Basic Protocol 1, step 15 (NS3 assay) or Basic Protocol 2, step 7 (Renilla luciferase assay). After background subtraction, the HCV replicon signal data (NS3 or luciferase) are converted into percentages of the untreated control and can be plotted in an X-Y plot along with the log drug concentration. The data are then fit to a three- or four-parameter dose response equation using regression software and the drug concentration eliciting 50% of the antiviral effect (EC 50 ) is calculated. In this particular example, the EC 50 is 2.6 log 10 μm or μm. Analyze data 15. Analyze initial velocity data to determine EC 50 values. First, determine the background signal of the assay: this is the average of initial NS3 velocity in the negative control wells (column 12 on the plate map, Fig C). This background level is then subtracted from the initial NS3 slope values for all of the drug titration wells (columns 1 through 11, Fig C). The final NS3 velocities are then converted into percentage inhibition relative to the untreated controls (row 11, Fig C, defined as 100%). Perform non-linear regression to fit the data and calculate the EC 50. Use Equation to fit the data; there are also other three- or four-parameter dose response equations that can also be used (see Equation for a four-parameter equation). In Equation , a is the maximum rate, x is the inhibitor concentration, b is the EC 50,andc is the Hill slope. Typical inhibition curves should be similar to the one in Figure a y = 1 + ( x / b ) c Equation The authors recommend using a commercial software package such as Prism 5 (Graphpad Software) or XLfit4 (IDBS) for this analysis. DETERMINATION OF ANTIVIRAL ACTIVITY (EC 50 VALUES) IN GENOTYPE 1a HCV REPLICONS USING AN END-POINT NS3 PROTEASE ASSAY An alternate protocol to the NS3 protease kinetic assay is to measure the absolute activity of NS3 protease in cell lysates as an end-point assay. The rationale for using an end-point assay is time conservation. Multiple plates can be processed very efficiently with little read time on the fluorimeter. However, it is critical to stop the end-point assay within the linear range of the NS3 protease reaction or the EC 50 results will not be accurate. This is due to NS3 protease level variation with total replicon levels in the cell, and replicon ALTERNATE PROTOCOL Current Protocols in Microbiology

8 level variation between cell lines and culture conditions. It is highly recommended to first run an NS3 kinetic assay to determine the linear range of substrate cleavage for the cells being tested and select a suitable time to stop the end-point assay. Additional Materials (also see Basic Protocol 1) NS3 substrate stop solution (see recipe) 1. Follow Basic Protocol 1, steps 1 through 9 (cell preparation, cell seeding in assay plates, and drug dilution), to seed and treat replicon cell assay plates. 2. On day 4, prepare fresh solutions for detecting NS3 protease activity in cell lysates. Prepare cell lysis/tr-fret-q NS3 substrate assay buffer by combining 5 cell culture lysis buffer, 5 M NaCl, and water to generate a final solution of 1 cell culture lysis buffer with 0.15 M NaCl. Dilute the 250 μm substrate stock to 0.15 μm with cell lysate assay buffer prior to use. Mix well. 3. Remove the medium from replicon assay plates and wash cells with 100 μl DPBS/well, as described in Basic Protocol 1, step Dispense 100 μl of cell lysis/tr-fret-q NS3 substrate assay buffer to each well of the 96-well cell plate. Shake the plate for 15 min at room temperature on a microplate shaker to facilitate complete lysis and allow the NS3 protease reaction to occur. The authors recommend carrying out Basic Protocol 1 first to determine the kinetic range of the NS3 assay for specific replicon cell lines. Depending on the level of NS3 protease in the cells, different reaction times may be optimal for the end-point assay. 5. Add 10 μl of NS3 substrate stop solution to each well and mix well. Once the NS3 protease reaction is stopped, plates can be kept for up to 4 hr at room temperature before reading fluorescence on a microplate reader. 6. Read absolute fluorescence using a fluorescence plate reader with the excitation wavelength set at 340 nm and emission wavelength set at 615 nm with a 200-μsec delay. Quantify fluorescence values at each inhibitor concentration using Workout 1.5 software (DAZDAQ). Fluorescence values can then be corrected for background fluorescence, converted into percentages relative to the untreated controls and nonlinear regression, and EC 50 calculation can then be performed as detailed in Basic Protocol 1, step 15. BASIC PROTOCOL 2 Screening of Hepatitis C Virus Inhibitors Using Replicon Cell Lines SCREENING RENILLA LUCIFERASE REPORTER REPLICONS USING A LUCIFERASE ASSAY (96-WELL FORMAT) The following protocol provides a method to assay the potency of inhibitors using a genotype 1a replicon that expresses the Renilla luciferase reporter gene. Renilla luciferase, which utilizes coelenterazine-based substrates, is increasingly utilized as a reporter gene in HCV cell culture systems due to its high signal-to-noise ratio and smaller gene size compared to firefly luciferase. Note that firefly and Renilla luciferases use different substrates. Consequently, if a genotype 1a replicon cell line expressing firefly luciferase is used, the protocol will need to be modified to incorporate a firefly luciferase substrate. Materials Genotype 1a HCV replicon cell line expressing the reporter gene Renilla luciferase (Robinson et al., 2010) Renilla luciferase assay system (Promega, cat. no. E2820) containing: Renilla luciferase assay lysis buffer (see recipe) Renilla luciferase assay reagent (see recipe) Current Protocols in Microbiology

9 1 Dulbecco s phosphate-buffered saline (DPBS) without calcium and magnesium (Cellgro, cat. no ) 37 C, 5% CO 2 humidified cell culture incubator 37 C water bath Microplate shaker Multi-channel pipettors (10 μl, 200 μl, 1000 μl, e.g., Rainin P10, P200, P1000) Luminescence plate reader (PerkinElmer Victor Light is used here, but there are additional instrument choices, e.g., Packard Top Count, PerkinElmer Envision, Biotek Synergy) Additional reagents and equipment for preparation of cells (see Basic Protocol 1) Prepare cells and antiviral dilution and set up assay 1. Set up HCV replicon cell plates (using a genotype 1a Renilla luciferase replicon cell line) with compound dilutions as described in Basic Protocol 1, steps 1 through 9. After completing Basic Protocol 1, step 9, proceed with step 2 below. These cells are available through Gilead Sciences provided that the requesting institution holds a valid agreement with Apath covering the use of the genotype 1a replicon. Renilla luciferase assay (day 4) 2. Prepare fresh solutions for detecting Renilla luciferase in cell lysates. a. Prepare Renilla luciferse assay lysis buffer by combining 5 Renilla luciferase assay lysis buffer and water to generate a final solution of 1 Renilla luciferase assay lysis buffer. Mix well. Use only the Promega Renilla luciferase assay lysis buffer provided with the Renilla luciferase assay system. Other lysis buffers may inhibit the assay. b. Prepare Renilla luciferase assay reagent by thawing Renilla luciferase assay buffer in a 37 C or room temperature water bath. Prepare an adequate volume of Renilla luciferase assay reagent to perform the desired number of Renilla luciferase assays. Add 1 vol of 100 Renilla luciferase substrate to 100 vol of Renilla luciferase assay buffer. For example, for a single 96-well plate, 10 ml of Renilla assay reagent is needed. Add 100 μl of Renilla assay substrate to 10 ml of Renilla assay buffer for a final concentration of Remove the medium by inverting the plate and decanting the liquid into a waste receptacle; then gently tap inverted plate onto paper towels to remove any remaining medium. Wash cells by adding 100 μl DPBS to each well and repeating the plate inverting step. 4. Dispense 40 μl of Renilla luciferase assay lysis buffer to each well of cell plate. Shake the plate for 15 min at room temperature on a microplate shaker to facilitate complete cell lysis. 5. Dispense 100 μl ofrenilla luciferase assay reagent into each well of the cell plates using a multichannel pipettor. 6. Read luminescence signal on microplate reader. If HCV replicon cell line has robust Renilla luciferase expression, the Renilla luciferase assay reagent can be added to multiple plates and a plate stacker can be utilized. If the cell line has a weak signal, it is recommended that plates be prepared and read individually to maximize signal. 7. Quantify expression of Renilla luciferase by HCV replicon as the luciferase signal above background (column 12) from the assay subtract the average luminescence Current Protocols in Microbiology

10 observed in column 12 from all other wells. Convert the resulting luciferase levels into percentages relative to the levels in the untreated controls (column 11, defined as 100%), and fit data to the dose response equation (Eqn ) as detailed in Basic Protocol 1, step 15. BASIC PROTOCOL 3 HIGH-THROUGHPUT (384-WELL) SCREENING OF NON-REPORTER REPLICON CELL LINES USING THE NS3 PROTEASE ASSAY This protocol describes a 384-well-based NS3 protease assay to measure the inhibitory effects of compounds on HCV replication at a higher throughput than a 96-well-based assay. Since NS3 protease is expressed in all HCV subgenomic replicon systems, this assay can be applied to any replicon cell line that expresses NS3 protease activity sufficient to be detected by the TR-FRET-Q substrate in a 384-well format. It is therefore applicable to a wide range of replicon systems including replicons of any genotype/subtype and can be used in replicons with or without other reporter genes. This 384-well protocol describes a multiplex assay to measure the antiviral potency and cytotoxicity of compounds in the same well. After 3 days of compound treatment, the viability of the replicon cells is determined using calcein-am. Calcein-AM is a non-fluorescent dye that is permeable to intact, live cells. Calcein-AM is hydrolyzed by intracellular esterases into calcein. This product (calcein) is fluorescent (excitation at 490 nm; emission at 520 nm) and is trapped within live cells. As a result, intact live cells will be labeled with a bright calcein fluorescent signal while dead cells will not. This method provides a noninvasive method to determine the cytotoxic effects of testing compounds (Papadopoulos et al., 1994). The cells used for the calcein assay are then processed using this NS3 protease assay or the Renilla luciferase assay (see Basic Protocol 4) to measure HCV replication levels. The protease assay used here is the same TR-FRET- Q assay introduced in Basic Protocol 1 but is optimized for the 384-well format multiplex assay. This protocol requires the capability of handling liquid on 384-well plates. Screening of Hepatitis C Virus Inhibitors Using Replicon Cell Lines Materials Genotype 1a HCV replicon cell line Replicon cell assay medium (see recipe) HCV NS3 protease inhibitor ITMN-191 (Selleck Chemicals, cat. no. S1183) Puromycin (Sigma Aldrich, cat. no. P7255) Test compounds 100% DMSO 5 mm calcein-am stock solution (Anaspec, cat. no ) 1 Dulbecco s phosphate buffered saline with Ca 2+ and Mg 2+ (DPBS) (Mediatech, cat. no CM) 5 cell lysis buffer (Promega, cat. no. PR-E1483) 5 M NaCl solution (Sigma Aldrich, cat. no. S6546) 250 μm TR-FRET-Q NS3 protease substrate stock solution (see recipe) NS3 substrate stop solution (see recipe) 384-well black polystyrene cell culture treated assay plates (Greiner Bio-one, cat. no ) 384-well polypropylene compound storage plates (Thermo Scientific, cat. no. 4341) 96-well polypropylene compound storage plates (Corning, cat. no. 3357) Dual-pod BioMek FX automation workstation equipped with a 384-multichannel pipettor and a span-8, 8-channel pipettor (Beckman Coulter) BioMek FX AP384 P30 tips (Beckman Coulter, cat. no ) 37 C, 5% CO 2, 85% humidity incubator Biotek ELX405 plate washer Current Protocols in Microbiology

11 MicroFlo Select bulk dispenser (Bio Tek Instruments) and 5-μl cassettes Envision plate reader (Perkin Elmer) Additional reagents and equipment for cell maintenance and preparation (see Basic Protocol 1) Maintain and prepare cells 1. Grow and prepare replicon cells for seeding in 384-well plates according to Basic Protocol 1, steps 1 to 3. Appropriate cell lines include non-reporter H-77 replicon cell lines available from Apath; however, genotype 1a cell lines expressing reporters can also be used (Robinson et al., 2010). 2. After completing Basic Protocol 1, step 3, centrifuge cell suspension 5 min at 233 g, room temperature, to gently pellet the cells at the bottom of the conical tube. Remove the supernatant and resuspend cells in replicon cell assay medium at a final concentration of cells/ml. The cell suspension must be more concentrated than for Basic Protocols 1 and 2. This is due to the smaller seeding volumes used for 384-well applications. Serially dilute test compounds 3. Dissolve ITMN-191, puromycin, and test compounds in 100% DMSO to prepare 10 mm stock solutions. As indicated in Basic Protocol 1, DMSO is the recommended solvent for test compounds and 10 mm stock concentration is preferred. However, due to solubility limitations on certain compounds, the optimum solvents used and the highest concentration achievable can be determined experimentally. 4. Transfer 100 μl per well of 10 mm drug stocks into a 96-well V-bottom source plate. Use a BioMek FX workstation to transfer 15 μl of starting stocks from the 96-well source plate to columns 3 and 13 of 384-well destination plates (Fig ). Transfer each test compound in four replicates using the span-8 pipettor. Therefore, each 384- well destination plate can hold up to eight test compounds for full dilutions. Fill the remaining wells of each 384-well destination plate with 10 μl DMSO except for columns 23 and 24. Transfer 10 μl per well of 100 μm HCV NS3 protease inhibitor ITMN-191 into column 23 as positive control for maximum antiviral effects (Seiwert et al., 2008). Transfer 10 μl per well of 10 mm puromycin into column 24 as positive control for maximal cytotoxic effects. Serially dilute 1:3 from columns 3 to 12 and from 13 to 20 of the 384-well destination plate using the 384-multichannel pipettor. To serially dilute, iteratively transfer 5 μl compound solution from each well into the next well containing 10 μl DMSO followed by mixing. To achieve optimal accuracy and precision for serial dilution, use the following robotic settings for mixing: 10-μl mixing volume; 5 mixing cycles; a dual mixing Z-height (aspirate at 0.5 mm from well bottom and dispense at 2 mm from well bottom); 80 μl/sec mixing speed. Use the same BioMek FX tips for each 384-well destination plate to minimize the time spent for serial dilution. After serial dilution, centrifuge the 384-well destination plates 60 sec at 233 g, room temperature, to ensure consistent well menisci. Depending on the estimated potency of test compounds, the 10 mm stock solution can be used directly or further diluted into 1 mm, 0.1 mm, etc., as starting concentrations of serial dilutions. The key to achieving high-quality serial dilutions is the mixing step. The BioMek FX workstation is used here for the serial dilution process. The robotic settings listed above were optimized experimentally for BioMek FX workstation and have shown to deliver overall coefficient of variation (CV) <10% and overall accuracy ratio 1:2.8 over ten-steps of 1:3 serial dilution. When different types of liquid handling instrument are Current Protocols in Microbiology

12 step 1: BioMek FX span-8 transfer 96-well source plate (10 mm or lower) 384-well destination plate step 2: BioMek FX serial dilution DMSO DMSO 100 M ITMN mm puromycin 384-well destination plate Figure Schematic illustration of 384-well serial dilution and plate map. As shown in the illustration, eight test compounds (100 μl each) are stored in column 1 of a 96-well source plate. The compound concentrations are 10 mm, depending on their estimated potency. In step 1, 15 μl of each compound is transferred in four replicates into column 3 or 13 of a 384-well destination plate using the span-8 pipettor of a BioMek FX workstation. The rest of the wells of the 384-well destination plate are filled with 10 μl DMSO (not illustrated in the figure) except for wells in columns 23 and 24. In step 2, three-fold serial dilutions are performed from columns 3 to 12 and from columns 13 to 22, using a 384-well pipettor of a BioMek FX workstation. A volume of 10 μl of 100 μm ITMN-191 is transferred into column 23 as a positive control for anti-replicon activity. A volume of 10 μl of 10 mm puromycin is transferred into column 24 as a positive control for cytotoxicity. Screening of Hepatitis C Virus Inhibitors Using Replicon Cell Lines used, it is recommended to optimize the four pipetting parameters experimentally using a fluorescein-based method (Barco et al., 2007). Plate replicon cells into 384-well assay plates 5. Plate the replicon cell suspension ( cells/ml) prepared in steps 1 and 2 into 384-well assay plates. Dilute the replicon cell suspension to cells/ml in replicon cell assay medium. To each well of a black polystyrene 384-well assay plate, add 90 μl of replicon cell assay medium containing 800 suspended replicon cells with a Biotek MicroFlo Workstation. Current Protocols in Microbiology

13 A RFU ( 10 3 ) Time (min) seeding density (cells per well) B NS3 protease activity (RFU/1000 per min) Seeding density (cells per well) 3000 B Luciferase activity (relative luminescent unit) 10, vehicle treated 0.5 M ITMN-191 treated Seeding density (cells per well) 3000 Figure Optimization of cell seeding density in 384-well-based NS3 protease assay and Renilla luciferase assay. (A) NS3 protease reaction progress curves at different seeding densities of replicon cells. On day 1, different densities of replicon cells were plated into a 384-well plate (n = 16 wells per seeding density). On day 4, the TRF signal from NS3 protease reaction was measured over 80 min. The dashed line indicates the 10-min point in the reaction progress curve, which is within the linear range of the reaction for all seeding densities. (B) A plot of NS3 protease activity versus seeding density. NS3 protease activity is expressed as the initial velocity of TRF signal production in the first 10 min of the reaction. NS3 protease activity increased linearly at seeding densities from 270 to 2700 cells/well. (C) A linear relationship was observed between Renilla luciferase activity and seeding cell densities from 360 to 2700 cells per well. The HCV RNA replication level was expressed as luciferase activity after 72 hr of growth from different seeding density in 384-well plate format. The assay signals were obtained from solvent-treated wells (0.44% DMSO) while assay background was obtained from 0.5 μm ITMN-191-treated wells. Each data point represents the mean of 16 replicates and standard deviation is indicated. Intracellular replicon levels are strongly influenced by the proliferation status of the cells. Due to the unique environment and reduced growth surface in the wells of a 384-well plate, it is important to optimize the seeding cell density to avoid over confluence after 72 hr of cell growth. It is recommended to perform a titration of seeding density to check the replicon level after 72 hr of cell growth. The optimal seeding density will be within a linear relationship between seeding density and HCV replicon signal (which can be measured by the NS3 assay). For the genotype 1a replicon cells used in this protocol, replicon levels show a linear relationship with seeding densities from 270 to 2700 cells per well (Fig A,B). A seeding density of 800 cells per well is used in this protocol. Black-colored assay plates are recommended since they are compatible with the calcein fluorescence signal in the cell viability assay. 6. Incubate the assay plates 15 min at room temperature and then incubate 3 hr in a 37 C, 5% CO 2 with 85% humidity before treating with test compounds. Incubating the assay plates 15 min at room temperature can prevent edge effects on the assay readout (Lundholt et al., 2003). Edge effects refers to a phenomenon in Current Protocols in Microbiology

14 cell-based assays where cells from the edge wells tend to behave differently than those from the inside wells. Treat replicon cells with test compounds 7. Transfer compounds from diluted 384-well dilution plates into the replicon cell assay plates. For compound transfer into assay plates, transfer 0.4 μl of compound solution from the compound serial dilution plates into assay plates on a Biomek FX workstation. To avoid DMSO solutions sinking into wells and directly contacting the cell monolayer, aspirate 10 μl of replicon cell assay medium prior to dispensing the drug dilutions into the final assay plate (final DMSO concentration in wells is 0.44%). Incubate assay plates for 3 days in a 37 C, 5% CO 2 85% humidity incubator. The compound concentrations in the final assay wells are 225-fold lower than those in serial dilution plates. A final DMSO concentration of 0.44% has no negative effects on cell viability or HCV replicon levels. The DMSO drug solutions are heavier than the replicon cell assay medium and can sink down to the cell monolayer and cause toxicity if the replicon cell assay medium is not aspirated into the liquid handler prior to dispensing the drug dilutions. Perform multiplexed calcein-am cell viability assay and NS3 protease assay The calcein-am cell viability assay is performed first (steps 8 through 14) followed immediately by the NS3 protease assay in the same plates (steps 15 through 18). Screening of Hepatitis C Virus Inhibitors Using Replicon Cell Lines Prepare an appropriate volume of 200 nm calcein-am assay solution. To prepare 500 ml of 200 nm calcein-am assay solution, add 20 μl of 5 mm calcein-am stock into 500 ml of 1 DPBS. A volume of 50 μl per well ( 20 ml per 384-well assay plate) is used. The required volume of calcein-am assay solution can be calculated based on the number of assay plates. Prepare this solution right before the assay. This solution is stable for 3 hr at room temperature. After 3 hr, calcein-am may hydrolyze and cause decreased fluorescence signal. 9. Prepare appropriate volume of NS3 protease assay solution: 1 luciferase cell culture lysis buffer/150 mm NaCl/100 nm TR-FRET-Q NS3 substrate. To prepare 100 ml of this solution, add 20 ml of 5 cell lysis buffer, 40 μl of 250 μm TR-FRET-Q NS3 protease substrate, and 3 ml of 5 M NaCl into 77 ml of water. A volume of 25 μl per well (10 ml per 384-well assay plate) is used. The required volume of solution can be calculated based on the number of assay plates. Prepare this solution right before the assay. Store the solution in the dark at room temperature until performing the assay. 10. Prepare an appropriate volume of NS3 substrate stop solution. NS3 substrate stop solution will later be added at 25 μl/well (10 ml required per 384-well assay plate). A 1% SDS solution is used to stop the NS3 protease reaction for an end-point read of the TRF signal generated from NS3 protease activity in the cell lysates. 11. Aspirate media from assay plates and wash four times with 100 μl/well 1 DPBS using a Biotek ELX405 plate washer. Residual assay media can interfere with the calcein-am assay readout. Four DPBS washes are sufficient to remove assay media from the wells. Current Protocols in Microbiology

15 12. Add 50 μl per well of calcein-am assay solution using a Biotek MicroFlo bulk dispenser with a 5-μl cassette. Place five plates before the assay plates to saturate the non-specific binding of calcein-am to the cassette tubing. Calcein-AM can be adsorbed into the tubing system of the bulk dispenser and result in an uneven delivery of calcein-am into the wells. Therefore, the first five plates usually give variable calcein-am assay signal from well to well. Dispensing into five plates before the assay plates resolves this problem. 13. Incubate assay plates 30 min at room temperature. Calcein-AM will permeate into intact, live cells and will be hydrolyzed by intracellular esterases to generate a fluorescent calcein signal. 14. Measure the calcein fluorescence signal using an Envision plate reader (excitation = 490 nm; emission = 520 nm). 15. Aspirate the calcein solution from the assay plates using a Biotek ELX405 workstation. 16. Add 25 μl per well NS3 protease assay solution into the assay plates using a Biotek MicroFlo workstation with a 5-μl cassette. Incubate the assay plates 10 min at room temperature. The cell lysis component in NS3 protease assay solution lyses the cells. NS3 protease will be released into the lysates and cleave the protease substrate, which results in the separation of europium chelate and fluorescence quencher QSY Stop the reaction by adding 25 μl per well of 1% SDS using a Biotek MicroFlo workstation with a 5-μl cassette. The NS3 protease reaction is stopped by the addition of an equal volume of 1% SDS (denatures NS3 protease and stops the reaction) and an end-point read of the TRF signal will be used to calculate the NS3 protease activity. Since the TRF signal generated from the NS3 protease reaction increases with time (a typical kinetic assay), it is important to stop the reaction within the linear range of the NS3 protease reaction progression. It is recommended to check the linear range of the NS3 protease enzymatic reaction at different seeding densities of replicon cells. Usually, the more cells seeded on day 1, the shorter the linear range in the NS3 protease reaction at day 4. As shown in Figure A, a seeding density of 800 cells per well will give a linear range from 0 to 10 min. Therefore, the NS3 protease reaction is stopped at 10 min for an end-point read of TRF signal. 18. Read the TRF assay signal (excitation = 340 nm using laser source; emission = 615 nm) on an Envision plate reader. Analyze data 19. The cytotoxicity effect is determined by the conversion of calcein-am to fluorescent products. Calculate percent cytotoxicity using Equation : XC M B % cytotoxicity or % inhibition = MD MB Equation where X C is the fluorescence signal from compound-treated well; M B is the average fluorescence signal (background signal) from puromycin-treated wells; and M D is the average fluorescence signal from DMSO-treated wells. The percent anti-hcv replication activity is determined by the TRF signal generated from the end-point NS3 protease enzymatic reaction. The percent inhibition on HCV replicon is calculated using Equation , where X C is the TRF signal from compound-treated well; M B is the average TRF signal (background signal) from Current Protocols in Microbiology

16 ITMN-191-treated wells; and M D is the average TRF signal from DMSO-treated wells. The cytotoxic concentration 50 (CC 50 ) value is the test compound concentration that caused a 50% decrease of cell viability. The anti-hcv efficacy concentration 50 (EC 50 ) value is the test compound concentration that caused a 50% decrease in HCV replication. Both CC 50 and EC 50 values are obtained using Pipeline Pilot 5.0 software package (Accelrys) by nonlinear regression of the experimental data using Equation : a d y = d + b x 1 + c Equation In this equation, y is the observed percent inhibition of HCV replicon or percent cytotoxicity at x concentration of compound; d is estimated response at zero compound concentration; a is estimated response at infinite compound concentration (maximal response); c is the mid-range concentration (CC 50 or EC 50 ); and b is the Hill slope factor. BASIC PROTOCOL 4 HIGH-THROUGHPUT (384-WELL) SCREENING OF RENILLA REPORTER REPLICON CELL LINES USING LUCIFERASE ASSAY With the availability of luciferase-encoding genotype 1a replicon cell lines, researchers may benefit from luciferase expression in a high-throughput assay format rather than the NS3-based assay described in Basic Protocol 3. Potential reasons to select the luciferase assay include the widespread commercial availability of luciferase reagents (versus the custom ordered NS3 substrate) and the desire to avoid artifacts based on compound autofluorescence, which can interfere with the TRF readout (certain classes of compounds are inherently auto-fluorescent). The protocol described here is a 384-well based multiplexed assay to measure the antiviral potency and cytotoxicity of test compounds in the same well. This assay is performed using a Huh7 cell line carrying a Renilla luciferase reporter based HCV genotype 1a replicon as described previously (Robinson et al., 2010). After 3 days of compound treatment, the viability of the replicon cells is determined using calcein-am as described in Basic Protocol 3. The same cells are then processed for luciferase activity as a marker for HCV replicon levels and thus the antiviral activity of test compounds. This protocol can be applied to any HCV replicon cell line with a stable expression of Renilla luciferase at a level similar to the HCV genotype 1a replicon cell line used in this protocol. The assay protocol described here requires the capability of performing liquid handling on 384-well plates. Screening of Hepatitis C Virus Inhibitors Using Replicon Cell Lines Materials HCV replicon cell line (e.g., human liver Huh7 cell line carrying a Renilla luciferase reporter carrying HCV genotype 1a replicon; Robinson et al., 2010) Dual-Glo luciferase buffer (Promega, cat. no. E298B) Dual-Glo Stop & Glo assay solution (Promega, cat. no. E314B) 384-well polypropylene compound storage plates (Thermo Scientific, cat. no. 4341) 384-well black polystyrene cell culture treated assay plates (Greiner Bio-one, cat. no ) Additional reagents and equipment for cell preparation and maintenance (see Basic Protocol 3) Current Protocols in Microbiology

17 Maintain and prepare cells 1. Maintain and prepare cells for antiviral/cytotoxicity assays as described in Basic Protocol 3, steps 1 and 2. Serially dilute test compounds 2. Serially dilute test and control compounds as described in Basic Protocol 3, steps 3 and 4. Plate replicon cells into 384-well assay plates 3. Seed replicon cells for antiviral/cytotoxicity assays as described in Basic Protocol 3, steps 5 and 6 (a total number of cells/well is seeded into 384-well assay plates). Treat replicon cells with test compounds 4. Add serially diluted drug solutions to replicon cell assay plates according to Basic Protocol 3, step 7. Perform multiplexed calcein-am cell viability assay and Renilla luciferase assay After 3 days of incubation with test compounds, the cells are processed for viability using the calcein-am cell assay (step 5), and the same plates are subsequently assessed for antiviral activity using Renilla luciferase (steps 6 through 9). 5. Measure the viability of cells by performing the calcein-am assay as described in Basic Protocol 3, steps 8 through After measuring calcein fluorescence on the Envision reader, aspirate the calcein solution from the assay plates using a Biotek ELX405 workstation. 7. Add 20 μl per well Dual-Glo luciferase buffer into the assay plates using a Biotek MicroFlo workstation with a 5-μl cassette. Incubate the assay plates 10 min at room temperature. Dual-Glo luciferase buffer contains cell lysis reagents to break the cells and release Renilla luciferase. A 10-min incubation is sufficient for complete cell lysis. 8. Add 20 μl per well Dual-Glo Stop & Glo assay solution into the assay plates using a Biotek MicroFlo workstation with a 5-μl cassette. Incubate the assay plates 10 min at room temperature. The Dual-Glo Stop & Glo assay solution contains ATP-Mg 2+ and a Renilla luciferase substrate for the Renilla luciferase reaction. Upon binding to the Renilla luciferase, this substrate can produce a glow-type luminescence signal with a half-life >2 hr. The prolonged half-life of the assay signal makes it suitable to process a large number of assay plates. 9. Read the luminescence assay signal on an Envision plate reader. Analyze data 10. Process data for the determination of cytotoxicity (CC 50 values) as described in Basic Protocol 3, step Determine the percent anti-hcv replication activity by the luminescence signal generated from the reporter Renilla luciferase of the HCV replicon. Calculate the percent inhibition on HCV replicon using Equation , where X C is the luminescence signal from compound-treated well; M B is average luminescence signal (background signal) from ITMN-191-treated wells; and M D is average luminescence signal from DMSO-treated wells. Then calculate EC 50 values by non-linear regression using Equation , as described in Basic Protocol 3, step Current Protocols in Microbiology