Abstract:
The present invention includes an assay useful for identifying inhibitors of Hepatitis C virus (HCV) activity. Particularly, the present invention is directed to a dual HCV assay useful for high throughput screening that quantifies both the amount of HCV RNA replication inhibitory activity associated with a test compound and the amount of cytotoxicity associated with that test compound. The present invention also includes compounds discovered using this assay, compositions containing such compounds and methods of treating Hepatitis C by the administration of such compounds. The present invention also includes reporter assays using enzymes associated with HCV RNA replication, as well as a cell line having ATTC Accession No. PTA-4583.

Description:
RELATED APPLICATIONS  
       [0001]    This application claims the benefit under 35 U.S.C. Section 119(e) of U.S. Provisional Patent Application No. 60/402,661 filed Aug. 12, 2002. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention includes assays useful for identifying inhibitors of Hepatitis C virus (HCV) activity. Particularly, the present invention includes a dual HCV assay useful for high throughput screening that quantifies both the amount of HCV RNA replication inhibitory activity associated with a test compound and the amount of cytotoxicity associated with the test compound. As such, an assay of the present invention permits the determination of both inhibitory activity associated with a test compound and selectivity of that test compound in a single well. The present invention also includes a reporter assay utilizing at least one enzyme associated with HCV RNA replication. The present invention also includes a cell line useful in assay of the present invention.  
         BACKGROUND OF THE INVENTION  
         [0003]    Hepatitis C virus (HCV) is the major etiological agent of 90% of all cases of non-A, non-B hepatitis (Dymock, B. W.  Emerging Drugs  6:13-42 (2001)). The incidence of HCV infection is becoming an increasingly severe public health concern with 2-15% individuals infected worldwide. While primary infection with HCV is often asymptomatic, most HCV infections progress to a chronic state that can persist for decades. Of those with chronic HCV infections, it is believed that about 20-50% will eventually develop chronic liver disease (e.g. cirrhosis) and 20-30% of these cases will lead to liver failure or liver cancer. As the current HCV-infected population ages, the morbidity and mortality associated with HCV are expected to triple.  
           [0004]    Known treatments for HCV infection include the use of interferon-α (IFN), which indirectly effects HCV infection by stimulating the host antiviral response. IFN treatment is largely ineffective, however, as a sustained antiviral response is produced in less than 30% of treated patients. Further, IFN treatment induces an array of side effects of varying severity in upwards of 90% of patients (e.g. acute pancreatitis, depression, retinopathy, thyroiditis). Therapy with a combination of IFN and ribavirin has provided a slightly higher sustained response rate, but has not alleviated the IFN-induced side effects.  
           [0005]    One research area of active interest includes the development of antiviral agents which inactivate virally encoded protein products essential for HCV viral replication. Examples of such agents include various tripeptide compounds, which act as selective HCV NS3 serine protease inhibitors. However, many of these compounds do not sufficiently inhibit HCV protease activity or do not have sufficient potency, and thus, may not provide optimal treatment of HCV-infected patients. Accordingly, there is an ongoing need for the development of HCV assays for the identification of agents effective for inactivating viral replication proteins.  
           [0006]    Known cell-based assays for screening compounds for HCV inhibitory activity rely upon the detection of viral RNA replication using RT-PCR (Ito et al.,  Hepatology  34(3):566-572 (2001); Bartenschlager R. and V. Lohman,  Antiviral Res.  52(1):1-17 (2001)). Such cell-based systems often yield variable results, making reproducibility a major problem and the use of such system for the screening of compounds impractical, particularly for use in high throughput screening (HTS). HCV assays which rely on the inhibition of viral enzymes essential for viral replication and which may be suitable for HTS are known (Bianchi et al.,  Analytical Biochemistry  237, 239-244 (1996); Taliani et al.,  Analytical Biochemistry  240, 60-67 (1996)), but such assays measure only in vitro activity.  
           [0007]    Accordingly, there exists a need for an accurate and reproducible cell-based HCV assays which permits the screening of compounds for HCV replication inhibitory activity. The present invention is directed towards such assays.  
         SUMMARY OF THE INVENTION  
         [0008]    The present invention includes a cell-based HCV assay which measures the inhibitory activity of compounds on HCV RNA replication. The present invention may include a dual assay useful for high throughput screening that quantifies both: (i) the amount of HCV RNA replication inhibitory activity associated with a test compound; and (ii) the amount of cytotoxicity associated with the test compound. Desirably, both steps are conducted in a single well. Assays of the present invention permit the determination of both the inhibitory activity as well as the selectivity of a test compound in a HTS.  
           [0009]    In one aspect, the present invention includes an assay for identifying a compound that inhibits HCV RNA replication. The assay comprising the steps of: (a) providing a cell which expresses at least one enzyme associated with HCV RNA replication; (b) contacting the cell with a test compound; (c) determining whether the test compound inhibits HCV RNA replication; and (d) determining whether the test compound is cytotoxic to the cell. The cell expressing at least one enzyme associated with HCV RNA replication may include a HCV replicon which is a polynucleotide having the nucleic acid sequence set forth in SEQ ID NO:1 and encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:2. Further, the HCV replicon may be the molecular construct set forth in FIG. 1. A cell useful in the present invention has the ATTC Accession No. PTA-4583.  
           [0010]    Both steps of an assay of the present invention are desirably conducted in a single well, or may be conducted in two or more wells. The enzyme associated with HCV RNA replication may be any enzyme associated with HCV RNA replication, and is desirably a protease, such as a serine protease. The serine protease is desirably NS3 protease. The protein may also be NS4A. The step of determining whether the test compound inhibits HCV RNA replication is desirably conducted by contacting the cell with a fluorescence substrate, and the step of determining whether the test compound is cytotoxic to the cell is desirably conducted by contacting the cell with an Alamar Blue solution.  
           [0011]    The present invention also includes compounds and pharmaceutical compositions containing such compounds identified by the inventive assay. Further, the present invention includes a method for treating hepatitis-C by administering to a mammalian species in need thereof a therapeutically effective amount of such a compound.  
           [0012]    In another aspect, the present invention includes an assay for identifying a compound that inhibits HCV RNA replication, which comprises the steps of: (a) providing a cell which expresses at least one enzyme associated with HCV RNA replication; (b) contacting the cell with a test compound; (c) contacting the cell with a compound which permits the determination of whether the test compound inhibits HCV RNA replication; and (d) contacting the cell with an indicator solution which permits the determination of whether the test compound is cytotoxic to the cell. The compound which permits the determination of whether the test compound inhibits HCV RNA replication is desirably a FRET peptide, and the indicator solution which permits the determination of whether the test compound is cytotoxic to the cell is desirably an Alamar Blue solution.  
           [0013]    In another aspect, the present invention includes an assay for identifying a compound that inhibits HCV RNA replication. The assay comprises the steps of: (a) providing a cell which expresses at least one enzyme associated with HCV RNA replication, the cell comprising a HCV replicon; (b) contacting the cell with a test compound; (c) contacting the cell with a FRET peptide for determining whether the test compound inhibits HCV RNA replication; and (d) contacting the cell with an indicator solution for determining whether the test compound is cytotoxic to the cell.  
           [0014]    In another aspect, the present invention includes a reporter assay for identifying a compound that modulates that activity of a gene of interest. The reporter assay, comprises the steps of: (a) providing an expression system, the expression system comprising (i) a cell and (ii) a construct comprising a promoter region associated with said gene of interest operably linked to an enzyme associated with HCV RNA replication; (b) contacting the expression system with a test compound; and (c) contacting the expression system with a compound capable of detecting expression of the enzyme associated with HCV RNA replication. The enzyme associated with HCV RNA replication is desirably NS3 protease, and the compound capable of detecting expression of the enzyme associated with HCV RNA replication is desirably a FRET peptide.  
           [0015]    In another aspect, the present invention includes a cell having ATCC Accession No. PTA-4583. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0016]    [0016]FIG. 1 shows the molecular construct of the HCV Replicon used in an assay of the present invention.  
         [0017]    [0017]FIG. 2 shows the nucleic acid sequence of the HCV Replicon used in an assay of the present invention.  
         [0018]    [0018]FIG. 3 shows the amino acid sequence of the HCV Replicon used in an assay of the present invention.  
         [0019]    [0019]FIG. 4 shows the 96-well layout used in an assay of the present invention.  
         [0020]    [0020]FIG. 5 shows the results of Interferon Titration in the HCV Replicon cell line used in an assay of the present invention.  
         [0021]    [0021]FIG. 6A shows an EC50 comparison of typical values determined by FRET, RT-PCR or Western analysis for titration of interferon in the HCV replicon cell line.  
         [0022]    [0022]FIG. 6B shows a Western immunoblot using an anti-NS3 protease serum for the determination of EC 50  of IFN-α.  
         [0023]    [0023]FIG. 7A shows the enzyme activity in each well after contact with test compounds.  
         [0024]    [0024]FIG. 7B shows the cytotoxicity activity in each well after contact with test compounds.  
         [0025]    [0025]FIG. 8 shows a graphical representation of the variation within an assay of the present invention.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0026]    The present invention includes a cell-based HCV assay for measuring the ability of compounds to inhibit HCV RNA replication. An assay of the present invention desirably include a first cytotoxicity assay step which measures the conversion of an indicator solution to a fluorescent product, to determine if a test compound is cytotoxic to a cell; and a second inhibition assay step, to determine if the test compound inhibits HCV RNA replication. Desirably, an assay of the present invention includes the use of cells transfected with a HCV replicon.  
         [0027]    The ability of the HCV replicon to replicate is highly dependent on the amounts or activity of host cell factors. Therefore, any slight toxicity may have significant effects on viral protein expression and ultimately on any assay which examines the effect of compounds on HCV replication. As such, the use of an indicator to assess cytotoxicity in an HCV replicon cell line in an assay of the present invention provides a significant advantage in the ability to address the issue of whether HCV inhibition is due to a specific compound-virus interaction or due to a subtle but toxic effect on the cellular replication machinery.  
         [0028]    Accordingly, the present invention includes a dual assay useful for HTS that quantifies both the amount of HCV RNA replication inhibitory activity associated with a test compound, and the amount of cytotoxicity associated with that test compound. The dual assay is desirably conducted in a single well. Assays of the present invention permit for the mass screening of compounds specifically directed towards HCV replication, and permit viral RNA as well as viral proteins to be produced at levels consistently detectable using standard immunological and molecular biology methods. These consistent levels are amendable for HTS of compounds specific for the HCV replicon since effects either toxic to the cell or specific to the replicon can be differentiated and quantitated.  
         [0029]    In an assay of the present invention, a first cytotoxicity assay step measures the conversion of an Alamar Blue solution to a fluorescent product while a second inhibition assay step that uses a fluorescence resonance energy transfer (FRET) protease substrate specifically measures the amount of HCV NS3 protease activity present and relates that activity to HCV RNA amounts. The first cytotoxicity assay step permits the determination of selectivity of the test compound under consideration for the cells in the assay. The use of Alamar Blue solution permits the assay steps to be run in the same well, as the Alamar Blue solution is non-lethal to the cells. An assay of the present invention has been validated and compared with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and western blot analysis using interferon-α, a known HCV inhibitor. An assay of the present invention yielded fifty-percent effective concentration (EC50) values of 1.9, 2.9 and 5.3 units for the western, FRET and qRT-PCR assays, respectively. Assays of the present invention are amenable for HTS to identify compounds which inhibit HCV RNA replication, providing a convenient and economical assay comparable to qRT-PCR.  
         [0030]    HCV is a plus (+) strand RNA virus which is well characterized, having a length of approximately 9.6 kb and a single, long open reading frame (ORF) encoding an approximately 3000-amino acid polyprotein (Lohman et al.,  Science  285:110-113 (1999), expressly incorporated by reference in its entirety). The ORF is flanked at the 5′ end by a nontranslated region that functions as an internal ribosome entry site (IRES) and at the 3′ end by a highly conserved sequence essential for genome replication (Lohman, supra). The structural proteins are in the NH 2 -terminal region of the polyprotein and the nonstructural proteins (NS) 2 to 5B in the remainder.  
         [0031]    In an assay of the present invention, a HCV replicon was used in a cell culture system and was made as set forth below in Materials and Methods. The HCV replicon was based on a full-length consensus genome cloned from viral RNA isolated from an infected human liver. As shown in the molecular construct set forth in FIG. 1, a HCV replicon useful in an assay of the present invention includes a neomycin (neo) selectable marker protein translated from the native HCV internal ribosome entry site (IRES) element and non-structural proteins translated by the IRES from encephalomyocarditis virus (Lohman, supra). The known viral specific enzymatic activities provided by the replicon include the protease (NS3) and activator of the protease (NS4A), helicase (NS3), ATPase (NS3) and RNA dependent RNA polymersase (NS5B). Expression of neo is solely dependent on active HCV RNA replication in cells, and the viral gene products NS3 to NS5B are believed to be essential for HCV RNA replication and are the primary targets for inhibitor identification. For purposes of the present invention, viral gene products which are “associated” with HCV RNA replication include any and all viral gene products believed to be essential for HCV RNA replication.  
         [0032]    Methods used to quantitate HCV can be applied to the replicon and include quantitative RT-PCR (qRT-PCR) for RNA levels and immunological methods for proteins such as ELISA (Rodriguez-Lopez et. al.,  J. Gen. Virol.  80:727-738 (1999), expressly incorporated by reference in its entirety) or Western analysis (Pietschmann et al.,  J. Virol.  75:1253-1264 (2001), expressly incorporated by reference in its entirety).  
         [0033]    An assay of the present invention consists of two parts. The first part is a cytotoxicity assay step which quantitates the amount of cytotoxicity associated with a test compound, as determined by the conversion of Alamar Blue dye. The second part is an inhibition assay step which quantitates the amount of NS3 protease activity associated with the test compound. Both measurements are then compared relative to control wells. This method provides a measure of cytotoxicity for each well and an indirect measure of HCV RNA levels. Inhibition of HCV RNA replication is expected to reduce the amount of viral proteins present, including NS3 protease. As such, inhibitory activity of test compounds on HCV RNA replication is indirectly measured by quantitating NS3 protease levels using a FRET assay. The results obtained with the FRET assay have been shown to be comparable to those obtained from qRT-PCR.  
         [0034]    The following section sets forth materials and methods used in the present invention, and which were utilized in the Example set forth hereinbelow.  
       Materials and Methods  
       [0035]    1. HCV Replicon Cell Line Preparation  
         [0036]    The HCV replicon cell line was isolated from colonies as described by Lohman et al. al. (Lohman, supra) and used for all experiments. The HCV replicon has the nucleic acid sequence set forth in FIG. 2 (EMBL Accession No.: AJ242652; SEQ ID NO:1), the coding sequence of which is from 1801 nt-7758 nt. The coding sequence encodes the polypeptide having the sequence set forth in FIG. 3 (SEQ ID NO:2).  
         [0037]    The cell line used in the present invention has been deposited as ATCC Accession No. PTA-4583 in the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 U.S.A. under the terms of the Budapest Treaty on the International Recognition of Deposits of Microorganisms for Purposes of Patent Procedure and the Regulations promulgated under this Treaty. Samples of the deposited material are and will be available to industrial property offices and other persons legally entitled to receive them under the terms of the Treaty and Regulations and otherwise in compliance with the patent laws and regulations of the United States of America and all other nations or international organizations in which this application, or an application claiming priority of this application, is filed or in which any patent granted on any such application is granted.  
         [0038]    The coding sequence of the published HCV replicon was synthesized by Operon Technologies, Inc. (Alameda, Calif.), and the full-length replicon was then assembled in plasmid pGem9zf(+) (Promega) using standard molecular biology techniques. The replicon consists of (i) the HCV 5′ UTR fused to the first 12 amino acids of the capsid protein, (ii) the neomycin phosphotransferase gene (neo), (iii) the IRES from encephalomyocarditis virus (EMCV), and (iv) HCV NS3 to NS5B genes and the HCV3′ UTR. Plasmid DNAs were linearized with ScaI and RNA transcripts were synthesized in vitro using the T7 MegaScript transcription kit (Ambion) according to manufacturer&#39;s directions.  
         [0039]    To generate cell lines, 4×10 6  Huh-7 cells (kindly provided by R. Bartenschlager and available from Health Science Research Resources Bank, Japan Health Sciences Foundation) were electroporated (GenePulser System, Bio-Rad) with 10 ug of RNA transcript and plated into 100-mm dishes. After 24 h, selective media containing 1.0 mg/ml G418 was added and media was changed every 3 to 5 days. Approximately 4 weeks after electroporation, small colonies were visible which were isolated and expanded for further analysis. These cell lines were maintained at 37° C., 5% CO 2 , 100% relative humidity in DMEM (Life Technologies #11965-084) with 110% heat inactivated calf serum (Sigma #F-2442), 100 U/ml of penicillin/streptomycin (Life Technologies #15140-122), Geneticin at 1 mg/ml (Life Technologies #10131-027). One of the cell lines which had approximately 3,000 copies of HCV replicon RNA/cell was used for development of the assay.  
         [0040]    Other HCV replicons, as well as different genotypes, are suitable for use in assays of the present invention, and it is to be understood that assays of the present invention are not limited to any particular HCV replicon or cell line created therefrom. For example, in addition to the HCV replicon described above, HCV replicons suitable for use in assays of the present invention include, but are not limited to, those available from Apath, LLC. Also, it is understood that modifications of such HCV replicons may be made such that the replicon is useful in assays of the present invention.  
         [0041]    2. RNA Detection  
         [0042]    HCV RNA detection was conducted using RT-PCR, according to the manufacturer&#39;s instructions, using a Gibco-BRL Platinum Quantitative RT-PCR Thermoscript One-Step Kit on a Perkin-Elmer ABI Prism Model 7700 sequence detector. The primers for TaqMan were selected for use following analysis of RNA sequences with Primer Express Software from ABI. Primers used for detection of the plus strand RNA were 131F-5′ GGGAGAGCCATAGTGGTCTGC 3′ (SEQ ID NO:3) and 231R-5′ CCCAAATCTCCAGGCATTGA 3′ (SEQ ID NO:4) which amplify the HCV 5′UTR from nucleotides 131 to 231. The probe used for detection, 5′ FAM-CGGAATTGCCAGGACGACCGG-BHQ1 3′ (SEQ ID NO:5) was obtained from Biosearch Technologies. RNA&#39;s were purified from 96-wells using the RNAeasy 96 kit from Qiagen.  
         [0043]    3. Western Analysis  
         [0044]    Experiments were done in duplicate. Western analysis was performed according to the instructions for Amershams Chemiluminescence Immunology Kit (NEL105 Renaissance) using a Molecular Dynamics Storm 860 phosphoimager and associated software. The primary and secondary antibody dilutions were at 1 to 5,000. Antisera was generated by immunizing rabbits with purified NS3 protease made from an  E. Coli  expression vector encoding the first 181 amino acids of HCV 1a NS3 with subsequent boosts.  
         [0045]    Bleeds were tested weekly and boosts continued until a positive signal on a control western was seen. Secondary antibody was a BioRad (#170-6515) Goat anti-Rabbit IgG HRP Conjugate. The protein samples for western analysis were from the same wells used for the FRET assay and were prepared by the addition of an equal volume of 2×SDS-PAGE buffer to the FRET assay mixture, heating and loading on a 10% gel for SDS-PAGE. Interferon alpha (IFN-α) was obtained from Sigma (#I-4276) and stored as recommended.  
         [0046]    4. FRET Assay Preparation  
         [0047]    To perform the HCV FRET screening assay, 96-well cell culture plates were used. The FRET peptide (Anaspec, Inc.) (Taliani et al.  Anal. Biochem.  240:60-67 (1996), expressly incorporated by reference in its entirety) contains a fluorescence donor, EDANS, near one end of the peptide and an acceptor, DABCYL, near the other end. The fluorescence of the peptide is quenched by intramolecular resonance energy transfer (RET) between the donor and the acceptor, but as the NS3 protease cleaves the peptide the products are released from RET quenching and the fluorescence of the donor becomes apparent.  
         [0048]    The assay reagent was made as follows: 5×cell Luciferase cell culture lysis reagent from Promega (#E153A) diluted to 1× with dH 2 O, NaCl added to 150 mM final, the FRET peptide diluted to 20 uM final from a 2 mM stock. Cells were trypsinized, placed into each well of a 96-well plate and allowed to attach overnight. The next day, the test compounds were added to columns 1 through 10; column 11 was media only, and column 12 contained a titration of interferon as a control (1000 units for A12, B12, 100 units for C12, D12, 10 units for E12, F12 and 1 unit for G12, H12). In addition, replicon cells in A12, B12 can be replaced, if desired, with naïve Huh-7 cells as a negative background control. The plates were then placed back in the incubator. FIG. 4 shows the layout for HTS of the replicon cell line in a 96-well plate. In FIG. 4, labels are as followed: “Screen” denotes wells with test compound; “1-HCV” denotes control replicon wells (100% activity), “Inhibited” denotes wells containing the highest amount of control inhibitor (100% inhibition), and was used to determine background for each plate; “Titration” denotes the titration of interferon, and was used as a sensitivity control. Units of interferon from the top of row 12 in duplicate are 1000, 100, 10, and 1.  
         [0049]    5. FRET Assay and Cytotoxicity Assay Steps  
         [0050]    Subsequent to addition of the test compounds described above (FRET Assay Preparation), at various times the plate was removed and Alamar Blue solution (Trek Diagnostics, #00-100) was added per well as a measure of cellular toxicity. After reading in a Cytoflour 4000 instrument (PE Biosystems), plates were rinsed with PBS and then used for FRET assay by the addition of 30 ul of the FRET peptide assay reagent described above (FRET Assay Preparation) per well. The plate was then placed into the Cytoflour 4000 instrument which had been set to 340 excite/490 emission, automatic mode for 20 cycles and the plate read in a kinetic mode. Typically, the signal to noise using an endpoint analysis after the reads was at least three-fold.  
         [0051]    Compound analysis was determined by quantification of the relative HCV replicon inhibition and the relative cytotoxicity values. To calculate cytoxicity values, the average Alamar Blue fluorescence signals from the control wells in row 11 (FIG. 4) were set as 100% non-toxic. The individual signals in each of the compound test wells were then divided by the average control signal and multiplied by 100% to determine percent cytotoxicity. To calculate the HCV replicon inhibition values, an average background value FRET signal was obtained from the two wells containing the highest amount of interferon at the end of the assay period. These numbers were similar to those obtained from naïve Huh-7 cells.  
         [0052]    The background numbers were then subtracted from the average FRET signal obtained from the control wells in row 11 (FIG. 4) and this number was used as 100% activity. The individual signals in each of the compound test wells were then divided by the averaged control values after background subtraction and multiplied by 100% to determine percent activity. EC 50  values for an interferon titration were calculated as the concentration which caused a 50% reduction in HCV RNA, HCV protein amounts or FRET activity. The two numbers generated for the compound plate, percent cytoxicity and percent activity were used to determine compounds of interest for further analysis.  
         [0053]    6. Calculation of Assay Variation  
         [0054]    The following formula was used to calculate the variation in the FRET assay. Z′ is a measure of the distance between the standard deviations for the signal versus the noise of the assay:  
           Z′= 1−((3* asds+ 3* asdb )/( as−ab ))  
         [0055]    Asds=standard deviation of the signal  
         [0056]    Asdb=standard deviation of the background  
         [0057]    As=average signal  
         [0058]    Ab=average background signal  
         [0059]    (Zhang et al.,  J. Biomolecular Screening  (4) 2:67-73 (1999), expressly incorporated by reference in its entirety).  
       EXAMPLE  
       [0060]    An assay of the present invention was prepared and conducted in the manner set forth above in Materials and Methods. The HTS assay was designed to indirectly measure RNA levels through the use of a specific NS3 protease fluorescence substrate which yields a fluorescent signal upon cleavage. To ensure that the NS3 protease substrate could only be cleaved by the NS3 protease and not by any cellular proteases present in the replicon cell lysates, the substrate was added to individual wells containing crude lysates made from either naive Huh-7 cells, HepG-2 cells or HeLa cells. The substrate was found to only yield a substantial increase in fluorescence in cells containing either the HCV replicon or in cells expressing the NS3 enzyme, indicating that the assay was specific for HCV protease.  
         [0061]    Prior to the FRET assay step, a solution of Alamar Blue was added to the same plates in a cytotoxicity assay step, allowing direct quantification of the level of toxicity in that well. Only compounds which show no apparent toxicity but significantly decrease the amount of NS3 protease activity were further analyzed for HCV inhibitory activity.  
         [0062]    In order to validate the FRET assay for HTS, the relationship between viral RNA levels and the amount of NS3 activity present was quantitated. One consideration of using the NS3 protease as a general indicator of RNA levels is that the t 1/2  life of the RNA compared to the protein may be substantially different (Lohman, supra). This could result in a substantial drop in RNA levels rather quickly compared to protein amounts. To compensate for this difference, the cells were exposed to interferon alpha (IFN-α), a known HCV inhibitor (Lauer G. M. and B. D. Walker,  N. Engl. J. Med.  345(1):41-52 (2001); Blight et al.,  Science,  290:1972-1974 (2000); Collier J. and R. Chapman,  BioDrugs,  15(4):225-238 (2001), each of which is expressly incorporated by reference in its entirety), for a period of days, allowing the cells to magnify the effect and let the amount of NS3 present decrease relative to controls.  
         [0063]    The validation of the assay was accomplished by the use of quantitative RT-PCR (qRT-PCR) for viral RNA levels, quantification of the amount of NS3 present by scanning of a Western blot for protein levels and measurement of NS3 protease activity using the FRET assay. The samples for these measurements were from 2 plates prepared the same day and treated at the same time with a titration of IFN-α. One plate was used for preparation of RNA for quantitative RT-PCR while the other plate was used for FRET. Samples from the same wells after the FRET assay were used for Western analysis. Compound plates were then used to ensure that the procedure was applicable under conditions of HTS.  
         [0064]    The results of a FRET assay with IFN-α titration following 96 hours of incubation are shown in FIG. 5 as a continuous kinetic graph. FIG. 5 shows the measurement of the increase in fluorescence of the HCV FRET peptide in the HCV cell line and the effect of exposure to various interferon concentrations. The units per ml of IFN-α used for the different wells are listed to the right of the pertinent graphs. The assay is linear over a period of 40 minutes. As seen in FIG. 5, in the absence of IFN-α, the FRET signal is increased with time and is linear for at least 30 minutes. A decrease in the rate of FRET activity is clearly evident in the graph with increasing IFN-α concentration. The titration was from 0.1 units to 1,000 units per milliliter with control wells containing IFN-α dilution buffer only.  
         [0065]    Calculations involved subtracting the final background fluorescence signal while using the control wells as 100% activity. These numbers from the linear range are required for determination of the IFN-α EC 50 . Similarly, RNA levels were measured by qRT-PCR while the amount of NS3 protein present in each well was quantitated by scanning a Western immunoblot. An EC 50  was determined for all three methods by normalizing to the controls for each measurement. FIG. 6A shows a comparison of typical values determined by FRET, RT-PCR or scanning of a western blot for titration of interferon in the HCV replicon cell line, and also shows values for quantification of NS3 protease specific bands (FIG. 6B) by phosphorimaging. Each value in FIG. 6A represents a well of a 96-well plate at a single interferon concentration relative to a control value. Data at the lowest concentration of interferon tended to contain more variation. FIG. 6B shows the Western immunoblot using an anti-NS3 protease serum for the determination of EC 50  of IFN-α.  
         [0066]    The results shown in FIG. 6A indicate EC 50  values (in units of IFN-α per milliliter) of 1.9 for the Western, 2.9 for the FRET and 5.3 for RT-PCR. These values are within 3-fold of one another and indicate equivalency between the assay methods. This demonstrates the utility of the FRET assay method for inhibitor titration in an assay of the present invention and provides a comparison of a HTS format to the conventional qRT-PCR method of HCV quantification.  
         [0067]    A random compound plate was used in a method test of both the Alamar Blue assay and the FRET HCV replicon assay steps. The results are presented in FIGS. 7A and 7B for both the FRET and Alamar Blue assay as diagrammed in FIG. 4. FIG. 7A shows the percentage of activity in each well following FRET readings and performing the calculations described above for the endpoint reading from cycle 21 of the FRET assay. In FIG. 7A, lower numbers represent less activity present and indicate that the HCV replicon is inhibited. Wells F2 and G5 (underlined and enlarged) indicate that the compounds present in these wells inhibited the HCV replicon approximately 73% and 99% respectively.  
         [0068]    [0068]FIG. 7B shows Alamar Blue readings from the random compound plate expressed as a measure of cytoxicity. Wells corresponding to F2 and G5 (underlined and enlarged) indicate that compound present in F2 shows very little toxicity while compound in G5 has substantial toxicity. Comparing the results of the FRET assay with the Alamar assay it is likely that the inhibition of the HCV replicon for G5 is due to a toxic mechanism while the inhibition due to compound in F2 is not toxic in this assay, suggesting the compound may be specific for HCV.  
         [0069]    In general, the majority of compounds did not cause a significant variation in either the FRET or Alamar Blue assay indicating acceptable results amenable to HTS. The FRET activity yielded a 12.7% standard deviation in wells containing control media (FIG. 7A, column 11). In the IFN-α control samples, a clear inhibition was observed, the EC 50  was close to or slightly lower than the lowest concentration of IFN-α used (FIG. 7A, column 11). The Alamar Blue measurements in this plate yielded a variation of 4% for the cytotoxicity measurements in wells containing control media (FIG. 7B, column 11). Approximately 18% cytotoxicity was observed in the wells with the highest concentration of IFN-α (1000 units, FIG. 7B, columns A12 and B12), but no apparent Alamar Blue staining change was seen at lower concentrations of IFN-α. In the compound test area, two compounds showed a noticeable reduction in FRET activity, down to 27% and 1% detectable activity, respectively, of the control level (FIG. 7A, columns F2 and G5).  
         [0070]    Inspection of the numbers and comparison of FIGS. 7A and 7B indicate a toxic compound is present in well G5 due to the decrease in FRET activity along with a corresponding decrease for the Alamar assay. Well F2, however, was seen to have a noticeable decrease in FRET activity without a corresponding decrease in the Alamar Blue measurement, indicating HCV replicon inhibition without measurable toxicity for this compound. Therefore, this compound was chosen for further evaluation.  
         [0071]    To confirm that the variation in the FRET assay would remain acceptable, 40 additional compound plates were used to quantitate the variation using a statistical analysis to measure the Z′ statistic (Materials and Methods). The Z′ statistic is a measure of the distance between the standard deviations for the signal versus the noise of the assay. This analysis was used since the signal to noise in the assay was usually only 3-fold which is less than the Alamar signal to noise of approximately 8-fold indicating less tolerance for variation in the assay. An assay is considered acceptable if the Z′ statistic is 0.5 or greater indicating acceptable signal to noise scatter in the plates.  
         [0072]    Forty plates were used to measure the standard deviations and the number distribution between the endpoint signal obtained for the controls and the signal obtained for the background. FIG. 8 shows a graphical representation of the averaged numbers from 40 separate compound plates used in the Z′ calculation. The numbers at a signal of approximately 500 are the readings from the wells containing 1000 units of interferon and are considered to have 0% FRET activity. The numbers at a signal of approximately 1500 are from wells containing buffer only and are considered as 100% FRET activity. The Z′ measurement calculates the distance as a fraction between the two number distributions in terms of the means of those distributions.  
         [0073]    Using this calculation, a Z′ of 0.62 was obtained indicating a plate to plate variation acceptable for HTS. In addition, this measurement can be used on individual plates to determine if the controls were acceptable validating the data for a particular plate.  
       Discussion  
       [0074]    Assays of the present invention may be conducted in a 96-well format, as demonstrated by the dose response curve generated by IFN-α and yields results comparable to qRT-PCR, and are amenable to an even greater degree of miniaturization, such as a 384 or smaller based cell culture assay.  
         [0075]    As illustrated in FIGS. 7A and 7B, assays of the present invention are capable of measuring toxicity associated with a test compound as well as inhibitory activity associated with the test compound in the same well, thereby providing a method to prioritize compounds according to their inhibitory profile versus HCV as well as according to their toxicity profile. The variation associated with such assays is also statistically acceptable, as illustrated in FIG. 8. The cytotoxicity assay reagents, such as Alamar Blue, are desirably easily removed and are not deleterious to the cells.  
         [0076]    Assays of the present invention have distinct advantages when compared to qRT-PCR or other methods in that assays of the present invention may take place in-situ in a detergent based crude cell lysate, which requires no further preparation prior to performing the assays. Assays of the present invention do not involve numerous manipulations to add or subtract reagents after addition of test compounds, and are desirably based on a viral protein which is required by the HCV replicon for replication. The FRET protease substrate peptide, which is resistant to cleavage by endogenous Huh-7 cellular proteases over the assay time period, is efficiently recognized by the replicon-based NS3 enzyme. Given that the original purpose of the substrate was to monitor the in-vitro cleavage (Taliani, supra) of this substrate by purified rather than crude enzyme, it is probable that the substrate can still be cleaved by the many different genotypes of HCV NS3, thereby providing greater utility.  
         [0077]    The present invention also includes reporter assays. Reporter assays of the present invention include the use of a HCV protease and FRET peptide combination. The FRET substrate is relatively resistant to Huh-7, HeLa and HepG2 cellular proteases, indicating that it is very specific for HCV protease and therefore likely resistant to cellular proteases in other cell types. Placement of the HCV NS3 protease in a mammalian or bacterial expression system, or in the context of other viruses, allows the FRET assay to provide a sensitive method to use the viral protein in a wider cell repertoire. Such a reporter system is useful in a similar manner to known luciferase/beta-galactosidase systems, and are useful for the measurement of protein production, promoter strength, cell viability or other combinations. Adaptation of this method of assay is also possible with other viral proteases, provided a suitable and specific assay substrate is synthesized. The present invention also includes a cell line having ATCC Accession No. PTA-4583.  
         [0078]    While the invention has been described in connection with specific embodiments therefore, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims. All references cited herein are expressly incorporated in their entirety.  
     
       
       
         1 
         
           
             5  
           
           
             1  
             7989  
             DNA  
             Artificial  
             
               HCV Replicon  
             
           
            1 

gccagccccc gattgggggc gacactccac catagatcac tcccctgtga ggaactactg     60 

tcttcacgca gaaagcgtct agccatggcg ttagtatgag tgtcgtgcag cctccaggac    120 

cccccctccc gggagagcca tagtggtctg cggaaccggt gagtacaccg gaattgccag    180 

gacgaccggg tcctttcttg gatcaacccg ctcaatgcct ggagatttgg gcgtgccccc    240 

gcgagactgc tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg    300 

gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacg aatcctaaac    360 

ctcaaagaaa aaccaaaggg cgcgccatga ttgaacaaga tggattgcac gcaggttctc    420 

cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct    480 

ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg    540 

acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca    600 

cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc    660 

tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga    720 

aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc    780 

cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc    840 

ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg    900 

ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct    960 

gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc   1020 

tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc   1080 

ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc   1140 

agcgcatcgc cttctatcgc cttcttgacg agttcttctg agtttaaaca gaccacaacg   1200 

gtttccctct agcgggatca attccgcccc tctccctccc ccccccctaa cgttactggc   1260 

cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg   1320 

ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct   1380 

aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca   1440 

gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg   1500 

aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct   1560 

gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa   1620 

tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt   1680 

atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa   1740 

aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgataatacc   1800 

atggcgccta ttacggccta ctcccaacag acgcgaggcc tacttggctg catcatcact   1860 

agcctcacag gccgggacag gaaccaggtc gagggggagg tccaagtggt ctccaccgca   1920 

acacaatctt tcctggcgac ctgcgtcaat ggcgtgtgtt ggactgtcta tcatggtgcc   1980 

ggctcaaaga cccttgccgg cccaaagggc ccaatcaccc aaatgtacac caatgtggac   2040 

caggacctcg tcggctggca agcgcccccc ggggcgcgtt ccttgacacc atgcacctgc   2100 

ggcagctcgg acctttactt ggtcacgagg catgccgatg tcattccggt gcgccggcgg   2160 

ggcgacagca gggggagcct actctccccc aggcccgtct cctacttgaa gggctcttcg   2220 

ggcggtccac tgctctgccc ctcggggcac gctgtgggca tctttcgggc tgccgtgtgc   2280 

acccgagggg ttgcgaaggc ggtggacttt gtacccgtcg agtctatgga aaccactatg   2340 

cggtccccgg tcttcacgga caactcgtcc cctccggccg taccgcagac attccaggtg   2400 

gcccatctac acgcccctac tggtagcggc aagagcacta aggtgccggc tgcgtatgca   2460 

gcccaagggt ataaggtgct tgtcctgaac ccgtccgtcg ccgccaccct aggtttcggg   2520 

gcgtatatgt ctaaggcaca tggtatcgac cctaacatca gaaccggggt aaggaccatc   2580 

accacgggtg cccccatcac gtactccacc tatggcaagt ttcttgccga cggtggttgc   2640 

tctgggggcg cctatgacat cataatatgt gatgagtgcc actcaactga ctcgaccact   2700 

atcctgggca tcggcacagt cctggaccaa gcggagacgg ctggagcgcg actcgtcgtg   2760 

ctcgccaccg ctacgcctcc gggatcggtc accgtgccac atccaaacat cgaggaggtg   2820 

gctctgtcca gcactggaga aatccccttt tatggcaaag ccatccccat cgagaccatc   2880 

aaggggggga ggcacctcat tttctgccat tccaagaaga aatgtgatga gctcgccgcg   2940 

aagctgtccg gcctcggact caatgctgta gcatattacc ggggccttga tgtatccgtc   3000 

ataccaacta gcggagacgt cattgtcgta gcaacggacg ctctaatgac gggctttacc   3060 

ggcgatttcg actcagtgat cgactgcaat acatgtgtca cccagacagt cgacttcagc   3120 

ctggacccga ccttcaccat tgagacgacg accgtgccac aagacgcggt gtcacgctcg   3180 

cagcggcgag gcaggactgg taggggcagg atgggcattt acaggtttgt gactccagga   3240 

gaacggccct cgggcatgtt cgattcctcg gttctgtgcg agtgctatga cgcgggctgt   3300 

gcttggtacg agctcacgcc cgccgagacc tcagttaggt tgcgggctta cctaaacaca   3360 

ccagggttgc ccgtctgcca ggaccatctg gagttctggg agagcgtctt tacaggcctc   3420 

acccacatag acgcccattt cttgtcccag actaagcagg caggagacaa cttcccctac   3480 

ctggtagcat accaggctac ggtgtgcgcc agggctcagg ctccacctcc atcgtgggac   3540 

caaatgtgga agtgtctcat acggctaaag cctacgctgc acgggccaac gcccctgctg   3600 

tataggctgg gagccgttca aaacgaggtt actaccacac accccataac caaatacatc   3660 

atggcatgca tgtcggctga cctggaggtc gtcacgagca cctgggtgct ggtaggcgga   3720 

gtcctagcag ctctggccgc gtattgcctg acaacaggca gcgtggtcat tgtgggcagg   3780 

atcatcttgt ccggaaagcc ggccatcatt cccgacaggg aagtccttta ccgggagttc   3840 

gatgagatgg aagagtgcgc ctcacacctc ccttacatcg aacagggaat gcagctcgcc   3900 

gaacaattca aacagaaggc aatcgggttg ctgcaaacag ccaccaagca agcggaggct   3960 

gctgctcccg tggtggaatc caagtggcgg accctcgaag ccttctgggc gaagcatatg   4020 

tggaatttca tcagcgggat acaatattta gcaggcttgt ccactctgcc tggcaacccc   4080 

gcgatagcat cactgatggc attcacagcc tctatcacca gcccgctcac cacccaacat   4140 

accctcctgt ttaacatcct ggggggatgg gtggccgccc aacttgctcc tcccagcgct   4200 

gcttctgctt tcgtaggcgc cggcatcgct ggagcggctg ttggcagcat aggccttggg   4260 

aaggtgcttg tggatatttt ggcaggttat ggagcagggg tggcaggcgc gctcgtggcc   4320 

tttaaggtca tgagcggcga gatgccctcc accgaggacc tggttaacct actccctgct   4380 

atcctctccc ctggcgccct agtcgtcggg gtcgtgtgcg cagcgatact gcgtcggcac   4440 

gtgggcccag gggagggggc tgtgcagtgg atgaaccggc tgatagcgtt cgcttcgcgg   4500 

ggtaaccacg tctcccccac gcactatgtg cctgagagcg acgctgcagc acgtgtcact   4560 

cagatcctct ctagtcttac catcactcag ctgctgaaga ggcttcacca gtggatcaac   4620 

gaggactgct ccacgccatg ctccggctcg tggctaagag atgtttggga ttggatatgc   4680 

acggtgttga ctgatttcaa gacctggctc cagtccaagc tcctgccgcg attgccggga   4740 

gtccccttct tctcatgtca acgtgggtac aagggagtct ggcggggcga cggcatcatg   4800 

caaaccacct gcccatgtgg agcacagatc accggacatg tgaaaaacgg ttccatgagg   4860 

atcgtggggc ctaggacctg tagtaacacg tggcatggaa cattccccat taacgcgtac   4920 

accacgggcc cctgcacgcc ctccccggcg ccaaattatt ctagggcgct gtggcgggtg   4980 

gctgctgagg agtacgtgga ggttacgcgg gtgggggatt tccactacgt gacgggcatg   5040 

accactgaca acgtaaagtg cccgtgtcag gttccggccc ccgaattctt cacagaagtg   5100 

gatggggtgc ggttgcacag gtacgctcca gcgtgcaaac ccctcctacg ggaggaggtc   5160 

acattcctgg tcgggctcaa tcaatacctg gttgggtcac agctcccatg cgagcccgaa   5220 

ccggacgtag cagtgctcac ttccatgctc accgacccct cccacattac ggcggagacg   5280 

gctaagcgta ggctggccag gggatctccc ccctccttgg ccagctcatc agctagccag   5340 

ctgtctgcgc cttccttgaa ggcaacatgc actacccgtc atgactcccc ggacgctgac   5400 

ctcatcgagg ccaacctcct gtggcggcag gagatgggcg ggaacatcac ccgcgtggag   5460 

tcagaaaata aggtagtaat tttggactct ttcgagccgc tccaagcgga ggaggatgag   5520 

agggaagtat ccgttccggc ggagatcctg cggaggtcca ggaaattccc tcgagcgatg   5580 

cccatatggg cacgcccgga ttacaaccct ccactgttag agtcctggaa ggacccggac   5640 

tacgtccctc cagtggtaca cgggtgtcca ttgccgcctg ccaaggcccc tccgatacca   5700 

cctccacgga ggaagaggac ggttgtcctg tcagaatcta ccgtgtcttc tgccttggcg   5760 

gagctcgcca caaagacctt cggcagctcc gaatcgtcgg ccgtcgacag cggcacggca   5820 

acggcctctc ctgaccagcc ctccgacgac ggcgacgcgg gatccgacgt tgagtcgtac   5880 

tcctccatgc ccccccttga gggggagccg ggggatcccg atctcagcga cgggtcttgg   5940 

tctaccgtaa gcgaggaggc tagtgaggac gtcgtctgct gctcgatgtc ctacacatgg   6000 

acaggcgccc tgatcacgcc atgcgctgcg gaggaaacca agctgcccat caatgcactg   6060 

agcaactctt tgctccgtca ccacaacttg gtctatgcta caacatctcg cagcgcaagc   6120 

ctgcggcaga agaaggtcac ctttgacaga ctgcaggtcc tggacgacca ctaccgggac   6180 

gtgctcaagg agatgaaggc gaaggcgtcc acagttaagg ctaaacttct atccgtggag   6240 

gaagcctgta agctgacgcc cccacattcg gccagatcta aatttggcta tggggcaaag   6300 

gacgtccgga acctatccag caaggccgtt aaccacatcc gctccgtgtg gaaggacttg   6360 

ctggaagaca ctgagacacc aattgacacc accatcatgg caaaaaatga ggttttctgc   6420 

gtccaaccag agaagggggg ccgcaagcca gctcgcctta tcgtattccc agatttgggg   6480 

gttcgtgtgt gcgagaaaat ggccctttac gatgtggtct ccaccctccc tcaggccgtg   6540 

atgggctctt catacggatt ccaatactct cctggacagc gggtcgagtt cctggtgaat   6600 

gcctggaaag cgaagaaatg ccctatgggc ttcgcatatg acacccgctg ttttgactca   6660 

acggtcactg agaatgacat ccgtgttgag gagtcaatct accaatgttg tgacttggcc   6720 

cccgaagcca gacaggccat aaggtcgctc acagagcggc tttacatcgg gggccccctg   6780 

actaattcta aagggcagaa ctgcggctat cgccggtgcc gcgcgagcgg tgtactgacg   6840 

accagctgcg gtaataccct cacatgttac ttgaaggccg ctgcggcctg tcgagctgcg   6900 

aagctccagg actgcacgat gctcgtatgc ggagacgacc ttgtcgttat ctgtgaaagc   6960 

gcggggaccc aagaggacga ggcgagccta cgggccttca cggaggctat gactagatac   7020 

tctgcccccc ctggggaccc gcccaaacca gaatacgact tggagttgat aacatcatgc   7080 

tcctccaatg tgtcagtcgc gcacgatgca tctggcaaaa gggtgtacta tctcacccgt   7140 

gaccccacca ccccccttgc gcgggctgcg tgggagacag ctagacacac tccagtcaat   7200 

tcctggctag gcaacatcat catgtatgcg cccaccttgt gggcaaggat gatcctgatg   7260 

actcatttct tctccatcct tctagctcag gaacaacttg aaaaagccct agattgtcag   7320 

atctacgggg cctgttactc cattgagcca cttgacctac ctcagatcat tcaacgactc   7380 

catggcctta gcgcattttc actccatagt tactctccag gtgagatcaa tagggtggct   7440 

tcatgcctca ggaaacttgg ggtaccgccc ttgcgagtct ggagacatcg ggccagaagt   7500 

gtccgcgcta ggctactgtc ccaggggggg agggctgcca cttgtggcaa gtacctcttc   7560 

aactgggcag taaggaccaa gctcaaactc actccaatcc cggctgcgtc ccagttggat   7620 

ttatccagct ggttcgttgc tggttacagc gggggagaca tatatcacag cctgtctcgt   7680 

gcccgacccc gctggttcat gtggtgccta ctcctacttt ctgtaggggt aggcatctat   7740 

ctactcccca accgatgaac ggggagctaa acactccagg ccaataggcc atcctgtttt   7800 

tttccctttt tttttttctt tttttttttt tttttttttt tttttttttt ttctcctttt   7860 

tttttcctct ttttttcctt ttctttcctt tggtggctcc atcttagccc tagtcacggc   7920 

tagctgtgaa aggtccgtga gccgcttgac tgcagagagt gctgatactg gcctctctgc   7980 

agatcaagt                                                           7989 

 
           
             2  
             1985  
             PRT  
             Artificial  
             
               HCV Replicon  
             
           
            2 

Met Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly 
1               5                   10                  15 

Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Arg Asn Gln Val Glu Gly 
            20                  25                  30 

Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys 
        35                  40                  45 

Val Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr 
    50                  55                  60 

Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp 
65                  70                  75                  80 

Gln Asp Leu Val Gly Trp Gln Ala Pro Pro Gly Ala Arg Ser Leu Thr 
                85                  90                  95 

Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala 
            100                 105                 110 

Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu 
        115                 120                 125 

Ser Pro Arg Pro Val Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu 
    130                 135                 140 

Leu Cys Pro Ser Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys 
145                 150                 155                 160 

Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met 
                165                 170                 175 

Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro 
            180                 185                 190 

Ala Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly 
        195                 200                 205 

Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr 
    210                 215                 220 

Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly 
225                 230                 235                 240 

Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly 
                245                 250                 255 

Val Arg Thr Ile Thr Thr Gly Ala Pro Ile Thr Tyr Ser Thr Tyr Gly 
            260                 265                 270 

Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile 
        275                 280                 285 

Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr Thr Ile Leu Gly Ile 
    290                 295                 300 

Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val 
305                 310                 315                 320 

Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn 
                325                 330                 335 

Ile Glu Glu Val Ala Leu Ser Ser Thr Gly Glu Ile Pro Phe Tyr Gly 
            340                 345                 350 

Lys Ala Ile Pro Ile Glu Thr Ile Lys Gly Gly Arg His Leu Ile Phe 
        355                 360                 365 

Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly 
    370                 375                 380 

Leu Gly Leu Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val 
385                 390                 395                 400 

Ile Pro Thr Ser Gly Asp Val Ile Val Val Ala Thr Asp Ala Leu Met 
                405                 410                 415 

Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys 
            420                 425                 430 

Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu 
        435                 440                 445 

Thr Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly 
    450                 455                 460 

Arg Thr Gly Arg Gly Arg Met Gly Ile Tyr Arg Phe Val Thr Pro Gly 
465                 470                 475                 480 

Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr 
                485                 490                 495 

Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val 
            500                 505                 510 

Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp 
        515                 520                 525 

His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp 
    530                 535                 540 

Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr 
545                 550                 555                 560 

Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro 
                565                 570                 575 

Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr 
            580                 585                 590 

Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn 
        595                 600                 605 

Glu Val Thr Thr Thr His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met 
    610                 615                 620 

Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly 
625                 630                 635                 640 

Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val 
                645                 650                 655 

Ile Val Gly Arg Ile Ile Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp 
            660                 665                 670 

Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ala Ser 
        675                 680                 685 

His Leu Pro Tyr Ile Glu Gln Gly Met Gln Leu Ala Glu Gln Phe Lys 
    690                 695                 700 

Gln Lys Ala Ile Gly Leu Leu Gln Thr Ala Thr Lys Gln Ala Glu Ala 
705                 710                 715                 720 

Ala Ala Pro Val Val Glu Ser Lys Trp Arg Thr Leu Glu Ala Phe Trp 
                725                 730                 735 

Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly 
            740                 745                 750 

Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe 
        755                 760                 765 

Thr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln His Thr Leu Leu Phe 
    770                 775                 780 

Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser Ala 
785                 790                 795                 800 

Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala Ala Val Gly Ser 
                805                 810                 815 

Ile Gly Leu Gly Lys Val Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala 
            820                 825                 830 

Gly Val Ala Gly Ala Leu Val Ala Phe Lys Val Met Ser Gly Glu Met 
        835                 840                 845 

Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro 
    850                 855                 860 

Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg His 
865                 870                 875                 880 

Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala 
                885                 890                 895 

Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu 
            900                 905                 910 

Ser Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Ser Leu Thr Ile 
        915                 920                 925 

Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile Asn Glu Asp Cys Ser 
    930                 935                 940 

Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp Ile Cys 
945                 950                 955                 960 

Thr Val Leu Thr Asp Phe Lys Thr Trp Leu Gln Ser Lys Leu Leu Pro 
                965                 970                 975 

Arg Leu Pro Gly Val Pro Phe Phe Ser Cys Gln Arg Gly Tyr Lys Gly 
            980                 985                 990 

Val Trp Arg Gly Asp Gly Ile Met  Gln Thr Thr Cys Pro  Cys Gly Ala 
        995                 1000                 1005 

Gln Ile  Thr Gly His Val Lys  Asn Gly Ser Met Arg  Ile Val Gly 
    1010                 1015                 1020 

Pro Arg  Thr Cys Ser Asn Thr  Trp His Gly Thr Phe  Pro Ile Asn 
    1025                 1030                 1035 

Ala Tyr  Thr Thr Gly Pro Cys  Thr Pro Ser Pro Ala  Pro Asn Tyr 
    1040                 1045                 1050 

Ser Arg  Ala Leu Trp Arg Val  Ala Ala Glu Glu Tyr  Val Glu Val 
    1055                 1060                 1065 

Thr Arg  Val Gly Asp Phe His  Tyr Val Thr Gly Met  Thr Thr Asp 
    1070                 1075                 1080 

Asn Val  Lys Cys Pro Cys Gln  Val Pro Ala Pro Glu  Phe Phe Thr 
    1085                 1090                 1095 

Glu Val  Asp Gly Val Arg Leu  His Arg Tyr Ala Pro  Ala Cys Lys 
    1100                 1105                 1110 

Pro Leu  Leu Arg Glu Glu Val  Thr Phe Leu Val Gly  Leu Asn Gln 
    1115                 1120                 1125 

Tyr Leu  Val Gly Ser Gln Leu  Pro Cys Glu Pro Glu  Pro Asp Val 
    1130                 1135                 1140 

Ala Val  Leu Thr Ser Met Leu  Thr Asp Pro Ser His  Ile Thr Ala 
    1145                 1150                 1155 

Glu Thr  Ala Lys Arg Arg Leu  Ala Arg Gly Ser Pro  Pro Ser Leu 
    1160                 1165                 1170 

Ala Ser  Ser Ser Ala Ser Gln  Leu Ser Ala Pro Ser  Leu Lys Ala 
    1175                 1180                 1185 

Thr Cys  Thr Thr Arg His Asp  Ser Pro Asp Ala Asp  Leu Ile Glu 
    1190                 1195                 1200 

Ala Asn  Leu Leu Trp Arg Gln  Glu Met Gly Gly Asn  Ile Thr Arg 
    1205                 1210                 1215 

Val Glu  Ser Glu Asn Lys Val  Val Ile Leu Asp Ser  Phe Glu Pro 
    1220                 1225                 1230 

Leu Gln  Ala Glu Glu Asp Glu  Arg Glu Val Ser Val  Pro Ala Glu 
    1235                 1240                 1245 

Ile Leu  Arg Arg Ser Arg Lys  Phe Pro Arg Ala Met  Pro Ile Trp 
    1250                 1255                 1260 

Ala Arg  Pro Asp Tyr Asn Pro  Pro Leu Leu Glu Ser  Trp Lys Asp 
    1265                 1270                 1275 

Pro Asp  Tyr Val Pro Pro Val  Val His Gly Cys Pro  Leu Pro Pro 
    1280                 1285                 1290 

Ala Lys  Ala Pro Pro Ile Pro  Pro Pro Arg Arg Lys  Arg Thr Val 
    1295                 1300                 1305 

Val Leu  Ser Glu Ser Thr Val  Ser Ser Ala Leu Ala  Glu Leu Ala 
    1310                 1315                 1320 

Thr Lys  Thr Phe Gly Ser Ser  Glu Ser Ser Ala Val  Asp Ser Gly 
    1325                 1330                 1335 

Thr Ala  Thr Ala Ser Pro Asp  Gln Pro Ser Asp Asp  Gly Asp Ala 
    1340                 1345                 1350 

Gly Ser  Asp Val Glu Ser Tyr  Ser Ser Met Pro Pro  Leu Glu Gly 
    1355                 1360                 1365 

Glu Pro  Gly Asp Pro Asp Leu  Ser Asp Gly Ser Trp  Ser Thr Val 
    1370                 1375                 1380 

Ser Glu  Glu Ala Ser Glu Asp  Val Val Cys Cys Ser  Met Ser Tyr 
    1385                 1390                 1395 

Thr Trp  Thr Gly Ala Leu Ile  Thr Pro Cys Ala Ala  Glu Glu Thr 
    1400                 1405                 1410 

Lys Leu  Pro Ile Asn Ala Leu  Ser Asn Ser Leu Leu  Arg His His 
    1415                 1420                 1425 

Asn Leu  Val Tyr Ala Thr Thr  Ser Arg Ser Ala Ser  Leu Arg Gln 
    1430                 1435                 1440 

Lys Lys  Val Thr Phe Asp Arg  Leu Gln Val Leu Asp  Asp His Tyr 
    1445                 1450                 1455 

Arg Asp  Val Leu Lys Glu Met  Lys Ala Lys Ala Ser  Thr Val Lys 
    1460                 1465                 1470 

Ala Lys  Leu Leu Ser Val Glu  Glu Ala Cys Lys Leu  Thr Pro Pro 
    1475                 1480                 1485 

His Ser  Ala Arg Ser Lys Phe  Gly Tyr Gly Ala Lys  Asp Val Arg 
    1490                 1495                 1500 

Asn Leu  Ser Ser Lys Ala Val  Asn His Ile Arg Ser  Val Trp Lys 
    1505                 1510                 1515 

Asp Leu  Leu Glu Asp Thr Glu  Thr Pro Ile Asp Thr  Thr Ile Met 
    1520                 1525                 1530 

Ala Lys  Asn Glu Val Phe Cys  Val Gln Pro Glu Lys  Gly Gly Arg 
    1535                 1540                 1545 

Lys Pro  Ala Arg Leu Ile Val  Phe Pro Asp Leu Gly  Val Arg Val 
    1550                 1555                 1560 

Cys Glu  Lys Met Ala Leu Tyr  Asp Val Val Ser Thr  Leu Pro Gln 
    1565                 1570                 1575 

Ala Val  Met Gly Ser Ser Tyr  Gly Phe Gln Tyr Ser  Pro Gly Gln 
    1580                 1585                 1590 

Arg Val  Glu Phe Leu Val Asn  Ala Trp Lys Ala Lys  Lys Cys Pro 
    1595                 1600                 1605 

Met Gly  Phe Ala Tyr Asp Thr  Arg Cys Phe Asp Ser  Thr Val Thr 
    1610                 1615                 1620 

Glu Asn  Asp Ile Arg Val Glu  Glu Ser Ile Tyr Gln  Cys Cys Asp 
    1625                 1630                 1635 

Leu Ala  Pro Glu Ala Arg Gln  Ala Ile Arg Ser Leu  Thr Glu Arg 
    1640                 1645                 1650 

Leu Tyr  Ile Gly Gly Pro Leu  Thr Asn Ser Lys Gly  Gln Asn Cys 
    1655                 1660                 1665 

Gly Tyr  Arg Arg Cys Arg Ala  Ser Gly Val Leu Thr  Thr Ser Cys 
    1670                 1675                 1680 

Gly Asn  Thr Leu Thr Cys Tyr  Leu Lys Ala Ala Ala  Ala Cys Arg 
    1685                 1690                 1695 

Ala Ala  Lys Leu Gln Asp Cys  Thr Met Leu Val Cys  Gly Asp Asp 
    1700                 1705                 1710 

Leu Val  Val Ile Cys Glu Ser  Ala Gly Thr Gln Glu  Asp Glu Ala 
    1715                 1720                 1725 

Ser Leu  Arg Ala Phe Thr Glu  Ala Met Thr Arg Tyr  Ser Ala Pro 
    1730                 1735                 1740 

Pro Gly  Asp Pro Pro Lys Pro  Glu Tyr Asp Leu Glu  Leu Ile Thr 
    1745                 1750                 1755 

Ser Cys  Ser Ser Asn Val Ser  Val Ala His Asp Ala  Ser Gly Lys 
    1760                 1765                 1770 

Arg Val  Tyr Tyr Leu Thr Arg  Asp Pro Thr Thr Pro  Leu Ala Arg 
    1775                 1780                 1785 

Ala Ala  Trp Glu Thr Ala Arg  His Thr Pro Val Asn  Ser Trp Leu 
    1790                 1795                 1800 

Gly Asn  Ile Ile Met Tyr Ala  Pro Thr Leu Trp Ala  Arg Met Ile 
    1805                 1810                 1815 

Leu Met  Thr His Phe Phe Ser  Ile Leu Leu Ala Gln  Glu Gln Leu 
    1820                 1825                 1830 

Glu Lys  Ala Leu Asp Cys Gln  Ile Tyr Gly Ala Cys  Tyr Ser Ile 
    1835                 1840                 1845 

Glu Pro  Leu Asp Leu Pro Gln  Ile Ile Gln Arg Leu  His Gly Leu 
    1850                 1855                 1860 

Ser Ala  Phe Ser Leu His Ser  Tyr Ser Pro Gly Glu  Ile Asn Arg 
    1865                 1870                 1875 

Val Ala  Ser Cys Leu Arg Lys  Leu Gly Val Pro Pro  Leu Arg Val 
    1880                 1885                 1890 

Trp Arg  His Arg Ala Arg Ser  Val Arg Ala Arg Leu  Leu Ser Gln 
    1895                 1900                 1905 

Gly Gly  Arg Ala Ala Thr Cys  Gly Lys Tyr Leu Phe  Asn Trp Ala 
    1910                 1915                 1920 

Val Arg  Thr Lys Leu Lys Leu  Thr Pro Ile Pro Ala  Ala Ser Gln 
    1925                 1930                 1935 

Leu Asp  Leu Ser Ser Trp Phe  Val Ala Gly Tyr Ser  Gly Gly Asp 
    1940                 1945                 1950 

Ile Tyr  His Ser Leu Ser Arg  Ala Arg Pro Arg Trp  Phe Met Trp 
    1955                 1960                 1965 

Cys Leu  Leu Leu Leu Ser Val  Gly Val Gly Ile Tyr  Leu Leu Pro 
    1970                 1975                 1980 

Asn Arg 
    1985 

 
           
             3  
             21  
             DNA  
             Artificial  
             
               Primers  
             
           
            3 

gggagagcca tagtggtctg c                                               21 

 
           
             4  
             20  
             DNA  
             Artificial  
             
               Primers  
             
           
            4 

cccaaatctc caggcattga                                                 20 

 
           
             5  
             21  
             DNA  
             Artificial  
             
               Probe  
             
           
            5 

cggaattgcc aggacgaccg g                                               21