Friday, July 10, 2009

Statins potentiate the in vitro anti-hepatitis C

Statins potentiate the in vitro anti-hepatitis C virus activity of selective hepatitis C virus inhibitors and delay or prevent resistance development



Hepatology July 2009


Leen Delang 1, Jan Paeshuyse 1, Inge Vliegen 1, Pieter Leyssen 1, Susan Obeid 1, David Durantel 2, Fabien Zoulim 2, Anne Op de Beeck 3, Johan Neyts 1 *

1Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium

2INSERM, U871, Lyon, France; Université Lyon 1, IFR62 Lyon-Est, Lyon, France; Hospices Civils de Lyon, Hôtel Dieu, Service d'hépatologie et de gastroentérologie, Lyon, France

3Virology Unit, Laboratory of Epigenetic of Cancer, Faculty of Medicine, ULB, Belgium




"In summary, the combination of statins with several selective HCV inhibitors results in a pronounced antiviral effect in cell culture and prevents or delays the emergence of drug variants resistant to a STAT-C inhibitor. Statins may have the potential to (1) increase the efficacy of current or future HCV therapy and (2) delay the development of resistance against STAT-C inhibitors."



"We report on the combined anti-HCV activity of mevastatin and simvastatin with IFN-a or with HCV NS5B polymerase or NS3 protease inhibitors. Mevastatin and simvastatin were selected for these combination experiments because these statins exhibited the strongest antiviral activity when used alone. The combined effect of mevastatin or simvastatin with IFN- or with selective HCV inhibitors was first studied in regular 3-day antiviral assays. All combinations resulted in an overall additive antiviral activity"



"The potential antiviral activity of statins was studied in patients chronically infected with HCV. Neither atorvastatin (after conventional 12-week therapy)[34] nor rosuvastatin[35] resulted in a reduction in viral load. The most plausible explanation for the discrepancy between the in vitro activity and the lack of clinical efficacy is, as suggested by the authors, that the plasma concentrations were likely below the levels employed in cell culture.[34] In a recently published study, fluvastatin was found to inhibit HCV RNA replication in HCV-infected patients.[36] The drug was well tolerated and, at relatively low doses (20-80 mg daily), resulted in a transient reduction in viral load (-0.5 to -1.75 log10, 2-5 weeks); higher doses of fluvastatin did not reduce viral load"

"There is today no obviously compelling evidence that statins, used in monotherapy, may result in a marked reduction in HCV load in chronically infected patients. Also, ribavirin exhibits a very limited antiviral activity when used in monotherapy."

"Possibly, the combination of a statin with the current standard therapy or with one or more potent STAT-C inhibitor(s) may result in a more than additive effect, as suggested by the clearance-rebound experiments reported in the present study. The potential use of statins in HCV-infected patients may thus lie in combination therapy."....."Concentrations of statins in the liver may therefore be more relevant than plasma concentrations."..... "In rats, the liver concentration of lovastatin is 15-fold to 18-fold higher than the concentration in blood and other tissues"



Abstract




Statins are 3-hydroxyl-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors used for the treatment of hypercholesterolemia. It was recently reported that statins inhibit in vitro hepatitis C virus (HCV) RNA replication.

We here report that, of five statins studied, mevastatin and simvastatin exhibit the strongest in vitro anti-HCV activity, lovastatin and fluvastatin have moderate inhibitory effects, and pravastatin is devoid of an antiviral effect.

A combination of statins with interferon-alpha (IFN-) or HCV nonstructural (NS)5B polymerase or NS3 protease inhibitors results in an additive antiviral activity in short-term (3 days) antiviral assays.

Neither statins, at a concentration of five-fold their median effective concentration (EC50) value, nor polymerase, protease inhibitors, or IFN-, at concentrations 10- or 20-fold their EC50 value, were able to clear cells from their replicon following four or six consecutive passages of antiviral pressure.

However, the combination of HCV polymerase or protease inhibitors with mevastatin or simvastatin resulted in an efficient clearance of the cultures from their replicon.

In colony formation experiments, mevastatin reduced the frequency or prevented the selection of HCV replicons resistant to the nonnucleoside inhibitor HCV-796.

Conclusion: A combination of specific HCV inhibitors with statins may result in a more profound antiviral effect and may delay or prevent the development of resistance to such inhibitors.



Hepatitis C virus (HCV) is a positive single-stranded RNA virus and a member of the Hepacivirus genus within the Flaviviridae family. Worldwide approximately 170 million people (or almost 3% of the global population) are chronically infected with HCV. Chronically infected patients are at increased risk of developing liver cirrhosis and hepatocellular carcinoma.[1] In Western countries, infection with HCV is the most common cause of liver transplantation. The current standard therapy for chronic hepatitis C consists of the combination of pegylated interferon alpha (IFN-a) and ribavirin. This therapy is only effective in 50% to 60% of infected patients and is associated with serious side effects.[2] There is thus an urgent need for more selective, potent, and better-tolerated therapies for chronic hepatitis C.



Most antiviral drugs that are currently in clinical trials are inhibitors of the viral polymerase or serine protease. The design of antiviral drugs that inhibit the function of the HCV protease and polymerase therefore appears to be logical. The first HCV nonstructural (NS)3/4A serine protease inhibitor to enter clinical trials was BILN-2061.[3] The most clinically advanced investigational inhibitors of the HCV protease are telaprevir (VX-950), boceprevir (SCH-503034), ITMN-191, and TMC-435350. Both nucleoside and nonnucleoside inhibitors of the HCV RNA-dependent RNA polymerase (RdRp) have been reported.[4] Nucleoside HCV polymerase inhibitors act as premature chain terminators following conversion to their 5-triphosphate metabolite by competition and incorporation in the viral genome. 2-C-methylcytidine was the first nucleoside HCV inhibitor to enter clinical studies. Development of this compound has been discontinued because of modest antiviral efficacy along with significant gastrointestinal side effects.[5] Various nonnucleoside inhibitor classes of the HCV RdRp, including benzimidazoles, thiophene-based carboxylic acids, benzothiadiazines, and others have been reported.[6] On the other hand, host factors that are essential for efficient viral replication may also be good antiviral targets. The non-immunosuppressive cyclophilin binding molecule Debio-025 is a potent inhibitor of HCV replication and has shown excellent efficacy in phase I and phase II clinical trials.[7]



HCV requires elements of the cholesterol and fatty-acid biosynthetic pathways for efficient replication.[8] Lipoprotein receptors (low-density lipoprotein [LDL] receptor, SR-BI receptor) have been reported to be involved in HCV entry. Nevertheless, the role of the LDL-receptor in HCV entry is still uncertain.[9] Cholesterol metabolism is also required for the assembly of very low-density lipoprotein (VLDL) particles. These lipoprotein particles have been shown to complex with HCV virions in serum. Statins, which are used to treat hypercholesterolemia, inhibit 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis in the liver. HMG-CoA reductase catalyzes the conversion of HMG-CoA to mevalonic acid. Besides their cholesterol-lowering effect, statins have been reported to exhibit antiviral activities against a variety of viruses. Antiviral activity was reported against human immunodeficiency virus (HIV)-1,[10-12] poliovirus,[13] cytomegalovirus,[14] and respiratory syncytial virus.[15] Recently, statins were shown to inhibit the replication of subgenomic HCV-1b replicons[16] and to suppress RNA replication of Japanese fulminant hepatitis-1 (JFH-1) HCV.[17] The precise mechanism of the anti-HCV activity of statins has not yet been defined. Recent studies suggest that the anti-HCV activity of statins may result from inhibition of geranylgeranylation of cellular proteins rather than the inhibition of cholesterol synthesis.[8][18] Geranylgeranylation is a posttranscriptional modification that covalently attaches geranylgeranyl to various cellular proteins to facilitate their membrane association. These geranylgeranyl groups are isoprenoids synthesized in the cholesterol biosynthesis pathway. More recently, FBL2 has been reported to be a host target for geranylgeranylation. Geranylgeranylation of FBL2 appears to be critical for HCV replication because the association between FBL2 and NS5A, an interaction that is a prerequisite for HCV replication, depends on geranylgeranylation of FBL2.[19]



A major concern for successful anti-HCV therapy is the rapid emergence of drug resistance to selective HCV inhibitors.[20][21] Combination therapy of drugs with a different mode of action will most likely be necessary to delay or prevent the development of drug resistance. We here report on the effect of combining statins with selective HCV antivirals.



Discussion



We report on the combined anti-HCV activity of mevastatin and simvastatin with IFN- or with HCV NS5B polymerase or NS3 protease inhibitors. Mevastatin and simvastatin were selected for these combination experiments because these statins exhibited the strongest antiviral activity when used alone. The combined effect of mevastatin or simvastatin with IFN-a or with selective HCV inhibitors was first studied in regular 3-day antiviral assays. All combinations resulted in an overall additive antiviral activity. An additive activity is to be expected with compounds that (likely) do not interfere with each others' metabolism or mechanism of action. Others reported recently that in vitro combinations of IFN-a with simvastatin, fluvastatin, or pitavastatin resulted in a synergistic activity.[16][17][29] It must be emphasized, however, that in these studies combinations were analyzed using the fractional inhibitory concentration (FIC) method and that minimal FIC values of 0.5 were observed, which suggests a subsynergistic rather than a synergistic activity. Pronounced synergistic activities should result in lower FIC values. For example, we reported earlier that mycophenolic acid, the active component of the immunosuppressive drug mycophenolate mofetil, markedly potentiate the antiherpes virus activities of acyclovir and other guanine-based nucleoside analogs and we explained the biochemical mechanism responsible for this potentiating effect. In this case, FIC values of about 0.1 were calculated.[31]



A short-term antiviral assay may not necessarily predict the antiviral effect of either single compounds or combinations thereof during long-term treatment. We therefore studied the anti-HCV activity of statins alone or in combination with either IFN-a, an HCV NS3 protease, or an HCV polymerase inhibitor in so-called clearance-rebound experiments. Replicon-containing cells were cultured for six consecutive passages in the presence of mevastatin alone or in combination with IFN-a or a selective HCV inhibitor and in the absence of neomycin selection pressure (clearance phase). Following every two passages (passages 2, 4, 6) a rebound condition was included. Despite the fact that no synergistic anti-HCV activity was observed in regular short-term antiviral combination assays, all statin-containing combinations were able to clear cells from their replicon. The combination of simvastatin with the benzofuran polymerase inhibitor HCV-796 did so after only two passages of combined drug pressure. Clearance-rebound experiments may possibly have predictive value for estimating the potential of drugs (or combinations thereof) to clear liver cells from replicating virus and may better mimic the real-life situation than a 3-day antiviral combination assay. Our data also indicate that regular short-term antiviral combination assays (in replicon-based systems) may not necessarily predict synergistic antiviral effects that develop following long-term culture. It may be important to further assess the potential value of such combination strategies in animal models. The human liver-uPA-SCID mouse may be well suited for this purpose.[32]



Our data also demonstrate that statins may delay or prevent the development of drug-resistant variants. These findings can be important, because it is known that the fidelity of the viral replication machinery of HCV is low, therefore enabling the virus to quickly develop resistance mutations for compounds targeting viral enzymes.[4] Antiviral compounds that target cellular factors generally select less readily drug-resistant variants than those inhibiting viral proteins because cellular factors are independent of the viral escape by way of genetic mutations caused by the RNA-dependent-RNA polymerase.[33]



Replicons lack viral structural proteins; therefore the antiviral effect of statins must be targeted at the viral replication complex. Statins inhibit biosynthesis of cholesterol, thereby upregulating the expression of LDL receptors at the cell membrane, leading to an increased uptake of LDL particles in hepatocytes. Because HCV infection is partially dependent on the LDL receptor, statin treatment may theoretically increase HCV infectivity. We observed that statins proved indeed less potent in the HCVcc model than in the subgenomic replicon model. Our preliminary experiments revealed, however, that (1) there was a tendency toward less efficient uptake of HCV pseudoparticles in hepatoma cells that had been treated with mevastatin (34% inhibition at a concentration of 10 M); (2) statin treatment did not markedly affect binding of HCVcc to hepatoma cells; and (3) there was a tendency toward less efficient internalization of HCVcc in cells that had been treated with statins (48% inhibition at a concentration of 10 uM mevastatin) (data not shown). It is thus rather unlikely that the reduced potency of statins in the infectious HCVcc system as compared to the replicon system can be explained by an increased binding/uptake of HCV in statin-treated cells.



The potential antiviral activity of statins was studied in patients chronically infected with HCV. Neither atorvastatin (after conventional 12-week therapy)[34] nor rosuvastatin[35] resulted in a reduction in viral load. The most plausible explanation for the discrepancy between the in vitro activity and the lack of clinical efficacy is, as suggested by the authors, that the plasma concentrations were likely below the levels employed in cell culture.[34] In a recently published study, fluvastatin was found to inhibit HCV RNA replication in HCV-infected patients.[36] The drug was well tolerated and, at relatively low doses (20-80 mg daily), resulted in a transient reduction in viral load (-0.5 to -1.75 log10, 2-5 weeks); higher doses of fluvastatin did not reduce viral load. The authors hypothesize that fluvastatin acts by binding to lipo-viro-particles (LVP) and that the result of this attachment would interfere with the ability of the virus to modulate the immune system of the host.[36] There is today no obviously compelling evidence that statins, used in monotherapy, may result in a marked reduction in HCV load in chronically infected patients. Also, ribavirin exhibits a very limited antiviral activity when used in monotherapy. The combination of ribavirin and (pegylated) IFN-a results, however, in a more than additive effect in HCV-infected patients. It has been suggested that a disturbance of the kinetics of viral replication brought about by IFN- is required to create a condition where a weak antiviral drug, such as ribavirin, can exert substantial antiviral activity.[37] Possibly, the combination of a statin with the current standard therapy or with one or more potent STAT-C inhibitor(s) may result in a more than additive effect, as suggested by the clearance-rebound experiments reported in the present study. The potential use of statins in HCV-infected patients may thus lie in combination therapy. Although plasma levels of statins are usually rather low (Cmax [ng/mL] based on a 40-mg oral dose; lovastatin: 10-20 ng/mL, pravastatin: 45-55 ng/mL, simvastatin: 10-34 ng/mL, fluvastatin: 448 ng/mL),[38] the replication of HCV occurs predominantly in the liver. Concentrations of statins in the liver may therefore be more relevant than plasma concentrations. Although the thesis that statin concentrations are likely much higher in the human liver than in plasma is widely accepted, there are, to the best of our knowledge, no studies published in which concentrations of statins in the human liver have been determined. In rats, the liver concentration of lovastatin is 15-fold to 18-fold higher than the concentration in blood and other tissues.[39-41] If one would assume that liver concentrations of statins in the human liver may also be 15-fold higher than in plasma, a very rough estimation would suggest concentrations of 0.75-15 uM in the human liver (depending on the pharmacokinetic profile of the specific statin). Statin doses as high as two-fold to eight-fold the currently used standard doses can be safely used to treat hypercholesterolemia.[42] Hence, higher dosing may be expected to result in liver concentrations that are sufficiently high to inhibit HCV replication, in particular when combined with STAT-C inhibitors. Because statins target cellular factors, the genetic barrier to resistance is expected to be (relatively) high (which is corroborated by our unpublished findings). Furthermore, because we here demonstrate that mevastatin delays or even prevents the development of HCV-796 escape mutants, the combination of STAT-C inhibitors with statins may also have the potential to delay or even to prevent resistance development in the clinical setting.



In summary, the combination of statins with several selective HCV inhibitors results in a pronounced antiviral effect in cell culture and prevents or delays the emergence of drug variants resistant to a STAT-C inhibitor. Statins may have the potential to (1) increase the efficacy of current or future HCV therapy and (2) delay the development of resistance against STAT-C inhibitors.



Results



Antiviral Activity of Statins in HCV Subgenomic Replicon Cells.



The effect of lovastatin, mevastatin, simvastatin, fluvastatin, and pravastatin on in vitro HCV replication was evaluated in genotype 1b Con1 HCV subgenomic replicons (Huh 5-2). Lovastatin, mevastatin, simvastatin, and fluvastatin inhibited HCV replicon replication (measured as luciferase signal) in a dose-dependent manner (Table 1, Fig. 1). These four statins were roughly equipotent (with EC50 values of 1.9, 1.3, 1.5, 1.8 uM), whereas pravastatin was devoid of an inhibitory effect on HCV replicon replication, as expected.[16][29] The anti-HCV activity of statins was not the result of a cytostatic effect; the 50% cytotoxic concentrations of lovastatin, mevastatin, simvastatin, and fluvastatin were, respectively, 60 uM, 34 uM, 32 uM, and 44 uM, thus resulting in selectivity indices of 32, 23, 21, and 24 (Table 1).



The anti-HCV activity was confirmed in Huh 9-13 replicon-containing cells by means of RT-qPCR (Table 1). Mevastatin and simvastatin exhibited the strongest anti-HCV activity, whereas fluvastatin and lovastatin had moderate inhibitory effects and pravastatin was, akin to the situation in Huh 5-2 replicon-containing cells, devoid of antiviral activity. The antiviral activity of statins, however, was less effective in the HCVcc model than in the subgenomic replicon model (Table 1).



It was next studied whether statins inhibit the replication of other members of the Flaviviridae family, i.e., flaviviruses (yellow fever virus 17P) and pestiviruses (bovine viral diarrhea virus). No activity was observed against these viruses nor against a selection of unrelated viruses (chikungunya virus, sindbis virus, coxsackie B4 virus, punta toro virus, para-influenza-3 virus, respiratory syncytial virus, vesicular stomatitis virus, influenza A virus [H1N1 and H3N2], influenza B virus, reovirus-1, vaccinia virus, herpes simplex virus 1 and 2).



Combination of Mevastatin or Simvastatin with Selective HCV Inhibitors or IFN-a.



Drug-resistant variants develop readily against most selective inhibitors of HCV replication. Combination therapy of drugs with different modes of action will most likely be necessary to delay or prevent the development of viral escape mutants. Therefore, the combined antiviral effects of mevastatin with either IFN-a or selective HCV polymerase inhibitors (R1479, GSK-4 benzothiadiazine and HCV-796) or a protease inhibitor (VX-950) were studied in checkerboard format. Combinations were analyzed by the method of Prichard and Shipman (Fig. 2).[28] Overall, the combined activity of mevastatin with IFN-a or with selective HCV polymerase and protease inhibitors was additive. Combinations of simvastatin with the various HCV inhibitors resulted also in an additive antiviral effect (data not shown).



HCV Replicon Clearance and Rebound.



The effect of statin-containing combinations was studied in clearance-rebound experiments. Huh 9-13 replicon containing cells were cultured for six consecutive passages in the presence of mevastatin or simvastatin, alone or in combination with IFN-a, BILN-2061, 2-C-methylcytidine, or HCV-796 (in the absence of neomycin pressure). IFN-a, BILN-2061, 2-C-methylcytidine, and HCV-796, at concentrations of 10- or 20-fold their EC50 values, already resulted after one passage in a pronounced decrease (about 2 log10) in HCV RNA content (Fig. 3A,B). For simvastatin and mevastatin, at a concentration of 5-fold their EC50 value, three passages of drug pressure were needed before the HCV RNA content of the cells dropped below 3% (simvastatin) or 1% (mevastatin) of the untreated control. To study whether the mono- and combination therapies were able to clear the cells from their replicon following six passages of antiviral pressure, the antiviral compounds were omitted from the culture medium and cells were cultured under the selective pressure of G418 for another three consecutive passages. In such a case that antiviral therapy is able to clear the replicon from the cultures, cells will not be able to proliferate when cultured in the presence of G418. Cells that still carry the replicon will be able to survive under these conditions. Although monotherapy with either statins or IFN-a, BILN-2061, 2-C-methylcytidine, or HCV-796 was able to reduce replicon levels by 2.5 to 5 log10 after passage six, none of the cultures were cleared from their replicon under these conditions. However, cultures that contained statins in combination with IFN-a or another specifically targeted antiviral therapy for HCV (STAT-C) inhibitor were completely cleared from their replicon. It was next studied at which passage these combinations resulted in complete clearance. The combination of 2-C-methylcytidine and simvastatin, the combination of IFN- and simvastatin, and the combination of BILN-2061 and mevastatin cleared cells from their replicon after four passages of antiviral pressure; the combination of the benzofuran HCV-796 and simvastatin was able to clear cells after only two passages of antiviral pressure (Fig. 3C).



Susceptibility of Various Drug-Resistant Replicons to Mevastatin.



It was studied whether statins retain their antiviral activity against HCV replicons that are resistant to several selective HCV inhibitors. To this end BILN-2061res, 2-C-methylcytidineres, and HCV-796res replicon-containing cells were used. Mevastatin proved equipotent against replicons resistant to the NS5B polymerase (2-C-methylcytidine, HCV-796) and NS3 protease (BILN-2061) inhibitors as against wild-type replicons (Table 2).



Combined Resistance Selection.



It was next studied whether statins may delay or prevent the emergence of escape variants against the nonnucleoside polymerase inhibitor HCV-796. Huh 9-13 replicon-containing cells were cultured in the presence of G418 and mevastatin (1, 5, or 10 uM), HCV-796 (20, 100, or 200 nM), or combinations of mevastatin and HCV-796. Following 3 weeks of culturing, cells were fixed and stained with methylene blue (Fig. 4). Mevastatin, at concentrations of 1, 5, or 10 uM, did not reduce replicon replication to sufficiently low levels to render the culture sensitive to G418. At a concentration of 20 M mevastatin (that is near to the cytostatic concentration of 34 M), replicon-containing cells did not survive in the presence of G418 (data not shown). Replicon-containing cells that were cultured in the presence of 100 or 200 nM HCV-796 (25- and 50-fold the EC50) went through a crisis after which colonies developed. The HCV-796-resistant phenotype of the colonies that developed in the presence of HCV-796 was confirmed (Table 3). Genotyping of these replicons revealed that mutation C445F was responsible for this drug-resistant phenotype (data not shown). In contrast, mevastatin escape mutants did not develop. Replicon from several conditions (1 uM mevastatin, 200 nM HCV-796, 1 uM mevastatin + 200 nM HCV-796, 5 uM mevastatin + 20 nM HCV-796) proved equally sensitive to mevastatin as wild-type replicon. On the other hand, replicon-containing cells that were cultured in the presence of 200 nM HCV-796 and 1 uM mevastatin were found to be resistant to HCV-796. Mutation M414I was responsible for this drug-resistant phenotype (data not shown).[30]



The average number of colonies formed for each condition in two independent experiments is presented in Table 4. When HCV-796 was combined with mevastatin, the frequency of colony formation was markedly reduced. For example, at an HCV-796 concentration of 100 nM and a mevastatin concentration of 5 uM, on average only two colonies (0.003% of control) developed. Mevastatin is thus able to delay (at concentrations of 1 uM and 5 uM) or prevent (at a concentration of 10 uM) the emergence of HCV-796-resistant replicon.



Materials and Methods






Cells and Replicon Constructs.



Huh 7 cells containing subgenomic HCV replicons I389luc-ubi-neo/NS3-3/5.1 (Huh 5-2) and I377/NS3-3/wt (Huh 9-13) have been described before.[22][23] Cells were cultured in Dulbecco's modified Eagle's Medium (DMEM; Gibco, Merelbeke, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (Integro, Zaandam, The Netherlands), 1× nonessential amino acids, 100 IU/mL penicillin (Gibco), 100 ug/mL streptomycin (Gibco), 250 g/mL Geneticin (G418; Gibco) for Huh 5-2 cells, and 1 mg/mL G418 for Huh 9-13 cells. Cell cultures were maintained at 37°C in an atmosphere of 5% CO2. Replicons resistant to the protease inhibitor BILN-2061, the nucleoside polymerase inhibitor 2-C-methylcytidine, or the nonnucleoside polymerase inhibitor HCV-796 were generated by selective pressure.



HCV Inhibitor Compounds.



Mevastatin, simvastatin, lovastatin, and pravastatin were purchased from Sigma-Aldrich (Bornem, Belgium). Fluvastatin was purchased from Cayman Chemical (Huissen, The Netherlands). Recombinant IFN-a 2b (Intron A) was purchased from Schering Plough (Kenilworth, NJ). HCV NS5B polymerase inhibitors 2-C-methylcytidine, R1479, benzothiadiazine GSK-4 and benzofuran HCV-796, and NS3 protease inhibitors VX-950 and BILN-2061 were synthesized as described before.[24]



Antiviral Assay with Huh 5-2 Cells or Huh 9-13 Cells.



Antiviral assays were performed as described earlier.[25] Briefly, cells were seeded at a density of 5 × 103 cells per well (for Huh 9-13 cells) or 6.5 × 103 cells per well (for Huh 5-2 cells) in a 96-well cell culture plate in complete DMEM. Following incubation of 24 hours at 37°C (5% CO2), serial dilutions of the test compounds in complete DMEM were added in a total volume of 100 L. For the Huh 5-2 cells, luciferase activity was determined after 3 days of incubation using the Steady-Glo luciferase assay system (Promega, Leiden, The Netherlands). For Huh 9-13 cells, replicon RNA levels were determined by a reverse transcription quantitative polymerase chain reaction (RT-qPCR).



Antiviral Assay with JFH-1/CS-N6 HCVcc.



Huh 7.5.1 cells were seeded at a density of 5 × 103 cells per well in a 96-well cell culture plate in complete DMEM. Following incubation of 24 hours at 37°C (5% CO2), serial dilutions of the test compounds in complete DMEM were added in a total volume of 100 uL. Cells were then inoculated with 100 L diluted culture media containing infectious HCV JFH-1/CS-N6 (2.7 × 105 HCV RNA copies).[26] Intracellular HCV RNA levels were determined after 3 days of incubation by RT-qPCR.



RT-qPCR.



RT-qPCR was performed as previously described.[25] Primers used for detection of HCV replicon RNA were: 5-CCG GCT ACC TGC CCA TTC-3 (forward primer), 5-CCA GAT CAT CCT GAT CGA CAA G-3 (reverse primer) and 5-FAM-ACA TCG CAT CGA GCG AGC ACG TAC-TAMRA-3 (probe). Primers used for detection of HCVcc RNA were: 5-ACG CAG AAA GCG CCT AGC CAT GGC GTT AGT A-3 (forward primer), 5-TCC CGG GGC ACT CGC AAG CAC CCT ATC AGG-3 (reverse primer) and 5-FAM-TGG TCT GCG GAA CCG GTG AGT ACA CC-TAMRA-3 (probe).



Cytostatic Assay.



Cytostatic assays were performed as previously described.[25] Briefly, cells were seeded at a density of 5 × 103 cells per well (for Huh 9-13 cells) or 6.5 × 103 cells per well (for Huh 5-2 cells) in a 96-well cell culture plate in complete DMEM. After 24 hours of incubation at 37°C serial dilutions of the test compounds in complete DMEM were added. After 3 days of incubation at 37°C, cell viability was determined using the MTS/PMS method (Promega).



Other Antiviral Assays.



Antiviral assays with herpes simplex virus-1 (KOS strain and TK- KOS ACVr), herpes simplex virus-2 (G strain) [Herpesviridae], vaccinia virus [Poxviridae], vesicular stomatitis virus [Rhabdoviridae], coxsackie virus B4 [Picornaviridae], respiratory syncytial virus, para-influenza-3 virus [Paramyxoviridae], reovirus-1 [Reoviridae], sindbis virus, chikungunya virus [Alphaviridae], influenza A virus (H1N1 and H3N2), influenza B virus [Orthomyxoviridae], Punta Toro virus [Bunyaviridae], yellow fever virus (YFV-17D), and bovine viral diarrhea virus (BVDV) [Flaviviridae] were carried out as reported.[27]



Combination Antiviral Assay.



The effects of drug-drug combinations were evaluated using the method of Prichard and Shipman.[28] In brief, the theoretical additive effect is calculated from the dose-response curves of individual compounds by the equation Z=X+Y(1-X) where X and Y represent the inhibition produced by the individual compounds and Z represents the effect produced by the combination of compounds. The theoretical additive surface is subtracted from the actual experimental surface, resulting in a horizontal surface that equals the zero plane when the combination is additive. A surface that lies higher than 20% above the zero plane indicates a synergistic effect of the combination and a surface lower than 20% below the zero plane indicates antagonism. The antiviral assays were performed in a similar way as described for Huh 5-2 cells except that the compound dilutions were added in a checkerboard format. Combination studies for each pair of compounds were performed in triplicate.



Clearance Rebound Assay.



Huh 9-13 cells were seeded at a density of 3 × 105 cells in complete DMEM with 1 mg/mL G418 in 25 cm2 T-flasks. After 24 hours of incubation at 37°C (5% CO2) cell culture medium was removed and replaced by complete DMEM without G418 containing either no antiviral compound or containing one or two antiviral compounds at a fixed concentration. Mevastatin and simvastatin were used at concentrations of 5-fold their effective concentration (EC)50 value, 2-C-methylcytidine, HCV-796, and IFN- at 10-fold their EC50 value, and BILN-2061 at 20-fold its EC50 value. Concentrations of particular compounds were selected such that the concentration was not cytostatic but still able to result in at least 2-log reduction in replicon replication following two passages of treatment. When 90% confluency was reached cells were trypsinized, after which 3 × 105 cells were seeded in a new 25 cm2 T-flask in complete DMEM with the same concentration of compound(s); 1.5 × 105 cells from each flask were lysed in RLT buffer. In total, cells were passaged six consecutive times in the presence or absence of compound(s) and in the absence of G418. Following every two passages (P2, P4, and P6), cells of each flask were passaged for three consecutive times in the presence of 1 mg/mL G418 (rebound phase). Again, 3 × 105 cells were seeded in new 25 cm2 T-flasks and 1.5 × 105 cells from each flask were lysed in RLT buffer. After collecting all samples, RNA was extracted and the replicon RNA content was measured by real-time qPCR.



Combined Resistance Selection.



Huh 9-13 cells were seeded at a density of 7.2 × 104 cells in a 12-well plate in complete DMEM containing 1 mg/mL G418 and in the presence of mevastatin (1 uM, 5 uM, and 10 uM) or HCV-796 (20 nM, 100 nM, and 200 nM) or a combination of both in a matrix format. When 90% confluency was reached cells were trypsinized, after which 7.2 × 104 cells were seeded in a new 12-well plate in complete DMEM with the same concentration of antiviral(s). After about 3 weeks of selection, HCV-796 resistant colonies developed. Cultures were either fixed with ethanol and stained with 1% methylene blue or expanded to obtain sufficient cells for subsequent phenotype and genotype characterization.



Sequence Analysis of the NS5B Gene.



Total cellular RNA was extracted using the RNeasy minikit (Qiagen, Venlo, The Netherlands) and subjected to RT-PCR using primers NS5B-F [5-TGCTTTGACTCAACGGTCAC-3] (corresponding to nucleotides 6649 to 6668 of accession number AJ242652) and NS5B-R [5-TGTAACCAGCAACGAACCAG-3] (7629 to 7648 of accession number AJ242652). Nucleotide sequences were determined by automated sequencing using BigDye terminator v. 3.1 (Applied Biosystems).

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