Phenotypic and Structural Analyses of Hepatitis C Virus NS3 Protease Arg155 Variants: SENSITIVITY TO TELAPREVIR (VX-950) AND INTERFERON α

Abstract

Telaprevir (VX-950) is a highly selective, potent inhibitor of the hepatitis C virus (HCV) NS3·4A serine protease. It has demonstrated strong antiviral activity in patients chronically infected with genotype 1 HCV when dosed alone or in combination with peginterferon alfa-2a. Substitutions of Arg155 of the HCV NS3 protease domain have been previously detected in HCV isolates from some patients during telaprevir dosing. In this study, Arg155 was replaced with various residues in genotype 1a protease domain proteins and in genotype 1b HCV subgenomic replicons. Characterization of both the purified enzymes and reconstituted replicon cells demonstrated that substitutions of Arg155 with these residues conferred low level resistance to telaprevir (<25-fold). An x-ray structure of genotype 1a HCV protease domain with the R155K mutation, in a complex with an NS4A co-factor peptide, was determined at a resolution of 2.5Å. The crystal structure of the R155K protease is essentially identical to that of the wild-type apoenzyme (Protein Data Bank code 1A1R) except for the side chain of mutated residue 155. Telaprevir was docked into the x-ray structure of the R155K protease, and modeling analysis suggests that the P2 group of telaprevir loses several hydrophobic contacts with the Lys155 side chain. It was demonstrated that replicon cells containing substitutions at NS3 protease residue 155 remain fully sensitive to interferon α or ribavirin. Finally, these variant replicons were shown to have reduced replication capacity compared with the wild-type HCV replicon in cells. Telaprevir (VX-950) is a highly selective, potent inhibitor of the hepatitis C virus (HCV) NS3·4A serine protease. It has demonstrated strong antiviral activity in patients chronically infected with genotype 1 HCV when dosed alone or in combination with peginterferon alfa-2a. Substitutions of Arg155 of the HCV NS3 protease domain have been previously detected in HCV isolates from some patients during telaprevir dosing. In this study, Arg155 was replaced with various residues in genotype 1a protease domain proteins and in genotype 1b HCV subgenomic replicons. Characterization of both the purified enzymes and reconstituted replicon cells demonstrated that substitutions of Arg155 with these residues conferred low level resistance to telaprevir (<25-fold). An x-ray structure of genotype 1a HCV protease domain with the R155K mutation, in a complex with an NS4A co-factor peptide, was determined at a resolution of 2.5Å. The crystal structure of the R155K protease is essentially identical to that of the wild-type apoenzyme (Protein Data Bank code 1A1R) except for the side chain of mutated residue 155. Telaprevir was docked into the x-ray structure of the R155K protease, and modeling analysis suggests that the P2 group of telaprevir loses several hydrophobic contacts with the Lys155 side chain. It was demonstrated that replicon cells containing substitutions at NS3 protease residue 155 remain fully sensitive to interferon α or ribavirin. Finally, these variant replicons were shown to have reduced replication capacity compared with the wild-type HCV replicon in cells. Hepatitis C virus (HCV) 3The abbreviations used are: HCV, hepatitis C virus; PI, protease inhibitor; IFN, interferon; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MES, 4-morpholineethanesulfonic acid. 3The abbreviations used are: HCV, hepatitis C virus; PI, protease inhibitor; IFN, interferon; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MES, 4-morpholineethanesulfonic acid. is an important human pathogen that causes chronic infection in a majority of patients after an initial, acute infection. It is estimated that about 170 million patients worldwide and ∼1% of the population in developed countries are chronically infected with HCV (1Wasley A. Alter M.J. Semin. Liver Dis. 2000; 20: 1-16Crossref PubMed Google Scholar). Chronic hepatitis C can lead to severe liver diseases, including fibrosis, cirrhosis, or hepatocellular carcinoma (2Kenny-Walsh E. Clin. 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Agents Chemother. 2005; 49: 4305-4314Crossref PubMed Scopus (110) Google Scholar) and remained as sensitive to IFN-α or ribavirin as the wild-type replicon in cell assays (28Lu L. Pilot-Matias T.J. Stewart K.D. Randolph J.T. Pithawalla R. He W. Huang P.P. Klein L.L. Mo H. Molla A. Antimicrob. Agents Chemother. 2004; 48: 2260-2266Crossref PubMed Scopus (153) Google Scholar, 30Lin C. Gates C.A. Rao B.G. Brennan D.L. Fulghum J.R. Luong Y-P. Frantz J.D Lin K. Ma S. Wei Y.-Y. Perni R.B. Kwong A.D. J. Biol. Chem. 2005; 280: 36784-36791Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 34Mo H. Lu L. Pilot-Matias T. Pithawalla R. Mondal R. Masse S. Dekhtyar T. Ng T. Koev G. Stoll V. Stewart K.D. Pratt J. Donner P. Rockway T. Maring C. Molla A. Antimicrob. Agents Chemother. 2005; 49: 4305-4314Crossref PubMed Scopus (110) Google Scholar). A highly sensitive, clonal sequencing method was recently used to identify telaprevir-related variants in patients dosed with telaprevir alone (35Sarrazin C. Kieffer T.L. Bartels D. Hanzelka B. Muöh U. Welker M. Wincheringer D. Zhou Y. Chu H-M. Lin C. Weegink C. Reesink H. Zeuzem S. Kwong A.D Gastroenterology. 2007; 132: 1767-1777Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). Substitutions of residue 36 (V36A/M), 54 (T54A), 155 (R155K/T), or 156 (A156S/T/V) were observed in some patients dosed with telaprevir alone. The selection of different groups of HCV protease variants seems to be associated with the pattern of antiviral response as well as the plasma exposure of telaprevir observed in these patients. In the current report, we carried out extensive studies of several variants with substitutions at Arg155 of the HCV NS3 protease domain, including enzymatic characterization, replicon cell studies, x-ray crystallography, and computational modeling. Our data demonstrate that NS3 protease variants at Arg155 confer low level resistance to telaprevir (<25-fold) in both enzyme and replicon cell assays. The change in three-dimensional structure was subtle when the Arg155 was replaced with a Lys. Computational modeling analysis suggests that the impact on binding of telaprevir to the HCV NS3 protease is not expected to be dramatic, which is consistent with the low level loss of sensitivity to telaprevir observed in enzyme and replicon cell assays. In addition, these variants have decreased replication capacity in replicon cells, which is consistent with the reduced in vivo fitness shown previously in telaprevir-dosed hepatitis patients (35Sarrazin C. Kieffer T.L. Bartels D. Hanzelka B. Muöh U. Welker M. Wincheringer D. Zhou Y. Chu H-M. Lin C. Weegink C. Reesink H. Zeuzem S. Kwong A.D Gastroenterology. 2007; 132: 1767-1777Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). Plasmid Construction—Substitutions of Arg155 of the HCV NS3 protease domain were introduced into four sets of plasmids for replicon cell, enzymatic, or x-ray crystallography characterizations. For studies using replicon stable cells, substitutions at NS3 residue 155 were engineered by site-directed mutagenesis into a genotype 1b subgenomic HCV replicon plasmid, pBR322-HCV-Neo-mADE. As described previously, pBR322-HCV-Neo-mADE is a second generation, high efficiency replicon plasmid that contains three adaptive mutations (27Lin C. Lin K. Luong Y.P. Rao B.G. Wei Y.Y. Brennan D.L. Fulghum J.R. Hsiao H.M. Ma S. Maxwell J.P. Cottrell K.M. Perni R.B. Gates C.A. Kwong A.D. J. Biol. Chem. 2004; 279: 17508-17514Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) and was derived from a Con1 strain subgenomic replicon, I377neo/NS3-3′/wt (GenBank™ accession number CAB46913) (36Lohmann V. Korner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Science. 1999; 285: 110-113Crossref PubMed Scopus (2474) Google Scholar). The codon change at residue 155 of the genotype 1b HCV replicon was as follows: Arg (CGG) to Lys (AAG), Thr (ACG), Ser (AGT), Met (ATG), Ile (ATC), or Gly (GGG). The same set of substitutions at Arg155 was then subcloned into pBR322-HCV-Luc-mADE (30Lin C. Gates C.A. Rao B.G. Brennan D.L. Fulghum J.R. Luong Y-P. Frantz J.D Lin K. Ma S. Wei Y.-Y. Perni R.B. Kwong A.D. J. Biol. Chem. 2005; 280: 36784-36791Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) for replication capacity assay in replicon cells using transient transfection. All constructs were confirmed by sequencing. For expression of protein used in enzymatic studies, HCV cDNA was amplified using reverse transcription-PCR from the viral RNA isolated from a genotype 1a HCV-infected patient who was enrolled in a phase 1b clinical study in which patients were treated with telaprevir (VX-950) alone (35Sarrazin C. Kieffer T.L. Bartels D. Hanzelka B. Muöh U. Welker M. Wincheringer D. Zhou Y. Chu H-M. Lin C. Weegink C. Reesink H. Zeuzem S. Kwong A.D Gastroenterology. 2007; 132: 1767-1777Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). A cDNA fragment encoding HCV NS3 residues Ala1–Ser181 from this patient (Fig. 1) (GenBank™ accession number AM489456) was then subcloned into a pBEV10 expression vector containing a C-terminal His6 tag. In each expression construct, NS3 protease residue Leu13 (codon CTC) was replaced with Lys (codon AAG) to improve the solubility of the protein. The HCV cDNA containing mutations at residue 155 of the NS3 protease domain was derived either by reverse transcription-PCR from serum samples of this patient (R155K) or constructed via site-directed mutagenesis (R155T, R155S, or R155I). The R155K and R155T variants were observed in this patient at either the end of the 14-day telaprevir dosing period or in the 7–10-day follow-up period after the last dose, whereas the R155S, R155I, R155M, and R155G variants were seen in other patients (35Sarrazin C. Kieffer T.L. Bartels D. Hanzelka B. Muöh U. Welker M. Wincheringer D. Zhou Y. Chu H-M. Lin C. Weegink C. Reesink H. Zeuzem S. Kwong A.D Gastroenterology. 2007; 132: 1767-1777Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). The codon change for residue 155 was as follows: Arg (AGG) to Lys (AAG), Thr (ACG), Ser (AGC), or Ile (ATC). To solve the x-ray crystal structure of R155K variant protease, a wild-type HCV-H strain (genotype 1a) cDNA fragment encoding NS3 residues Ala1–Ser181 was cloned into a different pBEV10 plasmid. The resulting expression construct encodes an NS3 protease flanked by a T7 tag at the N terminus and a His6 tag at the C terminus, similar to what has been previously described for a pET-based expression plasmid (20Kim J.L. Morgenstern K.A. Lin C. Fox T. Dwyer M.D. Landro J.A. Chambers S.P. Markland W. Lepre C.A. O'Malley E.T. Harbeson S.L. Rice C.M. Murcko M.A. Caron P.R. Thomson J.A. Cell. 1996; 87: 343-355Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). The substitution of Arg155 (AGG) with Lys (AAG) was generated via site-directed mutagenesis. Generation of Stable HCV Replicon Cells—Full-length HCV subgenomic replicon RNA was generated from ScaI-linearized DNA template using a MEGAscript T7 kit (Ambion, Austin, TX), treated with RNase-free DNase I in the kit to remove the DNA template, and purified by LiCl precipitation. RNA run-off transcripts were electroporated into naive Huh-7 cells, and G418-resistant HCV replicon cells were selected with 0.5 mg/ml G418 (Geneticin; Invitrogen) in Dulbecco’s modified minimal essential medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), 2 mm l-glutamine, 1× nonessential amino acids, and 100 units/ml penicillin plus 100 μg/ml streptomycin for 2–3 weeks. (All reagents were purchased from Invitrogen, except FBS, which was purchased from JRH Biosciences (Lenexa, KS)). The cells were split whenever they reached confluence. Replicon cells were then maintained in DMEM containing 10% FBS, 2 mm l-glutamine, 1× nonessential amino acids, and 0.25 mg/ml G418. Reverse transcription-PCR products amplified from total cellular RNA using SuperScript III One-Step reverse transcription-PCR System (Invitrogen) were sequenced to confirm the presence of mutations in the replicon cells. IC50 Determination of Antiviral Agents in the HCV Replicon Cell Assay—The IC50 values of antiviral agents were determined in a 48-h assay using HCV replicon cells as described before (27Lin C. Lin K. Luong Y.P. Rao B.G. Wei Y.Y. Brennan D.L. Fulghum J.R. Hsiao H.M. Ma S. Maxwell J.P. Cottrell K.M. Perni R.B. Gates C.A. Kwong A.D. J. Biol. Chem. 2004; 279: 17508-17514Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Briefly, 10,000 HCV replicon cells/well were plated in a tissue culture-treated 96-well plate in DMEM containing 10% FBS, 2 mm l-glutamine, 1× nonessential amino acids, and 0.25 mg/ml G418 and then incubated overnight at 37 °C in a humidified incubator with 5% CO2. The next day, the medium was replaced with DMEM containing 2% FBS, 2 mm l-glutamine, 1× nonessential amino acids, and serially diluted antiviral agents (final concentration of Me2SO is 0.5%). After a 48-h incubation with the antiviral agents, the replicon RNA levels were determined using the QuantiGene discover assay kit (Panomics, Inc., Fremont, CA) with an HCV-specific primer set. The IC50 values of the antiviral agents were calculated using a four-parameter curve fitting method in the Softmax Pro program (Molecular Devices Corp., Sunnyvale, CA). Three independent assays were conducted for each viral variant, and the mean and S.D. of the replicon IC50 values were calculated. The -fold change in sensitivity to anti-HCV agents was calculated by dividing the mean IC50 of the agent against each variant by that against the wild-type (mADE) replicon cells. Replication Capacity of HCV NS3 Protease Variants in Replicon Cells—T7 RNA run-off transcripts were generated from the ScaI-linearized pBR322-Luc-mADE plasmids and transfected into Huh7.5 cells (37Blight K.J. McKeating J.A. Rice C.M. J. Virol. 2002; 76: 13001-13014Crossref PubMed Scopus (953) Google Scholar) by electroporation as described above. Transfected cells were plated into DMEM plus 10% FBS and penicillin/streptomycin in a set of duplicated 96-well white clear bottom plates, and plates were incubated for 3 h (the first set) or 72 h (the second set). The cells were lysed with cell lysis buffer and kept frozen at -80 °C until they were thawed prior to measurement of the luciferase activity using a luciferase assay kit (Promega, Madison, WI). For any given replicon variant, a normalized luciferase signal was calculated by dividing the luciferase signal at 72 h postelectroporation with that at 3 h postelectroporation of the same replicon variant. The relative replication capacity of an NS3 protease variant is expressed as the percentage of the normalized luciferase signal of the mutant replicon compared with that of the wild-type replicon (as 100%) and that of a HCV polymerase null mutant (as 0%). Expression and Purification of the HCV NS3 Serine Protease Domain—HCV NS3 serine protease domain containing the wild-type (base-line) sequence of the patient (Fig. 1) or mutations (R155K, R155T, R155S, or R155I) were expressed from the corresponding pBEV10/HCV-3201/NS3181-His6 plasmids in BL21/DE3 Escherichia coli cells (Stratagene, La Jolla, CA) and purified as described before (27Lin C. Lin K. Luong Y.P. Rao B.G. Wei Y.Y. Brennan D.L. Fulghum J.R. Hsiao H.M. Ma S. Maxwell J.P. Cottrell K.M. Perni R.B. Gates C.A. Kwong A.D. J. Biol. Chem. 2004; 279: 17508-17514Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), with minor modifications. Briefly, freshly transformed cells were grown at 37 °C in a BHI medium (Difco) supplemented with 100 μg/ml carbenicillin to an optical density of 0.75 at 600 nm, followed by induction with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 5 h at 25 °C. All purification steps were performed at 4 °C as described before (27Lin C. Lin K. Luong Y.P. Rao B.G. Wei Y.Y. Brennan D.L. Fulghum J.R. Hsiao H.M. Ma S. Maxwell J.P. Cottrell K.M. Perni R.B. Gates C.A. Kwong A.D. J. Biol. Chem. 2004; 279: 17508-17514Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), with minor modifications. Briefly, cell paste was lysed in buffer A (50 mm HEPES (pH 8.0), 300 mm NaCl, 0.1% n-octyl-β-d-glucopyranoside, 5 mm β-mercaptoethanol, and 10% (v/v) glycerol) supplemented with 5 mm imidazole, using BugBuster Reagent (Novagen, Madison, WI), followed by centrifugation at 16,000 × g for 30 min. The clarified lysate was passed over pre-equilibrated TALON affinity resin (Clontech) and washed with 30 column volumes of buffer A plus 5 mm imidazole. The HCV NS3 proteins were eluted in buffer A containing 300 mm imidazole, concentrated, and loaded onto a Hi-Load 16/60 Superdex 200 column that was pre-equilibrated with buffer A. The appropriate fractions of purified HCV proteins were pooled and stored at -80 °C. The purity of these proteases was determined to be either over 90% (wild type and R155K) or around 80% (R155T and R155S) by SDS-PAGE with Coomassie Blue staining. The HCV-H NS3 serine protease domain, which contains the R155K variation and is fused to a T7 tag at the N termi