Abstract
VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3.4A protease inhibitor, BILN 2061. Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala(156) with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.
Highlights
hepatitis C virus (HCV) is an enveloped virus containing a single-stranded, positive polarity RNA that encodes a polyprotein precursor of ϳ3000 amino acids
We have previously shown that the A156S mutant is resistant to VX-950, but not to BILN 2061 (25)
Because there is no apparent overlap between the in vitro dominant resistance mutation profiles of VX-950 and BILN 2061, it is likely that a combination of VX-950 and BILN 2061 would suppress the appearance of their dominant resistance mutations
Summary
Plasmid Construction—An Escherichia coli expression plasmid containing a DNA fragment encoding Met1–Ser181 of the HCV NS3 protease (GenBankTM accession number CAB46913) of the HCV Con replicon I377neo/NS3-3Ј/wt (26) (renamed as pBR322-HCV-Neo in this study) followed by a C-terminal hexahistidine tag has been described previously (25). The cross-resistance mutations identified in this study were introduced into the pBR322-HCV-Neo-mADE replicon plasmid by site-directed mutagenesis as described previously (25). To determine the frequency of PI resistance mutations, the 1.7-kb RT-PCR products of HCV RNA from the PI-cross-resistant replicon cells were ligated into the TA cloning vector pCR2.1 (Invitrogen); multiple individual bacterial colonies were isolated for each time point; and the HCV NS3 protease coding region of the purified plasmid DNA was sequenced. Fitness of HCV NS31⁄74A Protease Inhibitor-resistant Mutants in Replicon Cells—T7 RNA runoff transcripts were generated from the ScaIlinearized pBR322-HCV-Luc-mADE plasmid with or without the PI resistance mutations and transfected into PI-cured Con subgenomic replicon cells by electroporation as described previously (26). The inhibitors VX-950 and BILN 2061 were docked into these mutant enzyme active sites to model the effect of the mutations on inhibitor binding
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