Abstract
Phenotypic and Structural Analyses of Hepatitis C Virus NS3 Protease Arg155 Variants: SENSITIVITY TO TELAPREVIR (VX-950) AND INTERFERON α
Highlights
Hepatitis C virus (HCV)3 is an important human pathogen that causes chronic infection in a majority of patients after an initial, acute infection
These results indicate that substitutions of NS3 Arg155 led to low level (Ͻ25-fold) resistance to telaprevir in hepatitis C virus (HCV) replicon cells, independent of the physical properties of the substituted residue, which include a positive charged residue (Lys), a hydrophilic residue (Thr or Ser), a hydrophobic residue (Ile or Met), or a residue that lacks a side chain (Gly)
The replicon IC50 of either IFN-␣ or ribavirin remained virtually the same for HCV replicon cells containing R155K, R155T, or R155M mutations compared with the wild-type replicon cells. These results suggest that combination with IFN-␣ with or without ribavirin could be a potential therapeutic strategy to suppress the emergence of HCV variants with substitutions at NS3 protease residue 155
Summary
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. 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. 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 [37] by electroporation as described above. The average enzymatic IC50(1 h) values Ϯ S.D. of telaprevir were determined for the purified wild type and for four variant HCV NS3 serine protease domains using the KK4A cofactor peptide and the FRET substrate in 3–5 independent experiments. The Vmax and Km values Ϯ S.D. of the wild type and two variant HCV NS3 protease domains using the KK4A cofactor peptide and the 5A/5B high pressure liquid chromatography substrate were determined in three independent experiments.
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