Phenotypic characterization of resistant Val36 variants of hepatitis C virus NS3-4A serine protease

Abstract

In patients chronically infected with hepatitis C virus (HCV) strains of genotype 1, rapid and dramatic antiviral activity has been observed with telaprevir (VX-950), a highly selective and potent inhibitor of the HCV NS3-4A serine protease. HCV variants with substitutions in the NS3 protease domain were observed in some patients during telaprevir dosing. In this study, purified protease domain proteins and reconstituted HCV subgenomic replicons were used for phenotypic characterization of many of these substitutions. V36A/M or T54A substitutions conferred less than eightfold resistance to telaprevir. Variants with double substitutions at Val36 plus Thr54 had approximately 20-fold resistance to telaprevir, and variants with double substitutions at Val36 plus Arg155 or Ala156 had >40-fold resistance to telaprevir. An X-ray structure of the HCV strain H protease domain containing the V36M substitution in a cocomplex with an NS4A cofactor peptide was solved at a 2.4-A resolution. Except for the side chain of Met36, the V36M variant structure is identical to that of the wild-type apoenzyme. The in vitro replication capacity of most variants was significantly lower than that of the wild-type replicon in cells, which is consistent with the impaired in vivo fitness estimated from telaprevir-dosed patients. Finally, the sensitivity of these replicon variants to alpha interferon or ribavirin remained unchanged compared to that of the wild-type.

FIG. 1.

Amino acid sequence of the NS3 protease domain of HCV isolates collected from patients who were enrolled in a 14-day clinical trial with telaprevir alone. The amino acid sequences of the NS3 protease domain (residues 1 to 181) of two genotype 1a isolates, HCV strains H and 3201 (GenBank accession number AM489456), and two genotype 1b isolates, HCV strains BK and 3111 (GenBank accession number AM489454), are aligned with the residue numbers indicated at the top. The four residues involved in telaprevir resistance are shown in boldface, and the catalytic triad residues of the HCV NS3 serine protease are underlined. The extra residues from the expression vector, a Met residue at the N terminus and an IEGRIHHHHHH sequence at the C terminus, are not shown.

FIG. 2.

(A) Superimposition of the X-ray structures of the wild-type and the V36M variant NS3 protease domains in a complex with the NS4A cofactor. The Cα atom traces of both the wild-type (in purple) and the V36M variant (in blue) proteases are shown as lines. Residue 36 is highlighted with a stick model (Val³⁶ in green and Met³⁶ in yellow). (B and C) Close-up view of the side chains of residue 36 and the other key residues in the wild-type NS3-4A protease (B) and the V36M variant protease (C). The catalytic triad (His⁵⁷, Asp⁸¹, and Ser¹³⁹) is shown in red. Residue 36 is highlighted either in green (Val³⁶ of the wild type) or in yellow (Met³⁶ of the V36M variant). Other key NS3 protease residues (Phe⁴³, Ile⁶⁴, and Trp⁸⁵) are shown in blue, and Ile²⁵ of the NS4A cofactor is shown in cyan. (D) Close-up view of the H bond between residues Thr⁵⁴ and Leu⁴⁴ in the wild-type NS3-4A protease. Thr⁵⁴ of the C1 β strand (in cyan) forms an H bond (shown as a dashed line) with the main-chain carbonyl of Leu⁴⁴ of the B1 β strand (in green). The locations of Phe⁴³ (in green) and Val³⁶ (in orange) are also shown. Nitrogen atoms are colored in blue, and oxygen atoms are colored in red.