Comparison of daclatasvir resistance barriers on NS5A from hepatitis C virus genotypes 1 to 6: implications for cross-genotype activity

Abstract

A comparison of the daclatasvir (DCV [BMS-790052]) resistance barrier on authentic or hybrid replicons containing NS5A from hepatitis C virus (HCV) genotypes 1 to 6 (GT-1 to -6) was completed using a replicon elimination assay. The data indicated that genotype 1b (GT-1b) has the highest relative resistance barrier and genotype 2a (GT-2a M31) has the lowest. The rank order of resistance barriers to DCV was 1b>4a≥5a>6a≅1a>2a JFH>3a>2a M31. Importantly, DCV in combination with a protease inhibitor (PI) eliminated GT-2a M31 replicon RNA at a clinically relevant concentration. Previously, we reported the antiviral activity and resistance profiles of DCV on HCV genotypes 1 to 4 evaluated in the replicon system. Here, we report the antiviral activity and resistance profiles of DCV against hybrid replicons with NS5A sequences derived from HCV GT-5a and GT-6a clinical isolates. DCV was effective against both GT-5a and -6a hybrid replicon cell lines (50% effective concentrations [EC50s] ranging from 3 to 7 pM for GT-5a, and 74 pM for GT-6a). Resistance selection identified amino acid substitutions in the N-terminal domain of NS5A. For GT-5a, L31F and L31V, alone or in combination with K56R, were the major resistance variants (EC50s ranging from 2 to 40 nM). In GT-6a, Q24H, L31M, P32L/S, and T58A/S were identified as resistance variants (EC50s ranging from 2 to 250 nM). The in vitro data suggest that DCV has the potential to be an effective agent for HCV genotypes 1 to 6 when used in combination therapy.

FIG 1

(A) Schematic diagram of a bicistronic JFH1 replicon with Renilla luciferase (Rluc) and neomycin resistance (neo^R) genes. The 5′ untranslated region (5′-UTR) is derived from the GT-1b Con1 strain, and the nonstructural genes and 3′-UTR are derived from the GT-2a JFH1 strain. Hybrid replicons were constructed by replacing the indicated BspEI restriction fragment with sequences from GT-5a-6, GT-5a-7, GT-5a-9, and GT-6a-16 clinical isolates or the pJ6CF infectious clone (GT-2a M31). (B) Alignment of the N-terminal 100 amino acids of the NS5A from GT-5a-6, GT-5a-7, and GT-5a-9 isolates and two GT-5a NS5A reference sequences (SA13, GenBank accession no. AF064490; EUH1480, GenBank accession no. Y13184). (C) Alignment of the N-terminal 100 amino acids of the NS5A from the GT-6a-16 isolate and two GT-6a NS5A reference sequences (EUHK2, GenBank accession no. Y12083; HK6a, GenBank accession no. HQ852465). (D) Alignment of the N-terminal 100 amino acids of the NS5A from the JFH1 strain and the pJ6CF (GT-2a M31) infectious clone. Amino acid identities are indicated with dots.

FIG 2

HCV replicon elimination with DCV. Huh-7 cells harboring GT-1 to -6 replicons were treated with the indicated concentrations of DCV for 14 days in the absence of G418. Cell cultures were maintained at subconfluency by splitting as described in Materials and Methods. After 14 days, DCV was removed, and a portion of the cell cultures were treated with medium containing G418 (500 μg/ml) for 2 weeks to allow cells that harbored the HCV replicon to form colonies. The colonies that formed were photographed. For GT-2a JFH, GT-2a M31, and GT-3a replicon cells, parallel cultures of cells were amplified for genotypic analysis. The replicon cell lines used in this study are as follows: 1b, Con1, and 1a, H77c (2, 13); 2a JFH, JFH-1 (AB047639); 2a M31 NS5A (aa 1 to 425), 2a infectious clone-pJ6CF (NC_009823.1); 3a NS5A (aa 1 to 429), HCV3a1 (JX944789); 4a NS5A, HCV4a-23 (JQ347515); 5a NS5A (aa 1 to 430), GT-5a-7 (KJ719453); and 6a NS5A (aa 1 to 431), GT-6a-16 (KJ719455).

FIG 3

GT-2a M31 replicon cells were treated for 3 or 7 days with the indicated concentration of a PI, MK-5172, in the absence or presence of 250 nM DCV and without G418. After either 3 or 7 days of treatment (D3 and D7, respectively), inhibitors were removed and cells were split and maintained in medium containing G418 (500 μg/ml) for 2 weeks to allow replicon-retaining cells to form colonies.