Tracking the evolution of multiple in vitro hepatitis C virus replicon variants under protease inhibitor selection pressure by 454 deep sequencing

Abstract

Resistance to hepatitis C virus (HCV) inhibitors targeting viral enzymes has been observed in in vitro replicon studies and during clinical trials. The factors determining the emergence of resistance and the changes in the viral quasispecies population under selective pressure are not fully understood. To assess the dynamics of variants emerging in vitro under various selective pressures with TMC380765, a potent macrocyclic HCV NS3/4A protease inhibitor, HCV genotype 1b replicon-containing cells were cultured in the presence of a low, high, or stepwise-increasing TMC380765 concentration(s). HCV replicon RNA from representative samples thus obtained was analyzed using (i) population, (ii) clonal, and (iii) 454 deep sequencing technologies. Depending on the concentration of TMC380765, distinct mutational patterns emerged. In particular, culturing with low concentrations resulted in the selection of low-level resistance mutations (F43S and A156G), whereas high concentrations resulted in the selection of high-level resistance mutations (A156V, D168V, and D168A). Clonal and 454 deep sequencing analysis of the replicon RNA allowed the identification of low-frequency preexisting mutations possibly contributing to the mutational pattern that emerged. Stepwise-increasing TMC380765 concentrations resulted in the emergence and disappearance of multiple replicon variants in response to the changing selection pressure. Moreover, two different codons for the wild-type amino acids were observed at certain NS3 positions within one population of replicons, which may contribute to the emerging mutational patterns. Deep sequencing technologies enabled the study of minority variants present in the HCV quasispecies population present at baseline and during antiviral drug pressure, giving new insights into the dynamics of resistance acquisition by HCV.

FIG. 1.

Structural formulae of TMC380765.

FIG. 2.

Summary overview of mutations at NS3 protease positions 43, 156, and 168 detected by population sequencing during selection experiments using 100 nM, 1,000 nM, and stepwise-increasing (0 to 1,000 nM) concentrations of TMC380765. It is important to note that not every individual mutation was observed in each individual experiment and that the majority were detected as mixtures of mutant and wild-type amino acids. Each individual selection protocol was performed at least twice.

FIG. 3.

Codon frequencies at six key amino NS3 acid positions (41, 43, 80, 155, 156, and 168; indicated by 1 to 6, respectively) of selection experiment with stepwise-increasing TMC380765 concentrations (A and B) or in the absence of NS3/4A inhibitor (C), as determined by 454 GS-FLX shotgun (A) or 454 GS-FLX amplicon (B and C) deep sequencing. Frequencies of individual amino acids were calculated as the number of sequence reads per specific codon divided by the total number of reads at that codon position. Dotted lines connect points where no intermittent data points were available. Alternative codon usage is indicated by an apostrophe ('). Based on 454 deep sequencing results of replicon control plasmid (data not shown), the limit of detection was set at 0.4%; therefore, single-codon frequencies below 0.4% were excluded from this analysis. However, percentages below 0.4% were considered significant if mutants were present in previous or subsequent samples.

FIG. 4.

Codon usage of amino acids detected by 454 deep sequencing during selection experiments at NS3 protease position D168. Two wild-type codons, GAC and GAT, coding for aspartate (D), are shown in the middle box. Boxes on the left and right show mutations detected that are one nucleotide change away from wild-type codons GAC and GAT (nucleotide changes resulting in amino acid changes are underlined).

FIG. 5.

Change in TMC380765 susceptibility (EC₅₀) and replication capacity (RC) values determined using a transient replicon assay with replicons harboring single or double mutations in the NS3 protease domain. Fold change in EC₅₀ = EC₅₀ mutant/EC₅₀ clone ET measured at 48 h. RC = (Luc48h_mut/Luc48h_ET)/(Luc4h_mut/Luc4h_ET), where Luc48h_mut or -ET and Luc4h_mut and -ET are luciferase levels of the mutant or the ET clone at 48 h and 4 h postelectroporation, respectively. Inverted triangles, diamonds, and upright triangles indicate amino acid changes detected primarily during low, middle-range, and high TMC380765 concentration selection, respectively. Double mutants are indicated by full circles.

FIG. 6.

Combined amino acid frequencies determined by clonal sequencing for mutations Q41R, F43S, Q80R, and A156G (A) (mutations with change in EC₅₀s < 15-fold) or A156V and D168V (B) (mutations with change in EC₅₀s > 500-fold) in experiment B. For every group, the frequency of genomes/clones harboring single or multiple mutations is shown.

FIG. 7.

Amino acid frequencies at a TMC380765 concentration of 20 nM (A), 560 nM (B), or 880 nM (C) detected by clonal sequencing. “wt” indicates wild-type sequences carrying no mutations at any of the six key NS3 protease amino acid positions (41, 43, 80, 155, 156, and 168). Sequences carrying single mutations are marked in gray. Sequences carrying multiple mutations are highlighted in color.