Substitutions at Hepatitis C Virus Genotypes 2-6 NS3 Residues 155, 156, or 168 Induce Complex Patterns of Protease Inhibitor Resistance

Various protease inhibitors (PIs) are currently becoming available for treatment of hepatitis C virus (HCV). For genotype 1, substitutions at NS3 protease positions 155, 156, and 168 are main determinants of PI resistance. For other genotypes, similar substitutions were selected during PI treatment but were not characterized systematically. To elucidate the impact of key PI resistance substitutions on genotypes 2-6, we engineered the substitutions R155A/E/G/H/K/Q/T, A156G/S/T/V, D/Q168A/E/G/H/N/V into HCV recombinants expressing genotype 2-6 proteases. We evaluated viral fitness and sensitivity to nine PIs (telaprevir, boceprevir, simeprevir, asunaprevir, vaniprevir, faldaprevir, paritaprevir, deldeprevir, and grazoprevir) in Huh7.5 cells. We found that most variants showed decreased fitness compared to original viruses. Overall, R155K-, A156G/S-, and D/Q168A/E/H/N/V-variants showed highest fitness; however, genotype 4 168-variants showed strong fitness impairment. Most variants tested were resistant to several PIs. Resistance levels varied significantly, depending on the specific substitution, genotype, and PI. For telaprevir and boceprevir, specific 155-, 156-, but not 168-variants proved resistant. For the remaining PIs, most genotype 2-, 4-, 5-, and 6-, but not genotype 3-variants, showed varying resistance levels. Overall, grazoprevir (MK-5172) had the highest efficacy against original viruses and variants.This is the first comprehensive study revealing the impact of described key PI resistance substitutions on fitness and PI resistance of HCV genotypes 2-6. In conclusion, the studied substitutions induced resistance to a panel of clinically relevant PIs, including newer PIs paritaprevir, deldeprevir, and grazoprevir. We discovered complex patterns of resistance, with the impact of substitutions varying from increased sensitivity to high resistance.