Running title : HCV genotype 2-6 protease inhibitor resistance 7 8

44 Various protease inhibitors (PIs) are currently becoming available for treatment of hepatitis C virus 45 (HCV). For genotype 1, substitutions at NS3 protease positions 155, 156, and 168 are main 46 determinants of PI resistance. For other genotypes, similar substitutions were selected during PI 47 treatment but were not characterized systematically. To elucidate the impact of key PI resistance 48 substitutions on genotypes 2-6, we engineered the substitutions R155A/E/G/H/K/Q/T, A156G/S/T/V, 49 D/Q168A/E/G/H/N/V into HCV recombinants expressing genotype 2-6 proteases. We evaluated viral 50 fitness and sensitivity to nine PIs (telaprevir, boceprevir, simeprevir, asunaprevir, vaniprevir, 51 faldaprevir, paritaprevir, deldeprevir, and grazoprevir) in Huh7.5 cells. We found that most variants 52 showed decreased fitness compared to original viruses. Overall, R155K-, A156G/S-, and 53 D/Q168A/E/H/N/V-variants showed highest fitness; however, genotype 4 168-variants showed strong 54 fitness impairment. Most variants tested were resistant to several PIs. Resistance levels varied 55 significantly, depending on the specific substitution, genotype, and PI. For telaprevir and boceprevir, 56 specific 155-, 156-, but not 168-variants proved resistant. For the remaining PIs, most genotype 2-, 4-, 57 5-, and 6-, but not genotype 3-variants, showed varying resistance levels. Overall, grazoprevir (MK58 5172) had the highest efficacy against original viruses and variants. 59 This is the first comprehensive study revealing the impact of described key PI resistance substitutions 60 on fitness and PI resistance of HCV genotypes 2-6. In conclusion, the studied substitutions induced 61 resistance to a panel of clinically relevant PIs, including newer PIs paritaprevir, deldeprevir, and 62 grazoprevir. We discovered complex patterns of resistance, with the impact of substitutions varying 63 from increased sensitivity to high resistance. 64