comparable fitness to wild type reference virus. However, since no crystallized structures are to date available for non-1 HCV proteases, the overall impact of such polymorphisms on the three-dimensional protein structure will need further investigations. It is important to mention that very recent data demonstrated a AMG-337 pan-genotypic activity of the second generation macrocyclic PI MK-5172, even against HCV-3 genotype. Furthermore, MK-5172 retained activity also against HCV-1 viral strains harbouring key first generation PI RAMs, thus providing a great opportunity for patients infected with all different HCV-genotypes, including those without virological response to previous regimens. Beside HCV-3, also other genotypes showed remarkable sequence differences from HCV-1b. Of particular interest were those genotype-specific amino acid 315706-13-9 biological activity variations affecting residues associated to macrocyclic and linear PIs-resistance or located in proximity of the PI-binding pocket. For instance, HCV-1a and HCV-1b consensus sequences showed different wild-type amino acids at 17/181 NS3- protease positions, including some associated with resistance, enhanced replication or compensatory effects if mutated. This amino acidic variability may potentially facilitate viral breakthrough and selection of specific resistant variants, that have been indeed observed consistently more frequently in patients infected with HCV-1a than HCV-1b, using both linear and macrocyclic PIs. All together, these results help explaining experimental and clinical observations, indicating that mutations appearing rapidly and frequently in PI-treated patients are actually those with a lower genetic barrier in the specific genotype/subtype considered. Indeed, in both telaprevir and/or boceprevir failing patients, the most common resistance mutations detected in HCV-1a infected patients were V36M, T54S, and R155K, whereas mutations T54A/S, V55A, A156S, and V170A were specifically developed in HCV-1b patients. Furthermore, classically the genetic barrier calculation is performed referring to the most prevalent wild-type codon found in each genotype. Nevertheless, as it appears clearly from Table 2 and Table 3, the variability of codon usage exists at high level even within the single genotypes. For instance, we found
