Compromising the achievement of a SVR and strongly increasing the risk of drugresistance development. The first PIs, have been developed on the basis of HCV-1 NS3protease structure and indeed showed reduced efficacy in clinical trials including other HCV-genotypes. For instance, the first PI BILN-2061 was found to be substantially less effective in individuals infected with HCV-2-3. Telaprevir also showed potent activity against HCV-1, less efficacy against HCV-2, and almost no efficacy against HCV-3-4-5 genotypes in vitro and in vivo. Similarly, recent in vitro results showed marked differences in susceptibility of different genotypes also to macrocyclic inhibitors, such as danoprevir, vaniprevir and TMC435. On the contrary, within 
a small pilot study, boceprevir monotherapy recently resulted in a 1.37 and 1.7 log HCV-RNA Axitinib 319460-85-0 reduction in HCV-2 and HCV-3 infected patients respectively, a decrease similar to that observed in HCV-1 subjects receiving the same monotherapy dose. Boceprevir also showed similar efficacy when tested in vitro against several isolates from HCV genotypes 2a, 3a, 5a, 6a, with less pronounced changes against HCV-3 than telaprevir or other macrocyclic PIs. Differences were also observed at the level of HCV-subtypes. Indeed, GSK2118436 during clinical trials, selection of resistant variants to firstgeneration PIs and viral breakthrough were observed consistently more frequently in patients infected with HCV-1a than HCV-1b, and drug-resistant-variants emerged at frequencies of 5 to 20% of the total virus population as early as the second day after the beginning of treatment when either boceprevir or telaprevir were used as monotherapy. Fourteen positions have been previously reported as involved in the development of major and minor PI-drug resistance mutations to either linear, macrocyclic or both classes of PIs. While for HCV-1a and HCV-1b the different antiviral activity, viral-breakthrough and selection of resistant-variants to telaprevir, boceprevir or danoprevir have been associated with nucleotide-variability at position 155, the reason of a lower efficacy of PIs in HCV-2-3-4 is still largely unknown. Considering these data, it is indeed conceivable that the genetic variability among HCV genotypes would have a great importance in HCV sensitivity to PIs, determining drug efficacy and even a different rate of selection of pre-existing resistant HCV variants. However, the characterization of HCV genetic variability at NS3 positions critical for PIs drug-resistance is still missing, especially in non-1 HCV genotypes. Therefore, the aim of this study was to define, at either nucleotide or amino acid level, the HCV-NS3 genetic variability, among all different HCV-genotypes and subtypes commonly spread worldwide, focusing attention on codons associated with development of resistance to either first and second generations PIs. Notably, among all HCV-genotypes, the more difficult-to-treat HCV-3 presented several polymorphisms at positions close to the PI-binding site, which probably might be related to the low antiviral efficacy of several PIs observed in vivo and in vitro against this genotype. In particular, different wild-type amino acids at positions 123 and 168 resulted in non-conservative changes of charge. In cocrystalized structures of PIs and HCV-1 NS3-protease, the negatively charged D168 forms strong salt bridges with positively-charged residues R123 and R155. It has been proposed that mutations at either positions 155 or 168 could disrupt this salt bridge and affect the interaction with PIs, potentially leading to drug-resistance.