With the polar uncharged Q168, and the replacement of R123 with the polar T123 can thus abrogate these key structural salt bridges, potentially altering the active site conformation of NS3 protease, and in turn impact the HCV-3 sensitivity to PIs. Furthermore, HCV-3, together with HCV-2-4-5 genotypes, also presented two minor RAMs as natural polymorphisms, known to confer low-level resistance to boceprevir and/or telaprevir in vitro. Interestingly, both residues 36 and 175 are located near the protease catalytic domain of HCV NS3, but not close to the boceprevir and telaprevir binding sites in their respective complexes with HCV NS3-NS4 protease. Probably, even if mutations at position 36 and 175 should not be directly involved in resistance to PIs, they can influence the viral replication capacity. For instance, viruses with mutations V36A/ L/M demonstrated a 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 pan-genotypic activity of the second generation macrocyclic PI MK-5172, even against HCV-3 MLN4924 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 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 NS3protease positions, including some associated with resistance, Rapamycin cost 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. On the other hand, according to our GBPM structural analysis, highly conserved 
NS3-protease positions among all HCV genotypes were those pivotal for enzyme functionality and stability, such as the catalytic-triad, the oxyanion hole at G137 and the residues involved in Zn2+ binding, and also comprised the majority of residues essential for boceprevir-binding. Interestingly, we also observed two highly conserved stretches encompassing NS3 positions 135-142 and 154-159 that could assist in the rational design of new HCV inhibitors with more favourable resistance profiles. A correlation among conserved NS3 amino acid residues and base-paired organization on the putative RNA secondary structure was also observed. Indeed, highly conserved positions at both amino acid and nucleotide levels were located in highly stable RNA paired stems. Probably, the requirement for base-pairing in these structures severely limits the number of “neutral” sites in the genome, constraining neutral HCV drift, since even synonymous mutations could potentially affect and disrupt the RNA-folding. Interestingly, in our predicted RNA structure model, the conserved codon for resistance-associated residue A156 was base-paired with the conserved codon for residue I153. The presence of RAMs at this position, associated to resistance to all linear and some macrocyclic PIs, did not perturb the overall RNA structural conformation.