Figure 1.
Amino acid sequence alignment of HCV genotypes 1–6 NS3 protease.
The secondary structural elements identified from the HCV-1 NS3 crystal structures are reported at the bottom of the sequences. The consensus sequence of HCV-1b NS3-protease is shown as a reference, colored according to the frequency rate of mutations observed in 1568 HCV-sequences. HCV sequences reported are the consensus sequences obtained from the datasets included in the analysis. Secondary structural elements are indicated as reported in ViralZone (http://viralzone.expasy.org/). Catalytic residues (H57-D81-S139) are underlined. Identical amino acids among all HCV genotypes are indicated with dots “•”.
Table 1.
Boceprevir interacting residues in the experimental HCV-1a NS3 protease-boceprevir complex model (PDB 2OC8).
Figure 2.
Conservation of HCV NS3 protease from PI-naïve HCV infected patients.
Panel (A) reports the molecular surface structure of HCV-1b NS3-protease, colored according to the frequency rate of mutations observed in all 1568 HCV-sequences. The catalytic-triad and residues located inside and in proximity of the hydrophobic-core of the NS3-protease are reported. Panels (B) and (C) show a co-crystalized boceprevir-HCV-1a protease structure, colored according to the amino acid conservation observed in all HCV-sequences.
Figure 3.
Predicted RNA secondary structure of HCV NS3 serine protease sequence.
The RNA secondary structure of HCV-1b genome was predicted by the Mfold program. The viral genome sequence modelled, was an isolate phylogenetically classified as HCV 1b (accession number: AJ000009). A very stable stem-loop, including codons for amino acid positions 145 to 163 (corresponding at NS3 Protease coding region), was reported into box.
Figure 4.
Predicted RNA secondary structure of HCV NS3 serine protease mutated at position 156.
The RNA structures and the relative free energy values were individually predicted by using Mfold software. The NS3-fragment extrapolated from viral genome modelled was considered, specifically analyzing the resistance mutations at position 156: A156S (A), A156T (B), A156V (C) and A156G (D). Codons for amino acid at position 156 and for those base-paired with position 156 are reported into panels.
Figure 5.
Calculated genetic-barrier for resistance-mutations.
Mutations reported in panel (A) are those for which the calculated genetic-barrier was not affected by inter-genotype variability. Histograms in panel below represent the calculated genetic-barrier score for RAMs at positions 155 (B), 36 (C), and 80 (D). The score was calculated by summing the number of transitions (score = 1) and transversions (score = 2.5) required for the generation of any degenerated codon associated with drug-resistance, starting from the predominant wild-type codon found in each HCV-genotype.
Table 2.
Codon variability at HCV NS3 positions associated with major drug resistance to PIs and its impact on the genetic barrier to drug resistance development in HCV-genotypes 1–6.
Table 3.
Codon variability at HCV NS3 positions associated with minor drug resistance to PIs and its impact on the genetic barrier to drug resistance development in HCV-genotypes 1–6.