HCV genotypes are differently prone to the development of resistance to linear and macrocyclic protease inhibitors

Abstract

Background: Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed whether specific HCV-genotypes are differently prone to develop resistance to linear and macrocyclic protease-inhibitors (PIs).

Methods: The study includes 1568 NS3-protease sequences, isolated from PI-naive patients infected with HCV-genotypes 1a (N = 621), 1b (N = 474), 2 (N = 72), 3 (N = 268), 4 (N = 54) 5 (N = 6), and 6 (N = 73). Genetic-barrier was calculated as the sum of nucleotide-transitions (score = 1) and/or nucleotide-transversions (score = 2.5) required for drug-resistance-mutations emergence. Forty-three mutations associated with PIs-resistance were analyzed (36A/M/L/G-41R-43S/V-54A/S/V-55A-Q80K/R/L/H/G-109K-138T-155K/Q/T/I/M/S/G/L-156T/V/G/S-158I-168A/H/T/V/E/I/G/N/Y-170A/T-175L). Structural analyses on NS3-protease and on putative RNA-models have been also performed.

Results: Overall, NS3-protease was moderately conserved, with 85/181 (47.0%) amino-acids showing <1% variability. The catalytic-triad (H57-D81-S139) and 6/13 resistance-associated positions (Q41-F43-R109-R155-A156-V158) were fully conserved (variability <1%). Structural-analysis highlighted that most of the NS3-residues involved in drug-stabilization were highly conserved, while 7 PI-resistance residues, together with selected residues located in proximity of the PI-binding pocket, were highly variable among HCV-genotypes. Four resistance-mutations (80K/G-36L-175L) were found as natural polymorphisms in selected genotypes (80K present in 41.6% HCV-1a, 100% of HCV-5 and 20.6% HCV-6; 80G present in 94.4% HCV-2; 36L present in 100% HCV-3-5 and >94% HCV-2-4; 175L present in 100% HCV-1a-3-5 and >97% HCV-2-4). Furthermore, HCV-3 specifically showed non-conservative polymorphisms (R123T-D168Q) at two drug-interacting positions. Regardless of HCV-genotype, 13 PIs resistance-mutations were associated with low genetic-barrier, requiring only 1 nucleotide-substitution (41R-43S/V-54A-55A-80R-156V/T: score = 1; 54S-138T-156S/G-168E/H: score = 2.5). By contrast, by using HCV-1b as reference genotype, nucleotide-heterogeneity led to a lower genetic-barrier for the development of some drug-resistance-mutations in HCV-1a (36M-155G/I/K/M/S/T-170T), HCV-2 (36M-80K-155G/I/K/S/T-170T), HCV-3 (155G/I/K/M/S/T-170T), HCV-4-6 (155I/S/L), and HCV-5 (80G-155G/I/K/M/S/T).

Conclusions: The high degree of HCV genetic variability makes HCV-genotypes, and even subtypes, differently prone to the development of PIs resistance-mutations. Overall, this can account for different responsiveness of HCV-genotypes to PIs, with important clinical implications in tailoring individualized and appropriate regimens.

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 “•”.

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.