Figure 5. Calculated genetic-barrier for resistance-mutations. Mutations reported in panel (A) are those for which the calculated geneticbarrier 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. doi:10.1371/journal.pone.0039652.g005
compensate impaired viral fitness of R155K mutation) had a reduced genetic barrier in HCV-1a (score = 1), but also in HCV-24 (score = 2.5), in comparison to HCV-1b (score = 3.5), HCV-3 (score = 5), and HCV-5-6 (score = 3.5). A large nucleotide and amino acid variability among HCVgenotypes was also found at position 170, where HCV-1a-2-3-5 showed an Isoleucine (I) as a predominant wild-type amino acid instead of the Valine (V) predominantly found in HCV-1b-4-6. As a consequence, HCV-1a-2-3-5 could preferably develop the major boceprevir RAM 170T (with genetic barrier score = 1), while HCV-1b-4-6 could favor the development of 170A (also in this case, score = 1) (Table 2). Also the two positions associated with major resistance to macrocyclic PI (80 and 168) were highly variable among genotypes (Fig. 1 and Fig. 5, panel D). In particular, at position 80, genotypes HCV-1a-1b-3-4-6 showed a Q as wild-type amino acid, and consequently the development of major Q80K and Q80R RAMs will potentially require just a nucleotide substitution (score = 2.5 and 1, respectively). On the contrary, HCV-2 harbored the minor RAM 80G as wild-type, and presented a calculated genetic barrier score of 1 for the development of 80R and of 2 for the development of 80K (two transitions). Lastly HCV-5 already had the major 80K as wild-type, and had a genetic barrier for major 80R development scored as 1. As already mentioned before, near all HCV-3 sequences analyzed showed Q168 as wild-type amino acid, instead of D168 (Fig. 1 and Table 2). As a consequence, HCV-3 showed an increased genetic barrier for the major variant 168V (score = 5 for HCV-3 versus 2.5 for all other HCV-genotypes), 168T (score = 5 versus 3.5), and 168A (score = 5 versus 2.5) and the minor variant 168I (score = 6 versus 3.5) and 168Y (score = 3.5 versus 2.5), 168N (score = 5 versus 1). Taken together, these results indicate that the high level of variability in codon usage among HCV-genotypes can favor genotype-specific pathways for resistance-mutations development. This can result in different responsiveness of HCV-genotypes to PIs and very rapid selection for specific resistance patterns for both linear and macrocyclic PIs.
Discussion
Analyzing more than 1500 HCV NS3-protease sequences, a high degree of genetic variability among all HCV-genotypes was ?found in PI-naive HCV-infected patients, with only 85/181 (47.0%) conserved amino acids. This genetic heterogeneity among genotypes translated into significant molecular and structural differences, making HCV-genotypes, and even subtypes, differently sensitive to PIs treatment and differently prone to the development of PI resistance-mutations, for both linear and macrocyclic compounds. Indeed, the linear PI telaprevir showed less efficacy against HCV-2, and almost no efficacy against HCV3-4-5 genotypes in vitro and in vivo [10,24?6], and similar results were also obtained for macrocyclic inhibitors, such as danoprevir, vaniprevir and TMC435 [10,24,26]. As a first consequence of HCV sequence heterogeneity, we observed that four resistance-mutations (80K/G and 36L-175L) were already present, as natural polymorphisms, in selected genotypes. In particular, the major RAM 80K (for macrocyclic compounds TMC435 and Asunaprevir) was detected in 41.6% of