Hepatitis C Virus Genetic Variability and the Presence of NS5B Resistance-Associated Mutations as Natural Polymorphisms in Selected Genotypes Could Affect the Response to NS5B Inhibitors

V C Di Maio; V Cento; C Mirabelli; A Artese; G Costa; S Alcaro; C F Perno; F Ceccherini-Silberstein

doi:10.1128/AAC.02386-13

. 2014 May;58(5):2781–2797. doi: 10.1128/AAC.02386-13

Hepatitis C Virus Genetic Variability and the Presence of NS5B Resistance-Associated Mutations as Natural Polymorphisms in Selected Genotypes Could Affect the Response to NS5B Inhibitors

V C Di Maio ^a, V Cento ^a, C Mirabelli ^a,^*, A Artese ^b, G Costa ^b, S Alcaro ^b, C F Perno ^a,^c, F Ceccherini-Silberstein ^a,^✉

PMCID: PMC3993261 PMID: 24590484

Abstract

Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed the NS5B polymerase genetic variability in circulating HCV genotypes/subtypes and its impact on the genetic barrier for the development of resistance to clinically relevant nucleoside inhibitors (NIs)/nonnucleoside inhibitors (NNIs). The study included 1,145 NS5B polymerase sequences retrieved from the Los Alamos HCV database and GenBank. The genetic barrier was calculated for drug resistance emergence. Prevalence and genetic barrier were calculated for 1 major NI and 32 NNI resistance variants (13 major and 19 minor) at 21 total NS5B positions. Docking calculations were used to analyze sofosbuvir affinity toward the diverse HCV genotypes. Overall, NS5B polymerase was moderately conserved among all HCV genotypes, with 313/591 amino acid residues (53.0%) showing ≤1% variability and 83/591 residues (14.0%) showing high variability (≥25.1%). Nine NNI resistance variants (2 major variants, 414L and 423I; 7 minor variants, 316N, 421V, 445F, 482L, 494A, 499A, and 556G) were found as natural polymorphisms in selected genotypes. In particular, 414L and 423I were found in HCV genotype 4 (HCV-4) (n = 14/38, 36.8%) and in all HCV-5 sequences (n = 17, 100%), respectively. Regardless of HCV genotype, the 282T major NI resistance variant and 10 major NNI resistance variants (316Y, 414L, 423I/T/V, 448H, 486V, 495L, 554D, and 559G) always required a single nucleotide substitution to be generated. Conversely, the other 3 major NNI resistance variants (414T, 419S, and 422K) were associated with a different genetic barrier score development among the six HCV genotypes. Sofosbuvir docking analysis highlighted a better ligand affinity toward HCV-2 than toward HCV-3, in agreement with the experimental observations. The genetic variability among HCV genotypes, particularly with the presence of polymorphisms at NNI resistance positions, could affect their responsiveness to NS5B inhibitors. A pretherapy HCV NS5B sequencing could help to provide patients with the full efficacy of NNI-containing regimens.

INTRODUCTION

The advent of direct antiviral agents (DAAs) has opened a new era for the treatment of hepatitis C virus (HCV) infection. Since 2011, telaprevir and boceprevir, two linear protease inhibitors, have been approved by both the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) as a new standard of care for first-line treatment of HCV genotype 1 (HCV-1), in association with pegylated alpha interferon (PEG-IFN) and ribavirin. Both of these combination treatments have shown improved sustained virological response (SVR) rates along with reduced treatment duration (1 –4). Despite the increase in SVR and their high potency, these two first approved protease inhibitors are still “fragile” drugs because of their low genetic barrier, extensive cross-resistance profile, and significant side effects. Therefore, additional DAAs that target different HCV proteins as well are currently in development (5 –8) (http://www.pipelinereport.org; http://www.natap.org; http://hcvadvocate.blogspot.ca). Among these new drugs, several are designed to target the HCV enzyme NS5B, which is the viral RNA-dependent RNA polymerase (RpRd), a key component of the HCV life cycle (9, 10). The success of polymerase inhibitors has been demonstrated extensively in phase I and II trials, and they are expected to play an important role in newer DAA combination therapy regimens (11). Currently, two classes of NS5B inhibitors, nucleos(t)ide inhibitors (NIs) and nonnucleoside inhibitors (NNIs), are at different stages of clinical development (5, 12). For instance, the NI sofosbuvir (GS-7977) has been recently approved by the FDA for the treatment of chronic HCV infection as a component of a combination antiviral treatment regimen (http://www.fda.gov/forconsumers/byaudience/forpatientadvocates/ucm377920.htm). Moreover, the NI mericitabine (R7128) and two NNIs, ABT-333 (NNI class 3 [NNI-3]) and deleobuvir (BI207127, an NNI-1), are already in phase III studies. One NI (VX-135) and several other NNIs are in phase II trials, such as two NNI-1s (TMC647055 and BMS-791325), two NNI-2s (lomibuvir [VX-222] and GS-9669), two NNI-3s (setrobuvir and ABT-072), and one NNI-4 (IDX375). Meanwhile, two NNIs, filibuvir and tegobuvir, previously in phase II trials, have been very recently discontinued (5, 8, 13). In particular, tegobuvir has been discontinued owing to limited efficacy, while discontinuation of filibuvir followed a Pfizer strategic review (5; http://www.hepatitiscentral.com/mt/archives/2013/03/new-strategy-shifts-pfizer-away-from-new-hep-c-drug.html).

NIs mime the natural substrates of the RNA-dependent RNA polymerase and are incorporated within the elongation of RNA, where they act as chain terminators. NIs generally show a high-intermediate potency, a pan-genotypic activity, and a medium-high barrier to resistance (5, 14). In contrast, NNIs are a heterogeneous group of antiviral compounds with intermediate potency, which bind to one of the 4 allosteric sites at the surface of the enzyme, inhibiting the formation of the elongation complex. NNIs show a low genetic barrier to resistance and a genotype-dependent activity (5, 14 –16). Furthermore, since NNIs bind distantly to the active site, resistant variants may occur easily, with high fitness, and more frequently than under NI treatments both in vitro and in vivo (12).

The poor fidelity of the HCV NS5B enzyme, as a result of its error-prone and absent proofreading activity, together with the high replication rate and strong selective pressure on the virus, leads to an extreme variability of HCV. Seven HCV genotypes (1 to 7, with 31 to 33% nucleotide difference) and more than 100 subtypes (a, b, and so on, with 20 to 25% nucleotide difference) have been so far characterized with distinct geographic distributions (17, 18). This high genetic variability represents an important obstacle in developing pan-genotypic antivirals and also is the cause of a potential baseline presence of drug-resistant variants as major or minor populations in HCV-infected persons. Because of the intrinsic fitness cost of escape mutants, patients with DAA-resistant predominant viral quasispecies have been rarely observed (19 –24). Recently, some studies analyzed in detail the prevalence of NI/NNI resistance variants among DAA-naive patients but, however, only in selected genotypes (22 –25). Furthermore, one study, starting from 236 HCV sequences among six genotypes, analyzed also the degree of conservation of the entire NS5B polymerase (26). However, a broad characterization of HCV genetic variability at NS5B positions critical for drug resistance to the current important NIs/NNIs is still missing, especially in a large number of genotype 1 and non-genotype 1 HCV genotypes and subtypes commonly spread worldwide. As we have shown previously for protease inhibitors (PIs) (27), it is conceivable that the genetic variability among HCV genotypes would have a great importance in HCV sensitivity also to polymerase inhibitors, determining drug efficacy and even a different tendency to develop NI/NNI resistance. Therefore, considering all these aspects, the aim of this study was to analyze the NS5B polymerase variability among a large number of HCV sequences from all genotypes. We evaluated also the impact of the genetic variability on the genetic barrier (defined as the number and type of nucleotide mutations required to overcome the drug pressure) for the development of substitutions causing drug resistance to NIs/NNIs currently clinically relevant (such as all those under investigation in clinical phase II and III trials).

MATERIALS AND METHODS

Study population.

This study includes 1,145 NS5B polymerase sequences (amino acids [aa] 1 to 591), derived from NI/NNI-naive patients infected with HCV genotypes 1a (n = 506), 1b (n = 379), 2 (n = 113), 3 (n = 14), 4 (n = 38), 5 (n = 17), and 6 (n = 77) and the new genotype 7, recently found in a few patients from Central Africa (n = 1), retrieved from the Los Alamos HCV sequence database and GenBank. To ensure the quality of the data, public sequences were excluded from the analysis if they (i) contained stop codons in the NS5B gene or (ii) contained ambiguities consisting of >2 bases per nucleotide position or >2 ambiguities per codon at individual drug resistance-associated positions.

HCV genotype was confirmed by phylogenetic analysis of the polymerase coding region, aligning all retrieved sequences versus 11 reference strains for the 7 different genotypes (GenBank accession numbers: HCV-1a, NC_004102; HCV-1b, AJ238799; HCV-2a, NC_009823; HCV-2b, D10988; HCV-2c, D50409; HCV-3a, NC_009824; HCV-3b, D49374; HCV-4, NC_009825; HCV-5, NC_009826; HCV-6, NC_009827; HCV-7, EF108306). The NS5B phylogenetic tree was estimated using the MEGA 5.0 package (28) by a neighbor joining approach, using the Kimura 2-parameter model, with a proportion of invariable sites and application of a gamma distribution.

Conservation analysis.

HCV variability at the level of NS5B polymerase protein was assessed by calculating the prevalence of the most common wild-type amino acid at each position of the NS5B gene. For this analysis, the complete alignment of 1,145 sequences from the 7 HCV genotypes was used. The amino acid conservation was defined as the percentage of sites with ≤1% amino acid variability. The information obtained was then used on the tridimensional polymerase protein structure, in order to visualize the localization of conserved and variable residues among HCV genotypes. Structural analysis of the NS5B protein was performed by PyMOL Molecular Graphics System 2002 according to the HCV-1b 2QE5 structure (29).

To analyze the prevalence of resistance variants among all the HCV sequences, a total of 21 positions related to 33 substitutions causing drug resistance to 14 NS5B polymerase inhibitors were analyzed (see Fig. 1). According to the level of resistance, NI/NNI resistance-associated variants have been divided into major (high-intermediate level of resistance) and minor (low level of resistance) variants (7, 30 –46). Only variants with prevalences of >1% and found in ≥2 patients were considered. The HCV-1b reference sequence (GenBank accession number AJ238799) was used for the definition of amino acid substitutions.

FIG 1 — Amino acid substitutions in HCV NS5B polymerase associated with resistance to NIs and NNIs. In the list are shown variants associated with resistance to investigational NIs and NNIs in phase II and III trials (30 –46). The number in the bar represents the amino acid position. The letter above the number refers to the wild-type amino acid (HCV-1b reference sequence, GenBank accession number AJ238799), and the letter(s) below the bars represents the resistance substitutions. Bold type represents the key major substitutions associated with the resistance phenotype (30 –46). For the drug with an asterisk, reported substitutions are associated with resistance only *in vitro*.

Genetic barrier calculation.

The genetic barrier for the evolution of each drug resistance substitution was calculated according to a model previously described for HIV, HBV, or HCV (47, 48). In summary, transitions (A↔G and C↔T) were assigned a score of 1 and transversions (A↔C, A↔T, G↔C, and G↔T) were assigned a score of 2.5, since transitions have been generally shown to occur for steric reasons on average 2.5 times more frequently than transversions (27, 47, 48). An algorithm was built using JavaScript to calculate the genetic barrier at each individual NS5B position associated with resistance. A total of 33 drug resistance substitutions were analyzed (the 1 major NI resistance variant, 282T; 13 major NNI resistance variants, 316Y, 414T/L, 419S, 422K, 423I/T/V, 448H, 486V, 495L, 554D, and 559G; and 19 minor NNI resistance variants, 316N, 368T, 421V, 426A/T, 445F, 448C, 452H, 482L/S, 494A, 495S/A/T/Q, 496S, 499A, 554S, and 556G) (Fig. 1).

The methodology used to estimate the genetic barrier for drug resistance development has been described elsewhere (27, 47). Briefly, due to the degeneration of genetic code, each NS5B amino acid associated with drug resistance can be encoded by more than one nucleotide codon. Therefore, starting from the wild-type codon detected in drug-naive patients, we calculated a numerical score by summing the number of nucleotide transitions and/or transversions required to generate a specific resistance substitution. As a result, we obtained different scores for each pathway of nucleotide mutations required to generate a specific resistance substitution. The minimal genetic barrier score for each drug resistance substitution analyzed was considered.

Docking calculations.

To obtain three-dimensional models for each NS5B genotype, we checked the existence of available experimental structures in the Protein Data Bank (PDB) (49). Since NS5B structures from different HCV genotypes were not available, the Prime program (Prime, version 3.3; Schrödinger, LLC, New York, NY, 2013) has been used to structurally predict 1a, 1b, 2a, 2b, 2c, 2k, 3a, 3b, 3i, and 3k genotype models, using as a template HCV-1a in the 3QGH (50) X-ray structure, with a final sequence identity greater than 90% in all models predicted. The following sequences with the indicated GenBank accession numbers have been used in the analysis: HCV-1a, NC_004102; HCV-1b, AJ238799; HCV-2a, NC_009823; HCV-2b, D10988; HCV-2c, D50409; HCV-2i, DQ155561; HCV-2k, AB031663; HCV-3a, NC_009824; HCV-3b D49374; HCV-3i, FJ407092; HCV-3k D63821. After the alignment procedure, carried out by means of ClustalW (51), we built our models using a knowledge-based method. Finally, an energy minimization procedure to fully relax each model has been performed, using MacroModel v.10.0 (Schrödinger, LLC, New York, NY, 2013) under the following conditions: OPLS_2005 (52) as force field, a Polake-Ribiere conjugated gradient (PRCG) algorithm, 20,000 iterations of minimization, and the GB/SA (53) water implicit model to take into account the solvation effect. The obtained models were adopted for quantum mechanics polarized ligand docking (QPLD) (54) molecular docking studies. For each NS5B structure, a grid box of 27,000 Å³ was centered on the ligand (N-cyclopropyl-6-[(3R)-3-{[4-(trifluoromethoxy)benzyl]carbamoyl}-4-{[4-(trifluoromethoxy)phenyl]sulfonyl}piperazin-1-yl]pyridazine-3-carboxamide) cocrystallized in the 3QGH X-ray model. With adoption of such a grid box, the molecular recognition of sofosbuvir against all the genotype models was carried out using the default parameters with no constraints. The initial docking stage was performed with Glide (Glide, version 5.9; Schrödinger, LLC, New York, NY, 2013) SP precision, generating 10 maximum poses per ligand. In order to test the accuracy and reliability of our docking approach, as reported in some different drug-target complexes (55 –60), we performed redocking simulations of the N-cyclopropyl-6-[(3R)-3-{[4-(trifluoromethoxy)benzyl]carbamoyl}-4-{[4-(trifluoromethoxy)phenyl]sulfonyl}piperazin-1-yl]pyridazine-3-carboxamide cocrystallized in the 3QGH NS5B complex under the same conditions mentioned above, obtaining a root mean square deviation value, calculated on all atoms, equal to 1.26 Å (data not shown).

All the sofosbuvir complexes generated were submitted to the induced-fit energy minimization, with a thermodynamic evaluation of the free energy of complexation under the same conditions of the target optimization procedure. Analysis of the results was carried out taking into account the thermodynamic estimation of the state equations (free energy, enthalpy, and entropy of complex formation) computed at 300 K, according to the MolInE (61) method.

RESULTS

NS5B genetic variability and natural resistance across HCV genotypes.

Overall, the NS5B protein was moderately conserved among all HCV genotypes, with 313/591 (53.0%) conserved amino acid residues. Few NS5B residues (83/591, 14.0%) were highly variable among HCV genotypes, showing ≥25.1% amino acid variability, and were mostly located on the surface of the NS5B protein (Fig. 2 and 3). According to the conventional partition of the NS5B domains (9), the inner palm domain (aa 188 to 227 and aa 287 to 370) was the most conserved region (60.5% domain conservation). The other two domains, fingers (aa 1 to 187 and aa 228 to 286) and thumb (aa 371 to 563), showed more variability than did the palm domain (50.0% and 53.4% conservation, respectively). In particular, the β-loop (residues 443 to 454), within the thumb domain, was highly variable among the different genotypes (41.7% conservation).

FIG 2 — Amino acid sequence alignment of HCV NS5B polymerase across genotypes 1 to 7. The sequence of HCV-1b NS5B polymerase (GenBank accession number AJ238799) is shown as a reference and is colored according to the frequency rate of substitutions observed in analyzing 1,145 HCV sequences of the 7 genotypes. Each HCV genotype reference sequence included in the analysis is also reported (GenBank accession numbers are as follows: HCV-1a, NC_004102; HCV-2a, NC_009823; HCV-3a, NC_009824; HCV-4, NC_009825; HCV-5, NC_009826; HCV-6, NC_009827; HCV-7, EF108306). Amino acids (aa) identical among all HCV genotypes are indicated with dots.

FIG 3 — Conservation of HCV NS5B polymerase sequences across genotypes 1 to 7. The figure reports the molecular surface structure of HCV-1b NS5B polymerase (Protein Data Bank [PDB] accession number 2QE5), colored according to the frequency rate of substitutions observed in all 1,145 HCV sequences. The residues representing the catalytic triad, the GDD motif, and those associated with NIs and NNIs resistance are reported. The residues with asterisks cannot be visualized on the molecular polymerase structure orientation.

In analysis of the reference sequence alignment at the level of the five important motifs (named A to E) within the palm region (62, 63), the A motif (residues 213 to 228), which includes the catalytic pocket of the enzyme (D220 to D225) with two essential residues for metal binding (D220 and T221), was highly conserved among all the seven genotypes. The B motif (residues 282 to 302), taking part in the sugar selection, was also highly conserved within HCV genotype 1, with some variability found at a few positions (285-286, 289, 293, 296-297, and 300) in other, non-HCV-1 genotypes. As expected, the GDD functional motif (G317-D318-D319) within the C motif was conserved among all seven different genotypes. Also, the D motif (residues 326 to 347), forming the core structure of the palm, and the E motif (residues 360 to 370), responsible for the interaction of palm with thumb, were highly conserved at the amino acid level within the HCV-1 genotype, and to a lesser extent within the other genotypes.

In analysis of all the 21 NI/NNI resistance-associated positions, 9 (42.8%) were conserved at the amino acid level (S282, S368, R422, Y448, Y452, P495, P496, G554, and D559) among the genotypes, while the other 12 (57.2%) were highly variable (C316, M414, L419, A421, M423, M426, C445, I482, A486, V494, V499, and S556) (Fig. 2 and 3).

Moreover, 9 NNI resistance variants (2 major, 414L and 423I; 7 minor, 316N, 421V, 445F, 482L, 494A, 499A, and 556G) were found as natural polymorphisms in selected genotypes (Fig. 4A). In particular, the primary setrobuvir resistance substitution 414L was frequently found in HCV-4 (n = 14/38, 36.8% prevalence). Similarly, 423I, reported to confer high resistance to the NNI-2 filibuvir, was found in all HCV-5 sequences (n = 17, 100%), and rarely also in HCV-1a (n = 9/506, 1.8%).

FIG 4 — Prevalence and calculated genetic barrier scores of NS5B resistance-associated variants. The histogram in panel A represents the prevalence of polymorphic resistance variants found in specific HCV genotypes. The relative class of compounds is also indicated for each substitution. The histograms in panels B and C represent the calculated genetic barrier for the development of NI/NNI resistance variants. In panel B, substitutions reported are those for which the genetic barrier was not affected by intergenotype variability and are characterized by low genetic barrier scores (one transition or one transversion). In panel C, the histogram represents the calculated genetic barrier score for major NNI resistance variants at positions 414, 419, and 422. 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.

Furthermore, other resistance variants, conferring a moderate-low level of resistance to different compounds of NNI-1 (421V and 499A), NNI-2 (482L and 494A), NNI-3 (556G), and NNI-4 (316N and 445F) classes (12), were found with high prevalence in several genotypes: 421V in 100% of HCV-3 and -6; >80% of HCV-2, -4, and -5; 12.0% of HCV-1a; and 4.0% of HCV-1b genotypes; 499A in 100% of HCV-5; >91% of HCV-1a, -2, -3, -4, and -6; and 10.0% of HCV-1b genotypes; 482L in 99 to 100% of HCV-2, -3, -4, and -6 genotypes; 494A in 100% of HCV-2, 5.2% of HCV-4, and 75.3% of HCV-6 genotypes; 556G in 8.2% of HCV-1b; 100% of HCV-2, -3, and -5; 97.4% of HCV-4; and 2.6% of HCV-6 genotypes; 316N in 35.6% of HCV-1b and 7.9% of HCV-4 genotypes; 445F in 100% of HCV-2, -3, -4, -5, and -6 genotypes (Fig. 4A). Interestingly, the minor NNI resistance substitutions 445F and 556G were found in the unique HCV-7 reference sequence available; similarly, the major NI resistance substitution 282T was found in the reference sequence of the HCV-4 genotype (Fig. 2).

Genetic barrier for NI and NNI resistance.

The genetic barrier for the development of NI/NNI resistance was analyzed on the data set of 1,144 NS5B polymerase sequences from the 6 genotypes (HCV-1a, -1b, -2, -3, -4, -5, and -6). Only variants with prevalences of >1% and found in ≥2 patients were considered (for this reason, the unique available sequence of HCV-7 was excluded in this analysis). Starting from each wild-type codon, each transition was assigned a score of 1 and each transversion was assigned a score of 2.5.

Regardless of HCV genotypes, the NI resistance variant 282T and 19/32 NNI resistance variants required only one substitution to be generated and were thus associated with low genetic barrier scores (Fig. 4B). In particular, the NI resistance substitution 282T requires only one transversion (score = 2.5) to be generated (Table 1). Among NNIs, 8 major resistance variants required only 1 nucleotide transition (423I/T/V, 448H, 486V, 495L, 554D, and D559G; score = 1) while 2 other major resistance variants required only one nucleotide transversion (316Y and 414L; score = 2.5) (Table 1 and Fig. 4B).

TABLE 1.

Codon variability at HCV NS5B positions associated with major drug resistance to NIs and NNIs and its impact on the genetic barrier to drug resistance development in HCV genotypes 1 to 6

Major position(s) associated with resistance to type of inhibitor	WT amino acid^a	WT codon	Proportion of WT and polymorphic codons in HCV genotype, n (%)^b							Resistance variant
										Group 1			Group 2
			1a (n = 506)	1b (n = 379)	2 (n = 113)	3 (n = 14)	4 (n = 38)	5 (n = 17)	6 (n = 77)	Variant	Codon^c	Minimal score^d	Variant	Codon^c	Minimal score^d
Nucleos(t)ide inhibitors
282	S	AGC	500 (98.8)	361 (95.2)	111 (98.2)	4 (28.6)	36 (94.7)	17 (100)	25 (32.5)	S282T	ACC	2.5
282		AGT	4 (0.8)	18 (4.8)	2 (1.8)	10 (71.4)	1 (2.6)	0 (0.0)	51 (66.2)	S282T	ACT	2.5
Nonnucleoside inhibitors
Class 1
486	A	GCC	469 (92.7)	5 (1.3)	0 (0.0)	0 (0.0)	12 (31.6)	1 (5.8)	0 (0.0)	A486V	GTC	1
486		GCT	33 (6.5)	347 (91.5)	26 (23.0)	0 (0.0)	7 (18.4)	15 (88.2)	5 (6.5)	A486V	GTT	1
486		GCA	4 (0.8)	9 (2.4)	86 (76.1)	0 (0.0)	14 (36.8)	0 (0.0)	31 (40.2)	A486V	GTA	1
486		GCG	0 (0.0)	18 (4.7)	1 (0.88)	13 (100)	5 (13.1)	0 (0.0)	2 (2.6)	A486V	GTG	1
486	G	GGA	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	20 (25.9)	G486V	GTA	2.5
486		GGG	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	19 (24.6)	G486V	GTG	2.5
495	P	CCA	29 (5.7)	252 (66.5)	27 (23.9)	0 (0.0)	4 (10.5)	0 (0.0)	23 (29.9)	P495L	CTA	1
495		CCG	477 (94.3)	125 (33)	2 (1.8)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	P495L	CTG	1
495		CCT	0 (0.0)	2 (0.5)	83 (73.4)	3 (21.4)	0 (0.0)	0 (0.0)	28 (36.3)	P495L	CTT	1
495		CCC	0 (0.0)	0 (0.0)	1 (0.9)	11 (78.6)	34 (89.5)	17 (100)	25 (32.5)	P495L	CTC	1
Class 2
419	L	CTG	290 (57.3)	10 (2.6)	0 (0.0)	0 (0.0)	0 (0.0)	13 (76.5)	0 (0.0)	L419S	TCG	2
419		CTA	10 (2)	40 (10.5)	0 (0.0)	0 (0.0)	0 (0.0)	3 (17.6)	0 (0.0)	L419S	TCA	2
419		TTA	3 (0.6)	199 (52.5)	1 (0.9)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	L419S	TCA	1
419		TTG	202 (39.9)	124 (32.7)	0 (0.0)	0 (0.0)	0 (0.0)	1 (5.9)	0 (0.0)	L419S	TCG	1
419	I	ATA	0 (0.0)	5 (1.3)	26 (23)	0 (0.0)	27 (71.1)	0 (0.0)	44 (57.1)	I419S	TCA	3.5
419		ATC	0 (0.0)	0 (0.0)	80 (70.8)	11 (78.6)	4 (10.5)	0 (0.0)	13 (16.9)	I419S	AGC	2.5
419		ATT	0 (0.0)	0 (0.0)	3 (2.6)	3 (21.4)	7 (18.4)	0 (0.0)	20 (26)	I419S	AGT	2.5
422	R	AGG	400 (79.0)	374 (98.7)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	R422K	AAG	1
422		AGA	106 (20.9)	2 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	R422K	AAA	1
422		CGA	1 (0.2)	0 (0.0)	6 (5.3)	0 (0.0)	1 (2.6)	0 (0.0)	1 (1.3)	R422K	AAA	3.5
422		CGG	1 (0.2)	2 (0.5)	79 (69.9)	0 (0.0)	2 (5.3)	17 (100)	3 (3.4)	R422K	AAG	3.5
422		CGC	0 (0.0)	0 (0.0)	25 (22.1)	12 (85.7)	29 (76.3)	0 (0.0)	32 (41.5)	R422K	AAA/G	6
422		CGT	0 (0.0)	0 (0.0)	3 (2.6)	2 (14.3)	6 (15.8)	0 (0.0)	41 (53.2)	R422K	AAA/G	6
423	M	ATG	493 (97.4)	379 (100)	113 (100)	14 (100)	38 (100)	0 (0.0)	77 (100)	M423I	ATA	1	M423T	ACG	1
423	I	ATA	6 (1.2)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	17 (100)	0 (0.0)				I423T	ACA	1
423		ATC	3 (0.6)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)				I423T	ACC	1
423	V	GTG	3 (0.6)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	V423I	ATA	2	V423T	ACG	2
423	M	ATG	493 (97.4)	379 (100)	113 (100)	14 (100)	38 (100)	0 (0.0)	77 (100)	M423V	GTG	1
423	I	ATA	6 (1.2)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	17 (100)	0 (0.0)	I423V	GTA	1
423		ATC	3 (0.6)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	I423V	GTC	1
423	V	GTG	3 (0.6)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Classes 3 and 4
316	C	TGC	33 (6.5)	231 (60.9)	29 (25.7)	13 (92.9)	28 (73.7)	2 (11.7)	76 (98.7)	C316Y	TAC	2.5
316		TGT	473 (93.5)	9 (2.4)	84 (74.3)	1 (7.1)	7 (18.4)	15 (88.2)	1 (1.3)	C316Y	TAT	2.5
316	N	AAC	0 (0.0)	130 (34.3)	0 (0.0)	0 (0.0)	3 (7.9)	0 (0.0)	0 (0.0)	N316Y	TAC	2.5
316		AAT	0 (0.0)	5 (1.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	N316Y	TAT	2.5
414	M	ATG	505 (99.8)	378 (99.7)	0 (0.0)	14 (100)	0 (0.0)	17 (100)	75 (97.4)	M414L	C/TTG	2.5	M414T	ACG	1
414	I	ATC	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	6 (15.8)	0 (0.0)	0 (0.0)	I414L	CTC	2.5	I414T	ACC	1
414	A	GCG	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (1.3)	A414L	C/TTG	3.5	A414T	ACG	1
414	T	ACG	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (1.3)	T414L	C/TTG	3.5
414	V	GTC	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	16 (42.1)	0 (0.0)	0 (0.0)	V414L	CTC	2.5	V414T	ACC	2
414		GTT	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (2.6)	0 (0.0)	0 (0.0)	V414L	CTT	2.5	V414T	ACT	2
414	Q	CAA	0 (0.0)	0 (0.0)	67 (59.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	Q414L	CTA	2.5	Q414T	ACA	5
414		CAG	0 (0.0)	0 (0.0)	45 (39.8)	0 (0.0)	1 (2.6)	0 (0.0)	0 (0.0)	Q414L	CTG	2.5	Q414T	ACG	5
414	L	CTA	0 (0.0)	0 (0.0)	1 (0.9)	0 (0.0)	1 (2.6)	0 (0.0)	0 (0.0)				L414T	ACA	3.5
414		CTC	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	6 (15.8)	0 (0.0)	0 (0.0)				L414T	ACC	3.5
414		CTG	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (5.3)	0 (0.0)	0 (0.0)				L414T	ACG	3.5
414		CTT	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	5 (13.2)	0 (0.0)	0 (0.0)				L414T	ACT	3.5
448	Y	TAC	498 (98.4)	351 (92.6)	80 (70.8)	14 (100)	38 (100)	1 (5.8)	70 (90.9)	Y448H	CAC	1
448		TAT	8 (1.6)	28 (7.4)	33 (29.2)	0 (0.0)	0 (0.0)	16 (94.1)	7 (9.1)	Y448H	CAT	1
554	G	GGT	54 (10.7)	317 (83.6)	5 (4.4)	9 (64.3)	1 (2.6)	2 (11.7)	5 (6.5)	G554D	GAT	1
554		GGA	1 (0.2)	5 (1.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	G554D	GAC/T	3.5
554		GGC	450 (88.9)	51 (13.5)	107 (94.7)	5 (35.7)	37 (97.4)	14 (82.3)	72 (93.5)	G554D	GAC	1
554		GGG	1 (0.2)	5 (1.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	G554D	GAC/T	3.5
559	D	GAC	505 (99.8)	378 (99.7)	108 (95.6)	14 (100)	35 (92.1)	17 (100)	77 (100)	D559G	GGC	1
559		GAT	1 (0.2)	0 (0.0)	4 (3.5)	0 (0.0)	3 (7.9)	0 (0.0)	0 (0.0)	D559G	GGT	1

Open in a new tab

The wild-type (WT) amino acid of HCV genotype 1b at each position associated with drug resistance is shown.

Data for the predominant wild-type codon for each genotype are shown in bold.

Codon for drug resistance variants requiring the lowest number of transitions/transversions starting from the wild-type or polymorphic codon detected in drug-naive patients.

Minimal numerical score obtained by summing the number of nucleotide transitions and/or transversions (scored as 1 and 2.5, respectively; see Materials and Methods) required to generate the specific drug resistance variant.

Furthermore, 9 minor NNI resistance variants also required only one substitution to be generated (426T, 448C, 452H, 495S, and 496S, score = 1; 368T, 482S, and 495A/T, score = 2.5) (Table 2 and Fig. 4B).

TABLE 2.

Codon variability at HCV NS5B positions associated with minor drug resistance to NIs and NNIs and its impact on the genetic barrier to drug resistance development in HCV genotypes 1 to 6

Minor position(s) associated with resistance to type of nonnucleoside inhibitor	WT amino acid^a	WT codon	Proportion of WT and polymorphic codons in HCV genotype, n (%)^b							Resistance variant
										Group 1			Group 2
			1a (n = 506)	1b (n = 379)	2 (n = 113)	3 (n = 14)	4 (n = 38)	5 (n = 17)	6 (n = 77)	Variant	Codon^c	Minimal score^d	Variant	Codon^c	Minimal score^d
Class 1
421	A	GCG	431 (85.2)	38 (10.0)	1 (0.9)	0 (0.0)	2 (5.3)	0 (0.0)	0 (0.0)	A421V	GTG	1
421		GCA	11 (2.2)	325 (85.7)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	A421V	GTA	1
421		GCT	0 (0.0)	1 (0.3)	8 (7.1)	0 (0.0)	1 (2.6)	2 (11.7)	0 (0.0)	A421V	GTT	1
421		GCC	0 (0.0)	0 (0.0)	3 (2.6)	0 (0.0)	0 (0.0)	0 (0.0)	54 (70.1)	A421V	GTC	1
421	V	GTG	60 (11.8)	2 (0.53)	3 (2.6)	14 (100)	31 (81.6)	0 (0.0)	14 (18.2)
421		GTA	1 (0.2)	12 (3.2)	0 (0.0)	0 (0.0)	2 (5.2)	1 (5.9)	9 (11.7)
421		GTC	0 (0.0)	0 (0.0)	77 (68.1)	0 (0.0)	0 (0.0)	1 (5.9)	0 (0.0)
421		GTT	0 (0.0)	0 (0.0)	21 (18.6)	0 (0.0)	2 (5.2)	13 (76.5)	0 (0.0)
421	T	ACG	2 (0.4)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	T421V	GTG	2
495	P	CCA	29 (5.7)	252 (66.5)	27 (23.9)	0 (0.0)	4 (10.5)	0 (0.0)	23 (29.9)	P495A	GCA	2.5	P495S	TCA	1
495		CCG	477 (94.3)	125 (33)	2 (1.8)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	P495A	GCG	2.5	P495S	TCG	1
495		CCT	0 (0.0)	2 (0.5)	83 (73.4)	3 (21.4)	0 (0.0)	0 (0.0)	28 (36.3)	P495A	GCT	2.5	P495S	TCT	1
495		CCC	0 (0.0)	0 (0.0)	1 (0.9)	11 (78.6)	34 (89.5)	17 (100)	25 (32.5)	P495A	GCC	2.5	P495S	TCC	1
495	P	CCA	29 (5.7)	252 (66.5)	27 (23.9)	0 (0.0)	4 (10.5)	0 (0.0)	23 (29.9)	P495Q	CAA	2.5	P495T	ACA	2.5
495		CCG	477 (94.3)	125 (33)	2 (1.8)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	P495Q	CAG	2.5	P495T	ACG	2.5
495		CCT	0 (0.0)	2 (0.5)	83 (73.4)	3 (21.4)	0 (0.0)	0 (0.0)	28 (36.3)	P495Q	CAA/G	5	P495T	ACT	2.5
495		CCC	0 (0.0)	0 (0.0)	1 (0.9)	11 (78.6)	34 (89.5)	17 (100)	25 (32.5)	P495Q	CAA/G	5	P495T	ACC	2.5
496	P	CCC	502 (99.2)	371 (97.9)	109 (96.4)	13 (92.9)	0 (0.0)	0 (0.0)	30 (39)	P496S	TCC	1
496		CCA	0 (0.0)	0 (0.0)	1 (0.9)	0 (0.0)	17 (44.7)	4 (23.6)	17 (22.1)	P496S	TCA	1
496		CCG	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	21 (55.3)	5 (29.4)	24 (31.1)	P496S	TCG	1
496		CCT	3 (0.6)	8 (2.1)	3 (2.7)	1 (7.1)	0 (0.0)	5 (29.4)	6 (7.8)	P496S	TCT	1
499	V	GTC	2 (0.4)	323 (85.2)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	V499A	GCC	1
499		GTG	0 (0.0)	0 (0.0)	8 (7.1)	0 (0.0)	2 (5.3)	0 (0.0)	0 (0.0)	V499A	GCG	1
499		GTT	2 (0.4)	1 (0.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	V499A	GCT	1
499	T	ACC	0 (0.0)	16 (4.2)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	T499A	GCC	1
499		ACG	0 (0.0)	0 (0.0)	2 (1.8)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	T499A	GCG	1
499		ACT	12 (2.4)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	T499A	GCT	1
499	A	GCC	21 (4.2)	33 (8.7)	0 (0.0)	1 (7.1)	1 (2.6)	0 (0.0)	60 (77.9)
499		GCT	468 (92.5)	5 (1.3)	1 (0.9)	9 (64.3)	1 (2.6)	17 (100)	11 (14.3)
499		GCG	1 (0.2)	0 (0.0)	102 (90.3)	3 (21.4)	34 (89.5)	0 (0.0)	5 (6.5)
Class 2
426	M	ATG	462 (91.3)	359 (94.7)	113 (100)	12 (85.7)	38 (100)	17 (100)	53 (68.8)	M426A	GCG	2	M426T	ACG	1
426	I	ATA	1 (0.2)	0 (0.0)	0 (0.0)	2 (14.3)	0 (0.0)	0 (0.0)	0 (0.0)	I426A	GCA	2	I426T	ACA	1
426	L	CTA	1 (0.2)	4 (1.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	L426A	GCA	3.5	L426T	ACA	3.5
426		CTG	11 (2.2)	6 (1.6)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	L426A	GCG	3.5	L426T	ACG	3.5
426		TTG	22 (4.3)	4 (1.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	L426A	GCG	3.5	L426T	ACG	3.5
426		TTA	4 (0.8)	2 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	L426A	GCA	3.5	L426T	ACA	3.5
426	T	ACG	0 (0.0)	2 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	T426A	GCG	1
426	A	GCG	0 (0.0)	2 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)				A426T	ACG	1
426	C	TGC	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	24 (31.2)	C426A	GCC	5	C426T	ACC	5
482	I	ATC	503 (99.4)	377 (99.4)	0 (0.0)	0 (0.0)	0 (0.0)	17 (100)	0 (0.0)	I482L	CTC	2.5	I482S	AGC	2.5
482		ATT	2 (0.4)	1 (0.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	I482L	CTT	2.5	I482S	AGT	2.5
482	L	CTC	1 (0.2)	1 (0.3)	87 (77)	14 (100)	37 (97.4)	0 (0.0)	75 (97.4)				L482S	ATC	2.5
482		CTG	0 (0.0)	0 (0.0)	25 (22.1)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)				L482S	ATA	3.5
482		CTT	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (2.6)	0 (0.0)	2 (2.6)				L482S	ATT	2.5
494	V	GTA	53 (10.5)	357 (94.2)	0 (0.0)	0 (0.0)	24 (63.2)	14 (82.3)	12 (15.6)	V494A	GCA	1
494		GTC	326 (64.4)	4 (1.0)	0 (0.0)	0 (0.0)	3 (7.9)	0 (0.0)	0 (0.0)	V494A	GCC	1
494		GTG	2 (0.4)	5 (1.3)	0 (0.0)	0 (0.0)	7 (18.4)	3 (17.7)	5 (6.5)	V494A	GCG	1
494		GTT	125 (24.7)	12 (3.2)	0 (0.0)	0 (0.0)	2 (5.3)	0 (0.0)	0 (0.0)	V494A	GCT	1
494	A	GCA	0 (0.0)	1 (0.3)	4 (3.5)	0 (0.0)	0 (0.0)	0 (0.0)	16 (20.8)
494		GCG	0 (0.0)	0 (0.0)	109 (96.5)	0 (0.0)	1 (2.6)	0 (0.0)	8 (10.4)
494		GCT	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (2.6)	0 (0.0)	26 (33.7)
494		GCC	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	8 (10.4)
494	I	ATC	0 (0.0)	0 (0.0)	0 (0.0)	4 (28.6)	0 (0.0)	0 (0.0)	0 (0.0)	I494A	GCC	2
494	C	TGC	0 (0.0)	0 (0.0)	0 (0.0)	8 (57.2)	0 (0.0)	0 (0.0)	0 (0.0)	C494A	GCC	5
494	M	ATG	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (2.6)	M494A	GCG	2
Class 3
368	S	TCC	373 (73.7)	365 (96.3)	2 (1.8)	8 (57.1)	38 (100)	0 (0.0)	43 (55.8)	S368T	ACC	2.5
368		TCT	127 (25.1)	8 (2.1)	0 (0.0)	1 (7.2)	0 (0.0)	0 (0.0)	1 (1.3)	S368T	ACT	2.5
368		TCA	3 (0.6)	4 (1.0)	110 (97.3)	5 (35.7)	0 (0.0)	16 (94.1)	19 (24.7)	S368T	ACA	2.5
368		TCG	3 (0.6)	1 (0.3)	1 (0.9)	0 (0.0)	0 (0.0)	1 (5.9)	11 (14.3)	S368T	ACG	2.5
368	P	CCC	0 (0.0)	1 (0.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	P368T	ACC	2.5
368	A	GCA	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	3 (3.9)	A368T	ACA	1
554	G	GGT	54 (10.7)	317 (83.6)	5 (4.4)	9 (64.3)	1 (2.6)	2 (11.7)	5 (6.5)	G554S	AGT	1
554		GGA	1 (0.2)	5 (1.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	G554S	AGT/C	3.5
554		GGC	450 (88.9)	51 (13.5)	107 (94.7)	5 (35.7)	37 (97.4)	14 (82.3)	72 (93.5)	G554S	TCC	2.5
554		GGG	1 (0.2)	5 (1.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	G554S	AGT/C	3.5
556	S	AGC	503 (99.4)	333 (87.9)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	41 (53.2)	S556G	GGC	1
556		AGT	0 (0.0)	6 (1.6)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	S556G	GGT	1
556	N	AAC	1 (0.2)	6 (1.6)	0 (0.0)	0 (0.0)	1 (2.6)	0 (0.0)	0 (0.0)	N556G	GGC	2
556	R	CGC	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	13 (16.9)	R556G	GGC	2.5
556	D	GAC	0 (0.0)	3 (0.8)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	21 (27.3)	D556G	GGC	1
556	G	GGC	2 (0.4)	31 (8.2)	113 (100)	14 (100)	37 (97.4)	17 (100)	2 (2.6)
Class 4
316	C	TGC	33 (6.5)	231 (60.9)	29 (25.7)	13 (92.9)	28 (73.7)	2 (11.7)	76 (98.7)	C316N	AAC	3.5
		TGT	473 (93.5)	9 (2.4)	84 (74.3)	1 (7.1)	7 (18.4)	15 (88.2)	1 (1.3)	C316N	AAT	3.5
	N	AAC	0 (0.0)	130 (34.3)	0 (0.0)	0 (0.0)	3 (7.9)	0 (0.0)	0 (0.0)
		AAT	0 (0.0)	5 (1.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
445	C	TGC	458 (90.5)	95 (25.1)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	C445F	TTC	2.5
445		TGT	47 (9.3)	281 (74.1)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	C445F	TTT	2.5
445	F	TTC	0 (0.0)	2 (0.5)	4 (3.5)	38 (100)	48 (62.4)	1 (5.9)	48 (62.4)
445	F	TTT	0 (0.0)	0 (0.0)	109 (96.5)	0 (0.0)	29 (37.6)	15 (88.2)	29 (37.6)
448	Y	TAC	498 (98.4)	351 (92.6)	80 (70.8)	14 (100)	38 (100)	1 (5.8)	70 (90.9)	Y448C	TGC	1
448		TAT	8 (1.6)	28 (7.4)	33 (29.2)	0 (0.0)	0 (0.0)	16 (94.1)	7 (9.1)	Y448C	TGT	1
452	Y	TAC	503 (99.4)	373 (98.4)	40 (35.4)	9 (64.3)	31 (81.6)	14 (82.4)	35 (45.4)	Y452H	CAC	1
452		TAT	1 (0.2)	0 (0.0)	73 (64.6)	5 (35.7)	7 (18.4)	3 (17.6)	42 (54.5)	Y452H	CAT	1
452	H	CAC	2 (0.4)	6 (1.6)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)

Open in a new tab

The wild-type (WT) amino acid of HCV genotype 1b at each position associated with drug resistance is shown.

Data for the predominant wild-type codon for each genotype are shown in bold.

Codon for drug resistance variant requiring the lowest number of transitions/transversions starting from the wild-type or polymorphic codon detected in drug-naive patients.

In contrast, the genetic variability among the six HCV genotypes had an impact on the calculation of the genetic barrier for the development of 3 other major NNI resistance substitutions (Fig. 4C; Table 1) and different minor NNI substitutions (Table 2). According to the different wild-type codon usage at position 414, a methionine (M) was observed as predominant wild-type amino acid in genotypes 1a, 1b, 3, 5, and 6, while genotypes 2 and 4 presented a glutamine (Q) or a valine (V), respectively (Table 1). In terms of genetic barrier, this difference translated into different scores for the development of 414T, a substitution known to confer high levels of resistance to both the NNI-3 inhibitors setrobuvir and ABT-333: HCV genotypes 1a, 1b, 3, 5, and 6 had a far lower genetic barrier (score = 1) than did HCV-2 (score = 5) and HCV-4 (score = 2). For the development of 422K, associated with high resistance to NNI-2 compounds (filibuvir, lomibuvir, and GS-9669), the genetic barrier was lower in genotypes 1a and 1b (score = 1) than in the other genotypes HCV-2 and -5 (score = 3.5) and HCV-3, -4, and -6 (score = 6) (Fig. 4C; Table 1).

Notably, in addition to differences between genotypes, a difference in genetic barrier score was observed also between HCV-1a and HCV-1b subtypes, for the development of the major NNI-2 resistance variant 419S (HCV-1a, score = 2; HCV-1b, score = 1; HCV-2 and -3, score = 2.5; HCV-4 and -6, score = 3.5; HCV-5, score = 1) (Table 1 and Fig. 4C).

Moreover, the HCV genetic variability also had an impact on the development of other minor NNI resistance variants (Table 2). For instance, according to the different wild-type codon usage at position 495, the potential development of the variant 495Q had a low genetic barrier in HCV-1a and -1b (score = 2.5) in comparison to HCV-2, -3, -4, -5, and -6 (score = 5). Also, for the development of the minor 554S substitution, HCV-1b and -3 had a lower genetic barrier (score = 1) than did HCV-1a, -2, -4, -5, and -6 (score = 2.5) (Table 2).

Docking analysis between NS5B from different HCV genotypes and sofosbuvir.

Sofosbuvir-NS5B docking analysis highlighted a better ligand affinity toward HCV-2 than toward the HCV-3 genotype. In particular, as shown in Fig. 5, sofosbuvir molecular recognition against HCV-1, HCV-2, and HCV-3 genotypes was associated with an increased consensus binding affinity toward HCV-2. Such a result was further emphasized by analyzing the free energy of complexation values of sofosbuvir toward the different considered subtypes of HCV genotypes. Moreover, redocking simulations, carried out in order to reproduce the binding mode of the cocrystallized inhibitor, allowed us to validate the adopted method (root mean square deviation = 1.26 Å; data not shown) and to use it as an effective predictive tool.

FIG 5 — Free energy of complexation (ΔG_bind) of sofosbuvir against the different subtypes of HCV genotypes. The diagram reports the average sofosbuvir affinities toward HCV-1 (1a + 1b), HCV-2 (2a + 2b + 2c + 2k), and HCV-3 (3a + 3b + 3i + 3k). All data are expressed in kcal/mol.

DISCUSSION

This study evaluated the NS5B polymerase variability in a large number of HCV genotypes and subtypes commonly spread worldwide and its impact on the genetic barrier for the development of NI/NNI resistance substitutions. Analyzing 1,145 HCV NS5B polymerase sequences, we found an intermediate level of conservation among all 7 HCV genotypes, with 313/591 (53.0%) conserved amino acid residues (with a variability of ≤1%). This level of amino acid conservation is quite similar to what has been observed in two other studies, where the complete sequence of NS5B from different HCV isolates of genotypes 1 to 6 has been analyzed (26, 64). In the first study, analyzing 48 sequences, the number of fully conserved residues was 263 out of 591 (44.5% conservation), with 51% of them present as three or more adjacent amino acids (64). The overall degree of NS5B conservation is similar also to what was observed for NS3 and NS5A proteins, where 47% and approximately 50% of amino acid residues were conserved among all HCV genotypes, respectively (27, 65).

The NS5B polymerase palm domain, which contains the active site with the catalytic triad, results in the most conserved region compared with the others, fingers and thumb, which allow higher variability. According to our structural analysis, highly conserved NS5B positions among all HCV genotypes are those pivotal for enzyme functionality and stability. In all studies (including the present), the active site residues D220, D225, G317, D318, and D319 are highly conserved among all the HCV genotypes (26, 64). The other catalytic pocket residues, R158, S367, R386, and R394, which interact with the triphosphate moiety of nucleoside triphosphates (NTPs) (66), are also highly conserved, even if the codon for R394 is mutated to arginine in the HCV-5 reference sequence. The residues E18, Y191, C274, Y276, and H502, which take part in primer-template interaction (67), were also highly conserved except for H502, which is changed to serine in the majority of HCV-2 sequences and to asparagine in HCV-7. Interestingly, the presence of long regions of uninterrupted conserved residues corresponding to positions 219 to 230, 342 to 352, 364 to 373, and 524 to 530 has been also observed among all studies (26, 64). This could assist in the rational design of new HCV inhibitors with more favorable resistance profiles.

As we observed previously for the NS3 protease (31 out 181 residues, 17%), few NS5B residues (83/591, 14.0%) were highly variable among HCV genotypes, showing ≥25.1% amino acid variability, and were mostly located on the surface of NS5B protein. These results are in agreement with a lower ability to tolerate amino acid substitutions within the active site, compared to variations within allosteric sites. Indeed, allosteric sites are localized at the surface of the enzyme, where amino acid changes can be generated without severely affecting the polymerase activity (7), allowing higher levels of amino acid variability across HCV genotypes and lower fitness cost.

Overall, 9 NNI resistance variants were found as natural polymorphisms, with a prevalence ranging from 2 to 100%, in selected genotypes. In particular, the palm primary substitution 414L, described to confer high resistance in vitro to the NNI-3 setrobuvir (ANA-598) (12), was frequently found in HCV-4 (36.8% prevalence). Similarly, the thumb primary substitution 423I, reported to confer high resistance to the NNI-2 filibuvir (39), was found in all HCV-5 sequences, and rarely also in HCV-1a (1.8% prevalence). All other 11 primary palm/thumb NNI resistance variants (316Y, 414T, 419S, 422K, 423T/V, 448H, 486V, 495L, 554D, and 559G) were not observed in DAA-naive patients or only very rarely (≤1%).

Interestingly, other variants, associated with low-intermediate levels of resistance to NNIs, were naturally found with a different prevalence among HCV genotypes, and even in subtypes 1a and 1b. In particular, within the palm region, the 316N substitution, conferring an intermediate level of resistance to the NNI-4 tegobuvir (5.2-fold decrease in susceptibility) (68), was commonly found in HCV-1b (35.6% prevalence) and in HCV-4 (7.9% prevalence). Similarly, the variant 556G, observed in patients in early clinical trials with ABT-333 (41), in our analysis was found in 8.2% of HCV-1b sequences; 100% of HCV-2, -3, and -5 sequences; 97.4% of HCV-4 sequences; and 2.6% of HCV-6 sequences and was completely absent in HCV-1a.

Finally, among the thumb substitutions, 499A, associated with resistance to the NNI-1 deleobuvir (BI207127), was found with very high prevalence in HCV-1a, -2, -3, -4, -5, and -6 (prevalence, >90%) and with a lower prevalence in HCV-1b (10%).

In contrast, no instance of primary NI resistance variant 282T was found among all HCV sequences analyzed, with the exception of the reference sequence of HCV-4 (strain Y11604), as already reported in two previous papers (69, 70). However, T282 is not a universal marker for genotype 4, as additional sequences of HCV-4 show the presence of S282 (69).

Overall, these results well explain the higher heterogeneity in sensitivity to NNIs observed with the different HCV genotypes (71, 72), while the need for high conservation within the active site of the polymerase gives reason for the potent antiviral activity of NIs against a broad spectrum of HCV genotypes (12). For instance, in the context of interferon (IFN)-free regimens, several clinical trials in patients infected with HCV-1 and non-HCV-1 genotypes (such as HCV-2, -3, -4, -5, and -6) are ongoing with sofosbuvir, while very few are currently ongoing with NNIs (such as with ABT-333, GS-9669, and BMS-791325, mainly with HCV-1 and HCV-4) (www.clinicaltrials.gov). The simple combination of sofosbuvir and ribavirin for 12 weeks appears to be extremely successful in naive HCV-1-, -2-, and -3-infected patients (though this combination, using such a short regimen, is insufficient to cure previous null responders) (73). Interestingly, very recently, in analysis of two randomized phase III studies (involving patients with chronic HCV genotype 2 or 3 infection for whom treatment with pegylated interferon was not an option), the response rates of sofosbuvir and ribavirin were lower among patients with HCV-3 than among those with HCV-2 (74), indicating clearly that HCV-2 and HCV-3 are different viruses and that probably the duration and type of treatment should be considered separately in patients infected with those genotypes. Notably, even if an experimental structure of NS5B ternary complex with sofosbuvir is not available, and our docking analysis of sofosbuvir with NS5B from different HCV genotypes is largely speculative, we highlighted a better ligand affinity toward HCV-2 than toward HCV-3, in agreement with the experimental observations.

Besides the genetic variability and natural presence of drug resistance substitutions in selected genotypes before treatment, another factor that can be associated with the probability of success of a DAA-based regimen is the genetic barrier for the development of resistance. This can be broadly defined as the number and type of nucleotide mutations required for the generation of a specific resistance substitution, starting from the wild-type genetic background of the virus (75). Previous studies have shown that genetic variability among HIV, HBV, or HCV genotypes can in some cases facilitate the development of specific resistance variants (27, 47, 48, 75 –77). For instance, we recently proposed that the high degree of HCV genetic variability makes HCV genotypes, and even subtypes, differently prone to the development of PI resistance substitutions, with important clinical implications for tailoring individualized and appropriate regimens (27).

We therefore analyzed also the genetic barrier for the development of the 32 NNI resistance variants (316Y/N, 368T, 414T/L, 419S, 421V, 422K, 423T/I/V, 426A/T, 445F, 448C/H, 452H, 482L/S, 486V, 494A, 495S/Q/L/A/T, 496S, 499A, 554D/S, 556G, and 559G) and the NI resistance variant 282T.

Regardless of HCV genotype, in our model, 10/13 major NNI resistance variants required only one nucleotide substitution to be generated (one transition, score = 1, 423I/T/V, 448H, 486V, 495L, 554D, and 559G; one transversion, score = 2.5, 316Y and 414L). Interestingly, these results are highly consistent with the experimental and clinical observations available so far. Indeed, the majority of these NNI resistance substitutions with a lower genetic barrier are found in patients failing their NNI-containing regimen. For instance, 495L, reported to confer the highest level of resistance in vitro to NNI-1 inhibitors (36, 78, 79), was also described in vivo in patients failing deleobuvir (BI207127) in combination with PEG-IFN plus ribavirin in genotype-1-infected patients (35, 36).

Similarly, the other two major substitutions, 316Y and 448H, with low genetic barriers were shown to strongly reduce, in vivo and in vitro, the viral susceptibility to different NNIs of both classes 3 and 4 (12). In particular, the 316Y variant, located near the polymerase active site, was described to confer resistance to NNI-4 and NNI-3 compounds, such as ABT-333 (12, 41, 80) and tegobuvir, along with substitutions at positions 448, 452, and 445 (44). The 448H variant also was found to be the first resistance substitution to emerge in HCV-1 patients failing tegobuvir in combination with PEG-IFN plus ribavirin (45).

Conversely, 3 other major NNI resistance variants (414T, 419S, and 422K) were associated with a different genetic barrier for their development among the six HCV genotypes. Interestingly, the major substitution 419S (associated with resistance to NNI-2s, filibuvir, lomibuvir, and GS9669) showed a different calculated score also between HCV-1a and HCV-1b subtypes (HCV-1a, score = 2; HCV-1b, score = 1; HCV-2 and -3, score = 2.5; HCV-4 and -6, score = 3.5; HCV-5, score = 1).

Finally, the major resistance variant S282T, selected in vitro by sofosbuvir and mericitabine (32, 33), deserves particular attention. Although this substitution requires only a single G-to-C transversion (score = 2.5) and is rarely seen in the clinic (if at all) (7, 75), because it alters the conformation of the enzyme catalytic site (81, 82), it severely compromises viral fitness among different HCV genotypes (32, 69, 83, 84), and biochemical data revealed deficiencies in de novo primed RNA synthesis by HCV NS5B (33). In this case, the deficit in viral fitness and the low frequencies of transversions over transitions within the diversity of viral quasispecies found by deep-sequencing analyses of HCV samples from treatment-naive patients represent an overall high barrier to the selection of 282T in vivo (75, 76). According to this observation, there is little in vivo evidence of drug resistance to NIs and, when detected, resistant variants with 282T rapidly revert to wild type as soon as the treatment is interrupted (85, 86).

Moreover, recent papers (76, 87), based on deep sequencing and single-genome analysis, showed that, specifically for HCV, the differences in frequencies of transitions and transversions are higher than what was previously proposed. These groups reported a range between 16 and 50. Our calculation model, instead, assigns a score of 1 for transitions and 2.5 for transversions, because it was based on an initial paper that addressed the issue of calculating the genetic barrier for development of drug resistance substitutions between subtypes (48). In this paper, it was reported that transitions (replacement of a purine by another purine or of a pyrimidine by another pyrimidine) are for steric reasons occurring on average 2.5 times more frequently than are transversions (replacement of a purine by a pyrimidine and vice versa). For this reason, it is important to note that our scores (1 and 2.5) are arbitrary values, and probably, the 2.5 score underestimates the actual experimental evidence of a much lower frequency for transversions (75).

Taken all together, our results, according to some previously reported studies (19, 24, 88, 89), suggest the utility of performing a genotypic resistance test before starting a DAA-containing regimen, especially in patients with advanced disease and/or previous nonresponse to PEG-IFN plus ribavirin.

The genetic variability among HCV genotypes represents indeed an important issue in the global approach for the management and treatment of HCV-related disease. Particularly for NNIs, the presence in DAA-naive patients of natural polymorphisms at NNI resistance positions in selected genotypes, together with a broad low genetic barrier for the development of resistance, impairs the development of antivirals within this class with a pan-genotype activity and a high barrier to resistance.

Therefore, the HCV genotypic resistance test can thus provide at the same time two important types of information for clinical management of patients with chronic HCV infection: a correct subtype assignment and detection of variants that are potential nonresponders to therapy, highlighting patients with a higher risk of failure.

ACKNOWLEDGMENTS

This work was supported by the Italian Ministry of Instruction, University and Research (MIUR) (Accordi di Programma 2011: RBAP11YS7K_001, Bandiera InterOmics Protocollo PB05 1°) and by the Aviralia Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We thank Fabio Mercurio, who developed the algorithm using JavaScript to calculate the genetic barrier at each individual amino acid position.

Footnotes

Published ahead of print 3 March 2014

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PERMALINK

Hepatitis C Virus Genetic Variability and the Presence of NS5B Resistance-Associated Mutations as Natural Polymorphisms in Selected Genotypes Could Affect the Response to NS5B Inhibitors

V C Di Maio

V Cento

C Mirabelli

A Artese

G Costa

S Alcaro

C F Perno

F Ceccherini-Silberstein

Abstract

INTRODUCTION