Significance of NS5B Substitutions in Genotype 1b Hepatitis C Virus Evaluated by Bioinformatics Analysis

Abstract

To evaluate the effects of HCV NS5B amino acid substitutions on treatment outcome in Ledipasvir (LDV)/Sofosbuvir (SOF) for Japanese patients with genotype 1b HCV infection, NS5B sequences were examined in i) seven patients experiencing virologic failure after LDV/SOF in real-world practice, ii) 109 SOF-naïve patients, iii) 165 patients enrolled in Phase-3 LDV/SOF trial. A218S and C316N were detected in all patients with viral relapse; the percentages of these substitutions in SOF-naïve patients were 64.2% and 55.0%, respectively. Genotype 1b HCV strains with NS5B-C316N mutation were located in the leaves different from those in which HCV strains without such substitutions were present on the phylogenetic tree. Structural modeling revealed that amino acid 218 was located on the surface of the NTP tunnel. Free energy analysis based on molecular dynamics simulations demonstrated that the free energy required to pass through the tunnel was larger for triphosphate SOF than for UTP in NS5B polymerase carrying A218S, but not in wild-type. However, no susceptibility change was observed for these substitutions to SOF in replicon assay. Furthermore, the SVR rate was 100% in patients enrolled the Phase-3 trial. In conclusion, NS5B A218S and C316N were detected in all patients who relapsed following LDV/SOF in real-world practice. These substitutions did not impact the overall SVR rate after LDV/SOF, however, further studies are needed to elucidate the impact of these substitutions.

Figure 1

Amino Acid Mutations in the NS5A and NS5B Region of HCV Strains Obtained at Virologic Failure in 7 Patients Receiving Ledipasvir (LDV) and Sofosbuvir (SOF).

Figure 2

Frequencies of Amino Acid Mutations in the NS5B Regions of Genotype 1b HCV among 109 Patients without Previous Sofosbuvir Administration.

Figure 3

Linkage Disequilibrium between Phenotype of aa218 and Phenotypes of aa207, aa316, aa464 in the NS5B Region of Genotype 1b HCV in 109 Sofosbuvir-Naïve Patients.

Figure 4

Linkage Disequilibrium between Phenotype of aa316 and Phenotypes of aa207, aa318, aa464 in the NS5B Region of Genotype 1b HCV in 109 Sofosbuvir-Naïve Patients.

Figure 5

Phylogenetic Analysis in the NS5B region of Genotype 1b HCV. The genetic distance was estimated using Kimura 2 parameters, and a phylogenetic tree was constructed based on the nucleotide sequences in the NS5B region using the maximum likelihood method adapted with best fit model (Kimura 2-parameter plus Gamma distributed with Invariant sites). The bootstrap values are indicated at each tree root. Sequences with A218 and C316 wild-type (n = 40), A218S mutant and C316 wild-type (n = 10), and A218S and C316N mutant HCV strains (n = 59) observed in 109 sofosbuvir (SOF)-naïve patients are shown in blue, orange and black, respectively. Also, HCV strains (n = 7) detected in patients experiencing virologic failure after SOF-based therapy are shown in red.

Figure 6

Three-Dimensional Structure of HCV-NS5B Polymerase and Location of the Mutant Amino Acids Seen in HCV Strains Obtained from Patients Showing Virologic Failure after LDV/SOF Therapy. (a) Three-dimensional structure of NS5B polymerase as shown using a cartoon model. The amino acids at aa207, aa218, aa316, and aa464 are shown in red. (b) Enlarged view of NS5B polymerase showing the distances among aa207, aa218, and aa316. (c) Structure of NS5B polymerase in which the surface of each amino acid is visualized. The manganese complexes at the polymerase active site are shown as yellow balls. (d) Structure after the deletion of the thumb domain shown in Fig. 2c. (e) Structure of NS5B polymerase showing the surface of the NTP tunnel. The NS5B polymerase structure following the deletion of the thumb domain was rotated 45-degrees; aa218 and aa316 are labeled in red, and the activity region consisting of 3 aspartic acids, (aa220, aa318 and aa319) is labeled in blue. (f) Circumstances of the NS5B-NTP tunnel with and without A218S and/or C316N mutations. The red and white areas show hydrophobic and hydrophilic characteristics, respectively.

Figure 7

Free Energy Profile Required for the Passage of Uridine Triphosphate (UTP) and Activated Sofosbuvir (SOF) Through the NTP Tunnel of NS5B Polymerase of Genotype 1b HCV. (a) NS5B polymerase manifesting the A207, A218, C316 and Q464 wild-types as constructed based on the nucleotide sequence of HCV strains obtained from baseline sera from a patient who achieved an SVR after ledipasvir (LDV)/SOF therapy. (b) Single mutation of C316N was added to the polymerase shown in Fig. 3a. (c) Single mutation of A218S was added to the polymerase shown in Fig. 3a. (d) Double mutations of A218S and C316N were added to the polymerase shown in Fig. 3a. (e) Quadruple mutations of A207T, A218S, C316N and Q464E mutations were added to the polymerase shown in Fig. 3a. (f) A218S mutation was reverted to A218 wild-type in the polymerase shown in Fig. 3e. (g) NS5B polymerase manifesting A207T, A218S, C316N and Q464E mutations constructed based on nucleotide sequences of HCV strains obtained at the time of virologic failure in patients receiving LDV/SOF therapy. (h) A218S mutation was reverted to A218 wild-type in the polymerase shown in Fig. 3f. Molecular dynamics simulations were performed 3 times for each situation, and the mean, maximal, and minimal values are show in figure.