Multiple mutations in hepatitis C virus NS5A domain II are required to confer a significant level of resistance to alisporivir

Abstract

Alisporivir is the most advanced host-targeting antiviral cyclophilin (Cyp) inhibitor in phase III studies and has demonstrated a great deal of promise in decreasing hepatitis C virus (HCV) viremia in infected patients. In an attempt to further elucidate the mechanism of action of alisporivir, HCV replicons resistant to the drug were selected. Interestingly, mutations constantly arose in domain II of NS5A. To demonstrate that these mutations are responsible for drug resistance, they were reintroduced into the parental HCV genome, and the resulting mutant viruses were tested for replication in the presence of alisporivir or in the absence of the alisporivir target, CypA. We also examined the effect of the mutations on NS5A binding to itself (oligomerization), CypA, RNA, and NS5B. Importantly, the mutations did not affect any of these interactions. Moreover, the mutations did not preserve NS5A-CypA interactions from alisporivir rupture. NS5A mutations alone render HCV only slightly resistant to alisporivir. In sharp contrast, when multiple NS5A mutations are combined, significant resistance was observed. The introduction of multiple mutations in NS5A significantly restored viral replication in CypA knockdown cells. Interestingly, the combination of NS5A mutations renders HCV resistant to all classes of Cyp inhibitors. This study suggests that a combination of multiple mutations in domain II of NS5A rather than a single mutation is required to render HCV significantly and universally resistant to Cyp inhibitors. This in accordance with in vivo data that suggest that alisporivir is associated with a low potential for development of viral resistance.

Fig 1

(A) NS5A sequences from different genotypes (GT) surrounding D320/Y321 residues, which are mutated to E320/N321 residues under alisporivir selection, were aligned using the ClustalW algorithm within MacVector (version 12.5.1). The sequences used for the alignment were based on those available in the NCBI database. The genotypes (and accession numbers) are GT1a (AF009606), GT1b (AJ238799), GT2a [J6] (AF177036), GT2a [JFH] (BAF34893), GT2b (AY232730), GT3 (NC_009824), GT4 (NC_009825), GT5 (NC_009826), and GT6 (NC_009827). (B) Secondary-structure predictions of the NS5A 300-350 segment are indicated as helical (h; blue), extended (e; red), turn (t; green), or undetermined (coil [c]; yellow). Predictions were made by using the web-based algorithms DSC, HNNC, MLRC, PHD, Predator, and SOPM, which are available at the NPSA website (http://npsa-pbil.ibcp.fr) (7). Positions 320 and 321, exhibiting alisporivir-resistant mutations, are highlighted in green. Sec.Cons., secondary structure consensus.

Fig 2

(A) Schematic diagram of the NS5A-YFP reporter subgenomic Con1 replicon. NPTII, neomycin phosphotransferase gene; EMCV IRES, encephalomyocarditis virus internal ribosomal entry site. (B) The YFP-positive cell population was enriched using the BD FACSAria sorter. (C) Con1-NS5A-YFP Huh-7.5.1 cells were visualized via confocal analysis for 5 days. (D) Con1-WT NS5A-YFP, Con1-WT D320E NS5A-YFP, Y321N Con1-WT, and D320E/Y321N NS5A-YFP Huh-7.5.1 cells (500,000) were exposed to increasing concentrations of alisporivir and analyzed for YFP content 3 days after drug treatment. The percentage of YFP-positive cells treated with the DMSO control was arbitrarily fixed at 100. Results are representative of 3 independent experiments. Error bars represent standard errors from triplicates. (E) Same as panel D, except that luciferase activity in cell lysates was quantified; the percentage of luciferase activity in cells treated with the DMSO control was arbitrarily fixed at 100. Error bars represent standard errors from triplicates. Statistical significance was measured between each mutant construct in relation to Con1-WT NS5A-YFP for the following drug concentrations: 32, 63, and 125 nM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 3

(A) GST-CypA (10 μg) was mixed with wild-type, D320E, Y321N, and D320E/Y321N NS5A Con1-His proteins (200 ng) for 3 h at 4°C in the presence or absence of increasing concentrations of alisporivir. Glutathione beads were added to the GST-CypA–NS5A mixture for 30 min at 4°C and washed. Bound material was eluted and analyzed by Western blotting using anti-His antibodies. Results are representative of two independent experiments. (B) Same as panel A, except that GST, GST-CypA, or GST-NS5B (10 μg) was mixed with wild-type, D320E, Y321N, and D320E/Y321N NS5A Con1-His proteins (200 ng) for 3 h at 4°C in the presence or absence of 1.25 μM alisporivir. (C) Same as panel A, except that GST or GST-NSS5A (10 μg) was mixed with wild-type, D320E, Y321N, and D320E/Y321N NS5A Con1-His proteins (200 ng) for 3 h at 4°C in the presence or absence of 1.25 μM alisporivir.

Fig 4

Recombinant wild-type and mutant NS5A-His proteins (10 nM) were incubated with ³²P-labeled 3′ UTR RNA (1 nM) for 30 min in binding buffer in the presence or absence of 1.25 μM alisporivir. Reaction mixtures were added to filters for binding. Filters were extensively washed, dried, and placed in a scintillation counter for radioactivity measurement. Results expressed as percentages of RNA bound are representative of two independent experiments. Error bars represent standard errors from triplicates.

Fig 5

Ten micrograms of in vitro-transcribed subgenomic wild-type, D320E, Y321N, or D320E/Y321N Con1 RNA was electroporated into parental or CypA-KD Huh7.5.1 cells. At the indicated time points, intracellular HCV RNA was analyzed via RT-qPCR and is presented as genome equivalents (GE) per microgram total RNA. CypA-KD Huh7.5.1 cells were treated with or without 1.25 μM alisporivir. These results are representative of three independent experiments. Error bars represent standard errors from triplicates. Statistical significance was measured between each mutant construct in relation to Con1-WT NS5A-YFP after 8 days postinfection. **, P < 0.01; ***, P < 0.001.

Fig 6

Empty, Con1 wild-type NS5A-YFP, Con1 D320E NS5A-YFP, Con1 Y321N, or Con1 D320E/Y321N NS5A-YFP Huh7.5.1 cells (500,000) were exposed to increasing concentrations of telaprevir or a panel of Cyp inhibitors, including CsA, alisporivir, NIM811, SCY-635, SfA, SfB, and BC556, and analyzed for YFP content 3 days after drug treatment. The percentage of YFP-positive cells treated with the DMSO control was arbitrarily fixed at 100. Results are representative of 3 independent experiments. Error bars represent standard errors from triplicates. EC₅₀s were calculated for each Cyp inhibitor. Statistical significance was measured between each mutant construct in relation to Con1-WT NS5A-YFP for the 500 nM drug concentration. **, P < 0.01; ***, P < 0.001.