Fig 1.
Characterization of purified NS5A.
(A) Coomassie staining of purified proteins. Recombinant His-tagged proteins were purified as described in Materials and Methods. 1 μg of NS5A-6HIS and NS5A-Y93H-6HIS were subjected to SDS-PAGE and the protein bands were visualized by Coomassie Blue stain. Molecular weights for protein standards are indicated. (B) Phosphorylation sites determined by mass spectrometry of purified NS5A-6HIS and NS5A-Y93H-6HIS. Phosphorylation sites identified in NS5A-6HIS are shaded green; sites identified in NS5A-Y93H-6HIS are shaded red; and sites identified in both proteins are shaded cyan. (C) Analytical ultracentrifugation analysis of NS5A-6HIS, NS5A-Y93H-6HIS, and NS5A-domain 1. For NS5A-6HIS and NS5A-Y93H-6HIS, analytical ultracentrifugation was performed in 25 mM Tris pH 7.5, 150 mM NaCl, 0.01% NaN3, 0.5 mM TCEP, 2 μg/ml Leupeptin, 0.02% C12E8, and 21.1% (v/v) D2O. Sedimentation velocity analysis was performed at 42,000 rpm for two protein concentrations (3.3 and 6.6 μM). For NS5A-domain 1, analytical ultracentrifugation was performed in 25 mM Tris pH 8.0, 250 mM NaCl, 10% glycerol, and 0.5% DMSO. Sedimentation velocity analysis was performed at 48,000 rpm for 4 protein concentrations (7.5, 12, 15, 30 μM). Representative traces are shown for each protein. (D) Circular dichroism spectroscopy of NS5A-6HIS and NS5A-Y93H-6HIS. Circular dichroism measurements of NS5A-6HIS or NS5A-Y93H-6HIS (10 μM) were carried out at 20°C. The average of 3 measurements is shown.
Fig 2.
Structures of NS5A inhibitors.
EC50 represents the 50% effective inhibitory concentration of HCV RNA replication in the Renilla luciferase replicon genotype 1b, Con1 cell line. EC50 for daclatasvir and BMS-Biotin (data not shown) were determined as previously described for ledipasvir [10].
Fig 3.
(A) Each reaction, in a final volume of 200 μl, contained 50 nM of purified NS5A-6HIS and the indicated concentration of 3H-LDV in the absence () or presence (○) of unlabeled LDV. Bound 3H-LDV was measured as described in Materials and Methods. Each data point represents the average of 4–7 assays. (B) Specific binding of 3H-LDV to NS5A-6HIS (●) vs. NS5A-Y93H-6HIS (
). Specific binding was defined as the difference between the amount of 3H-LDV bound in the absence (total binding) and presence (non-specific binding) of unlabeled LDV. Each data point represents the average of at least 3 assays.
Fig 4.
Competitive binding of 3H-LDV in the presence of unlabeled inhibitor.
Each reaction, in a final volume of 200 μl, contained 50 nM NS5A-6HIS, 30 nM 3H-LDV and the indicated concentration of unlabeled LDV (●) or DCV (). % Bound represents the amount of 3H-LDV bound relative to that in the control tube, which contained no unlabeled inhibitor. Each data point represents the average of at least 3 assays. Ki was calculated using the Cheng-Prusoff equation [30]. EC50 represents the 50% effective inhibitory concentration of HCV RNA replication in the Renilla luciferase genotype 1b, Con 1 replicon cell line.
Fig 5.
Co-precipitation of BMS-Biotin with NS5A and NS5A-Y93H.
(A) Genotype 1b, Con1 replicon cells (wild type or harboring the Y93H mutant of NS5A) were treated with 1μM DCV or 25 μM BMS-Biotin and incubated for 24 hr. Cells were harvested and the membrane fraction was solubilized with C12E8. Solubilized proteins were incubated with streptavidin agarose beads and co-precipitated material was subjected to SDS-PAGE. NS5A was visualized by immunoblot analysis using an anti-NS5A antibody. (B) Huh7-lunet cells were transfected with pcDNA3-NS5A or pcDNA3-NS5A-Y93H. 24 hr post transfection, cells were treated with the indicated amount of BMS-Biotin, incubated for 42 hr and processed as in (A).
Fig 6.
BMS-Biotin does not compete for LDV binding to NS5A.
(A) Huh7-lunet cells were transfected with pTK-NS5A. 24 hr post transfection, cells were treated for 42 hr with a fixed concentration of BMS-Biotin and increasing concentrations of LDV or DCV. Cells were harvested and the membrane fraction was solubilized with C12E8. Solubilized proteins were incubated with streptavidin agarose beads and co-precipitated material was subjected to SDS-PAGE. NS5A was visualized by immunoblot analysis using an anti-NS5A antibody. (B) The relative intensity of pelleted NS5A, normalized to total NS5A, in (A) was quantified by densitometry. % Bound represents the amount of NS5A in the pellet relative to that in the control lane, which contained no competitor. (C). Each reaction, in a final volume of 200 μl, contained 50 nM NS5A-6HIS, 30 nM 3H-LDV and the indicated concentration of unlabeled BMS-Biotin () or LDV (●). % Bound represents the amount of 3H-LDV bound relative to that in the control tube, which contained no unlabeled inhibitor. Each data point represents the average of at least 3 assays.