Characterization of HCV persistently-infected cultures.

<p>(A) Examples of the level of HCV(1b/2a)-infected cells <i>vs</i>. uninfected cells after 21 days with or without replication inhibitor/entry inhibitor combination therapy. The yellow cells are infected (<i>i.e.</i> stained with anti-NS5A Ab as described in the Materials and Methods) and the blue cells are uninfected. The pluses signify relative quantifications of the percentage of infected cells in each culture (see Materials and Methods). (B) Bar graph depicting quantification of infected <i>vs</i>. uninfected cells in the untreated HCV persistently-infected culture shown in Figure. 1A (see Materials and Methods). (C) Viability of HCV persistently-infected cells after 25 days of incubation (see Materials and Methods). (D) Extracellular HCV levels (log₁₀ RNA copies/ml) during an 18-day time course initiated 7 days post infection (average of 3 assays) (HCV(2a) levels (solid circles), HCV(1b/2a) levels (solid squares, dashed line)).</p

Reduction of HCV(1b/2a) levels by EI-1 alone and in combination with BILN-2061 or BMS-790052.

<p>HCV(1b/2a) persistently-infected cell cultures were treated with 5×EC₅₀ concentrations of the indicated HCV inhibitors for 20 days (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065273#pone-0065273-t001" target="_blank">Table 1</a> and Materials and Methods). HCV levels were normalized relative to the level of the DMSO control at each time point. This data is the average of 3 assays. Error bars represent standard deviation. Asterisks indicate statistically significant differences at day 20 from the DMSO day 20 time point (<i>t</i> test P≤0.05). DMSO (solid circles and solid line), EI-1 (pierced diamonds and dashed line), BILN-2061 (solid squares and dashed line), BMS-790052 (pierced squares and solid line), BILN-2061/EI-1 (diamonds and solid line),BMS-790052/EI-1 (solid hexagons and solid line), and BILN 2061/BMS-790052 (pierced diamonds and dotted line).</p

Reduction of HCV(2a) levels by anti-CD81 Ab alone and in combination with BILN-2061 or BMS-790052.

<p>HCV(2a) persistently-infected cell cultures were treated with 5×EC₅₀ concentrations of the indicated HCV inhibitors for 21 days (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065273#pone-0065273-t001" target="_blank">Table 1</a> and Materials and Methods). HCV levels were normalized relative to the level of the DMSO control at each time point. This data is the average of 3 assays. Error bars represent standard deviation. Asterisks indicate statistically significant differences at day 21 from the DMSO day 21 time point (<i>t</i> test P≤0.05). (A) DMSO (solid circles and solid line), anti-CD81 Ab (pierced circles and dashed line), BILN-2061 (solid squares and dashed-dot line), BMS-790052 (pierced squares and solid line), BILN-2061/anti-CD81 Ab (solid diamonds and solid line), BMS-790052/anti-CD81 Ab (solid hexagons and solid line), and BILN-2061/BMS-790052 (pierced diamonds and dashed line).</p

Summary of HCV(1b/2a) Levels After 21 Days of Treatment +/− Anti-CD81 Ab.

a<p>The percentage of infected cells was estimated after anti-NS5A Ab staining (see Materials and Methods).</p

Summary of Antiviral Assay Results for the HCV Inhibitors Used in this Work.

a<p>All values are reported in nM unless otherwise indicated.</p>b<p>The HCV(1b/2a) chimeric virus expresses HCV(1b) envelope proteins and HCV(2a) replicative proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065273#pone.0065273-Chan1" target="_blank">[26]</a>.</p>c<p>Not determined.</p

Reduction of HCV(1b/2a) levels by anti-CD81 Ab alone and in combination with BILN-2061 or BMS-790052.

<p>HCV(1b/2a) persistently-infected cell cultures were treated with 5×EC₅₀ concentrations of the indicated HCV inhibitors for 21 days (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065273#pone-0065273-t001" target="_blank">Table 1</a> and Materials and Methods). HCV levels were normalized relative to the level of the DMSO control at each time point. This data is the average of 3 assays. Error bars represent standard deviation. Asterisks indicate statistically significant differences at day 21 from the DMSO day 21 time point (<i>t</i> test P≤0.05). (A) DMSO (solid circles and solid line), anti-CD81 Ab (pierced circles and dashed line), BILN-2061 (solid squares and dashed line), BMS-790052 (pierced squares and solid line), BILN-2061/anti-CD81 Ab (solid diamonds and solid line), BMS-790052/anti-CD81 Ab (solid hexagons and solid line), and BILN 2061/BMS-790052 (pierced diamonds and dashed line).</p

Summary of HCV(1b/2a) Levels After 20 Days of Treatment +/− EI-1.

a<p>The percentage of infected cells was estimated after anti-NS5A Ab staining (see Materials and Methods).</p

Summary of HCV(2a) Levels After 21 Days of Treatment +/− Anti-CD81 Ab.

a<p>The percentage of infected cells was estimated after anti-NS5A Ab staining (see Materials and Methods).</p

Summary of the Resistance Mutations Observed After 3 weeks of HCV Inhibitor Treatment.

a<p>These resistance mutations were observed in 1/5 sequenced clones.</p>b<p>The HCV(1b/2a) chimeric virus expresses HCV(1b) envelope proteins and HCV(2a) replicative proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065273#pone.0065273-Chan1" target="_blank">[26]</a>.</p>c<p>Not determined.</p

Effect of 17-AAG and CsA on HCV replication and viability determined by intracellular esterase activity.

<p>Antiviral activity (measured using the Renilla luciferase encoded by the HCV replicon; gray) and cell viability (measuring through the cleavage of calcein-AM by intracellular esterases; black) were assessed as a function of dose in a three day assay for the following molecules: (A) the HCV polymerase inhibitor HCV-796, (B) the ribosomal inhibitor puromycin, (C) the cyclophilin inhibitor CsA, (D) the microtubule inhibitor colchicine, (E) the anti-metabolite gemcitabine, and (F) the HSP90 inhibitor 17-AAG.</p