7 research outputs found
Time until E<sub>max</sub> = 50% for lipid peroxidation sensitive and resistant HCV clones, measured by GLuc assay.
<p>Time until E<sub>max</sub> = 50% for lipid peroxidation sensitive and resistant HCV clones, measured by GLuc assay.</p
Kinetics of HCV inhibition in infected cell culture following addition of an NS5A inhibitor, monitored using different GLuc reporter viruses.
<p>(A) Inhibition of the gt1a LPO<sup>S</sup> virus H77S.3/GLuc2A by elbasvir. (B) Inhibition of the gt2a LPO<sup>R</sup> virus JFH-1/QL/GLuc2A by elbasvir. (C) Maximum % inhibition (E<sub>max</sub>) of GLuc activity at different time points after addition of elbasvir to Huh7.5 cells infected with different cell culture infectious HCV genomes. LPO<sup>S</sup> viruses H77S.3/GLuc2A (gt1a) and N.2/GLuc2A (gt1b) are plotted in red. LPO<sup>R</sup> viruses H77D/GLuc2A (gt1a), JFH-1/QL/GLuc2A (gt2a) or HJ3-5/GLuc2A (gt1a/2a chimera). (D) Inhibition of the gt1a LPO<sup>R</sup> virus H77D-GLuc2A by elbasvir. (E) Fitness of different virus genomes used in (C) at 72 h post electroporation. Data shown represents GLuc activity relative to GLuc activity at 6 hours post electroporation to normalize for transfection efficiency. The replication incompetent reporter viruses H77S/AAG/GLuc2A and JFH-1/GND/GLuc2A contain point mutations in the NS5B polymerase and are included as mock-transfection controls.</p
Differences in kinetics of virus inhibition in H77S.3- vs H77D–infected cells following addition of an NS5A inhibitor are not due to differences in NS5A sequence.
<p>(A) Diagram of the H77S.3 genome showing positions of the 12 amino acids that differ between H77S.3 and H77D. The two amino acid changes in the NS5A coding region (I2204S and D2416G) are highlighted. (B) Maximum % inhibition (E<sub>max</sub>) at different time points after addition of elbasvir to Huh7.5 cells infected with either H77S.3/GLuc2A, H77D/GLuc2A or H77S.3/GLuc2A carrying either the I2204S mutation alone (H77S.3/IS) or in combination with D2416G (H77S.3/IS/DG). (C) Fitness of different virus genomes used in (B) at 72 h post electroporation. Data shown represents GLuc activity relative to GLuc activity at 6 h post electroporation to normalize for transfection efficiency. The replication incompetent reporter virus H77S/AAG/GLuc2A contains point mutations in the NS5B polymerase and serves as a mock-transfection control.</p
Slower kinetics of RNA synthesis inhibition by an NS5A inhibitor for H77S.3 compared to H77D.
<p>(A) Inhibition of RNA synthesis measured in cultures of Huh7.5 cells infected with either H77S.3 (left panel) or H77D (right panel) following treatment with elbasvir. (B) Maximum % inhibition (E<sub>max</sub>) of RNA synthesis at different time points after addition of elbasvir to Huh7.5 cells infected with either H77S.3 or H77D. (C) Measurements of dsRNA foci per cell in Huh7.5 cells infected with either H77S.3 (left panel), H77D (middle panel) or mock-infected cells (right panel). Cells were either mock-treated or treated with 10x EC90 elbasvir for 12 or 24 h before being fixed and stained using a dsRNA specific antibody. Foci of dsRNA per cell were quantified using MetaMorph Image analysis software. Treatment groups were analyzed by Kolmogorov-Smirnoff test (n.s.: not significant, ** p = 0.003, ***p<0.001).</p
Results of the mathematical model fitted to data measuring the decline of GLuc-reporter activity from the H77S.3 or H77D variants under 10nM elbasvir treatment.
<p>Data and simulation using best fit parameter values are shown as ‘x’s and lines, respectively. The data and simulation shown here represent the 10nM elbasvir treatment from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006343#ppat.1006343.s005" target="_blank">S5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006343#ppat.1006343.s008" target="_blank">S8</a> Figs. Here, they are overlaid on the same graph to highlight the transient increase in GLuc activity observed at 4-8h following NS5A inhibitor addition to cell cultures infected with H77S.3/GLuc2A but not H77D/GLuc2A.</p
Schematics for intracellular HCV replication dynamics considered in this study and the corresponding mathematical model.
<p>We consider the dynamics of positive strand HCV RNAs (+RNAs) in the ER (T), in the membranous web (R) and in the lipid droplets (A), the replicase complex (C) and the extracellular <i>Gaussia</i> luciferase (GLuc) proteins (G). See text for detailed explanations.</p
Descriptions of parameters and their values in the mathematical model.
<p>Descriptions of parameters and their values in the mathematical model.</p