Effect of p7 on intracellular pH in HCV replicon-bearing cells.
Intravesicular pH was measured in vesicles isolated from HCV replicon bearing cells that were subjected to rapid alkalinization as shown for Fig. 2. (A) A single representative experiment is shown demonstrating the time course of intravesicular pH changes in vesicles isolated from full-length (FL), sub-genomic (SG) and cured full-length (CFL) HCV replicons. (B) The average steady state pH change at 180 s in response to extravesicular alkalinization was determined. Data is presented as mean ± SE. n = a minimum of 6 independent vesicle preparations for each condition. Inhibitor compounds were added immediately prior to the pH shift as in Fig. 4.* indicates P≤0.05 compared to control, ** indicates P≤0.05 compared to full-length. (C) Free solution calibration of LysoSensor Yellow/Blue DND-160. Fluorochrome (100 nM) was dissolved in HEPES solution and titrated to various pH values. Aliquots (750 µl) were placed in a 25 mm cover slip chamber and the fluorescence was measured using a Nikon TiE fluorescence microscope at excitation 380 emission 525 (wavelength 1) and excitation 340 emission 440 (wavelength 2). Ratio is presented as wavelength 1/wavelength 2. (D) Sub-genomic (SG), full-length (FL) and cured full-length (CFL) HCV replicon bearing cells were loaded with the ratiometric pH sensor, LysoSensor Yellow/Blue DND-160, and imaged at the 2 wavelength pairs used for the calibration in panel C. Fluorescence ratio images are presented where the fluorescence ratio at each pixel was mapped to a pseudocolor representation based on the calibration curve. (E) Mean vesicular pH as determined from the fluorescence ratio images in multiple experiments as in panel D is shown. Data is presented as mean ± SE of cytosolic fluorescence ratio with the corresponding pH scale indicated on the right. Data represents the average of 25 cells in each of 4 independent cell preparations with the mean of each individual cell preparation counting as n = 1.* indicates P≤0.05 compared to sub-genomic replicons. Scale bar represents 20 µm. (F) Presence of highly acidic organelles was assessed in HCV replicon-bearing cells by incubation for 30 min with LysoTracker Red DND-99 (100 nM). Nuclei were counterstained with DAPI. Excitation and emission wavelengths for LysoTracker Red were excitation 560 nm and emission 607 nm, represented as red, and excitation 380 nm and emission 440 nm for DAPI, represented as blue. Acidic organelles appear as red structures. Scale bar represents 20 µm. (G) Total LysoTracker cellular fluorescence for experiments conducted as in panel F is shown. Data are presented as mean ± SE of total cellular LysoTracker fluorescence. Data represents the average of at least 25 cells in a minimum of 3 independent cell preparations with the mean of each individual cell preparation counted as n = 1. * indicates P≤0.05 compared to sub-genomic replicons.
