Figure 1.
Preparation and pH indicator loading of p7-containing membrane vesicles.
HEK-293FT cells were transiently transfected with either FLAG-p7 or FLAG-p7KR, homogenized 24 h later, and subjected to an initial centrifugation at 3,000× g followed by ultracentrifugation of the supernatant at 120,000× g. (A) Western blot of the cytosolic extract (3k Sup), heavy membrane fraction (3k Pellet) and light membrane fraction (120k pellet) showing that FLAG-p7 (left panel) and FLAG-p7KR (right panel) were present in whole cell extracts as well as in the heavy membrane and light membrane fractions. Marker proteins were LAMP-2 (lysosomal/late endosomal protein), PDI (ER chaperone) and GRP75 (mitochondrial chaperone). (B) HPTS (100 nM solution) was titrated to varying pH values from 4.5 to 10.5 and fluorescence emission (F) at 520 nm was determined using excitation wavelengths of 450 and 405 nm. The ratio of F450/405 is presented as a function of pH. Dotted lines represent the linear range. (C) Frozen 120k vesicle pellet was thawed in the presence of HPTS and re-homogenized as described in Materials and Methods. The mixture was applied to a Bio-Gel P-10 size exclusion column and eluted with constant flow. The column void volume eluted at approximately fraction 28. The right panel shows both fluorescence and protein content of fractions over the whole column profile while the left panel shows results around the void volume only with a magnified ordinate. Solid symbols (•) represent fluorescence; bars indicate protein concentration. (D) Protocol was similar to that shown in C except that the vesicle prep was first exposed to Triton X-100 prior to gel filtration. In this experiment the void volume eluted approximately at fraction 15. Note the absence of a fluorescence peak associated with the eluted protein.
Figure 2.
Measurement of Intravesicular pH.
HPTS-loaded vesicle suspensions or HPTS solution without vesicles were subjected to rapid alkalinization by sudden injection of 10 µl of 50 mM KOH into a total volume of 100 µl. Fluorescence ratio (F450/F405) and corresponding pH was measured at 1 s intervals in a fluorescence plate reader. (A) Comparison of pH changes of HPTS in free solution (gray circles) vs. in suspensions of HPTS-containing vesicles (gray triangles). Free solution rapidly equilibrated to pH 8.0 while vesicles alkalinized more slowly and failed to equilibrate with the extravesicular solution pH during the observation period. Inclusion of DPX, a membrane impermeant fluorescence quenching agent (black triangles) did not affect intravesicular pH changes. (B) Effects of DPX on extracellular solution fluorescence. Absolute fluorescence, F450, was measured in HPTS solution before and after alkalinization. The presence of DPX (15 mM) completely quenched fluorescence of this fluorochrome. (C) Steady state pH values measured at 240 s from a series of experiments performed as in panel A. Data is presented as mean ± SE, n = 4 independent vesicle preparations. (D) Measurement of the effect of the protonophore FCCP on intravesicular pH changes. HPTS-loaded vesicles were incubated with or without FCCP (4 µM) added either immediately prior to (red line) or 4 min after (green line) the pH shift. FCCP had no effect on free solution pH as shown in the black and grey curves. (E) Steady state pH values as described for panel C. n = 4 independent membrane preparations. * indicates P≤0.05 compared to control vesicles.
Figure 3.
Conductive properties of HCV p7 proteins.
Vesicle pellets were isolated from HEK-293FT cells that were transfected with either genotype 1b p7 (J4 sequence), genotype 2a p7 (JFH1 sequence), the corresponding channel inactive mutants p7K33A/R35A (p7KR) and p7R33A/R35A (p7RR) or mock control vector. Vesicles were loaded with HPTS as described in Materials and Methods and subjected to rapid alkalinization by sudden injection of KOH at time zero according to the protocol shown in Fig. 2. Where indicated, FCCP (4 µM) was added immediately prior to the addition of KOH. (A) A single representative experiment is shown demonstrating the time course of intravesicular pH changes in control, p7- or mutant p7-expressing vesicles. (B) The average steady state pH change at 200 s was determined as in A, in response to extravesicular alkalinization. Data is presented as mean ± SE. n = a minimum of 6 independent vesicle preparations for each condition. * indicates P≤0.05 compared to control vesicles with no FCCP.
Figure 4.
Sensitivity of p7-associated pH changes to known viroporin inhibitors.
HPTS loaded vesicles were subjected to rapid alkalinization by sudden injection of KOH as described for Figs. 2 and 3. The H+ conductance induced by the J4/1b p7 (A) and JFH1/2a/p7 (B) was inhibited by several viroporin inhibitors. Data is presented for each of the two genotypes as mean ± SE of the steady state pH change observed in control, p7-expressing vesicles (WT) and in p7 containing vesicles in which inhibitor compounds (1 µM) were added immediately prior to the pH shift. Ama = amantadine, Rim = rimantadine and HMA = hexamethylene amiloride. n = a minimum of 3 independent vesicle preparations. * indicates P≤0.05 compared to control, ** indicates P≤0.05 compared to wild-type.
Figure 5.
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.
Figure 6.
p7 channel activity within infected cells.
(A) Huh-7.5 cells were infected with HJ3-5 virus (HCVcc) or electroporated with parental HJ3-5 or mutant HJ3-5(KR) HCV RNA and assayed after 3 days. Cells were subsequently incubated with LysoTracker Red DND-99 as described in Materials and Methods and viral replication was detected by immunostaining for core protein (green). Representative images showing both infected and non-infected cells are shown for each condition. Scale bar represents 20 µm. (B) Mean ± SE of total cellular LysoTracker fluorescence. Total magnitude of red fluorescence was calculated separately for non-infected cells and for cells infected with each of the different virus constructs as described for Fig. 5G. Data represents the average of at least 6 cells in each of 4 independent cell preparations with the mean of each individual cell preparation counted as n = 1. * indicates P≤0.05 compared to uninfected cells.
Figure 7.
Effects of viroporin inhibitors on intracellular vesicular pH and infectious virus production.
(A) The ability of inhibitors to affect p7 channel function in vivo was assessed in full-length replicon bearing cells loaded with LysoSensor Yellow/Blue DND-160. Amantadine or rimantadine were added directly to LysoSensor loaded cells and the cells were imaged after 5 min. Fluorescence ratio images are presented as for Fig. 5D and pH is represented by pseudocolor mapping as indicated. Scale bar represents 20 µm. (B) Average pH changes from a minimum of 3 experiments performed as in panel A. pH values were determined from fluorescence ratios by interpolation on the calibration curve as shown in Fig. 5. FL = full-length, SG = sub-genomic and CFL = cured full-length HCV replicons. (C) Comparison of the effect of various concentrations of amantadine on both intravesicular pH (data from panel B) and infectious virus present in the extracellular culture medium. For measurement of virus titers, Huh-7.5 cells were incubated with the indicated concentration of amantadine for 24 hours prior to collection of medium and tittering by the FFU assay as described in Materials and Methods. (D) Intra- and extracellular infectious virus production after amantadine treatment. Huh-7.5 cells were infected with HJ3-5 virus and treated with amantadine as described in Materials and Methods. Medium was collected for determination of infectious virus titer by FFU assay (Extracellular), and cell pellets were washed, disrupted by freeze-thaw and then assayed similarly (Intracellular). Bars represent the proportion of the total infectious virus yield that was present in each compartment.
Figure 8.
Bafilomycin A1 rescue of infectious virus production.
(A) Concentration dependence of bafilomycin A1 on intravesicular pH in Huh-7.5 cells. Cells were loaded with the single wavelength pH indicator, LysoSensor Green DND-189, and treated with various concentrations of bafilomycin A1 as indicated. The increase in vesicular pH was measured as a decrease in fluorescence intensity at Ex/Em 443/510. Each data point represents mean ±SE from a minimum of 10 cells. (B) HCVcc (MOI 1.0) was used to infect naïve Huh-7.5 cells in the presence of varying concentrations of bafilomycin A1. Cells were then fixed and immunostained for core protein 3 days later. Relative infectivity was determined by measuring the intensity of core immunostaining normalized to cell number (see methods). (C) Cytotoxicity of bafilomycin A1 in Huh7.5 cells. Cells were cultured in the presence of various concentrations of bafilomycin A1 for 72 hrs. Cell viability was determined using CellTiter-Blue Reagent (see methods). Data are presented as the number of live cells, with untreated (no bafilomycin A1) set to 100%. (D) Huh-7.5 cells were electroporated with parental HJ3-5, mutant HJ3-5(KR) or HJ3-5(Δp7) HCV RNA and allowed to grow for 96 h. The cells were then treated with 2.5 pM bafilomycin A1 for 24 h after which time, intra and extracellular virus was collected and used to infect naïve Huh-7.5 cells as described in Materials and Methods. Wild-type virus supernatants were diluted 10-fold, KR and Δp7 supernatants were undiluted. These cells were immunostained for core protein (green). Example images of the formation of FFUs in each condition are presented. The scale bar represents 200 µm. (E) Titers of intra- and extracellular virus production in the presence or absence of 2.5 pM bafilomycin were determined from 4 experiments conducted as in panel D. No FFU were detected in the KR virus in the absence of bafilomycin in any of the experiments. (F) Western blot for the presence of core protein in whole cell lysates prepared from Huh-7.5 cells electroporated with WT (HJ3-5), KR (HJ3-5(KR)) or Δp7 (HJ3-5(Δp7)) HCV RNA.
Figure 9.
Influenza M2 protein can trans-complement an HCV p7 channel mutant.
(A) Huh-7.5 cells were loaded with the ratiometric pH sensor LysoSensor Yellow/Blue DND-160 and imaged as described in Materials and Methods. Mean vesicular pH as determined from the fluorescence ratio in multiple experiments is shown. Data is presented as pH ± SE. Data represents the average of 20 cells in each of 3 independent cell preparations with the mean of each individual cell preparation counting as n = 1. * indicates P≤0.05 compared to empty vector. (B) Huh-7.5 cells were co-electroporated with mCherry-M2 or the channel inactive mCherry-M2(A30P) and one of three HCV RNAs: wild-type JFH-1, channel inactive JFH-1(RR) or polymerase null JFH-1(GND) HCV RNA as described in Materials and Methods. Supernatants were collected and assayed for infectious virus titer by FFU determination. Where indicated, cells were treated with 50 µM amantadine for the final 24 hrs prior to harvesting the medium. Data are represented as mean ± SE from three independent experiments.
Figure 10.
Intracellular infectious virus is acid sensitive.
Huh-7.5 cells were infected with JFH-1 virus. After five days, cell culture supernatants (extracellular virus) and freeze-thaw lysates (intracellular virus) were collected and diluted 1∶1 in MES/HEPES buffer to give final pH values of 7, 6, 5 and 4. After a ten min incubation at 37°C, the viral titers were determined by FFU assay. Data are represented as mean ± SE from three independent experiments.