Opening of size-selective pores in endosomes during human rhinovirus serotype 2 in vivo uncoating monitored by single-organelle flow analysis

Abstract

The effect of virus uncoating on endosome integrity during the early steps in viral infection was investigated. Using fluid-phase uptake of 10- and 70-kDa dextrans labeled with a pH-dependent fluorophore (fluorescein isothiocyanate [FITC]) and a pH-independent fluorophore (cyanine 5 [Cy5]), we determined the pHs of labeled compartments in intact HeLa cells by fluorescence-activated cell sorting analysis. Subsequently, the number and pH of fluorescent endosomes in cell homogenates were determined by single-organelle flow analysis. Cointernalization of adenovirus and 70-kDa FITC- and Cy5-labeled dextran (FITC/Cy5-dextran) led to virus-induced endosomal rupture, resulting in the release of the marker from the low-pH environment into the neutral cytosol. Consequently, in the presence of adenovirus, the number of fluorescent endosomes was reduced by 40% compared to that in the control. When human rhinovirus serotype 2 (HRV2) was cointernalized with 10-and 70-kDa FITC/Cy5-dextrans, the 10-kDa dextran was released, whereas the 70-kDa dextran remained within the endosomes, which also maintained their low pH. These data demonstrate that pores are generated in the membrane during HRV2 uncoating and RNA penetration into the cytosol without gross damage of the endosomes; 10-kDa dextran can access the cytosol through these pores. Whereas rhinovirus-mediated pore formation was prevented by the vacuolar ATPase inhibitor bafilomycin A1, adenovirus-mediated endosomal rupture also occurred in the presence of the inhibitor. This finding is in keeping with the low-pH requirement of HRV2 infection; for adenovirus, no pH dependence for endosomal escape was found with this drug.

FIG. 1.

Experimental setup for FACS analysis of intact cells and SOFA of cellular organelles. FITC/Cy5-dextran was internalized into HeLa cells in the absence of virus (left) and in the presence of virus for 20 min (right). Cells were cooled and washed, the fluorescence intensities of the two fluorophores were determined by FACS analysis of intact cells, and the FITC fluorescence/Cy5 fluorescence ratio was calculated. An aliquot of the control sample was used to obtain a calibration curve. The endosomal pH was equilibrated with buffers with known pHs in the presence of azide and ammonium acetate, and the fluorescence ratios of the fluorophores were plotted against the pHs. From this plot, the average pH of all labeled compartments in intact cells was derived. To selectively determine the pH (and the number) of the endocytic compartments, cells were homogenized and nuclei were removed by centrifugation to obtain a PNS. The PNS then was analyzed by SOFA.

FIG. 2.

SOFA and selection of gates. (A and B) The PNS from unlabeled HeLa cells was analyzed in SSC versus FSC mode (A) as well as in fluorescence mode (B). Regions corresponding to buffer and sheath fluid alone (A, dark gray area) and background fluorescence (B) were identified, and gates were set to exclude these events in the following experiments with labeled samples. (C to F) HeLa cells were labeled with 70-kDa FITC/Cy5-dextran for 20 min at 37°C, washed with ice-cold PBS, and homogenized, and the PNS was analyzed as described in Materials and Methods. (C) To differentiate large vesicles from small vesicles, an analysis window was created with the lower FSC value just above the maximum FSC value determined for sheath fluid alone. Large vesicles were defined as all events outside this window. (E and F) Distributions of large and small fluorescent vesicles (endosomes), respectively, determined with these gates. FITC- and Cy5-positive compartments were located outside the region corresponding to background fluorescence (squares at lower left). (D) Entire population of fluorescent vesicles (large plus small). The mean FITC fluorescence/Cy5 fluorescence ratio of the population of large endosomes was calculated, and the pH was determined by using a pH calibration curve. In the absence of virus, a mean pH of 5.8 was obtained.

FIG. 3.

Effect of bafilomycin A1 on endosomal pH as determined by FACS analysis and SOFA. HeLa cells were preincubated in the absence or in the presence of 200 nM bafilomycin A1 for 30 min at 37°C, transferred to fresh medium containing FITC/Cy5-dextran (70 kDa) without or with bafilomycin A1, and further incubated for 20 min. Cells were cooled and washed with PBS, and an aliquot was subjected to FACS analysis. The remaining cells were homogenized, and a PNS was prepared and analyzed by SOFA. The pH was calculated by using a pH calibration curve (see Materials and Methods). For SOFA, only large vesicles were considered. The mean and standard error of the mean for 13 experiments with eight measurements each are shown.

FIG. 4.

Ad5 ruptures endosomes regardless of the presence of bafilomycin A1. (A) FITC/Cy5-dextran (70 kDa) was internalized into HeLa cells in the absence of Ad5 (control) or in the presence of Ad5 (1,000 viral particles per cell) for 20 min at 37°C. The mean pH of labeled compartments in intact cells was calculated from the fluorescence ratio determined by FACS analysis by using a pH calibration curve. Data are the mean and standard error of the mean for 22 experiments with eight parallel determinations. (B) HeLa cells were preincubated without or with 200 nM bafilomycin A1 for 30 min at 37°C. FITC/Cy5-dextran was internalized in the absence of Ad5 (control) or in the presence of Ad5 for 20 min at 37°C without or with bafilomycin A1. Cells were homogenized, and a PNS was analyzed by SOFA. The number of large fluorescent endosomes is shown and is expressed as a percentage of the value for the respective control. Data are the mean and standard error of the mean for 16 individual experiments with eight parallel determinations. (C) The pH of fluorescent endosomes shown in panel B in the absence or in the presence of virus was calculated by using the pH calibration curve. Data are the mean and standard error of the mean for four experiments with eight parallel determinations.

FIG. 5.

HRV2 differentially affects the pHs of compartments labeled with 70- and 10-kDa dextrans. Internalization of the respective dextran was carried out in the absence of HRV2 (control) or in the presence of HRV2 at 1,000 TCID₅₀/cell at 34°C. (A) Data obtained with 70-kDa FITC/Cy5-dextran internalized for 20 min. (B) Data obtained with 10-kDa FITC/Cy5-dextran internalized for 5 and 20 min. The pH of labeled compartments in intact cells was calculated from the data obtained by FACS analysis (A and B). Data are the mean and standard error of the mean for three individual experiments with eight parallel determinations. (C) Cells from panels A and B (right panels) were homogenized, and the PNS was analyzed by SOFA. Data are expressed as a percentage of the value for the respective control and are the mean and standard error of the mean for three individual experiments, with each sample being measured eight times.

FIG. 6.

The pH of intact endosomes is unchanged by HRV2. Seventy-kilodalton FITC/Cy5-dextran was internalized into HeLa cells for 20 min in the absence of HRV2 (control) or in the presence of HRV2 at 34°C. Cells were homogenized, a PNS was prepared, and the endosomal pH was determined by SOFA. Each experiment was carried out four times, with each sample being measured eight times; data are the mean and standard error of the mean.

FIG. 7.

RNA release from HRV2 occurs at 20°C. (A) HeLa cells were preincubated in the absence or in the presence of 200 nM bafilomycin A1 for 30 min at 20°C. Next, 10-kDa FITC/Cy5-dextran was internalized for 120 min at 20°C (without or with the drug and without or with HRV2). Cells were washed, and the pH of labeled compartments in intact cells was determined by FACS analysis. (B) Subsequently, the cells were homogenized, and the number of fluorescent endosomes in the PNS was determined by SOFA. Data are the mean and standard error of the mean for three individual experiments, with each sample being measured eight times.

FIG. 8.

Schematic representation of endosomal penetration of HRV2 and release of markers with different molecular masses. Following HRV2 binding to its receptors and cointernalization of the fluid-phase marker, the cargo is delivered to early endosomes, where virus-receptor dissociation takes place. Receptors are recycled, whereas HRV2 is transported to late endosomes, where it undergoes conformational modifications at pHs below 5.6, accompanied by RNA release. Cointernalized dextran is released from the endosomes into the cytosol only if it is small enough (10 kDa) to pass through pores opened in the endosomal membrane by viral proteins. RNA-free B particles are finally delivered to and degraded in lysosomes.