Human rhinovirus type 2 uncoating at the plasma membrane is not affected by a pH gradient but is affected by the membrane potential

Abstract

The minor receptor group human rhinovirus type 2 enters host cells by endocytosis via members of the low-density-lipoprotein receptor family. In late endosomes, it undergoes a conformational change solely induced by a pH of < or =5.6, resulting in RNA transfer across the endosomal membrane into the cytoplasm. To determine potential driving forces of this process, we investigated whether RNA penetration might depend on the pH gradient and/or the membrane potential between the acidic endosome lumen and the neutral cytoplasm. Since these parameters are difficult to assess in endosomes, we took advantage of the possibility of inducing structural changes, RNA release, and consequently infection from the plasma membrane. To manipulate the pH gradient, cell-bound virus was exposed to membrane-permeant or -impermeant acidic buffers at 4 degrees C, and this was followed by a shift to 34 degrees C in medium containing bafilomycin to prevent RNA release from endosomes. To manipulate the plasma membrane potential, similar experiments were carried out, but these included K(+) diffusion potentials in the presence of the K(+) ionophore valinomycin. We demonstrated that infection does not depend on a pH gradient but is enhanced by plasma membrane hyperpolarization compared to plasma membrane depolarization.

FIG. 1.

Acetate buffer but not EPPS buffer at pH 5.3 decreases the pH_i. The time course of pH_i in HeLa cells was determined at 4°C via the pH-dependent fluorescence intensity ratio of BCECF. Fluorescence was excited at 440 nm (pH independent) and 490 nm (pH dependent) and read at 535 nm every other 10 min. The respective fluorescence intensity ratios were calculated, and the pH_i was deduced from a calibration curve. The pH_o was changed from pH 7.4 to pH 5.3 and vice versa by transferring the cells into the corresponding buffer. Where indicated, 12 μM nigericin was added (arrow). Without the ionophore, incubation in EPPS buffer (pH 5.3) did not significantly alter the pH_i for 60 min, whereas incubation in acetate buffer (pH 5.3) quickly equilibrated the pH_i and pH_o.

FIG. 2.

A pH gradient does not affect HRV2 uncoating and RNA translocation. (A) Scheme of incubation and infection conditions. Cells were preincubated with or without bafilomycin in MEM, washed, and transferred into infection medium with or without bafilomycin for incubation with HRV2 at 4°C; after 1 h, nonattached virus was washed away and the cells were transferred into HEPES buffer (pH 7.4), acetate buffer (pH 5.3) with bafilomycin, or EPPS buffer (pH 5.3) with bafilomycin and incubated for 1 h at 4°C. After transfer into infection medium, the cells were incubated at 34°C for 15 h to allow for replication. Except for in the control incubation in HEPES buffer (pH 7.4) bafilomycin was present throughout to prevent infection via the endosomal pathway. (B) Cells were stained with Hoechst dye, and viral proteins were visualized by indirect immunofluorescence microscopy. For each incubation condition, at least 50,000 cells were counted. Data are expressed as percentages of cells infected ± the standard errors of the means of three independent experiments. (C) Viral titers were determined at time zero (T = 0), and percentages of virus uncoated were calculated from the difference between infectious virus bound to the cells at pH 7.4 and the infectious virus remaining after the incubation with the pH 5.3 buffers. The means ± standard errors of the means of four independent experiments, each carried out in duplicate, are shown. (D) Infectious virus recovered at a T of 15 h. The means ± standard errors of the means from three independent experiments, each carried out in triplicate, are shown.

FIG. 3.

The plasma membrane potential can be manipulated by incubation in buffers with different K⁺ concentrations at pH 5.3 in the presence of valinomycin. (A) Confluent HeLa cells on coverslips were transferred from cold infection medium into high-K⁺ buffer (pH 5.3) or low-K⁺ buffer (pH 5.3) with or without valinomycin, containing 0.16 μCi (25 μM) [³H]TPP⁺, and incubated for 1 h at 4°C. (B) Effects of high and low extracellular K⁺ concentrations and valinomycin on the plasma membrane potential (ΨPM) of HeLa cells. Ki, intracellular potassium. (C) The amount of cell-associated [³H]TPP⁺ was determined by liquid scintillation counting and corrected for nonspecific binding of [³H]TPP⁺ to coverslips without cells. In the presence of valinomycin, the amount of cell-associated [³H]TPP⁺ was decreased in cells incubated in high-K⁺ buffer (pH 5.3) (indicative of membrane depolarization) and increased after incubation with low-K⁺ buffer (pH 5.3) (indicative for membrane hyperpolarization) compared to treatment with low-K⁺ buffer (pH 5.3) without valinomycin (set to 100%). Means ± standard errors of the means of three independent experiments, each carried out in quintuplicate, are shown.

FIG. 4.

Cellular protein synthesis is reduced by valinomycin. (A) HeLa cells were depleted of methionine and cysteine at 37°C, and this was followed by incubation in DMEM −Met/Cys containing 30 mM MgCl₂ at 4°C. Cells were then treated with the buffers as indicated, and 20 nM bafilomycin was added after 30 min. After transfer into DMEM −Met/Cys supplemented with 2% FCS at 34°C and incubation for 3 h, 1 μCi/ml [³²S]methionine-cysteine was added. After 12 h, cellular proteins were precipitated with TCA. (B) TCA-precipitable radioactivity was determined by liquid scintillation counting. The means ± standard errors of the means from quintuplicates of a typical experiment are shown.

FIG. 5.

The membrane potential affects HRV2 uncoating and RNA translocation. (A) HRV2 bound to the plasma membrane at 4°C was exposed to high-K⁺ buffer (pH 5.3) or low-K⁺ buffer (pH 5.3) in the presence of valinomycin. Bafilomycin was added and then present throughout. At time zero (T = 0), cells were shifted into infection medium and incubated for 15 h at 34°C. (B) The percentage of cells producing virus was identified via immunofluorescence microscopy and related to the total number of cells stained with Hoechst dye. The means ± standard errors of the means of four independent experiments, each carried out in duplicate, are shown. The data reveal a statistically significant difference (P ≤ 0.05; asterisk). (C) The viral titer was determined prior to (T = −60) and after (T = 0) treatment with high- and low-K⁺ buffers (pH 5.3), and the amount of virus that had uncoated was calculated from the difference between cell-bound infectious virus after incubation at pH 7.4 and that remaining after incubation at pH 5.3. (D) Infectious virus produced at 15 h after shifting to 34°C. The means ± standard errors of the means from two independent experiments, each carried out in triplicate, are shown. The difference was significant at a P value of ≤0.05 (asterisk).