Ionic strength- and temperature-induced K(Ca) shifts in the uncoating reaction of rotavirus strains RF and SA11: correlation with membrane permeabilization

Abstract

The hydrodynamic diameters of native rotavirus particles, bovine RF and simian SA11 strains, were determined by quasielastic light scattering. By using this method and agarose gel electrophoresis, the Ca(2+) dissociation constant, K(Ca), governing the transition from triple-layer particles (TLPs) to double-layer particles (DLPs), was shown to increase, at constant pH, as the temperature and/or the ionic strength of the incubation medium increased. We report the novel observation that, under physiological conditions, K(Ca) values for both RF and SA11 rotaviruses were well above the intracytoplasmic Ca(2+) concentrations of various cells, which may explain why TLP uncoating takes place within vesicles (possibly endosomes) during the entry process. A correlation between TLP uncoating and cell membrane permeabilization was found, as shown by the release of carboxyfluorescein (CF) from CF-loaded intestinal brush-border membrane vesicles. Conditions stabilizing the virion in the TLP form inhibited CF release, whereas conditions favoring the TLP-to-DLP transformation activated this process. We conclude that membrane permeabilization must be preceded by the loss of the outer-capsid proteins from trypsinized TLP and that physiological ionic strength is required for permeabilization to take place. Finally, the paper develops an alternative explanation for the mechanism of rotavirus entry, compatible with the Ca(2+)-dependent endocytic pathway. We propose that there must be an iterative process involving tight coupling in time between the lowering of endosomal Ca(2+) concentration, virion decapsidation, and membrane permeabilization, which would cause the transcriptionally active DLPs to enter the cytoplasm of cells.

FIG. 1.

Calcium concentrations above 300 μM prevent heat-induced uncoating of rotavirus strain RF in media of high ionic strength. TLPs suspended in the standard buffer supplemented with 50 mM NaCl and [Ca²⁺] ranging from 2 μM to 1 mM were treated as follows: (i) no treatment, standing at 20°C (Δ); (ii) heat treatment at 60°C for 90 s (▪); and (iii) treatment with 10 mM EGTA at 20°C (⋄). After each of these treatments, appropriate aliquots were used for particle size determination by quasielastic light scattering at 20°C. The results are presented as particle diameters ± standard deviations (n = 2 to 6 determinations per point) as a function of the logarithm of the [Ca²⁺] in the incubation medium. The upper and lower solid lines correspond, respectively, to the arithmetic means of the TLP and DLP diameters. With the heat-treated TLPs (▪) the two statistically different horizontal segments were found to be separated by a region (broken line) where particle size determination is impossible, so that no experimental points can be shown here. The explanation for this anomaly and further details are given in the text.

FIG. 2.

Calcium concentrations above 50 μM prevent heat-induced uncoating of rotavirus strain RF in media of low ionic strength. Analysis was by agar electrophoresis. TLPs were suspended in the standard buffer supplemented with 500 mM sorbitol and the indicated nominal calcium concentrations. Before analysis by agar gel electrophoresis at room temperature, the particles were incubated at 37°C for 5 min and then heated for an additional 90 s at 60°C (lanes b to g) or not heated (lanes a and h) as shown. TLPs and DLPs were at 1.9 and 2.4 μg of protein per well, respectively.

FIG. 3.

Effect of ionic strength, calcium concentration, and temperature on the TLP-to-DLP transition for the RF strain. Analysis was by agar electrophoresis. TLPs were pretreated for 5 min at the indicated temperatures in the standard buffer supplemented with either 500 mM sorbitol (Sorb) or a mixture of 100 mM sorbitol and 200 mM monovalent salt (either KSCN or Tris-Cl) in the presence of the indicated nominal calcium concentrations. TLPs and DLPs were at 1.6 and 1.2 μg of protein per well, respectively.

FIG. 4.

Effect of calcium concentration on rotavirus SA11 uncoating. Analysis was by agar electrophoresis. TLPs were pretreated for 15 min at 37°C in the standard buffer supplemented with 200 mM NaCl, 1 mM CaCl₂, and variable EGTA concentrations to obtain the indicated nanomolar concentrations of free Ca²⁺. For the [Ca²⁺] in the micromolar range, the particles were pretreated as described in Fig. 2. TLPs and DLPs were each at 1.4 μg of protein per well, respectively.

FIG. 5.

Effect of the calcium present in the membrane vesicle incubation medium on time-dependent rotavirus-induced CF release. CF-loaded vesicles were incubated at 37°C in the membrane buffer supplemented with 200 mM Tris-Cl in the absence (none) or the presence of micromolar concentrations of extra CaCl_2, as indicated. Purified TLPs, RF strain, kept at 4°C were added at time zero. The final vesicular and viral protein concentrations were 6 and 13 μg/ml, respectively.

FIG. 6.

Effect of the ionic strength of the incubation medium on CF release induced by either intact or predecapsidated TLPs. CF-loaded vesicles were incubated at 37°C in the membrane buffer supplemented with either 500 mM sorbitol (curves 3 and 4) or 100 mM sorbitol plus 200 mM Tris-Cl (curves 1 and 2). At time zero, TLPs, strain RF, were added either directly (curves 2 and 4) or after treatment with 10 mM EGTA (curves 1 and 3). The final vesicular and viral protein concentrations were 8.5 and 18.8 μg/ml, respectively.