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Journal of Virology, January 2002, p. 552-559, Vol. 76, No. 2
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.2.552-559.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Ionic Strength- and Temperature-Induced K_Ca Shifts in the Uncoating Reaction of Rotavirus Strains RF and SA11: Correlation with Membrane Permeabilization

Sandra Martin, Mathie Lorrot, Mounia Alaoui El Azher,^, and Monique Vasseur^*

Institut National de la Santé et de la Recherche Médicale, Unité 510, Faculté de Pharmacie, Université de Paris XI, 92296 Châtenay-Malabry, France

Received 28 June 2001/ Accepted 11 October 2001

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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺-dependent endocytic pathway. We propose that there must be an iterative process involving tight coupling in time between the lowering of endosomal Ca²⁺ concentration, virion decapsidation, and membrane permeabilization, which would cause the transcriptionally active DLPs to enter the cytoplasm of cells.

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* Corresponding author. Mailing address: Unité 510, INSERM, Faculté de Pharmacie, Université de Paris XI, 5, rue J.-B. Clément, 92296 Châtenay-Malabry, France. Phone: (33 1) 46 83 57 96. Fax: (33 1) 46 83 58 44. E-mail: monique.vasseur{at}cep.u-psud.fr.

Present address: Unité Associée Pasteur/INSERM, U485, Institut Pasteur, Paris, France.

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Journal of Virology, January 2002, p. 552-559, Vol. 76, No. 2
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.2.552-559.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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