Martin Sandra, Lorrot Mathie, El Azher Mounia Alaoui, Vasseur Monique
Institut National de la Santé et de la Recherche Médicale, Unité 510, Faculté de Pharmacie, Université de Paris XI, 92296 Châtenay-Malabry, France.
J Virol. 2002 Jan;76(2):552-9. doi: 10.1128/jvi.76.2.552-559.2002.
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.
通过准弹性光散射测定了天然轮状病毒颗粒、牛RF株和猴SA11株的流体动力学直径。使用该方法和琼脂糖凝胶电泳表明，在恒定pH值下，随着孵育介质温度和/或离子强度的增加，控制从三层颗粒（TLP）向双层颗粒（DLP）转变的Ca(2+)解离常数K(Ca)会升高。我们报告了一项新的观察结果，即在生理条件下，RF和SA11轮状病毒的K(Ca)值远高于各种细胞的胞质内Ca(2+)浓度，这可能解释了为什么在进入过程中TLP脱壳发生在囊泡（可能是内体）内。如从负载羧基荧光素（CF）的肠刷状缘膜囊泡中释放CF所示，发现TLP脱壳与细胞膜通透性之间存在相关性。稳定病毒体TLP形式的条件会抑制CF释放，而有利于TLP向DLP转变的条件会激活这一过程。我们得出结论，膜通透性必须先于胰蛋白酶处理的TLP外层衣壳蛋白的丢失，并且通透性发生需要生理离子强度。最后，本文对轮状病毒进入机制提出了另一种解释，与Ca(2+)依赖的内吞途径相符。我们提出，必须存在一个迭代过程，涉及内体Ca(2+)浓度降低、病毒体脱壳和膜通透性之间在时间上的紧密耦合，这将导致转录活性DLP进入细胞细胞质。
