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.Recent advances in cryoelectron microscopy and icosahedral image reconstruction techniques have provided a detailed view of the three-dimensional structure of rotavirus, a nonenveloped member of the Reoviridae family (25,29). Rotavirus has a triple-layered spherical structure. The core is composed of the VP2 innermost capsid protein containing a "subcore" formed by the genomic double-stranded RNA and minor proteins VP1 and VP3 (8, 9). The double-layered particle (DLP) is formed by the VP6 intermediate-capsid protein surrounding the core. Finally, the triple-layered particle (TLP) results from addition of the outermost shell formed by proteins VP7 and VP4. In the presence of trypsin, VP4 is cleaved in situ into two smaller proteins, VP5* and VP8*, conferring infectivity to the virus (12).The critical Ca 2ϩ concentration ([Ca 2ϩ ]) governing the TLP-to-DLP transformation has been shown to be strain dependent. By using agar electrophoresis, Ruiz et al. (22) have shown that complete loss of the outer-capsid proteins from TLPs (TLP decapsidation) takes place in the nanomolar range of [Ca 2ϩ ] (e.g., 600 nM for the RF strain and 10 to 20 nM for the SA11 strain) when a Ca-EGTA buffer containing 10 mM MOPS (morpholinepropanesulfonic acid) and 100 mM KCl at room temperature is used. Nevertheless, it should be emphasized...

Solar and Temperature Treatments Affect the Ability of Human Rotavirus Wa To Bind to Host Cells and Synthesize Viral RNA

“…Recently, several studies have probed the life cycle of rotavirus infection (43)(44)(45). Interestingly, some rotavirus strains (including human rotavirus Wa) undergo internalization via clathrin-mediated endocytosis to enter an endosomal compartment and then a late endosome, while other rotavirus strains can enter the host cell via a clathrin-independent process (43).…”

Impact of physicochemical parameters on in vitro assembly and disassembly kinetics of recombinant triple‐layered rotavirus‐like particles

“…The critical calcium concentration, at which VP7 is solubilized and DLP is formed, depends on the virus strain (e.g., bovine RF, SA11), ranging from 10 to 600 nM (Ruiz et al, 1996) and is directly related to rotavirus infection through the endocytic pathway (Ruiz et al, 1997). Moreover, it is known that, at constant pH, both temperature and ionic strength increase transition of TLP to DLP (Martin et al, 2002) and that this reaction can be inhibited with neutralizing antibodies against VP7 (Ludert et al, 2002).…”

Adenovirus Protein VI Mediates Membrane Disruption following Capsid Disassembly

“…First, the capsids of nonenveloped virions are assembled as immature stable structures that are rendered metastable upon proteolytic processing of capsid proteins (17). During infection, these mature metastable virions undergo conformational changes in response to external stimuli, including binding to cell receptor (46), proteolytic digestion (42,44), altered divalent cation concentrations (9,22,32), or acidification in endosomes (8,30,37). Conformational changes often expose hydrophobic residues that subsequently interact with and reorganize the membrane bilayer, enabling cytosolic translocation of the viral genome, transcription complex, or partially disassembled virus capsid.…”