Rotavirus interaction with isolated membrane vesicles.
To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.
Ruiz
M C
MC
Centre de Recherche sur l'Endocrinologie Moléculaire et le Développement, Centre Nationale de le Recherche Schientifique, Meudon, France.
Alonso-Torre
S R
SR
Charpilienne
A
A
Vasseur
M
M
Michelangeli
F
F
Cohen
J
J
Alvarado
F
F
D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000494:pharmacology; D002213:Capsid / Q000037:antagonists & inhibitors / Q000502:physiology; D036022:Capsid Proteins; D004848:Epithelium / Q000382:microbiology; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007082:Ileum / Q000382:microbiology; D066298:In Vitro Techniques; D007583:Jejunum / Q000382:microbiology; D008871:Microvilli / Q000382:microbiology*; D011188:Potassium / Q000494:pharmacology; D012401:Rotavirus / Q000187:drug effects / Q000472:pathogenicity* / Q000502:physiology; D012400:Rotavirus Infections / Q000209:etiology* / Q000382:microbiology; D013552:Swine; D013696:Temperature
D000911:Antibodies, Monoclonal; D036022:Capsid Proteins; D005452:Fluoresceins; C060339:VP4 protein, Rotavirus; C024098:6-carboxyfluorescein; D011188:Potassium; D002118:Calcium