ROTAVIRUS INTERACTION WITH ISOLATED MEMBRANE-VESICLES

To gain information about the mechanism of epithelial cell infection b y rotavirus, we studied the interaction of bovine rotavirus, RF strain , with isolated membrane vesicles from apical membrane of pig enterocy tes. Vesicles were charged with high (quenching) concentrations of eit her carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with p urified virus particles. Purified single-shelled particles and untryps inized double-shelled ones had no effect. Trypsinized double-shelled v irions 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 i soosmotic salt-free sorbitol media. Other membrane vesicle preparation s such as apical membranes of piglet enterocyte and rat placenta syncy tiotrophoblasts, basolateral membranes of pig enterocytes, and the und ifferentiated plasma membrane of cultured MA103 cells all gave qualita tively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release i s VP5. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ sol ubilization of the outer capsid of trypsinized double-shelled particle s changed release kinetics from sigmoidal to hyperbolic and was not in hibited by Ca2+. Our results indicate that membrane destabilization ca used by trypsinized outer capsid proteins of rotavirus leads to fluoro phore release. From the data presented here, a hypothetical model of t he interaction of the various states of the viral particles with the m embrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into t he host cell.