Visualization of Calcium Ion Loss from Rotavirus during Cell Entry

Abstract

Nonenveloped viruses penetrate into the cytosol of the cells that they infect by disrupting the membrane of an intracellular compartment. The molecular mechanisms of membrane disruption remain largely undefined. Functional reconstitution of infectious rotavirus particles (TLPs) from RNA-containing core particles (DLPs) and the outer layer proteins that deliver them into a cell makes these important pediatric pathogens particularly good models for studying nonenveloped virus entry. We report here how the use of a fluorescent Ca 2+ sensor, covalently linked to one of the viral proteins, allows us to establish, using live-cell imaging, the timing of Ca 2+ loss from an entering particle and other molecular events in the entry pathway. Specific Ca 2+ binding stabilizes many other viruses of eukaryotes, and Ca 2+ loss appears to be a trigger for steps in penetration or uncoating. The experimental design that we describe may be useful for studying entry of other viral pathogens.