Visualization of Calcium Ion Loss from Rotavirus during Cell Entry

Bound calcium ions stabilize many nonenveloped virions. Loss of Ca²⁺ from these particles appears to be a regulated part of entry or uncoating. The outer layer of an infectious rotavirus triple-layered particle (TLP) comprises a membrane-interacting protein (VP4) anchored by a Ca²⁺-stabilized protein (VP7). Membrane-coupled conformational changes in VP4 (cleaved to VP8* and VP5*) and dissociation of VP4 and VP7 accompany penetration of the double-layered inner capsid particle (DLP) into the cytosol. Removal of Ca²⁺in vitro strips away both outer layer proteins; we and others have postulated that the loss of Ca²⁺ triggers molecular events in viral penetration. We have now investigated, with the aid of a fluorescent Ca²⁺ sensor, the timing of Ca²⁺ loss from entering virions with respect to the dissociation of VP4 and VP7. In live-cell imaging experiments, distinct fluorescent markers on the DLP and on VP7 report on outer layer dissociation and DLP release. The Ca²⁺ sensor, placed on VP5*, monitors the Ca²⁺ concentration within the membrane-bound vesicle enclosing the entering particle. Slow (1-min duration) loss of Ca²⁺ precedes the onset of VP7 dissociation by about 2 min and DLP release by about 7 min. Coupled with our previous results showing that VP7 loss follows tight binding to the cell surface by about 5 min, these data indicate that Ca²⁺ loss begins as soon as the particle has become fully engulfed within the uptake vesicle. We discuss the implications of these findings for the molecular mechanism of membrane disruption during viral entry.IMPORTANCE 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 Ca²⁺ sensor, covalently linked to one of the viral proteins, allows us to establish, using live-cell imaging, the timing of Ca²⁺ loss from an entering particle and other molecular events in the entry pathway. Specific Ca²⁺ binding stabilizes many other viruses of eukaryotes, and Ca²⁺ 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.