Probing single-molecule dynamics in self-assembling viral nucleocapsids

All viruses on Earth rely on host cell machinery for replication, a process that involves a complex self-assembly mechanism. Our aim here is to scrutinize in real time the growth of icosahedral viral nucleocapsids with single-molecule precision. Using total internal reflection fluorescence microscopy, we probed the binding and unbinding dynamics of fluorescently-labeled capsid subunits on hundreds of immobilized viral RNA molecules simultaneously at each time point. A step-detection algorithm, combined with statistical analysis, allowed us to estimate microscopic quantities such as the equilibrium binding rate and mean residence time, which are otherwise inaccessible through traditional ensemble-averaging techniques. Additionally, we could estimate a set of rate constants modeling the growth kinetics from nonequilibrium measurements, and we observed an acceleration in growth caused by the electrostatic screening effect of monovalent salts. Single-molecule fluorescence imaging will be crucial for elucidating virus self-assembly at the molecular level, particularly in crowded, cell-like environments.