Probing Single-Molecule\nDynamics in Self-Assembling\nViral Nucleocapsids

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