Nonequilibrium Self-Assembly Dynamics of Icosahedral Viral Capsids Packaging Genome

Abstract

The simplest icosahedral viruses can be viewed as nanometer-scaled protein shells called capsids encasing the genome. The packaging of genome is a crucial step for the survival of viruses, and it has to occur in a fast and accurate fashion. Surprisingly, little is known about the nonequilibrium dynamics that lead to infectious virions from their building blocks. Cooperative and nucleation-growth pathways have been identified in coarse-grained simulations, but even for the simplest viruses, experiments have failed so far to provide a clear-cut description of these phenomena. Here, we probed the self-assembly dynamics of icosahedral viral capsids packaging RNA genome or synthetic polyelectrolyte using time-resolved small-angle X-ray scattering with synchrotron source. We used the cowpea chlorotic mottle virus, a single-stranded RNA plant virus, whose capsid (28 nm in diameter) can self-assemble from and disassemble into dimeric subunits through long-lived intermediate species. By tuning the subunit-genome interactions through ionic strength, we found out that subunits and genome formed amorphous nucleoprotein complexes (NPCs) of ∼30 nm in size via a cooperative pathway. These NPCs in turn relaxed into virions via a nucleation-growth pathway by strengthening the subunit-subunit interactions through pH. The binding of subunits on genome could be as fast as 30 ms and the structural relaxation into virions could last several hours. Mean number of bound subunits as a function of time, binding and activation energies as well as apparent diffusion coefficient were estimated and the equilibrium species were imaged by cryo-transmission electron microscopy. Surprisingly, substituting synthetic polyelectrolyte to genome lowered the energy barrier to filled capsids. We propose a schematic free energy landscape that accounts for the observed pathways. Our results further point to the NPCs as checkpoints in the assembly pathway.