Using time-resolved solution small-angle X-ray scattering (TR-SAXS), stopped-flow, and flow-through setups at ESRF ID02, the assembly process of VP1, the major caspid protein of the SV40 virus, with RNA or DNA to form virus-like particles (VLPs) was studied in msec temporal resolution. By mixing the nucleotides and the capsid protein virus-like particles formed within 35 msec in the case of RNA that formed T=1 particles (where T is the triangulation number) and within 15 seconds in the case of DNA that formed T=7 particles, similar to wt SV40. The structural changes leading to the particle formation were followed in detail by TR-SAXS measurements. The RNA data set, reduced to the concentration of capsid as a function of time, was fitted to master equations that accounted for each step in assembly. The assembly was faster than the diffusion limit and was induced by electrostatic interactions between the RNA (or DNA) and the VP1 capsid protein molecules. The RNA data well fit a two-state model.

                To explain these results, we postulate that the electrostatic surface of the growing VLP attracts reactants, reducing the dimensionality of diffusion to enhance reaction rates. At early steps in the assembly of a given particle, the RNA can act as an antenna to attract protein subunits. If the RNA, however, was the only antenna, the reaction would slow down as it proceeded, which we didn’t observe. In fact, we saw no evidence of accumulating intermediates. For the reaction to proceed a pace, there needs to be another antenna. The growing capsid has a negatively charged exterior that act as a spherical antenna. Thus, the area of the antenna became progressively larger as access to the reactant binding sites became smaller. This explanation has immediate applicability to numerous viruses and is particularly relevant to the crowded environment in a cell where localizing reactants and enhancing diffusion has obvious advantages (JACS 2012). More recently, the effect of salt and pH on the assembly reaction was studied by both experiments and simulations (ACS Nano 2020).