Application of band centrifugation to the study of the assembly of alfalfa mosaic virus

A band‐centrifugation method for the analysis of an assembly reaction of a simple virus from its RNA and protein is described. The experiment was carried out by sedimenting a band of viral RNA through a solution of depolymerized coat protein. The resulting radial distribution of the reaction products, followed as a function of time, was analyzed by a computer simulation of the series of reaction. This method is based on a numerical solution of the continuity equation for the sedimentation–diffusion process [Claverie, J.‐M., Dreux, H. & Cohen, R. (1975) Biopolymers 14, 1685–1700; Cohen, R. & Claverie, J.‐M. (1975) Biopolymers 14, 1701–1716]. A numerical method for the simulation of the chemical reaction is derived. From the simulated reaction series, equilibrium constants emerge for the successive addition of protein subunits to the growing nucleoprotein particle. The method is applied to the assembly of alfalfa mosaic virus. If the reaction between RNA and protein is carried out in 0.32M CsCl, pH 7.0, two stages during particle growth are resolved, each characterized by an equilibrium constant K. The determined values for K range from 5 × 105 to 3 × 106 l. mol−1. The existence of these two stages may have a structural implication in the assembly, as they likely represent an elongation and a termination stage. If the reaction is carried out under more favorable conditions (0.25M CsCl, pH 7.0), a kinetic constant of at least 105 l. mol−1 sec−1 is derived for each reaction step. Under these conditions the assembly appears to be completed within 1 min, which is too fast to detect distinct stages by band sedimentation.