The strength of attraction between capsid proteins (CPs) of cowpea chlorotic mottle virus (CCMV) is controlled by the solution pH. Additionally, the strength of attraction between CP and the single-stranded RNA viral genome is controlled by ionic strength. By exploiting these properties, we are able to control and monitor the in vitro co-assembly of CCMV CP and single-stranded RNA as a function of the strength of CP-CP and CP-RNA attractions. Using the techniques of velocity sedimentation and electron microscopy, we find that the successful assembly of nuclease-resistant virus-like particles (VLPs) depends delicately on the strength of CP-CP attraction relative to CP-RNA attraction. If the attractions are too weak, the capsid cannot form; if they are too strong, the assembly suffers from kinetic traps. Separating the process into two steps-by first turning on CP-RNA attraction and then turning on CP-CP attraction-allows for the assembly of well-formed VLPs under a wide range of attraction strengths. These observations establish a protocol for the efficient in vitro assembly of CCMV VLPs and suggest potential strategies that the virus may employ in vivo.
Keywords: cowpea chlorotic mottle virus; packaging; self-assembly; ssRNA; virus-like particle.