The Assembly Pathway of an Icosahedral Single-Stranded RNA Virus Depends on the Strength of Inter-Subunit Attractions

• Experiments elucidate in vitro self-assembly path for CCMV.
• pH and ionic strength control protein and protein–RNA interactions, respectively.
• Viral-size structures formed at pH 7 lack order due to weak protein interactions.
• Capsids are produced only after protein attraction is strengthened by lowering of pH.
• Molecular basis for efficient CCMV in vitro assembly suggests in vivo requirements.

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.

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