The Assembly Pathway of an Icosahedral Single-Stranded RNA Virus Depends on the Strength of Inter-Subunit Attractions |
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| Authors: | Rees F. Garmann Mauricio Comas-Garcia Ajaykumar Gopal Charles M. Knobler William M. Gelbart |
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| Affiliation: | 1 Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA;2 California NanoSystems Institute, and Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA |
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| Abstract: | 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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| Keywords: | CCMV, cowpea chlorotic mottle virus ssRNA, single-stranded RNA BMV, brome mosaic virus VLP, virus-like particle CP, capsid protein wt, wild-type EM, electron microscopy cryo-EM, cryo-electron microscopy ARM, arginine-rich motif DOL, density of labeling |
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