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

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• 作者：Rees F. Garmann ; Mauricio Comas-Garcia ; Ajaykumar Gopal ; Charles M. Knobler ; William M. Gelbart
• 关键词：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
• 刊名：Journal of Molecular Biology
• 出版年：6 March, 2014
• 年：2014
• 卷：426
• 期：5
• 页码：1050-1060
• 全文大小：1800 K

文摘

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鈥攂y first turning on CP-RNA attraction and then turning on CP-CP attraction鈥攁llows 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.