Garmann Rees F, Comas-Garcia Mauricio, Gopal Ajaykumar, Knobler Charles M, Gelbart William M
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA; California NanoSystems Institute, and Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
J Mol Biol. 2014 Mar 6;426(5):1050-60. doi: 10.1016/j.jmb.2013.10.017. Epub 2013 Oct 19.
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.
豇豆褪绿斑驳病毒（CCMV）衣壳蛋白（CPs）之间的吸引力强度受溶液pH值控制。此外，CP与单链RNA病毒基因组之间的吸引力强度受离子强度控制。通过利用这些特性，我们能够根据CP-CP和CP-RNA吸引力的强度来控制和监测CCMV CP与单链RNA的体外共组装。使用速度沉降和电子显微镜技术，我们发现抗核酸酶病毒样颗粒（VLPs）的成功组装微妙地取决于CP-CP吸引力相对于CP-RNA吸引力的强度。如果吸引力过弱，衣壳就无法形成；如果吸引力过强，组装就会陷入动力学陷阱。将该过程分为两个步骤——先开启CP-RNA吸引力，然后开启CP-CP吸引力——可以在广泛的吸引力强度范围内组装出结构良好的VLPs。这些观察结果建立了一种高效体外组装CCMV VLPs的方案，并提出了该病毒在体内可能采用的潜在策略。
