Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Virology. 2010 Feb 5;397(1):199-204. doi: 10.1016/j.virol.2009.10.050. Epub 2009 Nov 25.
The capsid of SV40 virion is comprised of 72 pentamers of the major capsid protein, VP1. We examined the synergism between pentamer-pentamer interaction and pentamer-DNA interaction using a minimal system of purified VP1 and a linear dsDNA 600-mer, comparing electrophoresis with electron microscopy and size exclusion chromatography. At low VP1/DNA ratios, large tubes were observed that apparently did not survive native agarose gel electrophoresis. As the VP1 concentration increased, electrophoretic migration was slower and tubes were replaced by 200 A diameter particles and excess free pentamer. At high VP1/DNA ratios, a progressively larger fraction of particles was similar to 450 A diameter virions. VP1 association with DNA is very strong compared to the concentrations in these experiments yet, paradoxically, stable complexes appear only at high ratios of VP1 to DNA. These data suggest a DNA saturation-dependent nucleation event based on non-specific pentamer-DNA interaction that controls assembly and the ultimate capsid geometry.
SV40 病毒衣壳由 72 个五聚体的主要衣壳蛋白 VP1 组成。我们使用纯化的 VP1 和线性 dsDNA 600 -mer 的最小系统，通过电泳、电子显微镜和分子筛层析比较了五聚体-五聚体相互作用和五聚体-DNA 相互作用的协同作用。在 VP1/DNA 比例较低时，观察到明显不能在天然琼脂糖凝胶电泳中存活的大管。随着 VP1 浓度的增加，电泳迁移速度变慢，管被 200Å 直径的颗粒和过量的游离五聚体取代。在高 VP1/DNA 比例下，越来越多的颗粒类似于 450Å 直径的病毒粒子。与这些实验中的浓度相比，VP1 与 DNA 的结合非常强，但矛盾的是，仅在 VP1 与 DNA 的高比例下才出现稳定的复合物。这些数据表明，基于非特异性五聚体-DNA 相互作用的 DNA 饱和依赖性成核事件控制着组装和最终衣壳的几何形状。
