Radtke Kerstin, Anderson Fenja, Sodeik Beate
Département de Pathologie et Biologie Cellulaire, Université de Montréal, 2900 Blvd. Édouard-Montpetit Montréal, QC, Canada.
Methods Mol Biol. 2014;1144:191-208. doi: 10.1007/978-1-4939-0428-0_13.
Since viruses are obligate intracellular parasites, viral particles, subviral structures, and viral proteins enlist the support of host proteins to foster intracellular transport, viral gene expression, replication, and evasion from antiviral host responses. We have devised a biochemical in vitro method to analyze specific interactions of cytosolic factors with capsids of herpes simplex virus and to characterize host proteins that specifically coprecipitate with different types of viral particles by immunoblotting, mass spectrometry, and immunoelectron microscopy. Our method bridges the gap between assays such as co-immunoprecipitation and yeast-two-hybrid approaches that determine direct binding between individual subunits of protein complexes and microscopy methods that analyze the dynamic interplay between intact viral particles and host factor complexes in intact cells. Our protocol can be extended to functional analyses of herpesvirus capsids and other viral structures with more complex host structures such as microtubule transport, genome uncoating at nuclear pores, or capsid envelopment at host membranes.
由于病毒是专性细胞内寄生虫，病毒颗粒、亚病毒结构和病毒蛋白需要宿主蛋白的支持来促进细胞内运输、病毒基因表达、复制以及逃避宿主的抗病毒反应。我们设计了一种体外生化方法，用于分析胞质因子与单纯疱疹病毒衣壳的特异性相互作用，并通过免疫印迹、质谱和免疫电子显微镜来鉴定与不同类型病毒颗粒特异性共沉淀的宿主蛋白。我们的方法填补了诸如免疫共沉淀和酵母双杂交方法等检测之间的空白，这些方法用于确定蛋白质复合物单个亚基之间的直接结合，而显微镜方法则用于分析完整细胞中完整病毒颗粒与宿主因子复合物之间的动态相互作用。我们的方案可以扩展到对疱疹病毒衣壳和其他具有更复杂宿主结构的病毒结构进行功能分析，如微管运输、核孔处的基因组脱壳或宿主膜处的衣壳包裹。
