Csorba Tibor, Burgyán József
Istituto di Virologia Vegetale, Consiglio Nazionale dell Ricerche, Torino, Italy.
Methods Mol Biol. 2011;721:245-52. doi: 10.1007/978-1-61779-037-9_15.
The host-virus interaction is a continuous coevolutionary race involving both host defence strategies and virus escape mechanisms. RNA silencing is one of the main processes employed by eukaryotic organisms to fight viruses. However, viruses encode suppressor proteins to counteract this antiviral mechanism. Virtually all plant viruses encode at least one suppressor. In spite of being highly diverse at the protein level, a large group of these proteins inhibit RNA silencing very similarly, by sequestration of double-stranded RNA or small-interfering RNA molecules, the central players of the pathway. The RNA binding capacity of virus suppressor proteins can be studied by the electrophoretic mobility shift assay method. Also known as gel retardation assay, gel mobility assay, gel shift assay or band shift assay, EMSA is an in vitro technique used to characterize protein:DNA or protein:RNA interactions. The method had been developed based on the observation that protein: nucleic acid complexes migrate slower through a non-denaturing polyacrylamide gel than the free nucleic acid fragments. Here, we provide a detailed protocol for the analysis of crucifer-infecting Tobacco mosaic tobamovirus (cr-TMV) silencing suppressor protein p122 RNA binding capacity.
宿主与病毒的相互作用是一场持续的共同进化竞赛,涉及宿主防御策略和病毒逃逸机制。RNA沉默是真核生物对抗病毒所采用的主要过程之一。然而,病毒编码抑制蛋白来对抗这种抗病毒机制。几乎所有植物病毒都编码至少一种抑制蛋白。尽管这些蛋白在蛋白质水平上高度多样,但其中一大类蛋白通过隔离双链RNA或小干扰RNA分子(该途径的核心参与者)来非常相似地抑制RNA沉默。病毒抑制蛋白的RNA结合能力可以通过电泳迁移率变动分析方法来研究。EMSA也被称为凝胶阻滞分析、凝胶迁移分析、凝胶位移分析或条带位移分析,是一种用于表征蛋白质与DNA或蛋白质与RNA相互作用的体外技术。该方法是基于蛋白质与核酸复合物在非变性聚丙烯酰胺凝胶中比游离核酸片段迁移得慢这一观察结果而开发的。在这里,我们提供了一个详细的方案,用于分析感染十字花科植物的烟草花叶烟草花叶病毒(cr-TMV)沉默抑制蛋白p122的RNA结合能力。