Department of Life and Environmental Sciences, University of Cagliari, Cittadella di Monserrato SS554, 09042 Monserrato, Cagliari, Italy.
Antiviral Res. 2012 Mar;93(3):354-63. doi: 10.1016/j.antiviral.2012.01.005. Epub 2012 Jan 25.
The Ebola viruses (EBOVs) VP35 protein is a multifunctional major virulence factor involved in EBOVs replication and evasion of the host immune system. EBOV VP35 is an essential component of the viral RNA polymerase, it is a key participant of the nucleocapsid assembly and it inhibits the innate immune response by antagonizing RIG-I like receptors through its dsRNA binding function and, hence, by suppressing the host type I interferon (IFN) production. Insights into the VP35 dsRNA recognition have been recently revealed by structural and functional analysis performed on its C-terminus protein. We report the biochemical characterization of the Zaire ebolavirus (ZEBOV) full-length recombinant VP35 (rVP35)-dsRNA binding function. We established a novel in vitro magnetic dsRNA binding pull down assay, determined the rVP35 optimal dsRNA binding parameters, measured the rVP35 equilibrium dissociation constant for heterologous in vitro transcribed dsRNA of different length and short synthetic dsRNA of 8bp, and validated the assay for compound screening by assessing the inhibitory ability of auryntricarboxylic acid (IC(50) value of 50μg/mL). Furthermore, we compared the dsRNA binding properties of full length wt rVP35 with those of R305A, K309A and R312A rVP35 mutants, which were previously reported to be defective in dsRNA binding-mediated IFN inhibition, showing that the latter have measurably increased K(d) values for dsRNA binding and modified migration patterns in mobility shift assays with respect to wt rVP35. Overall, these results provide the first characterization of the full-length wt and mutants VP35-dsRNA binding functions.
埃博拉病毒（EBOV）VP35 蛋白是一种多功能的主要毒力因子，参与 EBOV 的复制和逃避宿主免疫系统。EBOV VP35 是病毒 RNA 聚合酶的一个重要组成部分，它是核衣壳组装的关键参与者，通过其 dsRNA 结合功能拮抗 RIG-I 样受体，从而抑制先天免疫反应，因此抑制宿主 I 型干扰素（IFN）的产生。最近对其 C 末端蛋白进行的结构和功能分析揭示了 VP35 dsRNA 识别的见解。我们报告了扎伊尔埃博拉病毒（ZEBOV）全长重组 VP35（rVP35）-dsRNA 结合功能的生化特征。我们建立了一种新颖的体外磁性 dsRNA 结合下拉测定法，确定了 rVP35 最佳 dsRNA 结合参数，测量了 rVP35 对不同长度和 8bp 短合成 dsRNA 的异源体外转录 dsRNA 的平衡解离常数，并通过评估auryntricarboxylic 酸的抑制能力（IC（50）值为 50μg/mL）验证了该测定法用于化合物筛选。此外，我们比较了全长 wt rVP35 与 R305A、K309A 和 R312A rVP35 突变体的 dsRNA 结合特性，这些突变体先前被报道在 dsRNA 结合介导的 IFN 抑制中存在缺陷，结果表明后者的 dsRNA 结合 K（d）值显著增加，并且在迁移率变动分析中与 wt rVP35 相比迁移模式发生了改变。总的来说，这些结果首次对全长 wt 和突变体 VP35-dsRNA 结合功能进行了表征。
