Chen Ming Wei, Tan Yaw Bia, Zheng Jie, Zhao Yongqian, Lim Bee Ting, Cornvik Tobias, Lescar Julien, Ng Lisa Fong Poh, Luo Dahai
Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore.
School of Biological Sciences, Nanyang Technological University, Singapore.
Antiviral Res. 2017 Jul;143:38-47. doi: 10.1016/j.antiviral.2017.04.001. Epub 2017 Apr 5.
Chikungunya virus (CHIKV) is an important arboviral infectious agent in tropical and subtropical regions, often causing persistent and debilitating disease. The viral enzyme non-structural protein 4 (nsP4), as RNA-dependent RNA polymerase (RdRP), catalyzes the formation of negative-sense, genomic and subgenomic viral RNAs. Here we report a truncated nsP4 construct that is soluble, stable and purified recombinantly from Escherichia coli. Sequence analyses and homology modelling indicate that all necessary RdRP elements are included. Hydrogen/deuterium exchange with mass spectrometry was used to analyze solvent accessibility and flexibility of subdomains. Fluorophore-conjugated RNA ligands were designed and screened by using fluorescence anisotropy to select a suitable substrate for RdRP assays. Assay trials revealed that nsP4 core domain is conditionally active upon choice of detergent species, and carries out both primed extension and terminal adenylyltransferase activities. The polymerization assay can be further developed to screen for antiviral compounds in vitro.
基孔肯雅病毒（CHIKV）是热带和亚热带地区一种重要的虫媒病毒感染因子，常引发持续性且使人衰弱的疾病。病毒酶非结构蛋白4（nsP4）作为RNA依赖性RNA聚合酶（RdRP），催化负链、基因组和亚基因组病毒RNA的形成。在此，我们报告了一种截短的nsP4构建体，它可溶、稳定且能从大肠杆菌中重组纯化得到。序列分析和同源建模表明，所有必需的RdRP元件均已包含。利用氢/氘交换质谱法分析亚结构域的溶剂可及性和灵活性。设计并通过荧光各向异性筛选荧光团共轭RNA配体，以选择适合RdRP检测的底物。检测试验表明，nsP4核心结构域在选择去污剂种类时具有条件活性，并具有引发延伸和末端腺苷酸转移酶活性。聚合反应检测可进一步开发用于体外筛选抗病毒化合物。
