Lin Cheng-Wen, Lin Kuan-Hsun, Lyu Ping-Chiang, Chen Wei-June
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan, Taiwan, ROC.
Virus Res. 2006 Mar;116(1-2):106-13. doi: 10.1016/j.virusres.2005.09.002. Epub 2005 Nov 10.
Flavivirus NS2B-NS3 proteases are associated with neurovirulence, becoming an important target for insight into the virus-induced pathogenesis. In this study, a phage-displayed human brain cDNA library was used to detect possible interaction between brain proteins and the Japanese encephalitis virus (JEV) NS2B-NS3 protease. After six rounds of biopanning, eight high-affinity NS2B-NS3 protease-interacting phages were identified. Identified NS2B-NS3 protease-interacting brain proteins contained several repeats of the consensus motifs E(R/K)(R/K)K and G(R/K)(R/K) with the dibasic residues, being similar to the conserved cleavage sites among flavivirus proteases. In addition, three identified brain proteins (phage-24, 34, and 44) were predicted as the domain of trypsin inhibitor and basic region leucine zipper (bZIP) using the SMART genome search. Immunoprecipitation and cleavage of two brain fusion proteins (phage-24 and phage-46) by the NS2B-NS3 protease confirmed the specific interaction between identified brain proteins and the JEV NS2B-NS3 protease. Fluorogenic peptide substrate assays revealed dose-manner inhibitory effects of these two brain fusion proteins on the trans-cleavage activity of NS2B-NS3 protease. Moreover, in vitro signaling pathway assay revealed that the JEV NS2B-NS3 protease significantly inhibited the signaling pathway of activator protein 1(AP1), a member of the bZIP family. Our results provide an insight into the protein interaction network of the JEV NS2B-NS3 protease in human brain.
黄病毒NS2B-NS3蛋白酶与神经毒力相关，成为深入了解病毒诱导发病机制的重要靶点。在本研究中，利用噬菌体展示的人脑cDNA文库检测脑蛋白与日本脑炎病毒（JEV）NS2B-NS3蛋白酶之间的可能相互作用。经过六轮生物淘选，鉴定出八个与NS2B-NS3蛋白酶具有高亲和力的噬菌体。鉴定出的与NS2B-NS3蛋白酶相互作用的脑蛋白含有几个具有双碱性残基的共有基序E(R/K)(R/K)K和G(R/K)(R/K)的重复序列，类似于黄病毒蛋白酶之间的保守切割位点。此外，利用SMART基因组搜索预测，三个鉴定出的脑蛋白（噬菌体-24、34和44）为胰蛋白酶抑制剂和碱性区域亮氨酸拉链（bZIP）结构域。NS2B-NS3蛋白酶对两种脑融合蛋白（噬菌体-24和噬菌体-46）的免疫沉淀和切割证实了鉴定出的脑蛋白与JEV NS2B-NS3蛋白酶之间的特异性相互作用。荧光肽底物分析显示这两种脑融合蛋白对NS2B-NS3蛋白酶的反式切割活性具有剂量依赖性抑制作用。此外，体外信号通路分析显示，JEV NS2B-NS3蛋白酶显著抑制bZIP家族成员激活蛋白1（AP1）的信号通路。我们的结果为深入了解JEV NS2B-NS3蛋白酶在人脑中的蛋白质相互作用网络提供了线索。
