乙型脑炎病毒NS2b蛋白跨膜区在病毒复制过程中的功能研究

Japanese Encephalitis Virus (JEV) is a highly neurotropic virus that is harmful for public health. It is urgent to find out potential antiviral targets. Recently, the functions of viral membrane proteins during viral replication have attracted intensive interest. So far, either topology or the function of JEV-NS2b hydrophobic region in viral replication still remains unknown although it is well-known that it serves as a co-factor for NS3 protease through its hydrophilic region. Our recent study suggested that the amino acid mutation at position 102 in NS2b resulted in a significant decrease in viral replication while had little effect on the activity of NS3 protease, indicating NS2b may exert an unknown function during viral replication through its hydrophobic region. In this project, the membrane topology of NS2b will be resolved through bioinformatics analysis together with biochemical and cellular assays. The effects of the transmembrane domains of NS2b on protease activity, NS2b subcellular localization and viral replication will be investigated using the methods of reverse genetics and cell biology. On the basis of these results, the key points/sequences in NS2b transmembrane domains involved in viral replication as well as the genetic interaction partners with NS2b will be determined. This project aims to provide detailed information about the topology of NS2b and clarify the biological function of its transmembrane domains in viral replication for the development of NS2b-based antiviral drugs.

乙型脑炎病毒（JEV）是一种严重危害人类健康的神经嗜性病毒，寻找有效药靶势在必行。近年来病毒膜蛋白在病毒复制中的生物学功能日益受到人们关注。虽然NS2b膜蛋白依赖于其亲水区发挥NS3蛋白酶辅助因子的功能早已为人们所熟知，但其拓扑结构及疏水区生物学功能尚不清晰。我们近期研究发现NS2b疏水区102位氨基酸突变虽然对蛋白酶活性无影响，但显著降低病毒复制，提示其疏水区在病毒复制过程中发挥着其它未知的重要功能。本项目拟借助生物信息学预测、生化和细胞生物学等方法，全面解析JEV-NS2b拓扑结构。利用反向遗传学工具和细胞生物学手段，研究NS2b不同跨膜区突变及回复突变对蛋白酶活性、NS2b亚细胞定位及病毒复制的影响，确定NS2b上关键氨基酸位点及与其有遗传学相互作用的病毒因子。此项目旨在解析NS2b膜蛋白拓扑结构，阐明其跨膜区在病毒复制中的生物学功能，为抗病毒药物筛选提供新思路和潜在作用靶点。

乙型脑炎病毒(JEV)是一种严重危害人类健康的神经嗜性病毒，寻找有效药靶势在必行 。近年来病毒膜蛋白在病毒复制中的生物学功能日益受到人们关注。虽然NS2b膜蛋白依赖于其亲水区发挥NS3蛋白酶辅助因子的功能早已为人们所熟知，但其疏水区的生物学功能尚不清晰。我们前期的研究发现NS2b疏水区虽然对蛋白酶活性无影响，但其在病毒复制过程中发挥着其它未知的重要功能。.本项目利用反向遗传学工具和细胞生物学手段，研究NS2b不同跨膜区保守突变及回复突变对蛋白酶活性及病毒复制的影响，发现NS2b跨膜区在病毒复制和包装过程中都发挥重要作用；通过遗传筛选，发现NS2b和NS2a蛋白存在着相互作用，其相互作用可能在病毒包装和复制过程中都发挥作用。上关键氨基酸位点及与其有遗传学相互作用的病毒因子。.此项目证明NS2b跨膜区在病毒复制和包装中的发挥重要的生物学功能，为抗病毒药物筛选提供新思路和潜在作用靶点。