What is the week point of flavivirus? : Biological role of viral protein NS4a and 3' UTR of genomic RNA

日本脑炎病毒（Japaneseencephalitis virus，JEV）是引起人类急性脑膜脑炎的重要病原体，病死率约30%。本课题旨在阐明病毒非结构蛋白NS 4a和基因组RNA 3' UTR的生物学功能。(1)在这里，我们使用JEV-JaGAr 01株和培养的人细胞系KN 73、HepG 2和IMR 32。我们构建了几种NS 4a表达载体和含JEV-3' UTR的荧光素酶报告系统。NS 4a在细胞中的表达特征在野生型和突变型蛋白之间存在差异。在RNA的3'端存在突变序列时，荧光素酶活性显著降低。结果表明，NS 4a和3' UTR的突变影响了细胞中病毒蛋白合成的活性。(2)阐明黄病毒感染诱导的宿主基因表达在病毒致病机制中的作用是十分必要的。本研究利用DNA微阵列技术检测了JEV急性感染和持续感染细胞中宿主基因的表达。通过对人肝细胞系KN 73和神经母细胞瘤细胞系IMR 32的比较，证实了干扰素（IFN）相关基因的表达是决定病毒复制水平的关键。此外，我们实验室建立的JEV持续感染细胞系（JK 1）表明，与急性感染相比，IFN相关基因的表达相对较低。结合其他宿主基因的表达数据，JEV复制的特征可能受IFN、IFN相关基因和其他宿主基因的表达调控。

Japanese encephalitis virus (JEV) is the important human pathogen and causes acute meningioencephalitis, resulting in fatality rate of ca 30%. The aim of this project is to clarify the biological function of nonstructural viral protein NS4a and 3' UTR of genomic RNA.(1) Here we used JEV-JaGAr01 strain and the cultured human-cell lines, KN73, HepG2, and IMR32. We constructed several NS4a-expression vectors and the luciferase reporter system containing JEV-3' UTR. The feature of NS4a expression in the cells was different between wild and mutant proteins. Luciferase activity was remarkably decreased in the presence of mutant sequence at the 3' end of RNA. The results suggest that the mutation of NS4a and 3' UTR influences the activity for viral protein synthesis in the cells.(2) In the viral pathogenesity, it is essential to clarify host gene expression induced by flavivirus infection. Here we used DNA microarray (Affymetrix) method to examine host gene expression in JEV-infected cells including acute and persistent infection. From the comparison of human liver derived cell line KN73 and neuroblastoma cell line IMR32, we confirmed the expression of interferon (IFN) related genes was critical to decide viral replication level. In addition, JEV-persistently infected cell lines (JK1) which has been established in our laboratory indicated relatively low expression of IFN-related genes in comparison with the acute infection. Taken together with the expression data of other host genes, the feature of JEV replication seems to be regulated by the expression of IFN, IFN related genes and other host genes.