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

流行性乙型脑炎病毒(JEV)是人类的重要病原体，可引起急性脑膜脑炎，病死率约为30%。本课题的目的是阐明病毒非结构蛋白NS4a和基因组RNA 3‘端非编码区的生物学功能。(1)本研究以JEV-JaGAr01株和培养的人细胞系KN73、HepG2和IMR32为研究对象。我们构建了几个NS4a表达载体和含有JEV-3‘UTR的荧光素酶报告系统。野生型和突变型NS4a在细胞中的表达特征不同。在RNA3‘端存在突变序列时，荧光素酶活性显著降低。结果表明，NS4a和3‘UTR的突变影响了细胞内病毒蛋白合成的活性。(2)在病毒致病中，明确黄病毒感染诱导的宿主基因表达是必要的。在这里，我们使用DNA微阵列(Affymetrix)方法检测JEV感染细胞中宿主基因的表达，包括急性感染和持续感染。通过对人肝细胞系KN73和神经母细胞瘤细胞系IMR32的比较，我们证实干扰素相关基因的表达是决定病毒复制水平的关键。此外，本实验室建立的乙脑病毒持续感染细胞株(JK1)与急性感染相比，其干扰素相关基因的表达水平相对较低。综合其他宿主基因的表达数据，JEV的复制特征似乎受干扰素及其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.