JEV核糖体移码蛋白NS1`在病毒感染与传播中的功能及其机制研究

Encephalitic flaviruses such as Japanese encephalitis virus (JEV) and west nile virus (WNV) encode programmed -1 ribosomal frameshift (PRF) motif in the beginning of nonstructural gene NS2A that results in production of an additional nonstructural protein NS1` which for JEV consists of NS1 protein with 52 amino acid addition at the C terminus. The attenuated JEV vaccine strain SA14-14-2 carrying silent mutation from G to A at 66th nt of NS2A gene abolishing PRF and NS1` protein expression. Plotting the conservation at synonymous sites along the polyprotein coding sequence of field JEV isolates reveals strong conservation peaks at the 5' end of the sequence encoding the NS2A protein. Such conservation peaks are generally indicative of functionally important non-coding sequence elements. However, the biological importance of JEV PRF/NS1` protein remain to be elucidated. The present study planed to decipher the biological function and the underline mechanism of PRF or NS1` protein on JEV infection and inter host transmission. Firstly, to construction site mutant JEV strains based on virus reverse genetic technology and then to explore the phenotypic differences of animals and mosquito after infection of series JEV differential expression NS1` protein. The second, to explore the expression characteristic and regulation factors on JEV NS1` protein expression in different host cells. The third, to analysis the effect of NS1` protein on JEV enter into and multiplication in host cells. Finally to identify the molecular interact with JEV NS1` that located inside cytoplasm, on cell surface and in host serum the then to discover the biological function of the interaction. In total the present study aimed to clarify the biological significance of highly conserved gene region at the 5' end of the sequence encoding the JEV NS2A protein so as to provide new ideas for the development of new vaccines and antiviral drugs.

日本脑炎病毒（JEV）和西尼罗病毒（WNV）等致脑炎黄病毒在非结构蛋白NS2A编码基因5`端区域存在核糖体移码(PRF)基序，引发NS1`蛋白的表达，JEV NS1`为NS1蛋白C端增加52个氨基酸。JEV弱毒疫苗株SA14-14-2 NS2A 基因发生G66A的突变，不产生NS1`蛋白。JEV野毒株基因同义突变分析发现引发NS1`表达的NS2A 5`端基因区域非常保守，但其重要的生物学意义尚未被揭示。本项目拟探究差异表达NS1/NS1 `蛋白的JEV毒株感染仔猪、小鼠和蚊子的表型差异；揭示JEV 感染不同细胞NS1` 蛋白的表达特性以及NS1`蛋白对JEV感染细胞的影响；鉴定JEV NS1` 蛋白在细胞内、细胞表面和病毒感染宿主血液中的互作分子及其生物学功能；阐明JEV NS1`在病毒感染与传播的作用及其分子作用机制，从而为新型疫苗和抗病毒药物的研制提供新思路。

流行性乙型脑炎是由日本脑炎病毒（JEV）引起的一种蚊媒传播的人畜共患传染病，严重危害我国人畜健康。JEV分离株基因组同义突变分析发现引发NS1`表达的NS2A 5`端基因区域非常保守，表明其具有重要的生物学功能，尚未阐明。本研究通过反向遗传和基因点突变技术构建了差异表达NS1`的重组JEV，测定重组病毒在多个传代细胞和原代细胞中的增殖曲线，结果表明NS1`蛋白显著增强JEV在A549细胞、小鼠腹腔巨噬细胞和树突状细胞中的增殖。小鼠感染试验结果表明rJEV SA14-14-2 A66G株对2周龄仔的鼠神经毒力稍强于rJEV SA14-14-2株。重要的是，研究发现仔猪接种表达NS1`蛋白的rJEV SA14-14-2 A66G株后在口鼻分泌物中检测到病毒，在扁桃体和脑组织中检测到病毒阳性细胞，而rJEV SA14-14-2株在仔猪中呈一过性感染，口鼻分泌物、扁桃体和脑组织中未检测到病毒。因此，NS1`蛋白对于JEV在猪的感染与传播过程中发挥重要作用。JEV NS1`蛋白可以分泌表达，我们建立了检测猪血液中NS1`蛋白的双抗体夹心ELISA，可以用于猪群早期感染JEV的监测。在NS1`促进JEV感染的分子机制方面，我们发现JEV NS1`可抑制感染细胞中IFN-β的表达。有趣的是，我们还发现NS1`蛋白可通过上调细胞中磷脂酰丝氨酸受体蛋白TIM-1的表达促进JEV感染与增殖。本研究还发现干扰素刺激基因（ISG）产物C19orf66可以下调JEV感染细胞中NS1`的表达，并降低NS1`/NS1的比例，表明C19orf66可抑制JEV蛋白翻译过程中的核糖体-1移码。综上所述，本研究初步阐明了NS1`蛋白在JEV感染与传播中的重要作用与机理、揭示了JEV NS1`蛋白的表达调控途径，从而为流行性乙型脑炎新型疫苗和药物的研制以及防控策略的制定提供了新思路。