Heterogeneous Nuclear Ribonucleoprotein A2 Participates in the Replication of Japanese Encephalitis Virus through an Interaction with Viral Proteins and RNA
Heterogeneous Nuclear Ribonucleoprotein A2 Participates in the Replication of Japanese Encephalitis Virus through an Interaction with Viral Proteins and RNA
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DOI：
10.1128/jvi.00846-11
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发表时间：
2011-11-01
影响因子：
5.4
通讯作者：
Matsuura, Yoshiharu
中科院分区：
文献类型：
--
作者：
Katoh, Hiroshi;Mori, Yoshio;Matsuura, Yoshiharu
日本脑炎病毒（JEV）是一种蚊媒黄病毒，存在于猪和蚊子之间的人畜共患传播循环中。JEV引起中枢神经系统感染，在包括人类在内的死端宿主中具有高死亡率。许多研究表明，黄病毒核心蛋白不仅是核衣壳的组成部分，而且是重要的致病决定因子。在这项研究中，我们确定了异质核核糖核蛋白A2（hnRNP A2）作为结合伙伴的JEV核心蛋白的下拉纯化和质谱。在转染和感染的细胞中的相互免疫共沉淀分析证实了JEV核心蛋白和hnRNP A2之间的特异性相互作用。在JEV亚基因组复制子细胞中，JEV核心蛋白的表达诱导hnRNP A2的胞质滞留。小干扰RNA（siRNA）介导的hnRNP A2的敲低导致感染JEV的细胞中病毒RNA复制减少90%，并且通过表达siRNA抗性hnRNP A2突变体来消除减少。除了核心蛋白外，hnRNP A2还与JEV非结构蛋白5（其具有甲基转移酶和RNA依赖性RNA聚合酶活性）和负义JEV RNA的5 '非翻译区相关。在一步生长过程中，正链和负链JEV RNA的合成被hnRNPA2的敲低延迟。这些结果表明，hnRNPA2通过与病毒蛋白和RNA的相互作用在JEV RNA的复制中起重要作用。
Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is kept in a zoonotic transmission cycle between pigs and mosquitoes. JEV causes infection of the central nervous system with a high mortality rate in dead-end hosts, including humans. Many studies have suggested that the flavivirus core protein is not only a component of nucleocapsids but also an important pathogenic determinant. In this study, we identified heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) as a binding partner of the JEV core protein by pulldown purification and mass spectrometry. Reciprocal coimmunoprecipitation analyses in transfected and infected cells confirmed a specific interaction between the JEV core protein and hnRNP A2. Expression of the JEV core protein induced cytoplasmic retention of hnRNP A2 in JEV subgenomic replicon cells. Small interfering RNA (siRNA)-mediated knockdown of hnRNP A2 resulted in a 90% reduction of viral RNA replication in cells infected with JEV, and the reduction was cancelled by the expression of an siRNA-resistant hnRNP A2 mutant. In addition to the core protein, hnRNP A2 also associated with JEV nonstructural protein 5, which has both methyltransferase and RNA-dependent RNA polymerase activities, and with the 5'-untranslated region of the negative-sense JEV RNA. During one-step growth, synthesis of both positive-and negative-strand JEV RNAs was delayed by the knockdown of hnRNP A2. These results suggest that hnRNP A2 plays an important role in the replication of JEV RNA through the interaction with viral proteins and RNA.