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Abstract：
Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3\'-NCR of JEV genomic RNA containing the GQ sequence could bind NCL in vitro. The replication complex binding to the 3\'-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication.IMPORTANCEJapanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3\'-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding.
摘要：
日本脑炎病毒（JEV）是节肢动物传播的，正链黄病毒引起人类病毒性脑炎，病死率高。具有RNA依赖性RNA聚合酶活性的JEV非结构蛋白5(NS5)与病毒和宿主蛋白相互作用以构成复制复合物。我们已经将多功能蛋白Nucleolin（NCL）鉴定为几种与NS5相互作用的宿主蛋白之一。我们证明了JEVNS5与NCL在病毒感染的HeLa细胞中的相互作用和共定位。siRNA介导的NCL敲低表明它是有效病毒复制所必需的。重要的是,JEV在过表达外源NCL的细胞中生长到更高滴度，证明了它的病毒作用。我们证明了NS5与NCL的RRM和GAR域相互作用。我们表明，含有G-四链体（GQ）结构和GQ配体BRACO-19的NCL结合适体AS1411引起了对JEV复制的显着抑制。AS1411和BRACO-19在HeLa细胞中的抗病毒作用可以通过外源NCL的过表达来克服。我们证明了来自含有GQ序列的JEV基因组RNA的3'-NCR的合成RNA可以在体外结合NCL。病毒RNA合成需要与3'-NCR结合的复制复合物。复制复合物中存在的NCL可能会使基因组RNA中的GQ结构不稳定，从而促进复制复合体的移动,导致有效的病毒复制。重要日本脑炎病毒（JEV）在东南亚大部分地区和西太平洋地区流行，导致脑炎流行，病死率高。虽然组织培养来源的JEV疫苗是可用的，不存在抗病毒治疗。JEVNS5蛋白具有RNA依赖性RNA聚合酶活性。连同几种宿主和病毒蛋白，它构成了病毒复制所必需的复制复合体。了解NS5与宿主蛋白的相互作用可以帮助设计新的抗病毒药物。我们确定Nucleolin（NCL）是JEVNS5的关键宿主蛋白相互作用因子，在病毒复制中具有促病毒作用。NS5相互作用的NCL与JEVRNA的3'-NCR中的G-四链体(GQ)结构序列结合。这可以使复制复合物沿着基因组RNA的运动变得平滑，从而促进病毒复制。这项研究是关于NCL，宿主蛋白，通过GQ绑定帮助JEV复制。
