Role of host cell protein TDP2 as VPg unlinkase during picornavirus replication

这项研究的目的是揭示病毒在宿主细胞复制中的作用 最近被鉴定为小核糖核酸病毒家族的VPg解链酶的蛋白质。小核糖核酸病毒是 一种小的正链RNA病毒，可引起人类和动物的多种疾病， 对健康和经济产生了重大影响。这些病毒的重要性 超越其公共卫生影响;作为第一个哺乳动物病毒发现，研究 小核糖核酸病毒对病毒分子生物学产生了重大影响，并导致了重要的发现 与病毒的细胞受体、RNA病毒的感染性cDNA、内部核糖体相关 进入位点（IRES）驱动的翻译，哺乳动物细胞的抗病毒反应， 作为多蛋白前体合成的蛋白质，病毒的化学合成，以及许多 其他重要研究领域。在过去十年中出现的一个主要主题是， 这些具有非常有限的编码能力的病毒利用和/或修饰宿主细胞功能 完成它们的复制周期。其中一个功能是细胞活性，VPg解链酶， 从小核糖核酸病毒基因组RNA的5'末端去除小病毒肽（VPg）， 在病毒蛋白质合成开始之前或之后裂解蛋白质-核苷酸键。PI的 一个实验室最近从HeLa细胞中鉴定出VPg解链酶为酪氨酰-DNA 磷酸二酯酶2（TDP 2），一种参与DNA修复、细胞信号传导的宿主酶， 转录调控该提议旨在表征TDP 2/VPg解链酶的活性 在被肠道病毒（脊髓灰质炎病毒或柯萨奇病毒）感染的细胞中， 鼻病毒，以确定当这种活性下调时病毒复制如何受到影响 在感染期间。具体目标是：（i）下调TDP 2/VPg解连接酶活性以确定 其在小核糖核酸病毒复制中的功能，和（ii）定义细胞再定位途径， 小RNA病毒感染细胞中TDP 2/VPg解链酶的蛋白结合伴侣。拟议 实验计划将检验小核糖核酸病毒利用TDP 2/VPg解链酶的假设 活性调节病毒RNA的命运，以执行翻译中的特定功能，RNA 复制或组装成感染性病毒颗粒。拟议研究的结果可能 揭示了TDP 2/VPg解链酶活性作为针对人的抗病毒治疗的新靶点 鼻病毒、柯萨奇病毒、甲型肝炎病毒和其他小核糖核酸病毒， 人类疾病后遗症。

The goal of the proposed research is to uncover the roles(s) in viral replication of a host cell protein recently identified as being VPg unlinkase for the picornavirus family. Picornaviruses are small, positive-strand RNA viruses that cause a wide range of diseases in humans and animals, with significant health and economic repercussions. The importance of these viruses extends beyond their public health implications; as the first mammalian viruses discovered, the study of picornaviruses has had a major impact on viral molecular biology and led to important findings related to cellular receptors for viruses, infectious cDNAs for RNA viruses, internal ribosome entry site (IRES)-driven translation, anti-viral responses of mammalian cells, the maturation of proteins synthesized as polyprotein precursors, chemical synthesis of a virus, and a host of other important research areas. A major theme that has emerged during the last decade is how these viruses with a very limited coding capacity utilize and/or modify host cell functions to complete their replication cycles. One such function is the cellular activity, VPg unlinkase, that removes the small viral peptide (VPg) from the 5' end of picornavirus genomic RNAs by cleaving a protein-nucleotidyl bond prior to or after the onset of viral protein synthesis. The PI's laboratory has recently identified VPg unlinkase from HeLa cells as tyrosyl-DNA phosphodiesterase 2 (TDP2), a host enzyme involved in DNA repair, cell signaling, and transcriptional regulation. This proposal aims to characterize the activity of TDP2/VPg unlinkase in cells infected by enteroviruses (poliovirus or coxsackievirus) and by the closely related human rhinovirus to determine how viral replication is impacted when this activity is down-regulated during infection. The specific aims are: (i) Down-regulate TDP2/VPg unlinkase activity to define its function(s) in picornavirus replication, and (ii) Define the cellular relocalization pathway and protein-binding partners of TDP2/VPg unlinkase in picornavirus-infected cells. The proposed experimental plan will examine the hypothesis that picornaviruses employ TDP2/VPg unlinkase activity to regulate the fate of viral RNAs to carry out specific functions in translation, RNA replication, or assembly into infectious virus particles. Results from the proposed studies may reveal TDP2/VPg unlinkase activity as a novel target for anti-viral therapy directed at human rhinovirus, coxsackievirus, hepatitis A virus, and other picornaviruses responsible for significant human disease sequelae.