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

描述（由申请人提供）：拟议研究的目标是揭示最近被鉴定为小核糖核酸病毒家族VPg解联酶的宿主细胞蛋白在病毒复制中的作用。小核糖核酸病毒是一种小的正链RNA病毒，可在人类和动物中引起广泛的疾病，对健康和经济产生重大影响。这些病毒的重要性超出了它们对公共卫生的影响；作为第一个发现的哺乳动物病毒，小核糖核酸病毒的研究对病毒分子生物学产生了重大影响，并导致了与病毒的细胞受体、RNA病毒的感染性cdna、内部核糖体进入位点（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的成熟、病毒的化学合成以及许多其他重要研究领域相关的重要发现。在过去十年中出现的一个主要主题是这些编码能力非常有限的病毒如何利用和/或修改宿主细胞功能来完成其复制周期。其中一个功能是细胞活性，VPg解联酶，它通过在病毒蛋白质合成开始之前或之后切割蛋白质-核苷酸键，从小核糖核酸基因组rna的5'端去除小病毒肽（VPg）。PI的实验室最近从HeLa细胞中鉴定出VPg解联酶为酪氨酸-DNA磷酸二酯酶2 (TDP2)，这是一种参与DNA修复、细胞信号传导和转录调节的宿主酶。本研究旨在表征肠病毒（脊髓灰质炎病毒或柯萨奇病毒）和密切相关的人鼻病毒感染细胞中TDP2/VPg解联酶的活性，以确定当该活性在感染期间下调时如何影响病毒复制。具体目的是：(i)下调TDP2/VPg解联酶活性，以确定其在小核糖核酸病毒复制中的功能；（ii）确定小核糖核酸病毒感染细胞中TDP2/VPg解联酶的细胞重定位途径和蛋白质结合伙伴。提出的实验计划将检验小核糖核酸病毒利用TDP2/VPg解联酶活性来调节病毒RNA的命运，以在翻译，RNA复制或组装成感染性病毒颗粒中执行特定功能的假设。提出的研究结果可能揭示TDP2/VPg解联酶活性作为抗病毒治疗的新靶点，针对人鼻病毒，柯萨奇病毒，甲型肝炎病毒，以及其他造成严重人类疾病后遗症的小核糖核酸病毒。

DESCRIPTION (provided by applicant): 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 forthe 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.