Roles of the Nucleoprotein 3'-5' Exonuclease Domain in Arenavirus Biology

项目总结/摘要 哺乳动物肾病毒在世界各地的天然啮齿动物宿主中建立慢性感染， 人畜共患乳房病毒病可在其流行地区造成重要的公共卫生问题。 哺乳动物肾病毒可以破坏感染个体的先天免疫应答，从而损害免疫系统。 发展有效的抗病毒适应性免疫反应，促进不受限制的病毒 繁殖和相关的病理表现和疾病。淋巴细胞性乳房病毒 脉络丛脑膜炎病毒（LCMV）为我们提供了一个高度易操作的实验系统，以阐明病毒-宿主 免疫系统相互作用导致这些事件。我们已经证明LCMV核蛋白（NP）， 以及来自其他哺乳动物肾病毒的NP，抑制干扰素β（IFNβ）的产生，干扰素β是哺乳动物肾病毒感染的关键因素。 宿主对病毒感染的先天免疫防御。I型干扰素（IFN-I）的对抗活性 哺乳动物病毒NP的免疫抑制与NP抑制IRF 3和NF-kB活化的能力相关。沙粒病毒NP 抗IFN-1活性定位于含有功能性3 ′-5 ′核酸外切酶的NP的C-末端区域 （ExoN）结构域和重叠的IKKε相互作用结构域。设计了该应用中的实验 阐明以下问题：1）沙粒病毒NP是否能够抑制IFNβ产生的诱导 需要其与IKKε结合或其ExoN活性，或两者兼而有之;和，2）突变影响IKK ε的机制， NP-IKKε相互作用或NP的ExoN活性影响病毒适合度以及这是否导致LCMV的丧失 在自然宿主老鼠体内存活的能力为此，我们建议完成以下具体目标： 1.确定LCMV中NP介导的IFNβ诱导抑制是否需要NP-IKKε相互作用- 感染的细胞：我们将鉴定沙粒病毒NP内NP-IKKε相互作用所需的残基， 在功能上表征与NP-IKKε结合能力受损的NP突变体，包括其ExoN 抗IFNβ诱导的活性和能力。 2.评价沙粒病毒NP的ExoN活性对IFNβ诱导和病毒抑制的贡献 在缺乏功能性IFN-I系统的情况下的适应性：我们将进行突变功能研究，以确定 NP的ExoN活性是否是其抗IFN β活性和促进正常LCMV的严格要求 在不存在宿主细胞功能性IFN-1系统的情况下增殖。这些研究将审查的作用， NP病毒转录的ExoN活性和LCMV复制保真度。 3. NP的抗IFN-Ⅰ和ExoN活性在大肠杆菌毒力和自然携带状态建立中的作用 小鼠中的LCMV：我们将使用反向遗传学来产生携带不同类型的NP突变的rLCMV。 这些rLCMV将首先被确认在培养细胞中表现出其预测的表型，然后将被 在LCMV感染其天然宿主小鼠的背景下检查它们的体内表型特征。

Project Summary/Abstract Mammarenaviruses establish chronic infections in their natural rodent reservoirs across the world, and mammarenavirus zoonoses can pose important public health problems in their endemic regions. Mammarenaviruses can subvert the innate immune responses in infected individuals, thus compromising the development of an effective antiviral adaptive immune response, which facilitates unrestricted virus multiplication and associated pathological manifestations and disease. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) provides us with a highly tractable experimental system to elucidate virus-host immune system interactions contributing to these events. We have documented that LCMV nucleoprotein (NP), as well as NPs from other mammarenaviruses, inhibits production of interferon β (IFNβ), a key player in the host innate immune defense against viral infections. The type I IFN (IFN-I) counteracting activity of mammarenavirus NP correlated with the NP's ability to inhibit activation of IRF3 and NF-kB. Arenavirus NP's anti-IFN-I activity was mapped to the C-terminal region of NP that contains a functional 3'-5' exonuclease (ExoN) domain and an overlapping IKKε-interacting domain. The experiments in this application are designed to elucidate the following issues: 1) whether arenavirus NP's ability to inhibit induction of IFNβ production requires its binding to IKKε or its ExoN activity, or both; and, 2) the mechanisms by which mutations affecting NP-IKKε interaction or NP's ExoN activity affect virus fitness and whether this result in the loss of LCMV's ability to persist in its natural host, the mouse. To this end, we propose to complete the following specific aims: 1. Determine whether NP-IKKε interaction is required for NP-mediated inhibition of IFNβ induction in LCMV- infected cells: We will identify residues within arenavirus NP that are required for NP-IKKε interaction, and functionally characterize NP mutants impaired in their ability to associate with NP-IKKε, including their ExoN activity and ability to counteract induction of IFNβ. 2. Evaluate the contribution of the ExoN activity of arenavirus NP to inhibition of IFNβ induction and virus fitness in the absence of a functional IFN-I system: We will conduct mutation-function studies to determine whether the ExoN activity of NP is strictly required for its anti-IFNβ activity, and to promote normal LCMV multiplication in the absence of a host cell functional IFN-I system. These studies will examine the role of the ExoN activity of NP viral transcription and LCMV replication fidelity. 3. Roles of NP's anti-IFN-I and ExoN activities in virulence and the establishment of the natural carrier state of LCMV in mice: We will use reverse genetics to generate rLCMVs carrying the different types of NP mutations. These rLCMVs will be first confirmed to exhibit their predicted phenotypes in cultured cells, and then will be examined for their in vivo phenotypic features in the context of LCMV infection of its natural host, the mouse.