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

项目摘要/摘要 哺乳动物病毒在世界各地的天然啮齿动物宿主中建立慢性感染，以及 人兽共患病病毒可在其流行地区造成重大公共卫生问题。 哺乳动物病毒可以颠覆受感染个体的先天性免疫反应，从而危及 开发一种有效的抗病毒获得性免疫反应，促进无限制病毒 增殖及相关的病理表现和疾病。哺乳动物病毒淋巴细胞性 脉络膜脑膜炎病毒(LCMV)为我们提供了一个高度可操作的实验系统来阐明病毒宿主 免疫系统的相互作用促成了这些事件。我们已经证明了LCMV核蛋白(NP)， 以及来自其他哺乳动物病毒的NP，抑制干扰素β(干扰素β)的产生，干扰素是 宿主对病毒感染的先天免疫防御。I型干扰素(干扰素-I)中和作用 哺乳动物病毒NP与NP抑制IRF3和NF-kB激活的能力有关。新城疫病毒NP 抗干扰素-I的活性被映射到NP的C-末端区域，该区域含有一个功能性的3‘-5’核酸外切酶 (外显子)结构域和重叠的IKKε相互作用结构域。设计了此应用程序中的实验 为了阐明以下问题：1)病毒NP是否具有抑制干扰素β诱导产生的能力 需要它与IKKε或其外显子活性结合，或两者兼而有之；以及，2)突变影响 NP-IKKε相互作用或NP的外显子活性影响病毒适合性以及这是否导致LCMV的损失 在它的自然宿主--老鼠身上坚持的能力。为此，我们建议完成以下具体目标： 1.确定是否需要NP-IKKε相互作用来抑制NP介导的干扰素β诱导 感染细胞：我们将确定病毒NP中NP-IKKε相互作用所需的残基，以及 NP突变体与NP-IKKε结合能力受损的功能特征，包括其外显子 抗干扰素β诱导的活性和能力。 2.评价病毒NP外显子活性在抑制干扰素β诱导和病毒感染中的作用 在缺乏功能性干扰素-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的外显子活性是否是其抗干扰素β活性和促进正常巨噬细胞病毒的严格要求 在没有宿主细胞功能的干扰素-I系统的情况下进行增殖。这些研究将考察 NP病毒转录外显子活性与LCMV复制保真度。 3.NP的抗干扰素-I和外显子活性在毒力中的作用及天然载体状态的建立 小鼠的LCMV：我们将使用反向遗传学产生携带不同类型NP突变的rLCMV。 这些rLCMV将首先在培养细胞中证实其预测的表型，然后将 在LCMV感染其自然宿主小鼠的背景下，检查它们在体内的表型特征。

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.