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

核蛋白 3-5 核酸外切酶结构域在沙粒病毒生物学中的作用

• 批准号: 9901456
• 负责人: Luis Martinez-Sobrido
• 金额: $ 43.44万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9901456
• 关键词: Address; Affect; Antiviral Agents; Antiviral Therapy; Arenavirus; Binding; Biochemical; Biological Assay; Biology; C-terminal; Carrier State; Cells; Communicable Diseases; Cultured Cells; Development; Disease; Event; Exhibits; Exonuclease; Failure; Genes; Genetic Transcription; Human; IRF3 gene; Immune; Immune system; Immunologic Surveillance; Impairment; Individual; Infection; Innate Immune Response; Interferon Type I; Interferon-beta; Interferons; Knowledge; Life Cycle Stages; Link; Lymphocytic choriomeningitis virus; Maps; Mediating; Morbidity - disease rate; Mus; Mutation; NF-kappa B; Nuclear Translocation; Nucleoproteins; Pathogenicity; Pathologic; Pathology; Phenotype; Phosphodiesterase I; Phosphorylation; Phosphotransferases; Play; Production; Property; Public Health; Recombinants; Ribavirin; Rodent; Role; Structure; System; Testing; Therapeutic; Transcriptional Activation; Translational Research; Vaccines; Vero Cells; Viral; Viral Hemorrhagic Fevers; Virulence; Virus; Virus Diseases; Virus Replication; Zoonoses; adaptive immune response; base; biodefense; chronic infection; clinically significant; combat; design; experimental study; fitness; human pathogen; in vivo; mortality; mutant; neglect; novel; novel strategies; off-label use; prevent; response; reverse genetics; side effect; vaccine development; viral fitness; virus host interaction

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.

项目摘要/摘要 Mammammanaviruses在世界各地的天然啮齿动物储层中建立慢性感染，并且 哺乳动物病毒动物可以在其流行地区构成重要的公共卫生问题。 mammeranaviruses可以颠覆感染个体的先天免疫反应，从而损害 开发有效的抗病毒适应性免疫响应，该免疫力不受限制的病毒 繁殖和相关的病理表现和疾病。乳腺癌病毒淋巴细胞 脉络膜脑膜炎病毒（LCMV）为我们提供了一个高度可进行的实验系统，以阐明病毒宿主 免疫系统相互作用有助于这些事件。我们已经证明LCMV核蛋白（NP）， 以及来自其他哺乳动物病毒的NP，抑制了干扰素β（IFNβ）的产生， 主持人对病毒感染的先天免疫防御。 I型IFN（IFN-I）的抵消活动 乳腺癌NP与NP抑制IRF3和NF-KB激活的能力相关。 Arenavirus NP 将抗IFN-I活性映射到NP的C末端区域，该区域包含功能性3'-5'外切酶 （外显子）域和重叠的IKKε相互作用域。该应用程序中的实验是设计的 阐明以下问题：1）Arenavirus NP抑制IFNβ产生的能力是否能力 需要它与IKKε或外显子活性或两者的结合； 2）突变影响的机制 NP-IKKε相互作用或NP的外显子活性会影响病毒适应性，这是否导致LCMV的丧失 能够在其自然宿主中持续的鼠标。为此，我们建议完成以下特定目标： 1。确定NP-IKKε相互作用是否需要NP介导的IFNβ诱导在LCMV-中的抑制 受感染的细胞：我们将确定NP-IKKε相互作用所需的Araravirus NP中的残留物，并且 在功能上表征NP突变体与NP-IKKε相关的能力受损，包括其外显子 应对IFNβ诱导的活性和能力。 2。评估Arhavirus NP外显子活性对抑制IFNβ诱导和病毒的贡献 在没有功能性IFN-I系统的情况下，适合度：我们将进行突变功能研究以确定 NP的外显子活性是否严格用于其抗IFNβ活性并促进正常LCMV 在没有宿主细胞功能IFN-I系统的情况下乘法。这些研究将研究 NP病毒转录和LCMV复制保真度的外显子活性。 3。NP的抗IFN-I和外显子活动在病毒中的作用以及建立自然载体状态 小鼠中的LCMV：我们将使用反向遗传学生成带有不同类型NP突变的RLCMV。 这些RLCMV将首先确认在培养细胞中表现出其预测的表型，然后将是 在其自然宿主的LCMV感染中，检查了其体内表型特征。