Anti-interferon Activity of the Arenavirus Nucleoprotein

摘要 ARENAV的原型LCMV已被证明是研究抗病毒宿主的一块罗塞塔石碑 免疫反应，以及病毒免疫逃避和持续的机制。此外，有证据表明 表明LCMV在世界范围内分布广泛，是一种被忽视的具有临床意义的人类病原体。 此外，拉萨病毒和其他几种阿拉伯病毒会导致人类患出血热(HF)疾病。 宿主对控制病毒增殖的先天免疫反应失败是导致病毒诱导的原因之一 疾病。我们已经证明，LCMV和HF ArenaVirus的核蛋白(NP)可以抑制 转录因子IRF3的激活与I型干扰素(I型干扰素)的产生 豁免权。我们的长期目标是了解干扰素的分子基础和生物学后果 对抗与阿雷纳病毒NP相关的活性。为此，我们提出以下具体目标： 目的1.确定LCMV-NP的干扰素拮抗作用的遗传决定因素。 我们已经确定了LCMV-NP的两个C-末端结构域，它们对NP介导的IRF3抑制至关重要 和干扰素启动子的激活。我们假设这两个区域内的特定残基对 在NP的干扰素拮抗功能中的关键作用，并且这一功能可以与 NP在病毒复制中的作用。我们建议进行全面的突变函数分析 这些区域用于识别NP中取消其抗干扰素功能的单个氨基酸突变，而不是 影响其在病毒复制中的作用。 目的2.明确NP发挥干扰素拮抗作用的机制。我们已经证明了 LCMV-NP通过抑制IRF3的激活和核转位来抵消干扰素的反应。我们 假设LCMV-NP通过与参与激活的宿主细胞蛋白相互作用实现这一点 对IRF3进行了改进。我们将寻求蛋白质组学方法，包括亲和纯化方法和质量结合 鉴定与NP相互作用的细胞蛋白质的光谱程序。我们将使用生化和遗传技术 方法，结合功能分析，评估已识别的NP相互作用细胞的贡献 蛋白质对NP的干扰素拮抗活性。 目的3.评估NP的干扰素拮抗活性对病毒增殖和 发病过程中LCMV感染其自然宿主小鼠。我们将产生重组人 LCM病毒(rLCMV/NP*)，含有在病毒RNA合成中保持wt活性的NP突变形式 和感染性颗粒形成，但失去了抑制IRF3激活和干扰素产生的能力。我们 假设这些rLCMV/NP*将在非常早期诱导比wt LCMV更高水平的干扰素 导致病毒衰减的感染的可能性。为了测试这一点，我们将比较rLCMV/NP*和WT病毒 小鼠的感染：1)增殖和诱导干扰素产生和适应性T细胞的能力 免疫反应，2)诱导致命性LCM的能力，3)建立持久性的能力。

Abstract The prototypic arenavirus LCMV has proven to be a Rosetta stone for the investigation of the anti-viral host immune responses, and mechanisms of virus immune evasion and persistence. In addition, evidence indicates that LCMV, worldwide distributed, is a neglected human pathogen of clinical significance. Moreover, Lassa virus and several other arenaviruses cause hemorrhagic fever (HF) disease in humans. The failure of the host innate immune response to control virus multiplication contributes to arenaviralinduced disease. We have shown that the nucleoprotein (NP) of LCMV and HF arenaviruses inhibits activation of the transcriptional factor IRF3 and production of Type I interferon (IFN), a key player in innate immunity. Our long-term goal is to understand the molecular bases and biological consequences of the IFN counteracting activity associated with arenavirus NP. To this end we propose the following specific aims: Aim 1. Define the genetic determinants of LCMV-NP responsible for its IFN antagonistic function. We have identified two C-terminal domains of LCMV-NP that are critical for NP-mediated inhibition of IRF3 and IFN¿ promoter activation. We hypothesize that specific residues within these two regions play a critical role on the IFN antagonistic function of NP, and that this function can be segregated from the role of NP in virus replication. We propose to conduct a comprehensive mutation-function analysis of these regions to identify single amino acid mutations in NP that abrogate its anti-IFN function without affecting its role in virus replication. Aim 2. Define the mechanisms by which NP exerts its IFN antagonistic activity. We have shown that LCMV-NP counteracts the IFN response by inhibiting activation and nuclear translocation of IRF3. We hypothesize that LCMV-NP achieves this by interacting with host cell proteins involved in activation of IRF3. We will pursue proteomic approaches including affinity purification methods combined with mass spectrometry procedures to identify NP-interacting cellular proteins. We will use biochemical and genetic approaches, together with functional assays, to assess the contribution of identified NP-interacting cellular proteins to the IFN antagonistic activity of NP. Aim 3. Assess the contribution of the IFN antagonistic activity of NP to virus multiplication and pathogenesis during LCMV infection of its natural host, the mouse. We will generate recombinant LCM viruses (rLCMV/NP*) containing mutated forms of NP that retain wt activity in virus RNA synthesis and infectious particle formation but lost the ability to inhibit IRF3 activation and IFN production. We hypothesize that these rLCMV/NP* will induce higher levels of IFN than wt LCMV at very early times of infection resulting in virus attenuation. To test this we will compare rLCMV/NP* and WT virus infections of mice with respect to: 1) their ability to multiply and induce IFN production and adaptive T cell immune responses, 2) ability to induce lethal LCM, and 3) ability to establish persistence.