Anti-interferon Activity of the Arenavirus Nucleoprotein

摘要 原型沙粒病毒LCMV已被证明是研究抗病毒宿主的罗塞塔石碑 免疫应答以及病毒免疫逃避和持久性的机制。此外，证据 表明LCMV在世界范围内分布，是一种被忽视的具有临床意义的人类病原体。 此外，拉沙病毒和其他几种沙粒病毒在人类中引起出血热（HF）疾病。 宿主先天性免疫应答控制病毒增殖的失败有助于沙粒病毒诱导的 疾病我们已经表明，LCMV和HF沙粒病毒的核蛋白（NP）抑制 转录因子IRF 3的激活和I型干扰素（IFN）的产生，I型干扰素（IFN）是先天免疫的关键因素 免疫力我们的长期目标是了解干扰素的分子基础和生物学后果 与沙粒病毒NP相关的抵消活性。为此，我们提出以下具体目标： 目标1.确定LCMV-NP的遗传决定因素，负责其IFN拮抗功能。 我们已经鉴定了LCMV-NP的两个C-末端结构域，这两个结构域对于NP介导的IRF 3抑制至关重要。 和IFN β启动子激活。我们推测，这两个区域内的特定残基发挥了 关键作用的干扰素拮抗功能的NP，并认为这种功能可以分离， NP在病毒复制中的作用。我们建议进行全面的突变功能分析， 这些区域鉴定NP中的单个氨基酸突变，所述突变消除其抗IFN功能， 影响其在病毒复制中的作用。 目标2.确定NP发挥其IFN拮抗活性的机制。我们已经证明 LCMV-NP通过抑制IRF 3的活化和核转位来抵消IFN应答。我们 假设LCMV-NP通过与参与激活的宿主细胞蛋白相互作用来实现这一点 IRF 3的我们将继续研究蛋白质组学方法，包括亲和纯化方法和质谱联用。 光谱分析程序来鉴定NP相互作用的细胞蛋白。我们将使用生物化学和遗传学 方法，连同功能测定，以评估所确定的NP相互作用细胞的贡献， NP的IFN拮抗活性。 目标3.评估NP的IFN拮抗活性对病毒增殖的贡献， LCMV感染其自然宿主小鼠期间的发病机制。我们将重组 LCM病毒（rLCMV/NP*），含有在病毒RNA合成中保留wt活性的NP突变形式 和感染性颗粒形成，但失去了抑制IRF 3活化和IFN产生的能力。我们 假设这些rLCMV/NP* 在非常早的时间将诱导比wt LCMV更高水平的IFN 感染导致病毒衰减。为了验证这一点，我们将比较rLCMV/NP* 和WT病毒 感染小鼠：1）它们繁殖和诱导IFN产生和适应性T细胞的能力 免疫应答，2）诱导致死LCM的能力，和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.