Anti-interferon Activity of the Arenavirus Nucleoprotein

描述(申请人提供)：原型ArenaVirus LCMV已被证明是研究抗病毒宿主免疫反应以及病毒免疫逃避和持续机制的一块罗塞塔石碑。此外，有证据表明，全球分布的LCMV是一种被忽视的具有临床意义的人类病原体。此外，其他几种阿拉伯病毒会引起人类出血热(HF)疾病。宿主的先天免疫反应不能控制病毒的繁殖，从而导致病毒引起的疾病。我们已经证明LCMV和HF ARENAV的核蛋白(NP)抑制转录因子IRF3的激活和I型干扰素(IFN-I)的产生，I型干扰素是先天性免疫中的关键角色。我们的长期目标是了解与ArenaVirus NP相关的干扰素中和活性的分子基础和生物学后果。为此，我们提出了以下具体目标：目的1.确定LCMV-NP具有干扰素拮抗功能的基因决定因素。我们已经确定了LCMV-NP的两个C-末端结构域，它们对NP的抗干扰素-I活性至关重要。我们假设这两个区域中的特定残基在NP的干扰素拮抗功能中起着关键作用，并且这一功能可以与NP在病毒复制和感染后代产生中的作用分开。我们建议对这些区域进行全面的突变功能分析，以确定NP中的单个氨基酸突变，这些突变取消了NP的抗干扰素功能，而不影响NP在病毒增殖中所需的其他作用。目的2.明确NP发挥干扰素拮抗作用的机制。我们已经证明，LCMV-NP通过抑制IRF3的激活和核转位来中和干扰素反应。我们假设LCMV-NP通过与参与IRF3激活的宿主细胞蛋白相互作用来实现这一点。我们将采用蛋白质组学方法，包括亲和纯化方法和质谱学程序来鉴定与NP相互作用的细胞蛋白质。我们将使用生化和遗传学方法，结合功能分析，评估已识别的与NP相互作用的细胞蛋白对NP的干扰素拮抗活性的贡献。目的3.探讨NP的干扰素拮抗活性在其自然宿主小鼠体内LCMV感染过程中对病毒增殖和致病作用的影响。我们将产生含有NP突变形式的重组LCM病毒(rLCMV/NP*)，这些NP在病毒RNA合成和感染性颗粒形成中保持wt活性，但失去抑制IRF3激活和干扰素产生的能力。我们假设，与LCMV/WT感染的细胞相比，rLCMV/NP*感染的细胞将表现出更高水平的IRF3诱导的基因表达，包括干扰素的产生。我们预测，这将导致rLCMV/NP*的生长特性改变，从而在急性和持续感染期间影响LCMV-宿主相互作用的结果。公共卫生关注性几种阿拉伯病毒引起人类出血热(HF)疾病，全球分布的原型性阿拉伯病毒LCMV被认为是一种被忽视的具有临床意义的人类病原体。此外，武器化形式的阿拉伯病毒作为生物恐怖主义的媒介构成严重威胁。目前还没有获得许可的抗ArenaVirus疫苗，目前的抗ArenaVirus治疗仅限于使用利巴韦林，利巴韦林只有部分疗效，而且往往与严重副作用有关。有证据表明，宿主的先天免疫反应不能控制病毒的复制在病毒引起的疾病中起着关键作用。我们已经证明LCMV和HF ArenaV的核蛋白(NP)抑制转录因子IRF3的激活和I型干扰素(IFN)的产生，I型干扰素是先天性免疫中的关键角色。我们的长期目标是了解与ArenaVirus NP相关的干扰素中和活性的分子基础和生物学后果。这一知识应该会推动更好的抗病毒战略的发展，以对抗人类致病Arena病毒。

DESCRIPTION (provided by applicant): 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, several other arenaviruses cause hemorrhagic fever (HF) disease in humans. The failure of the host innate immune response to control virus multiplication contributes to arenaviral-induced 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-I), 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 the anti-IFN-I activity of NP. 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 and production of infectious progeny. 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 other roles of NP required for arenavirus multiplication. 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 rLCMV/NP*-infected cells will exhibit higher levels of IRF3-inducible gene expression, including IFN production, compared to LCMV/WT-infected cells. We predict this to result in altered growth properties of rLCMV/NP* with consequences for the outcome of LCMV-host interaction during acute and persistent infection. PUBLIC HEALTH RELEVANCE Several arenaviruses cause hemorrhagic fever (HF) disease in humans, and the worldwide-distributed prototypic arenavirus LCMV is being considered as a neglected human pathogen of clinical significance. Moreover, weaponized forms of arenaviruses pose a serious threat as agents of bioterrorism. No licensed anti-arenavirus vaccines are available, and current anti-arenavirus therapy is limited to the use of ribavirin, which is only partially effective and often associated with severe side effects. Evidence indicates that a failure of the host innate immune response to control virus multiplication plays a critical role in arenaviral- induced 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. This knowledge should fuel the development of better antiviral strategies to combat human pathogenic arenaviruses.