Arenavirus Subversion of Dendritic Cells

描述（由申请人提供）：树突状细胞（DC）在自然感染和疫苗接种期间启动先天性和适应性免疫应答中发挥关键作用。两个主要的DC亚群，浆细胞样DC和常规DC，已经在人类和小鼠中被描述，并发挥独特的和重叠的免疫功能。在靶向DC的病毒中，沙粒病毒作为致命的人类病原体受到极大关注，并被NIAID认为是A类病原体。重要的是，沙粒病毒感染和DC的破坏是其抑制免疫系统、战胜感染宿主和引起疾病的能力的基础，但这一过程的分子基础仍然知之甚少。 沙粒病毒核蛋白（NP）参与病毒复制、转录、装配和抑制I型干扰素（IFN-I），一组重要的抗病毒细胞因子。我们发现，在小鼠体内感染过程中，原型沙粒病毒LCMV克隆13有效地感染了浆细胞样和常规DC。此外，IFN-I的产生在绝大多数感染的DC中被抑制。我们假设沙粒病毒NP的上述功能是通过劫持关键宿主蛋白质来实现的，这些蛋白质最终能够在DC中成功地实现病毒生命周期和/或DC功能损伤。 我们的目标是鉴定与沙粒病毒NP相互作用并有助于病毒生长和/或免疫逃避的DC蛋白。为此，我们将首先通过质谱鉴定LCMV感染的浆细胞样和常规DC中的NP结合配偶体，然后验证这些与其他沙粒病毒NP的物理相互作用。接下来，我们将敲除NP相互作用蛋白，以研究它们对沙粒病毒在DC内复制的重要性以及它们在病毒诱导的DC功能损伤中的作用。这些实验将在R21阶段进行，预计将提供沙粒病毒利用的宿主因子的短列表，以使其能够在DC中进行生产性复制和/或体外禁用DC功能。在该项目的R33阶段，将分别用LCMV克隆13（感染DC并抑制免疫系统）或Pichinde沙粒病毒（导致仓鼠严重出血热）感染具有选定NP相互作用蛋白遗传改变的小鼠或仓鼠。将监测病毒生长、免疫应答和存活率。这些研究将评估靶向选定DC蛋白以改变沙粒病毒体内感染过程的可能性。 我们研究的短期影响将是鉴定在沙粒病毒复制和/或破坏DC中发挥生物学意义作用的分子决定因素，并且可以操纵这些分子决定因素以改变小动物模型中感染的结果。我们研究的长期影响将是将这些新知识转化为人类感染沙粒病毒（可能是其他DC嗜性病毒）的新型治疗方式。 公共卫生相关性：树突状细胞在宿主防御感染中发挥重要作用。我们建议解开DCs被沙粒病毒破坏的分子机制，沙粒病毒包括在婴儿中引起致命的出血热或神经系统疾病以及在免疫功能低下的成人中引起致命症状的病毒。

DESCRIPTION (provided by applicant): Dendritic Cells (DCs) play key roles in the initiation of innate and adaptive immune responses during natural infections and vaccination. Two major subsets of DCs, plasmacytoid and conventional DCs, have been described in humans and mice, and exert unique and overlapping immune functions. Among viruses that target DCs, Arenaviruses are of great interest as lethal human pathogens and considered category A agents by NIAID. Importantly, arenavirus infection and subversion of DCs is fundamental for their ability to suppress the immune system, outcompete the infected host and cause disease, but the molecular bases of this process remain poorly understood. The arenavirus nucleoprotein (NP) participates in viral replication, transcription, assembly and suppression of type I interferons (IFN-I), a group of important anti-viral cytokines. We found that plasmacytoid and conventional DCs are productively infected by the prototypic arenavirus LCMV Clone 13 during in vivo murine infection. Furthermore, IFN-I production is inhibited in the vast majority of infected DCs. We hypothesize that the aforementioned functions of arenavirus NPs are achieved by hijacking key host proteins that ultimately enable a successful viral life cycle in DCs and/or DC functional impairment. Our goal is to identify the DC proteins that interact with arenavirus NP and contribute to viral growth and/or immune-evasion. For that, we will first identify NP binding partners in LCMV infected plasmacytoid and conventional DCs by Mass Spectrometry and then validate these physical interactions with other arenavirus NPs. Next, we will knockdown the NP interacting proteins to investigate their importance for arenavirus replication within DCs as well as their role in virus-induced DC functional impairment. These experiments will be performed during the R21 phase and are expected to provide a short-list of host factors that are exploited by arenaviruses to enable their productive replication in DCs and/or disable DC functions in vitro. During the R33 phase of this project, mice or hamsters with genetic alteration of selected NP interacting proteins will be infected with LCMV Clone 13 (infects DCs and suppresses the immune system) or Pichinde arenavirus (causes severe hemorrhagic fever in hamsters), respectively. Viral growth, immune responses and survival will be monitored. These studies will assess the likelihood of targeting selected DC proteins to change the course of an arenavirus infection in vivo. The short-term impact of our studies will be the identification of molecular determinants that play biologically meaningful roles in Arenavirus replication and/or subversion of DCs and that can be manipulated to change the outcome of the infection in small animal models. The long-term impact of our studies will be to translate this new knowledge into novel therapeutic modalities in human infections with arenaviruses and may be other DC-tropic viruses. PUBLIC HEALTH RELEVANCE: Dendritic Cells play fundamental roles in host defenses against infections. We propose to unravel the molecular mechanisms by which DCs are subverted by Arenaviruses, which encompass viruses that cause fatal hemorrhagic fevers or neurological disorders in infants and lethal symptoms in immuno-compromised adults.