Arenavirus Subversion of Dendritic Cells

描述(申请人提供)：在自然感染和疫苗接种过程中，树突状细胞(DC)在启动先天和获得性免疫反应中发挥关键作用。树突状细胞的两个主要亚群，浆细胞样树突状细胞和常规树突状细胞，已经在人类和小鼠中被描述，并发挥着独特的和重叠的免疫功能。在以DC为靶点的病毒中，ArenaVirus作为致命的人类病原体引起了极大的兴趣，NIAID认为是A类病毒。重要的是，ArenaVirus感染和颠覆DC是其抑制免疫系统、击败受感染宿主并导致疾病的基础，但这一过程的分子基础仍然知之甚少。阿雷诺病毒核蛋白(NP)参与病毒的复制、转录、组装和抑制I型干扰素(IFN-I)，干扰素是一类重要的抗病毒细胞因子。我们发现，在小鼠体内感染的过程中，浆细胞样树突状细胞和常规树突状细胞会被原型的阿雷诺病毒LCMV Clone 13高效感染。此外，在绝大多数受感染的DC中，干扰素-I的产生受到抑制。我们假设ArenaVirus NPs的上述功能是通过劫持关键宿主蛋白来实现的，这些宿主蛋白最终使DC中的病毒生命周期成功和/或DC功能受损。我们的目标是确定与ArenaVirus 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学确定LCMV感染的血浆细胞样树突状细胞和常规DC中的NP结合伙伴，然后验证这些与其他ArenaVirus NPs的物理相互作用。接下来，我们将敲除NP相互作用的蛋白，以研究它们对树突状细胞内ArenaVirus复制的重要性以及它们在病毒诱导的DC功能损害中的作用。这些实验将在R21阶段进行，预计将提供ArenaVirus利用的宿主因子的简短列表，以实现其在DC中的生产性复制和/或在体外禁用DC功能。在该项目的R33阶段，具有选定NP相互作用蛋白基因改变的小鼠或仓鼠将分别感染LCMV Clone 13(感染DC并抑制免疫系统)或Pichinde arenaVirus(导致仓鼠严重出血热)。将监测病毒的生长、免疫反应和存活率。这些研究将评估靶向选定的DC蛋白以改变体内ArenaVirus感染过程的可能性。我们研究的短期影响将是确定在ArenaVirus复制和/或DC的颠覆中发挥生物学意义的分子决定因素，并且可以操纵这些决定因素来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一新知识转化为人类感染ArenaVirus和可能是其他嗜DC病毒的新治疗方式。

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.