Role of NLRX1 in dengue virus infection

描述（由申请人提供）：登革热病毒是地球上最流行的人类虫媒病毒，每年感染约5000万至1亿人。尽管存在这种公共卫生负担，但目前没有获得许可的疫苗或治疗方法来治疗登革热。众所周知，宿主I型干扰素反应在限制登革热病毒的复制和传播方面发挥着关键作用，因此登革热已经进化出了阻断感染细胞中干扰素信号传导但不产生干扰素的对策。NLR(核苷酸结合域和leucine-rich-repeat-containing或nod样受体)家族的蛋白质已被确认为发起者和监管者的先天免疫反应各种病原体包括一些病毒。NLRX1是NLR家族的新成员，已被证明是mavs介导的抗病毒信号的负调节因子，研究表明，在病毒感染的情况下，敲低NLRX1的表达会导致干扰素的产生增加。然而，迄今为止，NLRX1在登革热病毒感染中的作用尚未得到探讨。此外，由于缺乏合适的小动物模型来研究登革热的发病机制和宿主对感染的免疫反应，登革热病毒的研究受到限制。那些已经开发出来的技术在研究人类对感染的先天免疫反应方面能力有限。本提案的目的是利用体外和体内系统研究NLRX1在人类对各种相关登革热病毒感染的先天免疫反应中的作用。目的1将研究NLRX1在登革热感染的人细胞中的功能作用。在这个目的中，我们将敲除人单核细胞中的NLRX1，以确定NLRX1在登革热感染期间如何调节mavs介导的抗病毒信号。这也将在受体介导与抗体增强感染的背景下进行检查。目的2将建立一种新型的人源化登革热病毒感染小鼠模型，以研究人的免疫反应。我们将用人免疫细胞和人肝细胞培养Rag2-gC双敲除（DKO）小鼠（AFC8-hu HSC/Hpe小鼠），以检测DENV病的内脏性和人对感染的免疫反应。目的3将利用这种人源化DENV疾病小鼠模型来评估人类NLRX1在DENV感染的先天免疫反应和疾病进展中的作用。我们将在移植到AFC8-hu HSC/Hpe小鼠的人细胞中使用shRNA敲低NLRX1，并将DENV感染与NLRX1水平不变的小鼠进行比较。综上所述，本研究结果将为DENV感染的先天免疫反应提供重要信息，并为研究DENV发病机制和人类免疫反应提供一种新的小动物模型。

DESCRIPTION (provided by applicant): Dengue virus is the most prevalent human arbovirus on earth, infecting an estimated 50-100 million individuals annually. Despite this public health burden, there are currently no licensed vaccines or therapeutics to treat dengue. The host type I interferon response is known to play a critical role in limiting replication and spread of dengue virus, and as such dengue has evolved countermeasures by which it blocks interferon signaling, but not production, in infected cells. The NLR (nucleotide binding domain and leucine-rich-repeat-containing or NOD-like receptor) family of proteins has been identified as initiators and regulators of innate immune responses to a wide range of pathogens including some viruses. NLRX1, a new member of the NLR family, has been shown to act as a negative regulator of MAVS-mediated antiviral signaling, and studies have indicated that knockdown of NLRX1 expression leads to increased interferon production in the presence of viral infection. To date, however, the role of NLRX1 in dengue virus infection has not been explored. Additionally, dengue virus research has been limited by the lack of suitable small animal models in which to study dengue pathogenesis and host immune responses to infection. Those that have been developed are limited in their ability to study human innate immune responses to infection. The goal of this proposal is to examine the role of NLRX1 in the human innate immune response to a variety of relevant dengue virus infections using both in vitro and in vivo systems. Aim 1 will examine the functional role of NLRX1 in dengue- infected human cells. In this aim we will knockdown NLRX1 in human monocytic cells to determine how NLRX1 modulates MAVS-mediated antiviral signaling during infection with dengue. This will also be examined in the context of receptor-mediated vs. antibody-enhanced infection. Aim 2 will establish a novel humanized mouse model of viscerotropic dengue virus infection in which to study human immune responses. We will generate Rag2-gC double knockout (DKO) mice humanized with human immune cells and human liver cells (AFC8-hu HSC/Hpe mice) in order to examine viscerotropic DENV disease and human immune responses to infection. Aim 3 will utilize this humanized mouse model of DENV disease to assess the role human NLRX1 plays in innate immune response to and disease progression of DENV infection. We will use shRNA knockdown of NLRX1 in human cells engrafted into AFC8-hu HSC/Hpe mice and compare DENV infection with that of mice with unaltered NLRX1 levels. Taken together the results of this proposal will provide critical information about innate immune responses to DENV infection and provide a novel small animal model in which to study DENV pathogenesis and human immune responses.