Novel Pathogen Recognition Pathways and Control of Hepatitis A Virus

尽管疫苗和抗病毒治疗取得了进展，但肝脏的病毒感染仍然在世界范围内引起大量的人类发病率和死亡率。甲型肝炎病毒（HAV）是一种正链RNA病毒，属于小核糖核酸病毒科肝病毒属，是急性肝炎的常见病因，也是NIAID的B类优先病原体。我们先前发表的研究表明，HAV以一种非常隐蔽的方式感染肝脏，尽管在肝细胞内经过几周的时间进行了稳健的复制，但几乎没有引起I/III型干扰素（IFN）刺激的基因表达。感染后3-4周急性肝损伤的发生以促炎细胞因子的显著增加为标志，同时病毒特异性抗体的最初出现和与感染消退相关的主要CD 4 + T细胞应答。我们在这个项目中的目标是更好地了解HAV是如何被先天免疫系统识别的，以及这如何有助于最终控制感染。拟议的研究建立在我们最近发现的基础上，即HAV，传统上被认为是一种无包膜病毒，从感染的肝细胞中释放出来，并在完全被宿主膜包裹的感染者血液中循环。这些新的包膜HAV病毒粒子（eHAV）是完全感染性的，但对中和抗体完全耐受。我们的初步数据显示，含有纯化的eHAV的梯度级分独特地诱导人pDC分泌IFN-α并刺激THP-1细胞的促炎细胞因子和NLRPS炎性体依赖性IL-1B表达，而高度浓缩的标准无包膜病毒粒子则不诱导。在目标1中，我们将确定当与HAV感染的细胞共培养时激活pDC的宿主因子需求，以及与核心B合作的pDC激活微泡的RNA和蛋白质组成。我们还将定量甲型肝炎中pDC向肝脏的募集。目标2将研究eHAV如何被THP-1细胞和原代人单核细胞/巨噬细胞感知，并与项目2和3合作，确定导致HAV炎性小体激活的信号来源。这些研究将利用核心C的独特能力，提供纯化的重组NLR，用于定量体外研究其作为病原体相关分子模式的直接受体的潜力。目的3将明确先天性免疫应答和HAV激活肝细胞炎性小体的分子基础。这些研究通过利用独特的生物化学能力来研究NLR作为新型病原体识别受体（PRR）的作用，应用尖端的定量蛋白质组学方法来鉴定参与人类病毒感测和控制的范式转变信号传导途径，检查人类对NIAID类别B优先病原体的反应中PRR途径之间的串扰，通过PRR研究病毒配体到抑制位点的细胞内运输，并最大限度地利用机会用原代人体材料验证实验结果。

Virus infections of the liver continue to cause substantial human morbidity and mortality worldwide despite advances in vaccines and antiviral therapy. Hepatitis A virus (HAV), a positive-strand RNA virus classified within the genus Hepatovirus of the family Picornaviridae, is a common cause of acute hepatitis and a Category B NIAID Priority Pathogen. Our previously published research reveals that HAV infects the liver in an extraordinarily stealthy fashion, evoking little type l/lll interferon (IFN)-stimulated gene expression despite robust replication within hepatocytes over a period of several weeks. The onset of acute hepatic injury 3-4 weeks after infection is marked by impressive increases in pro-inflammatory cytokines simultaneous with the initial appearance of virus-specific antibodies and a predominantly CD4+ T cell response that correlates with resolution ofthe infection. Our goal in this project is to better understand how HAV is recognized by the innate immune system and how this may contribute to ultimate control ofthe infection. The proposed studies build on our recent discovery that HAV, classically considered a non-enveloped virus, is released from infected hepatocytes and circulates in the blood of infected humans completely enveloped in host membranes. These novel 'enveloped HAV virions (eHAV) are fully infectious but completely resistant to neutralizing antibodies. Our preliminary data show that gradient fractions containing purified eHAV uniquely induce human pDCs to secrete IFN-a and stimulate pro-inflammatory cytokine and NLRPS inflammasomedependent IL-1B expression by THP-1 cells, while highly concentrated, standard, non-enveloped virions do not. In Aim 1, we will determine host factor requirements for activation of pDCs when co-cultured with HAV infected cells, and the RNA and protein composition of pDC-activating microvesicles in collaboration with Core B. We will also quantify pDC recruitment to the liver in hepatitis A. Aim 2 will study how eHAV is sensed by THP-1 cells and primary human monocyte/macrophages, and in collaboration with Projects 2 and 3 identify the origin of signals leading to inflammasome activation by HAV. These studies will exploit unique capabilities of Core C to provide purified recombinant NLRs for quantitative in vitro studies of their potential as direct receptors of pathogen-associated molecular patterns. Aim 3 will define innate immune responses and the molecular basis of inflammasome activation by HAV in hepatocytes. These studies support the goals of this multi-project U19 application by leveraging unique biochemical capabilities to investigate the role of NLRs as novel pathogen recognition receptors (PRRs), applying cutting-edge quantitative proteomic approaches to identify paradigm-shifting signaling pathways involved in sensing and control of human viruses, examining cross-talk between PRR pathways in the human response to a NIAID Category B Priority Pathogen, investigating intracellular trafficking of a viral ligand to the site of engagment by a PRR, and taking maximal advantage of opportunities to validate experimental findings with primary human materials.