Novel Pathogen Recognition Pathways and Control of Hepatitis A Virus

尽管疫苗和抗病毒疗法取得了进展，但肝脏的病毒感染继续在世界范围内造成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，属于细小病毒科肝炎病毒属，是引起急性肝炎的常见原因，是B类NIAID的优先病原体。我们之前发表的研究表明，甲型肝炎病毒以一种异常隐蔽的方式感染肝脏，尽管在几周的时间里在肝细胞内强劲复制，但仍能引起由L/11干扰素刺激的小型基因表达。急性肝损伤发生在感染后3-4周，伴随着病毒特异性抗体的最初出现，促炎症细胞因子显著增加，与感染的缓解相关的主要是CD4+T细胞反应。我们在这个项目中的目标是更好地了解甲型肝炎病毒是如何被先天性免疫系统识别的，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的发现基础上，即传统上被认为是无包膜病毒的甲型肝炎病毒从受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的血液中循环。这些新型的包膜甲型肝炎病毒粒子(EHAV)是完全具有传染性的，但完全对中和抗体具有抵抗力。我们的初步数据显示，含有纯化的eHAV的梯度组分独特地诱导人pDC分泌干扰素-α，并刺激THP-1细胞的促炎细胞因子和NLRPS炎性依赖的IL-1B的表达，而高度浓缩的标准无包膜病毒粒子则不能。在目标1中，我们将确定与甲型肝炎病毒感染细胞共培养时激活pDC所需的宿主因素，以及与Core B合作激活PDC激活微囊的RNA和蛋白质组成。我们还将量化甲型肝炎患者肝组织中PDC的募集情况。目标2将研究THP-1细胞和原代人类单核/巨噬细胞如何感知eHAV，并与项目2和3合作确定导致甲型肝炎病毒激活炎症小体的信号来源。这些研究将利用核心C的独特能力来提供纯化的重组NLR，用于体外定量研究它们作为病原体相关分子模式的直接受体的潜力。目的3明确先天免疫反应和甲型肝炎病毒激活肝细胞炎性小体的分子基础。这些研究支持这一多项目U19应用的目标，方法是利用独特的生化能力来研究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确定涉及感知和控制人类病毒的范式转换信号通路，检查人类对NIAID B类优先病原体的反应中PRR通路之间的串扰，调查PRR对病毒配基的细胞内运输，以及最大限度地利用机会用原始人类材料验证实验结果。

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.