Control of Viral Pathogenesis by Regulation of 2-5A Levels

描述（由申请方提供）：病毒逃避或拮抗宿主I型IFN应答的能力在病毒嗜性和疾病发病机制中起重要作用。然而，病毒阻碍组织特异性宿主防御导致病毒诱导的病理学的精确细胞和分子机制仍然难以捉摸。冠状病毒，小鼠肝炎病毒（MHV），在其天然宿主中引起急性肝炎，并为理解病毒与肝细胞的相互作用提供了有用的模型。MHV蛋白ns 2阻断巨噬细胞中IFN诱导的和有效的抗病毒2 '，5'-寡腺苷酸（2-5A）合成酶（OAS）-RNase L途径，并促进肝炎的发展。ns 2蛋白和相关的病毒（轮状病毒VP 3）和哺乳动物（AKAP 7）蛋白是具有2 '，5'-磷酸二酯酶（PDE）活性的2 H磷酸酯酶，其降解2-5A，RNA酶L的激活剂。我们的长期目标是探索病毒和宿主2 '，5'-PDE在抗病毒天然免疫中的作用及其对病毒发病机制的影响。我们在此的总体假设是，巨噬细胞中的OAS-RNase L途径在保护宿主免于发展病毒性肝炎中至关重要。我们的具体目标是：（1）确定肝巨噬细胞中的RNase L信号传导是否（枯否细胞，KC）限制病毒进入肝实质并诱导肝炎，我们将比较KC、肝内皮细胞和肝细胞的原代培养物中MHV复制和OAS-RNase L途径的活化，产生具有RNase L基因的细胞类型特异性缺失的小鼠，并使用野生型和ns 2突变型MHV评估肝炎的发展。(2)为了确定ns 2相关的病毒和宿主2 '，5'-PDE的作用，我们将研究它们对2-5A水平、病毒复制、细胞凋亡和IFN-γ诱导的影响;并鉴定ns 2相关的病毒和宿主2 '，5'-PDE的小分子抑制剂。 病毒2 '，5'-PDE（ns 2和VP 3）和细胞2 '，5'-PDE（AKAP 7）。(3)为了确定和比较细胞和病毒2 ′，5 ′-PDE的“前病毒”和RNA酶L“拮抗剂”活性，我们将通过确定MHV复制复合物和ns 2和细胞AKAP 7的亚细胞定位来研究2 ′，5 ′-PDE介导的对MHV的保护的潜在机制;产生表达不同病毒和细胞2 ′，5 ′-PDE的嵌合病毒，并测定它们拮抗RNA酶L和诱导肝炎的能力。此外，为了确定在持续感染期间控制2-5A水平对病毒发病机理的影响，将用2 '，5'-PDE的抑制剂处理病毒感染的小鼠。我们提出的研究将调查一种新的模式的干扰素抗病毒反应的控制病毒感染，扩展到肝炎以外的病毒介导的病理在其他器官系统的广泛影响。预期结果包括将KC定义为防止病毒进入肝实质的“看门人”，并定义不同病毒和细胞2 '，5'-PDE的前病毒和RNA酶L拮抗剂作用。预计进一步了解病毒和I型IFN信号之间的竞争，特别是OAS-RNase L途径，将有助于开发或完善人类病毒性肝炎和其他病毒性疾病的治疗策略。

DESCRIPTION (provided by applicant): The ability of viruses to evade or antagonize the host type I IFN response plays an important role in viral tropism and disease pathogenesis. However, the precise cellular and molecular mechanisms by which viruses impede tissue specific host defenses leading to virus-induced pathology remain elusive. The coronavirus, mouse hepatitis virus (MHV), causes acute hepatitis in its natural host and provides a useful model for understanding virus interaction with liver cells. The MHV protein ns2 blocks the IFN-inducible and potent antiviral 2',5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L pathway in macrophages and facilitates the development of hepatitis. The ns2 protein and related viral (rotavirus VP3) and mammalian (AKAP7) proteins are 2H phosphoesterases with 2',5'-phosphodiesterase (PDE) activities that degrade 2-5A, the activator of RNase L. Our long-term objective is to probe the role of viral and host 2',5'-PDEs in antiviral innate immunity and its effect on viral pathogenesis. Our overall hypothesis herein is that the OAS-RNase L pathway in macrophages is critical in protecting the host from developing viral hepatitis. Our Specific Aims are: (1) To determine if RNase L signaling in liver macrophages (Kupffer cells, KC) restricts virus from entering the liver parenchyma and inducing hepatitis, we will compare MHV replication and activation of the OAS-RNase L pathway in primary cultures of KC, liver endothelial cells and hepatocytes, generate mice with cell type specific deletions of the RNase L gene and assess the development of hepatitis using wild type and ns2 mutant MHV. (2) To determine the roles of ns2 related viral and host 2',5'-PDEs, we will study their effects on 2-5A levels, viral replication, apoptosis and IFN-¿ induction; and identify small molecule inhibitors of viral 2',5'-PDEs (ns2 and VP3) and cellular 2',5'-PDE (AKAP 7) by high throughput screening. (3) To determine and compare the "proviral" and RNase L "antagonist" activities of cellular and viral 2',5'-PDEs, we will investigate the mechanisms underlying 2',5'-PDE mediated protection to MHV by determining subcellular location of MHV replication complexes and ns2 and cellular AKAP7; generate chimeric viruses expressing different viral and cellular 2',5'-PDEs and determine their ability to antagonize RNase L and induce hepatitis. In addition, to determine effects of controlling 2-5A levels on viral pathogenesis during ongoing infections, virus-infected mice will be treated with inhibitors of 2',5'-PDEs. Our proposed studies will investigate a novel mode of regulation of the IFN antiviral response with broad implications for the control of viral infections that extend beyond hepatitis to virus mediated pathologies in other organ systems. Expected results include defining the role of KC as "gatekeeper" that prevents viruses from entering into the liver parenchyma and defining proviral and RNase L antagonist roles of diverse viral and cellular 2',5'-PDEs. It is anticipated that further understanding of the competition between virus and type I IFN signaling, particularly the OAS-RNase L pathway, will aid in the development or refinement of therapeutic strategies for human viral hepatitis and other viral diseases.