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

描述(由申请人提供)：病毒逃避或对抗宿主I型干扰素反应的能力在病毒趋向性和疾病发病机制中发挥着重要作用。然而，病毒阻碍组织特异性宿主防御导致病毒诱导的病理的确切的细胞和分子机制仍然不清楚。冠状病毒，小鼠肝炎病毒(MHV)，在其自然宿主中引起急性肝炎，为了解病毒与肝细胞的相互作用提供了有用的模型。MHV蛋白NS2阻断了巨噬细胞中干扰素诱导的、有效的抗病毒2‘，5’-寡腺苷合成酶-核糖核酸酶L途径，促进了肝炎的发生发展。NS2蛋白和相关的病毒(轮状病毒VP3)和哺乳动物(AKAP7)蛋白是具有2‘，5’-磷酸二酯酶(PDE)活性的2H磷酸酯酶，它能降解核糖核酸酶L的激活剂2-5A。我们的长期目标是探讨病毒和宿主2‘，5’-PDE在抗病毒先天免疫中的作用及其在病毒发病机制中的作用。我们在此的总体假设是，巨噬细胞中的oas-核糖核酸酶L通路在保护宿主免受病毒性肝炎的影响中起着至关重要的作用。我们的具体目标是：(1)为确定肝巨噬细胞(Kupffer cell，KC)中的核糖核酸酶L信号是否限制病毒进入肝实质而引发肝炎，我们将比较MHV在KC原代培养、肝内皮细胞和肝细胞中的复制和激活情况，建立RNaseNS2基因的细胞型特异性缺失小鼠，并利用野生型和突变型MHV评估肝炎的发生发展。(2)为了确定NS2相关病毒和宿主2‘，5’-PDE的作用，我们将研究它们对2-5A水平、病毒复制、细胞凋亡和干扰素-β诱导的影响；并鉴定小分子抑制物。 病毒2‘，5’-PDE(NS2和VP3)和细胞2‘，5’-PDE(AKAP 7)的高通量筛选。(3)为了检测和比较细胞和病毒2‘，5’-PDE的“前病毒”和核糖核酸酶L“拮抗剂”的活性，我们将通过确定MHV复制复合体和NS2和细胞AKAP7的亚细胞定位来研究2‘，5’-PDE介导MHV保护的机制；构建表达不同病毒和细胞2‘，5’-PDE的嵌合病毒并测定其拮抗核糖核酸酶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水平对病毒发病的影响，将用2‘，5’-PDE的抑制剂治疗病毒感染的小鼠。我们建议的研究将探索一种新的干扰素抗病毒反应调节模式，对控制病毒感染具有广泛的意义，病毒感染从肝炎延伸到其他器官系统的病毒介导的病理。预期结果包括将KC定义为防止病毒进入肝实质的“把关人”角色，以及定义不同病毒和细胞2‘，5’-PDE的前病毒和核糖核酸酶L拮抗剂的角色。预计进一步了解病毒和I型干扰素信号之间的竞争，特别是OAS-核糖核酸酶L通路，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.