A new mechanism of antiviral activity of 2'-5' Oligoadenylate Synthetase 1

摘要 这项建议旨在通过干扰素刺激基因(ISG)确定一种新的抗病毒活性机制 寡腺苷合成酶1(OAS1)。宿主的先天免疫反应是由对非我的感觉启动的 病毒核酸，主要通过I型和III型干扰素(干扰素)介导。干扰素诱导细胞因子的表达 数百个ISG，其中许多抑制受感染细胞中的病毒复制，保护未受感染的邻近细胞， 并塑造适应性免疫反应，以清除病毒感染。OAS是属于ISG的一个家族 进化上古老的核苷酸转移酶家族(NTase)。OAS的典型抗病毒机制 蛋白质涉及2‘-5’-寡腺苷的酶合成，导致核糖核酸酶L的下游激活 并导致蛋白质合成的抑制。然而，多糖的抗病毒作用机制 人类和小鼠基因组中存在的具有酶活性和非活性的OAS亚型尚不清楚。 我们已经发现，一种特定的OAS1亚型可以增强多种抗病毒蛋白的翻译，包括 CGAS和IRF1。这种OAS1亚型(OAS1 P46)增强翻译，不依赖于它的酶活性和 我们的初步结果表明，OAS1 P46通过 结合各自的mRNA。在人类中，OAS1 P46是由于C-端的另一种剪接事件而产生的。 OAS1基因的末端。该选择性剪接位点的自然多态(rs10774671，A/G) 调节P46的表达，并与多种病毒感染的疾病严重程度有关。vbl.使用 我们证明了编码P46的rs10774671 G等位基因也增强了 T细胞中IRF1蛋白的表达。我们还提供了多种证据，表明功能等价性 在OAS1 P46和小鼠同源基因Oas1b(没有NTase活性)之间，Oas1b类似地影响西尼罗河病毒的易感性 在活体内。基于这些观察，我们假设OAS1的特定亚型调节先天免疫。 通过独特的不依赖于NTase活性的机制对病毒作出反应。 这项提案的目标是确定OAS1如何增强特定蛋白质的翻译，以及其后果 新发现的OAS1在抗病毒、先天免疫和获得性免疫方面的功能。我们的三个独立的 目的：(1)确定OAS1介导的翻译调控的分子机制。 生化和细胞生物学方法；(2)确定介导OAS1抗病毒作用的细胞靶点 使用几个基因缺陷的细胞；以及(3)通过使用一种新的 生成了Oas1b敲入小鼠。在这项研究完成后，我们应该建立一个新的抗病毒范例 OAS蛋白的活性可能导致调节OAS1-cGAS-I型干扰素轴的治疗策略。

ABSTRACT This proposal aims to define a new mechanism of antiviral activity by an Interferon stimulated gene (ISG) Oligoadenylate Synthetase 1 (OAS1). The host innate immune response is initiated by the sensing of non-self viral nucleic acid, and largely mediated through type I and III interferons (IFN). IFNs induce expression of hundreds of ISGs, many of which inhibit virus replication in infected cells, protect uninfected neighboring cells, and shape the adaptive immune response to clear virus infection. OASs are a family of ISG that belongs to an evolutionarily ancient family of nucleotidyl-transferases (NTase). The canonical antiviral mechanism of OAS proteins involves the enzymatic synthesis of 2'-5'-oligoadenylates, causing downstream activation of RNase L and leading to the inhibition of protein synthesis. However, the mechanisms of antiviral activity of multiple enzymatically active and inactive OAS isoforms present in human and mouse genomes are not yet clear. We have found that a specific OAS1 isoform enhances the translation of multiple antiviral proteins, including cGAS and IRF1. This OAS1 isoform (OAS1 P46) enhances translation independent of its enzymatic activity and RNase L. Our preliminary results suggest that OAS1 P46 enhances the translation of specific proteins through binding respective mRNAs. In humans, OAS1 P46 is generated due to an alternative splicing event at the C- terminal of the OAS1 gene. A naturally occurring polymorphism (rs10774671, A/G) at this alternative splice site regulates P46 expression, and has been associated with disease severity to multiple virus infections. Using primary human hematopoietic cells, we demonstrate that rs10774671 G allele coding for P46 also enhances IRF1 protein expression in T cells. We also provide multiple evidences suggesting the functional equivalence between OAS1 P46 and a mouse ortholog, Oas1b (no NTase activity), which similarly affects WNV susceptibility in vivo. Based on these observations, we hypothesize that specific isoforms of OAS1 modulate innate immune responses against viruses through unique NTase activity-independent mechanisms. The goal of this proposal is to determine how OAS1 enhances specific protein translation, and the consequences of this newly identified function of OAS1 on the antiviral innate and adaptive immunity. Our three independent Aims are to: (1) Determine the molecular mechanism of OAS1-mediated translational regulation through biochemical and cell biology approaches; (2) Define the cellular targets mediating the antiviral effect of OAS1 using several gene-deficient cells; and (3) Define the in vivo role of Oas1b during virus infection using a newly generated Oas1b knock-in mouse. Upon completion of this study, we should establish a new paradigm of antiviral activity by OAS proteins that may lead to therapeutic strategies modulating this OAS1-cGAS-type I IFN axis.