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

摘要 该建议旨在通过干扰素刺激基因（ISG）来定义抗病毒活性的新机制 寡腺苷酸合成酶1（OAS 1）。宿主的先天性免疫反应是通过感知非我启动的。 病毒核酸，并且主要通过I型和III型干扰素（IFN）介导。干扰素诱导 数以百计的ISG，其中许多抑制病毒在感染细胞中复制，保护未感染的邻近细胞， 并形成适应性免疫反应以清除病毒感染。OAS是ISG的一个家族， 进化上古老的核苷酸转移酶家族（NTase）。OAS的经典抗病毒机制 蛋白质涉及2 '-5'-寡腺苷酸的酶促合成，引起RNA酶L的下游活化 并导致蛋白质合成的抑制。然而，多种抗病毒活性的机制 目前还不清楚人和小鼠基因组中存在的酶活性和无活性的OAS同种型。 我们已经发现，一种特定的OAS 1亚型增强了多种抗病毒蛋白的翻译，包括 cGAS和IRF 1。这种OAS 1同种型（OAS 1 P46）增强翻译，与其酶活性无关， 核糖核酸酶属我们的初步结果表明，OAS 1 P46通过以下途径增强特定蛋白的翻译： 结合各自的mRNA。在人类中，OAS 1 P46是由于C- OAS 1基因的末端。在该可变剪接位点天然存在的多态性（rs 10774671，A/G） 调节P46的表达，并与多种病毒感染的疾病严重程度相关。使用 在原代人造血细胞中，我们证明了编码P46的rs 10774671 G等位基因也增强了 T细胞中的IRF 1蛋白表达。我们还提供了多个证据表明功能等同 OAS 1 P46和小鼠直系同源物Oas 1b（无NTase活性）之间存在差异，其同样影响WNV易感性 in vivo.基于这些观察，我们假设OAS 1的特异性亚型调节先天免疫， 通过独特的非NTase活性依赖性机制对病毒的反应。 该提案的目标是确定OAS 1如何增强特定蛋白质翻译，以及其后果 OAS 1在抗病毒先天免疫和适应性免疫中的新功能。三个独立的 目的是：（1）确定OAS 1介导的翻译调控的分子机制， 生物化学和细胞生物学方法;（2）确定介导OAS 1抗病毒作用的细胞靶点 使用几种基因缺陷细胞;（3）使用新的基因缺陷细胞， Oas 1b基因敲入小鼠。这项研究完成后，我们应该建立一个新的抗病毒范例 通过OAS蛋白的活性，可能导致调节该OAS 1-cGAS-I型IFN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.