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

2-5寡腺苷酸合成酶1抗病毒活性的新机制

基本信息

批准号：
10299611
负责人：
Michael S Diamond
金额：
$ 60.38万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-20 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10299611
关键词：
3&apos Splice Site Affect Alleles Alternative Splicing Antiviral resistance Automobile Driving Binding Biochemical Biological Assay C-terminal Cell Shape Cells Cellular biology Code DNA Viruses Data Diamond Event Family Family member Flavivirus GTP-Binding Protein alpha Subunits, Gs Genes Genetic Polymorphism Goals Grant Hematopoietic Host Defense Human Human Genome IRF1 gene Immune response In Vitro Infection Innate Immune Response Interferon Type I Interferons Knock-in Knock-in Mouse Knowledge Laboratories Laboratory mice Lead Length Ligase Maps Mediating Mediator of activation protein Messenger RNA Molecular Mus Mutate Natural Immunity Nature Nucleic Acids Organ Orthologous Gene Play Predisposition Property Protein Family Protein Isoforms Protein Synthesis Inhibition Proteins Proteomics RNA Viruses RNA immunoprecipitation sequencing Reporter Ribonucleases Role Scheme Severity of illness Shapes Signal Transduction Site T-Lymphocyte Terminator Codon Testing Therapeutic Transferase Translational Regulation Translations Untranslated Regions Up-Regulation Vaccinia virus Viral Viral Pathogenesis Virus Virus Diseases Virus Replication West Nile viral infection West Nile virus adaptive immune response adaptive immunity antiviral drug development base cell type cellular targeting experimental study in vivo knockout gene loss of function member mouse genome oligoadenylate promoter protein expression response

项目摘要

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.

抽象的该提案旨在定义干扰素刺激基因（ISG）抗病毒活性的新机制寡腺苷酸合成酶 1 (OAS1)。宿主先天免疫反应是通过感知非自身来启动的。病毒核酸，主要通过 I 型和 III 型干扰素 (IFN) 介导。干扰素诱导表达数百个 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 ISG 的一个家族，属于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，导致下游 RNase L 激活并导致蛋白质合成受到抑制。然而，多种抗病毒活性的机制人类和小鼠基因组中存在的酶活性和非活性 OAS 亚型尚不清楚。我们发现特定的 OAS1 亚型可增强多种抗病毒蛋白的翻译，包括 cGAS 和 IRF1。这种 OAS1 同工型 (OAS1 P46) 增强翻译，与其酶活性无关，并且 RNase L。我们的初步结果表明，OAS1 P46 通过以下方式增强特定蛋白质的翻译：结合各自的mRNA。在人类中，OAS1 P46 是由于 C-位点的选择性剪接事件而产生的。 OAS1 基因的末端。该选择性剪接位点天然存在的多态性（rs10774671，A/G）调节 P46 表达，并与多种病毒感染的疾病严重程度相关。使用在人类原代造血细胞中，我们证明编码 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 OAS1 P46 和小鼠直系同源物 Oas1b（无 NTase 活性）之间的关系，后者同样影响 WNV 易感性体内。基于这些观察，我们假设 OAS1 的特定异构体调节先天免疫通过独特的 NTase 活性独立机制来对抗病毒。该提案的目标是确定 OAS1 如何增强特定蛋白质翻译及其后果新发现的 OAS1 在抗病毒先天免疫和适应性免疫中的功能。我们三个独立目的是：（1）通过以下方式确定OAS1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) 明确介导OAS1抗病毒作用的细胞靶点使用几种基因缺陷细胞； (3) 使用新的方法定义 Oas1b 在病毒感染过程中的体内作用生成 Oas1b 敲入小鼠。这项研究完成后，我们应该建立抗病毒的新范式 OAS 蛋白的活性可能导致调节 OAS1-cGAS-I 型 IFN 轴的治疗策略。