Antagonism of Innate Immunity By Picornaviruses

小核糖核酸病毒对先天免疫的拮抗作用

• 批准号: 8646879
• 负责人: VINCENT R RACANIELLO
• 金额: $ 39.8万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8646879
• 关键词: 3' Untranslated Regions; Address; Alien; Amino Acids; Animal Viruses; Antiviral Agents; Antiviral Response; Antiviral Therapy; Attenuated; Brain; Cell Line; Cells; Central Nervous System Infections; Code; Cultured Cells; DNA; Defect; Disease; Encephalomyocarditis virus; Family Picornaviridae; Genes; Genetic Programming; Genome; Goals; Hela Cells; Human; Human poliovirus; Immune; Infection; Interferon Receptor; Interferons; Investigation; Laboratories; Lead; Lytic; Modeling; Mus; Muscle; Mutation; Natural Immunity; Neuraxis; Neuroglia; Neurons; Nucleic Acids; Oral Poliovirus Vaccine; Paralysed; Pathogenesis; Peptide Hydrolases; Phenotype; Poliomyelitis; Polioviruses; Proteins; Reagent; Recombinants; Research; Resistance; Role; Specificity; Spinal Cord; Starvation; Stress; System; Temperature; Therapeutic; Tissues; Transgenic Mice; Tropism; Viral; Virulence; Virus; Virus Diseases; Virus Replication; Work; aspergillopepsin II; base; cytokine; insight; irradiation; microbial; mutant; neuroblastoma cell; neurotropic virus; neurovirulence; novel; overexpression; pathogen; poliovirus receptor; prevent; programs; protein function; relating to nervous system; research study; response; stem

DESCRIPTION (provided by applicant): All cells possess genetic programs that respond to various stresses, such as starvation, temperature, irradiation, and infection. Those programs that have evolved to repel cellular invaders recognize and respond to alien nucleic acids and other microbial products. One such program consists of innate immune defenses, which are activated by viral infections, leading to the synthesis of interferon (IFN). IFNs induce an antivirl state by inducing the expression of IFN-stimulated genes (ISGs). IFNs are vital to the antiviral response, yet we do not understand how the 1000-plus genes encoding ISG products establish an antiviral state. There is little known about the mechanisms of action of ISGs, and their target specificity. Experiments in this application utilize a well-characterized human pathogen, poliovirus, to identify inhibitory ISG products and study their mechanism of action. The ability of poliovirus to replicate in cultured cells treated with IFN is abolished by a single amino acid change in the viral 2Apro proteinase. Insertion of the 2Apro coding sequence into the genome of the IFN-sensitive encephalomyocarditis virus (EMCV) confers the ability to replicate in the presence of IFN. Our hypothesis is that the viral 2Apro protein antagonizes the antiviral activity of one or more ISG products. To address this hypothesis, we will first identify ISG products that inhibit poliovirus replication and their mechanism of action (aim 1). We will determine if the 2Apro protein antagonizes the activity of these ISG products, and identify the mechanism. Passage of recombinant EMCV expressing poliovirus 2Apro in high levels of IFN leads to selection of viral mutants with higher resistance to IFN. Mutations responsible for this phenotype will be identified, and the mechanisms by which they enhance replication in the presence of the cytokine will be determined. Non-neuronal cells of mice are protected from poliovirus infection by the ISG response. However, poliovirus replicates well in the brain and spinal cord, leading to muscle paralysis. A hypothesis to explain these observations is that neuronal tissues do not mount a protective ISG response, allowing poliovirus replication. Replication of the IFN-sensitive poliovirus 2Apro mutant Y88L in the human neuroblastoma cell line SK-N-SH is relatively insensitive to IFN, suggesting that these cells may be used as a model for understanding why the IFN response in the central nervous system (CNS) does not impair poliovirus replication. In aim 2 we will elucidate the differences in the antiviral state between HeLa and SK-N-SH cells. We will determine if the ISG products identified in aim 1 are induced in SK-N-SH cells in response to viral infection. If the ISG products are not induced we will determine if their overexpression protects the neuronal cells from viral infection. The results of the proposed experiments will identify inhibitory ISGs and their mechanisms of action. This information will provide new insights into the IFN-induced antiviral state, and may provide novel targets for enteroviral therapeutics.

描述（由申请人提供）：所有细胞都具有对各种应激（例如饥饿、温度、辐射和感染）做出反应的遗传程序。那些已经进化来击退细胞入侵者的程序能够识别外来核酸和其他微生物产物并对其做出反应。其中一项程序包括先天免疫防御，该防御由病毒感染激活，导致干扰素 (IFN) 的合成。干扰素通过诱导干扰素刺激基因（ISG）的表达来诱导抗病毒状态。 IFN 对于抗病毒反应至关重要，但我们不了解编码 ISG 产品的 1000 多个基因如何建立抗病毒状态。人们对 ISG 的作用机制及其靶点特异性知之甚少。本应用中的实验利用一种已充分表征的人类病原体脊髓灰质炎病毒来鉴定抑制性 ISG 产物并研究其作用机制。的能力 脊髓灰质炎病毒在经 IFN 处理的培养细胞中复制的能力因病毒 2Apro 蛋白酶中单个氨基酸的变化而被消除。将 2Apro 编码序列插入 IFN 敏感性脑心肌炎病毒 (EMCV) 的基因组中，使其能够在 IFN 存在的情况下进行复制。我们的假设是病毒 2Apro 蛋白拮抗一种或多种 ISG 产品的抗病毒活性。为了解决这一假设，我们将首先确定抑制脊髓灰质炎病毒复制的 ISG 产品及其作用机制（目标 1）。我们将确定 2Apro 蛋白是否拮抗这些 ISG 产物的活性，并确定其机制。在高水平 IFN 中表达脊髓灰质炎病毒 2Apro 的重组 EMCV 的传代导致选择对 IFN 具有更高抗性的病毒突变体。将鉴定导致该表型的突变，并确定它们在细胞因子存在下增强复制的机制。 ISG 反应可保护小鼠的非神经元细胞免受脊髓灰质炎病毒感染。然而，脊髓灰质炎病毒在大脑和脊髓中复制良好，导致肌肉瘫痪。解释这些观察结果的一个假设是，神经元组织不会产生保护性 ISG 反应，从而允许脊髓灰质炎病毒复制。 IFN 敏感的脊髓灰质炎病毒 2Apro 突变体 Y88L 在人神经母细胞瘤细胞系 SK-N-SH 中的复制对 IFN 相对不敏感，这表明这些细胞可用作理解中枢神经系统 (CNS) 中 IFN 反应的模型。 ）不会损害脊髓灰质炎病毒的复制。在目标 2 中，我们将阐明 HeLa 和 SK-N-SH 细胞之间抗病毒状态的差异。我们将确定目标 1 中确定的 ISG 产物是否在 SK-N-SH 细胞中诱导响应病毒感染。如果 ISG 产物没有被诱导，我们将确定它们的过度表达是否可以保护神经元细胞免受病毒感染。拟议实验的结果将确定抑制性 ISG 及其作用机制。这些信息将为 IFN 诱导的抗病毒状态提供新的见解，并可能为肠道病毒治疗提供新的靶点。