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: 8402767
• 负责人: VINCENT R RACANIELLO
• 金额: $ 39.66万
• 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/8402767
• 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; 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; receptor; 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. PUBLIC HEALTH RELEVANCE: Interferons are powerful antiviral proteins, but how they function to prevent virus infections is not well understood. We propose to use poliovirus, a well characterized virus, to identify the products of the interferon system that directly block virus replication. The results could lead to new antiviral therapies for a wide range of virus infections

描述（由申请人提供）：所有细胞都具有对各种应激（如饥饿、温度、辐射和感染）作出反应的遗传程序。这些程序已经进化到排斥细胞入侵者，识别并回应外来核酸和其他微生物产物。一个这样的程序包括先天免疫防御，这是由病毒感染激活，导致干扰素（IFN）的合成。IFN通过诱导IFN刺激基因（ISG）的表达来诱导抗病毒状态。干扰素对抗病毒反应至关重要，但我们不明白编码ISG产物的1000多个基因如何建立抗病毒状态。关于ISG的作用机制及其靶点特异性知之甚少。本申请中的实验利用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 脊髓灰质炎病毒在用IFN处理的培养细胞中复制的能力被病毒2Apro蛋白酶中的单个氨基酸改变所消除。将2Apro编码序列插入干扰素敏感性脑心肌炎病毒（EMCV）的基因组中，赋予了在干扰素存在下复制的能力。我们的假设是病毒2Apro蛋白拮抗一种或多种ISG产物的抗病毒活性。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（目的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。表达脊髓灰质炎病毒2Apro的重组EMCV在高水平IFN中的传代导致对IFN具有更高抗性的病毒突变体的选择。将鉴定导致这种表型的突变，并确定它们在细胞因子存在下增强复制的机制。小鼠的非神经元细胞通过ISG应答而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，导致肌肉麻痹。解释这些观察结果的一个假设是，神经元组织不产生保护性ISG反应，从而允许脊髓灰质炎病毒复制。在人神经母细胞瘤细胞系SK-N-SH中复制IFN敏感的脊髓灰质炎病毒2Apro突变体Y88 L对IFN相对不敏感，这表明这些细胞可用作理解为什么中枢神经系统（CNS）中的IFN应答不损害脊髓灰质炎病毒复制的模型。在目标2中，我们将阐明HeLa和SK-N-SH细胞之间的抗病毒状态的差异。我们将确定目的1中鉴定的ISG产物是否在SK-N-SH细胞中响应于病毒感染而被诱导。如果ISG产物没有被诱导，我们将确定它们的过表达是否保护神经元细胞免受病毒感染。所提出的实验的结果将确定抑制性ISG及其作用机制。这些信息将提供新的见解干扰素诱导的抗病毒状态，并可能提供新的肠道病毒治疗的目标。 干扰素是一种强大的抗病毒蛋白质，但它们如何预防病毒感染尚不清楚。我们建议使用脊髓灰质炎病毒，一个很好的特点，病毒，以确定干扰素系统的产品，直接阻止病毒复制。这一结果可能会导致新的抗病毒治疗广泛的病毒感染