A quantitative proteomics approach to understand viral immune evasion strategies

了解病毒免疫逃避策略的定量蛋白质组学方法

• 批准号: 7847650
• 负责人: MICHELE E HARDY
• 金额: $ 17.8万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7847650
• 关键词: 5 year old; Accident and Emergency department; Acute; Adenoviruses; Affect; Amino Acids; Antiviral Agents; Antiviral Response; Attention; Attenuated Live Virus Vaccine; Attenuated Vaccines; Bioinformatics; Bypass; Cell Fractionation; Cells; Child; Computer Systems Development; Country; DNA Binding; Data; Databases; Developed Countries; Developing Countries; Diarrhea; Drug Delivery Systems; Effectiveness; Family suidae; Gene Expression; Gene Targeting; Genes; Hospitalization; Host Defense; Immune; Immune response; Infection; Interferon Type I; Interferons; Intervention; Label; Laboratories; Lead; Learning; Licensing; Mediating; Mining; Morbidity - disease rate; Nonstructural Protein; Nutritional status; Outpatients; Parasitic infection; Pathway interactions; Pattern; Phosphotransferases; Prophylactic treatment; Proteins; Proteomics; Recombinants; Regulation; Relative (related person); Research; Resistance; Resolution; Rotavirus; Rotavirus Infections; Rotavirus NSP1 protein; SH2D3A gene; Severities; Signal Pathway; Signal Transduction; Signaling Molecule; Stable Isotope Labeling; Staging; System; Technology; Testing; Vaccines; Viral; Viral Gastroenteritis; Viral Genome; Viral Proteins; Virus; Virus Diseases; Virus Replication; Visit; Vomiting; cellular targeting; clinical efficacy; design; drug development; gel electrophoresis; human IRF3 protein; interferon regulatory factor-3; multicatalytic endopeptidase complex; novel strategies; pre-clinical; protein function; public health relevance; response; tandem mass spectrometry; transcription factor; ubiquitin ligase; virus host interaction

DESCRIPTION (provided by applicant): Research into mechanisms utilized by viruses to evade cellular antiviral responses is receiving increased attention. These pathways and the key players, both viral and host, have garnered such focus because of the potential to develop attenuated vaccines consisting of viruses with weakened evasion strategies. Inducers of type I interferons (IFN) typically are targets of innate immune antagonists. The complexity of the pathways suggests there is much to learn about the regulation of innate immune signals and the viral mechanisms that have evolved to escape these responses. Rotaviruses are the major cause of acute viral gastroenteritis in children under 5 years of age. Two attenuated vaccines recently have been licensed for use in some countries, yet it remains unknown how poor nutritional status or concurrent parasitic infections will affect responses to the vaccines. Thus it is important to understand how the innate antiviral response is triggered in rotavirus infected cells and what viral proteins function to modulate this first line of host defense. An understanding of virus-host interactions at the early stages of infection will establish the basics for new approaches to enhance the innate response in ways that bypass viral evasion strategies. We identified an interaction between nonstructural protein NSP1 and IFN-regulatory factor 3 (IRF3), a transcription factor required for IFN2 expression. NSP1 targets IRF3 for proteasome degradation and down-regulates the IFN response. New data suggest NSP1 of a porcine rotavirus strain targets a different substrate and may inhibit activation of NF:B. Together, the data suggest rotavirus antagonizes multiple signaling molecules important in induction and effector functions of IFN. Systems development studies of this application will test the hypothesis that rotavirus inhibits IFN responses at multiple steps, and that inhibition is mediated primarily by NSP1. Our proteomics approach predicts that convergent signaling pathways will be revealed by quantitation of changes in relative protein abundance in rotavirus infected and NSP1-expressing cells. We will employ stable isotope labeling of amino acids in culture (SILAC) followed by high resolution chromatographic separation and tandem mass spectrometry. We will perform quantitative proteomic analyses of rotavirus infected cells in the context of the IFN response. SILAC technology will be used to label proteins in cells infected with rotavirus or treated with IFN and cell lysates will be analyzed by LC-tandem-MS. We will investigate mechanisms of NSP1-mediated resistance to IFN by defining target substrates and pathways affected by substrate inhibition. NSP1 will be expressed by a recombinant adenovirus in SILAC labeled cells. Changes in protein abundance will be analyzed to define NSP1 substrates and determine how these proteins integrate into antiviral networks. PUBLIC HEALTH RELEVANCE: Rotavirus infections are the major viral cause of acute vomiting and diarrhea in children under 5 years of age. These viruses are responsible for significant morbidity in developed countries with an estimated 2.7 million cases, ~600,000 doctor, outpatient, and emergency room visits, and 70,000 hospitalizations annually in the U.S. In developing countries, approximately 2 million children die of dehydrating diarrhea every year and nearly half of these are due to rotavirus infection. The initial cellular response to virus infection is induction of a specific pattern of gene expression regulated by interferon (IFN). Expression of IFN and IFN-regulated genes, if successful, result in establishment of an antiviral state that restricts virus replication and spread, while simultaneously initiating recruitment of the adaptive immune responses. Most, if not all viruses have evolved mechanisms to escape this response. Rotavirus protein NSP1 interferes specifically with activation of cellular protein IRF3 that is required for IFN expression. We recently discovered an additional mechanism by which NSP1 expression inhibits the IFN response. The studies described in this application propose a quantitative proteomic analysis of rotavirus infected cells and NSP1 expressing cells to determine changes in relative protein abundance potentially associated with induction of the IFN response. Understanding these pathways and the mechanisms by which viruses evade the innate immune response may lead new approaches to attenuated vaccines or antiviral drug targets specifically designed to bypass viral evasion strategies.

描述（由申请人提供）：对病毒用来逃避细胞抗病毒反应的机制的研究正受到越来越多的关注。这些途径和关键参与者（病毒和宿主）已经引起了如此多的关注，因为有可能开发出由逃避策略减弱的病毒组成的减毒疫苗。 I 型干扰素 (IFN) 诱导剂通常是先天免疫拮抗剂的靶标。这些途径的复杂性表明，关于先天免疫信号的调节以及为逃避这些反应而进化的病毒机制，还有很多东西需要了解。轮状病毒是5岁以下儿童急性病毒性胃肠炎的主要原因。两种减毒疫苗最近已在一些国家获得使用许可，但尚不清楚营养状况不佳或并发寄生虫感染将如何影响对疫苗的反应。因此，了解轮状病毒感染细胞中如何触发先天抗病毒反应以及哪些病毒蛋白发挥调节宿主第一道防线的作用非常重要。了解感染早期阶段的病毒与宿主的相互作用将为新方法奠定基础，以绕过病毒逃避策略的方式增强先天反应。我们确定了非结构蛋白 NSP1 和 IFN 调节因子 3 (IRF3) 之间的相互作用，IRF3 是 IFN2 表达所需的转录因子。 NSP1 靶向 IRF3 进行蛋白酶体降解并下调 IFN 反应。新数据表明猪轮状病毒株的 NSP1 靶向不同的底物并可能抑制 NF:B 的激活。总之，数据表明轮状病毒拮抗多种信号分子，这些信号分子对 IFN 的诱导和效应功能很重要。该应用的系统开发研究将检验轮状病毒在多个步骤中抑制 IFN 反应的假设，并且该抑制主要由 NSP1 介导。我们的蛋白质组学方法预测，通过定量轮状病毒感染细胞和表达 NSP1 的细胞中相对蛋白质丰度的变化，将揭示汇聚信号通路。我们将采用稳定同位素标记培养物中的氨基酸 (SILAC)，然后进行高分辨率色谱分离和串联质谱分析。我们将在 IFN 反应的背景下对轮状病毒感染的细胞进行定量蛋白质组分析。 SILAC 技术将用于标记感染轮状病毒或用 IFN 处理的细胞中的蛋白质，并通过 LC-串联 MS 分析细胞裂解物。我们将通过定义靶底物和受底物抑制影响的途径来研究 NSP1 介导的 IFN 抗性机制。 NSP1 将由重组腺病毒在 SILAC 标记的细胞中表达。将分析蛋白质丰度的变化，以确定 NSP1 底物并确定这些蛋白质如何整合到抗病毒网络中。公共卫生相关性：轮状病毒感染是 5 岁以下儿童急性呕吐和腹泻的主要病毒原因。这些病毒在发达国家造成严重发病，估计每年有 270 万例病例，约 600,000 例医生、门诊和急诊室就诊，在美国每年有 70,000 例住院患者。在发展中国家，每年约有 200 万儿童死于脱水腹泻，其中近一半是由于轮状病毒感染造成的。细胞对病毒感染的最初反应是诱导受干扰素 (IFN) 调节的特定基因表达模式。干扰素和干扰素调节基因的表达如果成功，会导致建立限制病毒复制和传播的抗病毒状态，同时启动适应性免疫反应的招募。大多数（如果不是全部）病毒都进化出了逃避这种反应的机制。轮状病毒蛋白 NSP1 特异性干扰 IFN 表达所需的细胞蛋白 IRF3 的激活。我们最近发现了 NSP1 表达抑制 IFN 反应的另一种机制。本申请中描述的研究提出了对轮状病毒感染细胞和 NSP1 表达细胞进行定量蛋白质组学分析，以确定可能与 IFN 反应诱导相关的相对蛋白质丰度的变化。了解这些途径和病毒逃避先天免疫反应的机制可能会带来新的方法来开发减毒疫苗或专门设计用于绕过病毒逃避策略的抗病毒药物靶点。