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）：对逃避细胞抗病毒反应的病毒所使用的机制的研究正在受到越来越多的关注。这些途径和主要参与者（无论是病毒还是宿主），由于有潜力开发由逃避策略减弱的病毒组成的疫苗而赢得了这种重点。 I型干扰素（IFN）的诱导者通常是先天免疫拮抗剂的靶标。途径的复杂性表明，有关先天免疫信号的调节以及已进化以逃避这些反应的病毒机制的调节。轮状病毒是5岁以下儿童急性病毒胃肠炎的主要原因。最近有两种衰减的疫苗已在某些国家使用许可，但尚不清楚营养状况或同时寄生虫感染将如何影响对疫苗的反应。因此，重要的是要了解如何在轮状病毒感染细胞中触发先天抗病毒反应以及哪种病毒蛋白功能来调节宿主防御的第一线。在感染的早期阶段对病毒宿主相互作用的理解将建立新方法的基础知识，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与IFN调节因子3（IRF3）之间的相互作用，这是IFN2表达所需的转录因子。 NSP1靶向IRF3用于蛋白酶体降解并下调IFN响应。新数据表明，猪轮状病毒菌株的NSP1靶向不同的底物，并可能抑制NF：b的激活。共同数据表明，轮状病毒对IFN的诱导和效应函数重要的多个信号分子拮抗。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，并且该抑制作用主要由NSP1介导。我们的蛋白质组学方法预测，通过定量感染轮状病毒和表达NSP1的细胞中相对蛋白丰度的变化来揭示收敛信号通路。我们将在培养物（SILAC）中采用稳定的同位素标记，然后使用高分辨率色谱分离和串联质谱。我们将在IFN反应的背景下对轮状病毒感染细胞进行定量蛋白质组学分析。 SILAC技术将用于在感染轮状病毒或用IFN和细胞裂解液治疗的细胞中标记蛋白质，将通过LC-Tandem-MS分析。我们将通过定义受底物抑制影响的靶标底物和途径来研究NSP1介导的对IFN的抗性。 NSP1将由SILAC标记的细胞中的重组腺病毒表示。将分析蛋白质丰度的变化，以定义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。这些病毒是估计有270万例病例，约60万名医生，门诊和急诊就诊的发达国家的重大发病率，每年在美国的发展中国家每年有70,000个住院治疗，大约有200万儿童每年死亡，几乎每年脱水，其中近一半是由于rotavirus的感染而导致的。对病毒感染的初始细胞反应是诱导由干扰素调节的基因表达的特定模式（IFN）。 IFN和IFN调节的基因的表达，如果成功，会导致建立限制病毒复制和扩散的抗病毒状态，同时同时启动适应性免疫反应的募集。大多数（如果不是所有的病毒）都有发展的机制来逃避这种反应。轮状病毒蛋白NSP1特别干扰了IFN表达所需的细胞蛋白IRF3的激活。我们最近发现了一种额外的机制，该机制通过该机制抑制IFN响应。该应用中描述的研究提出了对轮状病毒感染细胞和NSP1表达细胞的定量蛋白质组学分析，以确定与诱导IFN反应有关的相对蛋白质丰度的变化。了解这些途径以及病毒逃避先天免疫反应的机制可能会导致新方法减弱疫苗或抗病毒药靶标，专门设计用于绕过病毒逃避策略。