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(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。这些途径和关键角色，无论是病毒还是宿主，都得到了这样的关注，因为有可能开发由具有弱化逃避策略的病毒组成的减毒疫苗。I型干扰素的诱导物通常是天然免疫拮抗剂的靶标。这些途径的复杂性表明，关于先天性免疫信号的调节以及为逃避这些反应而进化的病毒机制，还有很多需要了解的地方。轮状病毒是5岁以下儿童急性病毒性胃肠炎的主要原因。最近，两种减毒疫苗已获准在一些国家使用，但目前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，重要的是要了解轮状病毒感染细胞中先天抗病毒反应是如何触发的，以及病毒蛋白是如何调节这第一道宿主防线的。对感染早期病毒-宿主相互作用的了解将为以绕过病毒逃避策略的方式增强固有反应的新方法奠定基础。我们确定了非结构蛋白NSP1和干扰素调节因子3(IRF3)之间的相互作用，IRF3是IFN2表达所需的转录因子。NSP1针对IRF3进行蛋白酶体降解，并下调干扰素的反应。新的数据表明，猪轮状病毒株的NSP1靶向不同的底物，可能抑制NF：B的激活。总之，这些数据表明，轮状病毒拮抗多种在干扰素的诱导和效应功能中重要的信号分子。这一应用的系统开发研究将检验轮状病毒在多个步骤抑制干扰素反应的假设，并且抑制主要由NSP1介导。我们的蛋白质组学方法预测，通过量化轮状病毒感染和表达NSP1的细胞中相对蛋白质丰度的变化，将揭示收敛的信号通路。我们将使用培养中氨基酸的稳定同位素标记(SILAC)，然后进行高分辨率色谱分离和串联质谱分析。我们将在干扰素反应的背景下对轮状病毒感染的细胞进行定量的蛋白质组学分析。SILAC技术将用于标记感染轮状病毒或经干扰素治疗的细胞中的蛋白质，细胞裂解产物将用LC-Tandem-MS进行分析。我们将通过定义靶底物和底物抑制影响的途径来研究NSP1介导的干扰素耐药的机制。NSP1将通过重组腺病毒在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。在发达国家，这些病毒是导致严重发病率的主要原因，在美国，估计每年有270万病例，约60万名医生、门诊部和急诊室就诊，7万人住院。在发展中国家，每年约有200万儿童死于脱水腹泻，其中近一半是由轮状病毒感染造成的。细胞对病毒感染的最初反应是诱导由干扰素(干扰素)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，将导致建立一种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。大多数病毒，如果不是所有的病毒，都进化出了逃避这种反应的机制。轮状病毒蛋白NSP1特异性地干扰干扰素表达所需的细胞蛋白IRF3的激活。我们最近发现了NSP1表达抑制干扰素反应的另一种机制。本申请中描述的研究建议对轮状病毒感染细胞和表达NSP1的细胞进行定量蛋白质组学分析，以确定可能与诱导干扰素反应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导致新的方法，以获得专门设计来绕过病毒逃避策略的减毒疫苗或抗病毒药物靶标。