A quantitative proteomics approach to understand viral immune evasion strategies

描述（由申请人提供）：对病毒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（包括病毒和宿主）已经获得了这样的关注，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（IFN）的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进化来逃避这些反应的病毒机制，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获准在一些国家使用，但营养状况差或并发寄生虫感染将如何影响对疫苗的反应仍不清楚。因此，重要的是要了解先天性抗病毒反应是如何在轮状病毒感染的细胞中触发的，以及哪些病毒蛋白质起调节宿主防御的第一道防线的作用。在感染的早期阶段，对病毒-宿主相互作用的理解将为新方法建立基础，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NSP 1和IFN-调节因子3（IRF 3），IFN 2表达所需的转录因子之间的相互作用。NSP 1靶向IRF 3进行蛋白酶体降解并下调IFN应答。新的数据表明猪轮状病毒株的NSP 1靶向不同的底物，并可能抑制NF：B的活化。总之，这些数据表明轮状病毒拮抗多种信号分子在IFN的诱导和效应功能中很重要。本申请的系统开发研究将测试轮状病毒在多个步骤抑制IFN应答并且抑制主要由NSP 1介导的假设。我们的蛋白质组学方法预测，收敛信号通路将揭示轮状病毒感染和NSP 1表达细胞的相对蛋白质丰度的变化定量。我们将采用稳定同位素标记的氨基酸在文化（SILAC），然后高分辨率色谱分离和串联质谱。我们将在IFN应答的背景下对轮状病毒感染的细胞进行定量蛋白质组学分析。SILAC技术将被用来标记蛋白质的细胞感染轮状病毒或治疗与IFN和细胞裂解物将进行分析，通过LC-串联-MS。我们将研究NSP 1介导的抗干扰素的机制，通过定义目标底物和受底物抑制的途径。NSP 1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NSP 1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。这些病毒导致发达国家的显著发病率，在美国每年估计有270万例病例，约60万名医生，门诊和急诊室就诊，以及7万例住院治疗。在发展中国家，每年约有200万儿童死于脱水性腹泻，其中近一半是由于轮状病毒感染。对病毒感染的初始细胞应答是诱导由干扰素（IFN）调节的特定基因表达模式。IFN和IFN调节基因的表达，如果成功的话，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应答的募集。大多数（如果不是所有的话）病毒都进化出了逃避这种反应的机制。轮状病毒蛋白NSP 1特异性干扰IFN表达所需的细胞蛋白IRF 3的活化。我们最近发现了NSP 1表达抑制IFN应答的另一种机制。本申请中描述的研究提出了对轮状病毒感染的细胞和表达NSP 1的细胞进行定量蛋白质组学分析，以确定可能与IFN应答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，可能会导致新的方法，以减毒疫苗或抗病毒药物靶点，专门设计绕过病毒逃避策略。

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.