CMV DISRUPTION OF IFN SIGNAL TRANSDUCTION

CMV 干扰干扰素信号转导

• 批准号: 6458355
• 负责人: Daniel D Sedmak
• 金额: $ 29.48万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6458355
• 关键词: JAK kinase; MHC class I antigen; MHC class II antigen; antigen presentation; biological signal transduction; complementary DNA; cytokine receptors; cytomegalovirus; enzyme activity; enzyme inhibitors; gene expression; genetic library; interferons; mutant; phosphorylation; plasmids; protein degradation; receptor binding; site directed mutagenesis; tissue /cell culture; transfection; ubiquitin; virus genetics; virus infection mechanism; virus replication

Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in transplant recipients. The majority of HCMV- associated morbidity and mortality is the result of the spread of persistent virus. The long term goal of our laboratory is to understand at the host, cellular, and molecular level the mechanisms by which HCMV persists, to develop novel strategies for blocking HCMV persistence, and ultimately reduce the incidence of HCMV disease in transplantation. A critical means by which HCMV establishes a persistent infection is to evade host innate and adaptive immune responses. Recently, we have discovered that one of these key immunoevasion strategies is the blockade of interferon (IFN) stimulated responses in HCMV infected fibroblasts and endothelial cells. HCMV blocks IFN-gamma and IFN-alpha/beta stimulated signal transduction, IFN-stimulated transcription factor activation, and IFN- stimulated gene expression in infected cells. Analyses of the JAK/STAT signal transduction pathway reveals that protein levels of JAK1, a tyrosine kinase required for activation of IFN-gamma and IFN- alpha/beta signal transduction, are decreased in HCMV infected cells. The focus of this proposal is to determine the molecular mechanism for the HCMV-mediated decrease in JAK1 expression, identify the HCMV gene that decreases JAK1 expression and disrupts IFN signal transduction, and investigate the significance of IFN-blocking activity in mutant HCMV strains lacking this capability. In Specific Aim I, we will test the hypothesis that JAK1 protein levels are decreased by a proteasome-dependent process in HCMV infected cells. We will analyze the mechanism for the degradation of JAK1 proteins in infected cells and the upstream events prior to JAK1 degradation including phosphorylation, ubiquitination, and IFN-receptor binding. Moreover, the role of suppressors of cytokine signaling (SOCS) proteins in blocking the kinase activity of JAK1 and mediating JAK1 degradation will be investigated. In Aim II, we test the hypothesis that HCMV encodes a gene product that mediates the decrease of JAK1 levels thereby blocking IFN signal transduction in infected cells. 2C4 cells, which are stably transfected with an IFN-responsive promoter linked to the cell surface CD2 glycoprotein, will be utilized to expression clone the HCMV gene with IFN signal transduction blocking activity. In Aim III, we will test the hypothesis that HCMV-mediated IFN-blocking activity is critical for HCMV gene expression, replication, and reducing MHC-based antigen presentation. To test this hypothesis, we will generate a mutant HCMV strain lacking IFN-blocking activity and investigate HCMV gene expression, replication, and MHC based antigen presentation in the presence of IFNs.

人类巨细胞病毒（HCMV）是移植受者发病率和死亡率的重要原因。大多数HCMV相关的发病率和死亡率是持续病毒传播的结果。我们实验室的长期目标是在宿主，细胞和分子水平上了解HCMV持续存在的机制，以制定阻断HCMV持久性的新型策略，并最终降低HCMV疾病在移植中的发生率。 HCMV建立持续感染的一种关键手段是逃避宿主先天和适应性免疫反应。最近，我们发现这些关键的免疫避免策略之一是对HCMV感染的成纤维细胞和内皮细胞中干扰素（IFN）刺激反应的阻塞。 HCMV阻止IFN-gamma和IFN-Alpha/beta刺激信号转导，IFN刺激的转录因子激活以及感染细胞中IFN刺激基因表达。对HCMV感染的细胞中JAK/Stat信号转导途径的分析表明，JAK1的蛋白水平是激活IFN-GAMMA和IFN-Alpha/beta信号转导所需的酪氨酸激酶。该提案的重点是确定HCMV介导的JAK1表达降低的分子机制，确定降低JAK1表达的HCMV基因并破坏IFN信号转导，并研究IFN阻滞活性在突变HCMV缺乏这种能力中的重要性。在特定目标I中，我们将测试以下假设：HCMV感染细胞中蛋白酶体依赖性过程降低了JAK1蛋白水平。我们将分析JAK1降解之前的JAK1蛋白在JAK1降解之前降解的机制，包括磷酸化，泛素化和IFN受体结合。此外，将研究细胞因子信号传导（SOCS）蛋白在阻断JAK1的激酶活性和介导JAK1降解中的作用。在AIM II中，我们检验了HCMV编码介导JAK1水平降低的基因产物的假设，从而阻止了受感染细胞的IFN信号转导。 2C4细胞用与细胞表面CD2糖蛋白相关的IFN响应启动子稳定转染的2C4细胞将用于表达具有IFN信号传输阻断活性的HCMV基因。在AIM III中，我们将检验以下假设：HCMV介导的IFN阻断活性对于HCMV基因表达，复制和减少基于MHC的抗原呈递至关重要。为了检验该假设，我们将生成缺乏IFN阻断活性的突变HCMV菌株，并研究IFNS存在的HCMV基因表达，复制和基于MHC的抗原表现。