Systems-biology Approaches for Decoding Persistent Coxsackievirus B3 Infection

描述(申请人提供)：柯萨奇病毒B3(CVB3)是导致儿童和年轻人慢性心脏炎症和心力衰竭的主要原因。持续的CVB3感染与成熟病毒无关，而是与其RNA基因组在心肌细胞中的潜伏表达有关。我们的初步观察表明，CVB3RNA的存在扰乱了心肌细胞对炎症刺激的正常反应，这可能对建立慢性炎症状态很重要。这项建议的目的是为了消除慢性感染CVB3后心肌细胞发生的分子“重新布线”。我们的假设是，CVB3RNA的持续表达扰乱了心肌细胞对促炎刺激的转录后信号反应。在R21阶段，我们将开发高通量的生化方法，在系统水平上测量信号和转录的稳定性。在R33阶段，这些检测将被结合来检测表达CVB3RNA并受促炎症细胞因子刺激的培养心肌细胞的转录后信号处理。这些数据最终将作为计算系统模型的基础，该模型将信号事件与基因表达和转录稳定性联系起来。目的R21阶段的第一步是建立定量的、系统水平的生物检测方法，以测量慢性感染CVB3的心肌细胞中的激酶-磷酸酶的活性。目的建立一种实时、多重的R21期RNA稳定性分析方法，用于分析心肌细胞中核因子？B转录本的半抑制因子调控。R33阶段的目的#3是询问由促炎细胞因子触发的细胞内动力学，并建立一个预测的、数据驱动的系统模型，该模型捕捉CVB3诱导的心肌细胞重新连接。长期目标是使用该模型提出新的干预措施，以纠正持续CVB3感染患者的转录后信号处理。

DESCRIPTION (provided by applicant): Coxsackievirus B3 (CVB3) is a leading cause of chronic heart inflammation and heart failure in children and young adults. Persistent CVB3 infection is not associated with the mature virus but instead with latent expression of its RNA genome in cardiomyocytes. Our preliminary observations suggest that the presence of CVB3 RNA disrupts how cardiomyocytes normally respond to inflammatory stimuli, which may be important for establishing a chronically inflamed state. The objective of this proposal is to defin the molecular "rewiring" that takes place in cardiomyocytes upon chronic infection with CVB3. Our hypothesis is that persistent expression of CVB3 RNA disrupts the posttranscriptional signaling response of cardiomyocytes to proinflammatory stimuli. During the R21 phase, we will develop high-throughput biochemical methods to measure signaling and transcript stability at the systems level. In the R33 phase, these assays will be combined to examine posttranscriptional signal processing in cultured cardiomyocytes expressing CVB3 RNA and stimulated with proinflammatory cytokines. The data will ultimately serve as the basis for a computational-systems model that connects signaling events to gene expression and transcript stability. Aim #1 of the R21 phase is to develop quantitative, systems-level bioassays to measure kinase-phosphatase activation in cardiomyocytes chronically infected with CVB3. Aim #2 of the R21 phase is to develop a real-time, multiplex RNA stability assay for profiling half-lif regulation of NF-?B transcripts in cardiomyocytes. Aim #3 of the R33 phase is to interrogate intracellular dynamics triggered by proinflammatory cytokines and build a predictive, data-driven systems model that captures CVB3-induced cardiomyocyte rewiring. The long-term goal is to use the model to propose novel interventions that can correct posttranscriptional signal processing in patients with persistent CVB3 infection.