Systems-biology Approaches for Decoding Persistent Coxsackievirus B3 Infection

描述（由申请人提供）：柯萨奇病毒B3（CVB 3）是儿童和年轻人慢性心脏炎症和心力衰竭的主要原因。持续性CVB 3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，CVB 3 RNA的存在破坏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。该提案的目的是定义慢性感染CVB 3后心肌细胞中发生的分子“重新布线”。我们的假设是CVB 3 RNA的持续表达破坏了心肌细胞对促炎刺激的转录后信号反应。在R21阶段，我们将开发高通量生化方法来测量系统水平的信号传导和转录稳定性。在R33阶段，将结合这些试验来检查表达CVB 3 RNA并用促炎细胞因子刺激的培养心肌细胞中的转录后信号处理。这些数据最终将成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。R21阶段的目标#1是开发定量的、系统水平的生物测定，以测量慢性感染CVB 3的心肌细胞中的激酶-磷酸酶活化。R21阶段的目标#2是开发一种实时、多重RNA稳定性测定，用于分析NF-？心肌细胞中的B转录物。R33阶段的目标#3是询问由促炎细胞因子触发的细胞内动力学，并建立一个预测性的数据驱动系统模型，该模型捕获CVB 3诱导的心肌细胞重新布线。长期目标是使用该模型提出新的干预措施，可以纠正持续CVB 3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.