How Feedback Circuitry Drives Phenotype Switching in a Human Herpesvirus

描述（由申请人提供）：本研究培训计划的长期目标是了解转录反馈回路如何调节哺乳动物系统中的表型。具体目标是了解人类巨细胞病毒（CMV）（一种疱疹病毒和重要的人类病原体）的转录电路如何控制病毒的不同复制表型（CMV快速裂解成纤维细胞，持续感染内皮细胞，在上皮细胞中缓慢复制，并在CD 34+祖细胞中潜伏）。驱动这些不同病毒表型（特别是潜伏期）的机制仍然未知。然而，在感染和潜伏再激活时表达的第一个病毒回路是CMV的自动调节“主回路”，这是一个交叉的正反馈和负反馈回路，称为主要立即早期（MIE）回路。已知MIE回路启动病毒转录级联并驱动随后的病毒复制。我假设MIE电路也可能控制CMV的不同复制表型，包括潜伏期。为了了解MIE反馈如何作为调节进入和退出不同复制表型的遗传开关发挥作用，我将利用一种耦合的实验和理论方法来定量表征不同细胞类型中的MIE电路组件，动力学，反馈强度，协同性和电路噪声结构。如果MIE电路控制CMV的不同复制表型，则这些实验测量和并行数学建模研究预计将在不同细胞类型中产生不同的MIE反馈动力学（例如脉冲vs.稳定振荡vs.固定状态）。具体目标是：（1）表征不同细胞类型中隔离MIE电路组件的反馈动态和功能（即在病毒感染的背景之外）;（2）在野生型CMV感染期间将MIE反馈结构和噪声结构映射到完整的完整MIE回路中的复制表型（即在病毒感染的背景下）;和（3）阐明鼠模型感染期间的体内MIE反馈动力学。该项目将利用单细胞显微镜、遗传编码荧光探针和自动图像分析的最新进展来量化活单细胞中的MIE反馈动力学。我将获得整个动物和单细胞体内成像技术的培训，以及最近开发的用于探测单细胞转录反馈结构的基因表达噪声频率分析。最后，我还将接受病毒分子生物化学、病毒诱变和病毒“重组工程”技术的培训。该项目将通过提供哺乳动物转录自动调节的新概念模型和定量分析反馈回路如何驱动哺乳动物表型的新实验系统来推动新一代的理论模型。

DESCRIPTION (provided by applicant): The long-term goal of this research-training proposal is to understand how transcriptional feedback circuits regulate phenotype in mammalian systems. The specific goal is to understand how the transcriptional circuitry of human cytomegalovirus (CMV), a herpesvirus and important human pathogen, controls the virus's diverse replication phenotypes (CMV rapidly lyses fibroblasts, persistently infects endothelial cells, slowly replicates in epithelial cells, and enters latency in CD34+ progenitor cells). The mechanisms driving these diverse viral phenotypes (especially latency) remain unknown. However, the first viral circuit expressed upon infection and latent reactivation is CMV's auto-regulatory "master circuit", a crossed positive- and negative-feedback circuit called the Major Immediate-Early (MIE) circuit. The MIE circuit is known to initiate the viral transcriptional cascade and drive subsequent viral replication. I hypothesize that the MIE circuit might also control CMV's diverse replication phenotypes, including latency. To understand how MIE feedback might function as a genetic switch regulating entry into and exit from different replicative phenotypes, I will utilize a coupled experimental & theoretical approach to quantitatively characterize MIE circuit components, dynamics, feedback strength, cooperativity, and circuit noise structure in diverse cell types. If the MIE circuit controls CMV's diverse replication phenotypes, these experimental measurements, and the parallel mathematical modeling studies, are expected to yield different MIEfeedback dynamics in diverse cell types (e.g. pulses vs. stable oscillations vs. fixed states). The specific aims are: (1) to characterize the feedback dynamics and function of isolated MIE circuit components in diverse cell-types (i.e. outside the context of viral infection); (2) to map MIE feedback architecture & noise structure to replication phenotype in the full, intact MIE circuit during wild-type CMV infection (i.e.inside the context of viral infection); and (3) to elucidate MIE feedback kinetics in-vivo during infection of a murine model. This project will capitalize on recent advancesin single-cell microscopy, genetically encoded fluorescent probes, and automated image-analyses to quantify MIEfeedback kinetics in live single cells. I will gain training in whole-animaland single-cell in-vivo imaging techniques, and recently developed gene expression noise frequencyanalysesfor probing transcriptional feedback architecture in single-cells. Finally, I will also be trained in viral molecular biochemistry, viral mutagenesis, and viral "recombineering" techniques. This project should drive a new generation of theoretical models by providing a new conceptual model for mammalian transcriptional auto-regulationand a new experimental system for quantitativelyanalyzinghow feedback circuitry drives mammalian phenotype.