How Feedback Circuitry Drives Phenotype Switching in a Human Herpesvirus

描述(由申请人提供)：这项研究-培训计划的长期目标是了解转录反馈电路如何调节哺乳动物系统的表型。我们的具体目标是了解人类疱疹病毒和重要病原体-人巨细胞病毒(CMV)的转录电路如何控制病毒的各种复制表型(CMV快速溶解成纤维细胞，持续感染内皮细胞，在上皮细胞中缓慢复制，并在CD34+祖细胞中进入潜伏期)。驱动这些不同病毒表型(特别是潜伏期)的机制仍不清楚。然而，在感染和潜伏重新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。我推测MIE回路也可能控制CMV的不同复制表型，包括潜伏期。为了了解MIE反馈如何作为基因开关调节不同复制表型的进入和退出，我将利用实验和理论耦合的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、协作性和电路噪声结构。如果MIE电路控制CMV的不同复制表型，这些实验测量和并行的数学建模研究有望在不同的细胞类型中产生不同的MIE反馈动力学(例如，脉冲与稳定振荡与固定状态)。其具体目的是：(1)表征不同细胞类型(即病毒感染环境外)中分离的MIE回路组件的反馈动力学和功能；(2)在野生型CMV感染期间(即在病毒感染环境中)，将MIE反馈结构和噪声结构映射到完整、完整的MIE回路中的复制表型；以及(3)阐明在体内感染小鼠模型期间的MIE反馈动力学。该项目将利用单细胞显微镜、基因编码的荧光探针和自动图像分析方面的最新进展来量化活的单细胞中的MIE反馈动力学。我将接受全动物和单细胞体内成像技术方面的培训，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、病毒突变和病毒“重组工程”技术方面的培训。该项目将为哺乳动物转录自动调节提供一个新的概念模型，并为定量分析反馈电路如何驱动哺乳动物的表型提供一个新的实验系统，从而推动新一代理论模型的发展。

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.