Stochastic Gene Expression Effects in a Model Retrovirus

逆转录病毒模型中的随机基因表达效应

• 批准号: 6970261
• 负责人: DAVID V SCHAFFER
• 金额: $ 27.09万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6970261
• 关键词: Lentivirus; T lymphocyte; amidohydrolases; chromatin; clone cells; confocal scanning microscopy; enzyme activity; eukaryote; flow cytometry; gene expression; genetic models; genetic regulation; genetic transcription; green fluorescent proteins; host organism interaction; human immunodeficiency virus 1; model design /development; polymerase chain reaction; statistics /biometry; virus genetics; virus integration

DESCRIPTION (provided by applicant): In this proposal we seek to perform a rigorous experimental and computational analysis of the dynamics of gene expression in a lentiviral model of HIV-1. The lentiviral system is derived from HIV-1 but leaves the Tat-mediated positive feedback loop controlling transcriptional activity intact. This feedback loop motif is a common gene regulatory architecture across all organisms and cellular subsystems thus measurements and conclusions derived from this study will apply broadly. Based on physical chemical principles and simple models of the system, we hypothesize (and show initial experimental results) that gene expression from this system is highly stochastic and that the Tat-mediated feedback is activated sporadically such that after the virus infects its target cell and integrates into the host genome, there may be significant periods of time elapsed before viral expression reaches the point where reproduction can occur and then propagate. This time could be enough to allow an activated T-cell to transition to its memory state thereby trapping the lentivirus in an inactive form until such time as that memory cell is reactivated. Thus, it is possible that this hypothesized noise is large enough to contribute to the formation of the latent pool of virus that makes HIV-1 so hard to treat. However, there is little experimental evidence of this possibility and further it has never been rigorously shown that mammalian gene expression is significantly stochastic. We therefore set out a program for quantitatively measuring the integration-point dependent kinetics of the key steps in this lentiviral, HIV-1 based, autoactivated gene expression system. We use specially designed viral constructs with fluorescent markers of expression and quantitative microscopy techniques to tease out all the parameters for a model of the eukaryotic gene expression model. We use the models to estimate the role of each of the steps in the process from integration point, to transcription initiation, to elongation control by tar, to Tat production/ degradation, to Tat/tar interaction in generating noise in the expression process and allowing delays until viral production. We also use the comparison of models to data to determine which parameters are not constrained by optimizations and thus direct measurements. The resultant experimentally-validated model of mammalian viral gene expression will be a resource for others studying the kinetics and role of noise in eukaryotic cellular processes.

描述（由申请人提供）：在本提案中，我们寻求对 HIV-1 慢病毒模型中基因表达的动态进行严格的实验和计算分析。慢病毒系统源自 HIV-1，但保留了控制转录活性的 Tat 介导的正反馈环。这种反馈环基序是所有生物体和细胞子系统的常见基因调控架构，因此从本研究中得出的测量和结论将广泛适用。基于物理化学原理和系统的简单模型，我们假设（并显示了初步实验结果）该系统的基因表达是高度随机的，并且 Tat 介导的反馈被偶尔激活，这样在病毒感染其目标细胞后，当病毒整合到宿主基因组中时，在病毒表达达到可以发生繁殖然后传播的程度之前可能需要相当长的一段时间。这个时间可能足以让激​​活的 T 细胞转变到其记忆状态，从而将慢病毒捕获为非活动形式，直到该记忆细胞重新激活。因此，这种假设的噪音可能足够大，足以促成潜在病毒库的形成，从而使 HIV-1 变得难以治疗。然而，几乎没有实验证据证明这种可能性，而且从未严格证明哺乳动物基因表达具有显着的随机性。因此，我们制定了一个计划，用于定量测量这种慢病毒、基于 HIV-1 的自激活基因表达系统中关键步骤的整合点依赖性动力学。我们使用专门设计的带有表达荧光标记的病毒构建体和定量显微镜技术来梳理出真核基因表达模型的所有参数。我们使用模型来估计过程中每个步骤的作用，从整合点到转录起始，到焦油的延伸控制，到 Tat 产生/降解，到 Tat/焦油相互作用在表达过程中产生噪音并允许延迟直到病毒产生。我们还使用模型与数据的比较来确定哪些参数不受优化限制，从而确定哪些参数不受直接测量的限制。由此产生的经过实验验证的哺乳动物病毒基因表达模型将为其他研究噪声在真核细胞过程中的动力学和作用的人提供资源。