Quantitative Analysis of Cellular Signaling in Viral Latency

描述(由申请人提供)：潜伏的艾滋病毒是彻底根除患者艾滋病毒的最大障碍。然而，潜伏期的分子基础仍不清楚。利用HIV-1 TAT介导的正反馈环的逆转录病毒模型，教授的实验室。Schaffer(发起人)和Arkin(共同发起人)表明，具有单一病毒整合的受感染Jurkat细胞的克隆群体可以迅速启动病毒基因表达，或表现出类似于潜伏感染的长时间低基因表达。我们假设，这些漫长的随机转录和翻译延迟可以使病毒保持在不活跃的状态足够长的时间，使宿主T细胞转化为记忆T细胞，从而使病毒固化为潜伏状态，这是一个关于HIV潜伏期如何建立的全新假说。我正在使用实验和计算技术来探索核因子-kB是如何影响随机基因表达和潜伏期的，核因子-kB是HIV LTR启动子的关键转录调节因子。我的中心假设是1)在单个T细胞中，NF-kB在HIV LTR上的相互作用的波动改变了基础转录速率，并导致了激活或潜伏时间的决定；2)有了适当的核因子-kB激活强度和持续时间，有可能在没有毒性的情况下清除潜伏池。这些假设将通过一系列组织成两个特定目标的实验来检验。在特定的目标I中，我将确定HIV LTR启动子上的NF-kB相互作用中的细胞间异质性是否有助于HIV基因表达的随机性。为了做到这一点，我将使用分子生物学技术收集定量数据，以扰乱受感染Jurkat细胞中的NF-kB LTR结合和核浓度，然后使用这些数据来计算模拟HIV潜伏期的决定。在特定的目标II中，我将使用这个依赖于核因子-kB的HIV基因表达的计算模型来设计治疗抗潜伏期的策略，然后实验测试疗效。这种分子病毒学和计算生物学的结合有望在设计针对重大生物医学问题的疗法方面取得进展。 与公共卫生相关：潜伏的艾滋病毒，一种“隐藏”在宿主细胞中的具有复制能力的病毒池，是治愈艾滋病毒感染患者的重要障碍，然而，科学家们仍然不知道潜伏时间是如何建立的。我正在建立一个数学模型，以研究艾滋病毒如何导致某些细胞的主动感染和其他细胞的潜伏感染。然后，我将使用这个模型来帮助设计抗潜伏期药物策略，以清除细胞群体中的潜在感染。

DESCRIPTION (provided by applicant): Latent HIV is the most significant barrier to complete eradication of HIV from a patient. However, the molecular basis of latency remains unknown. Using a retroviral model of the HIV-1 Tat-mediated positive feedback loop, the laboratories of Profs. Schaffer (sponsor) and Arkin (co-sponsor) showed that clonal populations of infected Jurkat cells with a single viral integration can either rapidly initiate viral gene expression or exhibit long periods of low gene expression analogous to a latent infection. We hypothesize that these long stochastic transcriptional and translational delays could maintain the virus in an inactive state long enough for the host T cell to convert to a memory T cell and thereby solidify the virus into a latent state, a fundamentally new hypothesis for how HIV latency is established. I am using experimental and computational techniques to probe how NF-kB, a key transcriptional regulator of the HIV LTR promoter, influences stochastic gene expression and latency. My central hypotheses are 1) that fluctuations in the interaction of NF-kB at the HIV LTR in single T cells alters the basal transcription rate and leads to the activation or latency decision; and 2) that with an appropriate strength and duration of NF-kB activation, it is possible to purge the latent pool without toxicity. These hypotheses will be tested through a set of experiments organized into two specific aims. In Specific Aim I, I will determine if cell-to-cell heterogeneity in NF-kB interactions at the HIV LTR promoter contributes to stochasticity in HIV gene expression. To do this, I will collect quantitative data using molecular biology techniques to perturb NF-kB LTR binding and nuclear concentrations in infected Jurkat cells, and then use these data to computationally simulate the HIV latency decision. In Specific Aim II, I will use this computational model of NF-kB-dependent HIV gene expression to design therapeutic anti-latency strategies, and then experimentally test efficacy. This blend of molecular virology and computational biology promises to make progress in designing therapies for a major biomedical problem. PUBLIC HEALTH RELEVANCE: Latent HIV, a pool of replication-competent virus that "hides" in host cells, is the significant barrier curing HIV-infected patients, however, scientists still do not know how latency is established. I am building a mathematical model to investigate how HIV can lead to active infection in some cells and latent infection in other cells. I will then use this model to help design anti-latency drug strategies to purge latent infections from a cell population.