Quantitative Analysis of Cellular Signaling in Viral Latency

描述（由申请人提供）：潜伏性HIV是从患者体内完全根除HIV的最重要障碍。然而，潜伏期的分子基础仍然未知。使用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（申办方）和Arkin（共同申办方）表明，具有单一病毒整合的受感染Jurkat细胞的克隆群体可以快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，这些长时间的随机转录和翻译延迟可以使病毒保持足够长的非活性状态，以便宿主T细胞转化为记忆T细胞，从而将病毒固化为潜伏状态，这是关于HIV潜伏期如何建立的一个全新假设。我正在使用实验和计算技术来探测NF-kB（HIV LTR启动子的关键转录调节因子）如何影响随机基因表达和潜伏期。我的中心假设是：1）单个T细胞中HIV LTR处NF-kB相互作用的波动改变了基础转录速率，并导致激活或潜伏决定; 2）通过适当的NF-kB激活强度和持续时间，可以清除潜伏池而不产生毒性。这些假设将通过一组分为两个具体目标的实验进行测试。在具体目标I中，我将确定HIV LTR启动子处NF-κ B相互作用的细胞间异质性是否有助于HIV基因表达的随机性。为此，我将使用分子生物学技术收集定量数据，以干扰受感染的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潜伏期决定。在Specific Aim II中，我将使用NF-kB依赖的HIV基因表达的计算模型来设计治疗性抗潜伏策略，然后通过实验测试疗效。这种分子病毒学和计算生物学的融合有望在设计治疗一个主要的生物医学问题方面取得进展。 公共卫生相关性：潜伏的HIV，一种“隐藏”在宿主细胞中的具有复制能力的病毒库，是治愈HIV感染患者的重要障碍，然而，科学家们仍然不知道潜伏期是如何建立的。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活跃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药物策略，以清除细胞群中的潜伏感染。

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.