2.3 Microfluidics to probe regulation & treatment of HIV latency in single cells

尽管对HLV-1转录调控所涉及的分子组分有广泛的了解，但迫切需要了解这些组分如何动态地相互作用，形成一个调节开关，在单细胞水平上介导HIV-1潜伏期的建立、维持和重新激活。我们的长期目标是开发干预措施，以抑制或清除潜伏的艾滋病毒宿主，或者指示所有复制的艾滋病毒进入潜伏状态。这个特定项目的目标是定量分析HIV潜伏期背后的基因调节电路，并量化药物在不同原代细胞潜伏期模型中对HIV基因调节电路的影响。我们这个项目的基本原理是，HIV基因表达的单细胞定量成像将使我们能够阐明控制原代细胞潜伏期的基因调控电路，就像我们能够阐明Jurkat细胞中HIV潜伏期电路的关键方面一样。我们率先使用单细胞成像方法来研究随机噪声在HIV基因表达中的作用，我们的分析已经阐明，HLV-1通过TAT利用转录正反馈来放大HIV基因表达的随机波动，从而在前病毒潜伏期和活跃感染之间建立一个概率分子“开关”(Weinberger等人)。单元格2005)。 宿主SirT1抑制TAT反馈中的波动(Weinberger&Shenk，PLoS Biology 2007)，并且在J-Lat潜伏期模型中，操纵反馈强度可以使受感染的细胞偏向维持潜伏期而不是重新激活(Weinberger等人)。《自然遗传学》，2008)。我们将使用微流控方法和HIV基因表达的定量时移成像来定量比较不同候选药物在不同原代细胞模型中诱导潜伏基因表达的单细胞水平的影响。由此产生的分析将使不同的候选药物能够定量地比较它们对单细胞基因表达的影响，并将指导合作实验室内其他项目的分子分析。

Despite extensive knowledge of the molecular components involved in HlV-1 transcriptional regulation, there is a critical need to understand how these components interact kinetically to form a regulatory 'switch' that mediates establishment, maintenance, and reactivation of HIV-1 latency at the single-cell level. Our long-term goal is to develop interventions to inhibit or purge latent HIV reservoirs, or alternatively, direct all replicating HIV to enter a latent state. The objective of this particular project is to quantitatively analyze the gene-regulatory circuitry underlying HIV latency and to quantify the effects of drugs on HIV's gene-regulatory circuitry in different primary-cell latency models. Our rationale for this project is that quantitative single-cell imaging of HIV gene expression will enable us to elucidate the gene-regulatory circuitry that controls latency in primary cells just as we were able to eluciate key aspects of the HIV latency circuitry in Jurkat cells. We pioneered the use of single-cell imaging approaches to study the role of stochastic noise in HIV gene expression and our analysis has elucidated that HlV-1 utilizes transcriptional positive feedback via Tat to amplify stochastic fluctuations in HIV gene expression and thereby establish a probabilistic molecular 'switch' between proviral latency and active infection (Weinberger et al. Cell 2005). that host SirT1 dampens fluctuations in Tat feedback (Weinberger & Shenk, PLoS Biology 2007), and that manipulating the feedback strength, in the J-lat latency model, can bias infected cells towards maintaining latency and not reactivating (Weinberger et al. Nature Genetics, 2008). We will employ microfluidic approaches and quantitative time-lapse imaging of HIV gene-expression to quantitatively compare the single-cell level effects of diverse drug candidates upon induction of latent gene expression in different primary-cell models. The resulting analysis will allow different drug candidates to be quantitatively compared in terms of their effect on gene expression in single cells and will guide molecular analyses in other projects within the collaboratory.