Determinants of reservoir contraction and expansion in vivo, ex vivo, and in vitro

抗病毒治疗（ART）的出现揭示了CD4+ T细胞中的治疗抗性库 在停止抗逆转录病毒治疗后能够为HIV病毒血症补充能量。这个水库是一个主要的障碍， 治愈艾滋病病毒感染。我们最近的研究表明，固有的水库衰减是 比以前认识到的更快。这种衰变由于一种相反的力而被掩盖， 导致前病毒克隆扩增。前病毒克隆扩张背后的驱动力仍然存在 神秘的，但整合到致癌基因的内含子可能发挥作用。总体目标：在 建议，我们剖析了油藏收缩（目标1）和扩张（目标2）的驱动因素。我们 方法将是扰动这两种力，并研究由此产生的影响。我们将使用前病毒， 整合位点和RNA测序，以了解扰动这些力量如何影响 前病毒的遗传组成、它们的扩展倾向和它们的表达。我们的方法 是在几个个体中进行大规模的深度测序，而不是大样本量。我们 我相信我们有趣的结果证实了我们故意限制样本量的决定， 更深层次的序列信息。通过这种方法，我们最近提供了 前所未有的深度和阐明了以前未知的选择压力。设计与 方法：在目标1中，我们通过测量水库收缩来分离免疫清除的作用 在体内和体外。我们还剖析了HIV蛋白通过突变 个体在用条形码病毒感染CD4 T细胞之前打开阅读框。在目标2中， 我们通过使用一种新的方法，剖析了克隆扩张的驱动因素，包括艾滋病毒驱动的细胞分裂。 条形码病毒。我们还使用纵向整合位点分析比较了克隆率， 扩张以及精英控制者中扩张的前病毒克隆的“特征”， 和慢性感染者进行抗逆转录病毒治疗。 我们的建议的前提主要是基于我们的工作表明，有两个 平衡力量，导致（1）通过病毒细胞毒性和免疫原性收缩 清除和（2）通过克隆增殖的前病毒扩增。我们的重要性 该提案包括，它可能有助于越来越多的证据表明，水库更明显， 比以前意识到的。我们设想这项工作可以导致增强免疫的方法， 间隙或目标拼接以减小储层尺寸。

The advent of antiviral therapy (ART) revealed a treatment-resistant reservoir in CD4+ T cells capable of refueling HIV viremia when ART is stopped. This reservoir is a major barrier to achieving a cure for HIV infection. Our recent work suggests the inherent reservoir decay is more rapid than previously recognized. This decay is obscured due to an opposing force that results in proviral clonal expansion. The driving forces behind proviral clonal expansion remain mysterious, but integration into introns of oncogenes may play a role. Overall objective: In this proposal, we dissect the drivers of reservoir contraction (Aim 1) and expansion (Aim 2). Our approach will be to perturb both forces and to study the resulting effects. We will use proviral, integration site, and RNA sequencing to understand how perturbing these forces affects the genetic make-up of proviruses, their propensity to expand, and their expression. Our approach is to perform massive deep sequencing in a few individuals rather than large sample size. We believe our intriguing results validate our deliberate decision to limit sample size to obtain deeper sequence information within each individual. With this approach, we recently provided unprecedented depth and elucidated previously unknown selection pressures. Design and Methods: In Aim 1, we isolate the role of immune clearance by measuring reservoir contraction in vivo and in vitro. We also dissect the cytotoxicity induced by HIV proteins by mutating individuals Open Reading Frames before infecting CD4 T cells with a barcoded virus. In Aim 2, we dissect the drivers of clonal expansion, including HIV-driven cell division, by using a barcoded virus. We also use longitudinal integration site analysis to compare the rate of clonal expansion as well as the “character” of the expanded proviral clones in elite controllers, acutely and chronically infected individuals on ART. The premise of our proposal is largely based on our work showing that there are two counterbalancing forces that cause (1) proviral contraction through viral cytotoxicity and immune clearance and (2) proviral expansion through clonal proliferation. The significance of our proposal includes that it may contribute to growing evidence that the reservoir is more visible than previously realized. We envision this work could lead to approaches that enhance immune clearance or target splicing to reduce reservoir size.