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

抗病毒疗法(ART)的出现揭示了CD4+T细胞中存在耐药库 当ART停止时，能够补充艾滋病毒病毒血症。这个水库是一个主要障碍 实现艾滋病毒感染的治愈。我们最近的研究表明，储集层固有的衰变是 比之前认识到的更快。由于相反的力量，这种衰变被遮挡住了 导致前病毒克隆性扩张。前病毒克隆扩张背后的驱动力仍然存在 神秘的，但整合到癌基因内含子中可能起到作用。总体目标：在此 在此基础上，我们剖析了水库收缩(目标1)和扩张(目标2)的驱动因素。我们的 方法将是扰乱这两种力量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，以了解干扰这些力如何影响 前病毒的基因组成，它们的扩张倾向，以及它们的表达。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。我们 相信我们耐人寻味的结果证实了我们刻意限制样本大小以获得 每个个体都有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不为人知的选择压力。设计和 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。我们还通过突变剖析了HIV蛋白诱导的细胞毒性。 个体在用条形码病毒感染CD4T细胞之前打开阅读框。在目标2中， 我们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，通过使用一个 条形码病毒。我们还使用纵向整合部位分析来比较克隆比率 扩展以及扩展的前病毒克隆在精英控制器中的“特征”，尖锐地 以及接受抗逆转录病毒治疗的慢性感染者。 我们提案的前提在很大程度上是基于我们的工作，表明有两个 通过病毒的细胞毒性和免疫来平衡导致(1)前病毒收缩的力量 清除和(2)克隆增殖的前病毒扩增。我们的重要意义在于 这项提议包括，它可能有助于提供越来越多的证据，证明水库更明显 比之前意识到的要多。我们设想这项工作可以带来增强免疫力的方法。 间隙或目标拼接，以减小储油罐尺寸。

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.