Unveiling the chromosomal address of intact HIV clones to provide insights into persistence

揭示完整 HIV 克隆的染色体地址，以提供对持久性的见解

• 批准号: 9790462
• 负责人: Una T O'Doherty
• 金额: $ 25.18万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-08 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9790462
• 关键词: Address; Antiviral Therapy; CD4 Positive T Lymphocytes; Categories; Cell Proliferation; Cell division; Cells; Clonal Expansion; Clone Cells; Cytotoxic T-Lymphocytes; DNA; Data; Databases; Event; Exons; Genes; Genomics; HIV; HIV Infections; Human Genome; Immune Targeting; Individual; Introns; Lead; Length; Literature; Methodology; Methods; Mind; Modeling; Monitor; Oncogenes; Play; Positioning Attribute; Proliferating; Proteins; Proviruses; RNA; RNA Splicing; Reporting; Resistance; Role; Signal Transduction; Stretching; T-Cell Proliferation; T-Lymphocyte; Techniques; Testing; Time; Toxic effect; Transcript; Viral; Viremia; Work; base; driving force; falls; gene cloning; genetic element; human DNA; immune clearance; innovation; insight; integration site; pressure; protein expression

The advent of antiviral therapy (ART) revealed a treatment-resistant reservoir in CD4+ T cells capable of refueling HIV viremia when treatment is stopped. This reservoir is a major barrier to achieving a cure for HIV infection. Recent work suggests reservoir decay on ART is slower in some individuals due to proliferation of T cells containing intact proviruses since identical intact sequences are predominant after many years on ART in roughly half of subjects. These identical sequences likely represent clones of cells that have proliferated. The driving forces behind proviral clonal expansion remain mysterious, but integration into introns of oncogenes likely plays a role. To study this question, we combine proviral sequencing with integration site sequencing. Our innovation is recognizing that to properly assign proviruses we need longer stretches of HIV DNA than typically provided by current methodologies. We propose a new method to clone integration sites that is based on long-range PCR techniques previously utilized by our group. In Aim 1 we propose to develop a method that will capture unique junctions that are created in proviruses with large deletions. These deleted proviruses retain splicing ability and fall into two broad categories, those with strong and those with weak potential to express HIV proteins. In Aim 2, we propose a method to clone the integration site of intact proviruses which requires amplifying longer stretches of HIV DNA that are contiguous with the human DNA. With this in mind, we describe a systematic approach to identify the chromosomal address of the intact proviruses including intact proviral clones. We hypothesize that intact proviral clones are generally in introns and that this placement within the intron plays an important role in clonal expansion as it permits splicing of HIV to downstream exons of oncogenes. This in turn provides a new target for HIV eradication strategies. The premise of our proposal is largely based on preliminary data from our group showing that there are two counterbalancing forces that cause (1) proviral contraction through immune clearance and (2) proviral expansion through clonal proliferation after splicing to a downstream oncogene. We ask in this proposal if the same two forces act on both defective and intact replication-competent proviruses. The significance of our proposal include that it may contribute to growing evidence that the reservoir is more visible than previously realized. Our work suggests that perturbing these two forces by either enhancing immune clearance or targeting splicing or downstream exons of splicing may reduce reservoir size. We envision this work could lead to a larger study to understand if dysfunctional cytotoxic T cells have a role in intact proviral clonal expansion.

抗病毒疗法（ART）的出现显示CD4+ T细胞中的抗治疗库 停止治疗时能够加毒HIV病毒血症。该水库是 实现治愈艾滋病毒感染的方法。最近的工作表明，关于艺术的水库衰变在较慢 由于完全完整的T细胞的增殖，某些个体由于完整 在大约一半的受试者的艺术中，序列主要是序列。这些 相同的序列可能代表已经增殖的细胞的克隆。驱动力 前病毒克隆膨胀的背后仍然神秘，但整合到癌基因的内含子中 可能起作用。为了研究这个问题，我们将病毒测序与整合位点结合在一起 测序。我们的创新是认识到，要正确分配规定，我们需要更长的时间 HIV DNA的拉伸比当前方法通常提供的。我们提出了一个新的 基于先前使用的远程PCR技术的克隆集成站点的方法 由我们的小组。在AIM 1中，我们建议开发一种将捕获独特连接的方法 是在具有较大删除的Profirus中创建的。这些已删除的预科病毒保留剪接能力 并属于两个广泛的类别，那些具有强大的人，并且表达潜力较弱的人 HIV蛋白。在AIM 2中，我们提出了一种克隆完整预科病毒集成位点的方法 这需要扩大与人类连续的HIV DNA的更长的伸展 脱氧核糖核酸。考虑到这一点，我们描述了一种系统的方法来识别染色体地址 完整的原病毒，包括完整的病毒克隆。我们假设该完整的病毒 克隆通常是内含子中的，并且内含子内的位置起着重要作用 在克隆扩张中，它允许将HIV拼接到癌基因的下游外显子。反过来 为消除艾滋病毒策略提供了一个新的目标。我们的提议的前提在很大程度上是 基于我们小组的初步数据，表明有两个平衡力 原因（1）通过免疫清除和（2）通过 剪接到下游癌基因后的克隆增殖。我们在此提案中问是否相同 两种力对有缺陷和完整的复制能力病毒作用。的意义 我们的建议包括，它可能有助于越来越多的证据表明水库更可见 比以前意识到的。我们的工作表明，这两种力量通过 增强免疫清除或靶向剪接或剪接下游外显子可能会减少 储层尺寸。我们设想这项工作可能会导致更大的研究，以了解功能失调是否功能失调 细胞毒性T细胞在完整的前病毒克隆扩张中起作用。