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.

抗病毒治疗（ART）的出现揭示了CD4+ T细胞中的治疗抗性库 能够在治疗停止时补充HIV病毒血症。这个水库是一个主要的障碍， 治愈艾滋病病毒感染。最近的研究表明，在 一些个体由于含有完整前病毒的T细胞增殖， 在大约一半的受试者中，经过多年的ART治疗后，序列占主导地位。这些 相同的序列可能代表已经增殖的细胞的克隆。的驱动力 前病毒克隆扩增的背后仍然是个谜，但整合到癌基因的内含子中， 可能扮演了一个角色。为了研究这个问题，我们将前病毒测序和整合位点结合起来， 测序我们的创新是认识到，要正确分配前病毒， HIV DNA的延伸比通常由当前方法提供的要多。我们提出了一个新 基于先前使用的远程PCR技术的克隆整合位点的方法 我们的团队。在目标1中，我们提出开发一种方法，该方法将捕获独特的连接， 是在前病毒中产生的，有很大的缺失。这些删除的前病毒保留剪接能力 分为两大类，那些有很强的和那些有弱的表达潜力的人， HIV蛋白。在目标2中，我们提出了一种克隆完整前病毒整合位点的方法 这需要扩增更长的HIV DNA片段， DNA.考虑到这一点，我们描述了一个系统的方法来确定染色体地址 包括完整的前病毒克隆。我们假设完整的前病毒 克隆通常位于内含子中，内含子中的这种位置在 在克隆扩增中，因为它允许HIV剪接到癌基因的下游外显子。这反过来 为艾滋病毒根除战略提供了新的目标。我们的建议的前提主要是 根据我们小组的初步数据，有两种力量在制衡 引起（1）通过免疫清除的前病毒收缩和（2）通过免疫清除的前病毒扩张， 剪接到下游癌基因后的克隆增殖。我们在本提案中询问是否相同 两种力作用于有缺陷的和完整的复制能力的前病毒。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，水库更明显， 比以前意识到的。我们的工作表明，通过以下两种方式来扰动这两种力， 增强免疫清除或靶向剪接或剪接的下游外显子可以减少 水库规模我们设想这项工作可能会导致一个更大的研究，以了解如果功能失调， 细胞毒性T细胞在完整的前病毒克隆扩增中起作用。