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

抗病毒疗法(ART)的出现揭示了CD4+T细胞中存在耐药库 能够在治疗停止时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。最近的研究表明，ART的储层腐烂速度较慢 一些个体由于T细胞的增殖而含有完整的前病毒，自完全一样完整 在艺术研究多年后，序列在大约一半的受试者中占据主导地位。这些 相同的序列很可能代表了已经增殖的细胞的克隆。驱动力 原克隆扩张的背后仍然是个谜，但整合到癌基因的内含子中 可能起到了一定的作用。为了研究这一问题，我们将前病毒测序与整合位点相结合 测序。我们的创新是认识到，要正确分配前病毒，我们需要更长的时间 比目前方法通常提供的艾滋病毒DNA片段更多。我们提出了一个新的 一种基于先前使用的远程聚合酶链式反应技术的克隆整合位点的方法 被我们这群人。在目标1中，我们建议开发一种方法，该方法将捕获 是在删除较多的原文件中创建的。这些被删除的前病毒保留了剪接能力 分为两大类，一类是表达能力强的，另一类是表达能力弱的 HIV蛋白质。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 这需要扩增与人类相邻的较长的HIV DNA片段 DNA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 完整的前病毒包括完整的前病毒克隆。我们假设完整的前病毒 克隆通常在内含子中，内含子中的这种位置起着重要的作用 克隆扩增，因为它允许艾滋病毒与癌基因下游外显子的剪接。这又反过来 为根除艾滋病毒战略提供了新的目标。我们提案的前提很大程度上是 根据我们小组的初步数据显示，有两种制衡力量 这导致(1)通过免疫清除的前病毒收缩和(2)通过免疫的前病毒扩张 剪接到下游癌基因后的克隆性增殖。我们在这份提案中询问是否同样 两种力量同时作用于有缺陷和有完整复制能力的前病毒。的重要意义。 我们的建议包括，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。我们的工作表明，通过任何一种方式干扰这两种力量 增强免疫清除或靶向剪接或剪接的下游外显子可能会减少 水库大小。我们设想这项工作可能会导致一项更大规模的研究，以了解功能障碍 细胞毒性T细胞在完整的前病毒克隆性扩增中起作用。

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.