Role of clonal expansion in HIV-1 persistence

克隆扩增在 HIV-1 持久性中的作用

• 批准号: 10222530
• 负责人: Ya-Chi Ho
• 金额: $ 54.41万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-17 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10222530
• 关键词: Antigens; Blood specimen; CD27 Antigens; CD4 Positive T Lymphocytes; Cell Line; Cell Proliferation; Cells; Clinical; Clonal Expansion; Clone Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Competence; DNA Integration; Development; Distal; Event; FUS-1 Protein; Fluorescent in Situ Hybridization; Frequencies; Gene Expression; Genes; Genetic Transcription; Genome; Goals; HIV Genome; HIV-1; Individual; Length; Leukapheresis; Libraries; Location; Malignant Neoplasms; Maps; Measures; Mediating; Methods; Modeling; Oncogenes; Pattern; Population; Proviruses; RNA; RNA Sequences; RNA Splicing; Reporter; Rest; Role; Sampling; Site; Sorting - Cell Movement; Spliced Genes; System; T-Lymphocyte; Time; Variant; Viral; antiretroviral therapy; cancer gene expression; clinically relevant; established cell line; gene cloning; gene interaction; in vivo; innovation; integration site; memory CD4 T lymphocyte; novel; overexpression; prevent; promoter; prospective; single-cell RNA sequencing; site-specific integration; transcriptome

Project summary Despite effective antiretroviral therapy (ART), HIV-1 persists in memory CD4+ T cells as the major barrier to cure. It was recently proposed that HIV-1 can drive the aberrant proliferation of the infected cells through integration into cancer-related genes. The clonally expanding latent reservoir, if present, hampers HIV-1 eradication efforts and should be targeted specifically. However, it remains unclear whether HIV-1 proviruses integrated into cancer-related genes are intact or defective, and how HIV-1 may drive the clonal expansion through integration into cancer-related genes. Several challenges prevent the study of HIV-1 persistence. First, the rarity of HIV-1 infected cells and the lack of reliable markers which can distinguish cells containing inducible HIV-1 (~1-10 per million resting CD4+ T cells) from cells containing defective HIV-1 (~100-1000 per million resting CD4+ T cells) and uninfected cells makes HIV-1-specific analysis difficult. Second, transcriptome analysis of bulk CD4+ T cells from HIV-1-infected individuals captures mostly the transcriptome of HIV-1 uninfected cells. Third, methods studying HIV-1 integration sites disrupt the HIV-1 genome, while methods studying HIV-1 full-length sequences and replication competence exclude HIV-1 integration sites from amplification. To this end, we developed the innovative, cutting-edge HIV-1 RNA SortSeq which identifies cells containing inducible HIV-1 for single cell RNAseq analysis and the integration site of inducible HIV-1. From blood samples obtained from virally suppressed individuals, we identified HIV-1-host chimeric RNA which depicts inducible HIV-1 RNA and HIV-1 integration sites at the same time. Further, we identified three patterns of HIV-1-host interactions: 1) read-through transcription, 2) host RNA splicing into HIV-1 RNA, creating novel transcription variants encoding a host-HIV-1 fusion protein, and 3) HIV-1 RNA splicing into host RNA, indicating HIV-1 driven host (cancer- related) gene expression. We hypothesize that HIV-1 which are integrated into cancer-related genes may drive the proliferation of the infected cells and promote HIV-1 persistence through HIV-1-host RNA interactions. Our goal is to examine whether HIV-1 integration into cancer-related genes causes clonal expansion (Aim 1) and to identify the mechanisms of HIV-1-driven proliferation (Aim 2). In Aim 1, we will obtain blood samples from virally suppressed individuals at different time points to determine whether inducible HIV-1 which are integrated into cancer-related genes undergo clonal expansion using HIV-1 RNASortSeq. We will determine whether HIV-1 integration into cancer-related gene changes the host cell transcriptome at the single cell level. We will examine the contribution of T cell activation, antigen-driven proliferation and homeostatic proliferation in HIV-1 clonal expansion. In Aim 2, we will use our established cell line model to examine HIV-1- host RNA interactions and clonal expansion upon HIV-1-specific and integration site-specific stimulations. Overall, we will examine HIV-1 persistence and clonal expansion at the single cell level, which will facilitate the development of a more effective HIV-1 cure strategies targeting the clonally expanding latent reservoir.

项目总结 尽管进行了有效的抗逆转录病毒治疗(ART)，但HIV-1仍然存在于记忆中的CD4+T细胞中，成为治愈的主要障碍。 最近有人提出HIV-1可以通过整合来驱动感染细胞的异常增殖。 与癌症相关的基因。克隆扩大的潜在宿主，如果存在的话，阻碍了根除HIV-1的努力 而且应该是特别有针对性的。然而，目前尚不清楚HIV-1前病毒是否整合到 癌症相关基因是完整的还是有缺陷的，以及HIV-1如何通过整合驱动克隆扩张 与癌症相关的基因。一些挑战阻碍了对HIV-1持久性的研究。第一，HIV-1病毒的罕见 感染的细胞以及缺乏可靠的标记来区分含有可诱导的HIV-1的细胞(~1-10% 从含有HIV-1缺陷的细胞中提取)(每百万个静止的CD4+T细胞中约100-1000个) 而未感染的细胞使HIV-1特异性分析变得困难。第二，大量CD4+T细胞的转录组分析 从HIV-1感染者身上获取的大部分是HIV-1未感染细胞的转录组。三、方法 研究HIV-1整合位点会破坏HIV-1基因组，而研究HIV-1全长序列的方法 复制能力将HIV-1整合位点排除在扩增范围之外。为此，我们开发了 创新、尖端的HIV-1 RNA SortSeq，用于识别含有单个可诱导HIV-1的细胞 细胞RNAseq分析和可诱导的HIV-1整合位点。从从病毒中获得的血液样本 在抑制的个体中，我们鉴定了HIV-1宿主嵌合RNA，它描述了可诱导的HIV-1 RNA和 同时整合HIV-1病毒的地点。此外，我们确定了HIV-1与宿主相互作用的三种模式：1) 通读转录，2)宿主RNA拼接到HIV-1 RNA中，创建编码 宿主-HIV-1融合蛋白，以及3)HIV-1RNA拼接到宿主RNA中，表明HIV-1驱动的宿主(癌症- 相关)基因表达。我们推测，整合到癌症相关基因中的HIV-1可能 通过HIV-1宿主RNA驱动感染细胞增殖并促进HIV-1持续存在 互动。我们的目标是检查HIV-1整合到癌症相关基因是否会导致克隆 扩大(目标1)，并确定艾滋病毒-1驱动的扩散的机制(目标2)。在目标1中，我们将获得 在不同时间点采集病毒抑制个体的血液样本，以确定可诱导的HIV-1 整合到癌症相关基因中的基因利用HIV-1 RNASortSeq进行克隆扩增。我们会 确定HIV-1整合到癌症相关基因是否改变宿主细胞转录组 细胞水平。我们将研究T细胞活化、抗原驱动的增殖和体内平衡的作用 在HIV-1克隆扩增中增殖。在目标2中，我们将使用我们建立的细胞系模型来检测HIV-1- 宿主RNA在HIV-1特异性和整合位点特异性刺激下的相互作用和克隆扩展。 总体而言，我们将在单细胞水平上检查HIV-1的持久性和克隆性扩张，这将有助于 针对克隆扩大的潜伏宿主制定更有效的HIV-1治疗策略。