Role of clonal expansion in HIV-1 persistence

克隆扩增在 HIV-1 持久性中的作用

基本信息

批准号：
9766189
负责人：
Ya-Chi Ho
金额：
$ 62.24万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
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/9766189
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 site-specific integration transcriptome transcriptome sequencing

项目摘要

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.

项目概要尽管抗逆转录病毒疗法 (ART) 有效，但 HIV-1 仍然存在于记忆 CD4+ T 细胞中，成为治愈的主要障碍。最近有人提出，HIV-1可以通过整合驱动受感染细胞的异常增殖转化为癌症相关基因。克隆性扩张的潜伏病毒库（如果存在）会阻碍 HIV-1 根除工作并且应该有针对性。然而，目前尚不清楚 HIV-1 原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转化为癌症相关基因。一些挑战阻碍了 HIV-1 持久性的研究。一、HIV-1的稀有性感染细胞和缺乏可靠的标记来区分含有诱导型 HIV-1 的细胞（每个细胞约 1-10 个）百万静息 CD4+ T 细胞）来自含有缺陷 HIV-1 的细胞（每百万静息 CD4+ T 细胞约 100-1000 个）未感染的细胞使得 HIV-1 特异性分析变得困难。二、批量CD4+T细胞的转录组分析来自 HIV-1 感染者的检测主要捕获了 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之外。为此，我们开发了创新、尖端的 HIV-1 RNA SortSeq，可识别含有可诱导 HIV-1 的细胞细胞 RNAseq 分析和诱导型 HIV-1 的整合位点。从病毒获得的血液样本中抑制个体，我们鉴定了 HIV-1 宿主嵌合 RNA，它描述了可诱导的 HIV-1 RNA 和 HIV-1同时整合位点。此外，我们还确定了 HIV-1 与宿主相互作用的三种模式：1) 通读转录，2) 宿主 RNA 剪接成 HIV-1 RNA，创建新的转录变体编码宿主-HIV-1融合蛋白，以及3) HIV-1 RNA剪接成宿主RNA，表明HIV-1驱动的宿主（癌症- 相关）基因表达。我们假设整合到癌症相关基因中的 HIV-1 可能通过 HIV-1 宿主 RNA 驱动受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中是否会导致克隆扩展（目标 1）并确定 HIV-1 驱动的增殖机制（目标 2）。在目标 1 中，我们将获得不同时间点的病毒抑制个体的血液样本，以确定是否可诱导 HIV-1 整合到癌症相关基因中的病毒使用 HIV-1 RNASortSeq 进行克隆扩增。我们将确定 HIV-1 整合到癌症相关基因中是否会改变宿主细胞转录组细胞水平。我们将研究 T 细胞激活、抗原驱动的增殖和稳态的贡献 HIV-1 克隆扩增中的增殖。在目标 2 中，我们将使用我们建立的细胞系模型来检查 HIV-1- HIV-1 特异性和整合位点特异性刺激下的宿主 RNA 相互作用和克隆扩增。总体而言，我们将在单细胞水平上检查 HIV-1 的持久性和克隆扩增，这将有助于针对克隆扩展的潜伏病毒库制定更有效的 HIV-1 治疗策略。