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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.

项目摘要尽管有效的抗逆转录病毒疗法（ART），HIV-1仍然存在于记忆性CD 4 + T细胞中，成为治愈的主要障碍。最近提出HIV-1可以通过整合驱动感染细胞的异常增殖转化为癌症相关基因。如果存在的话，克隆扩大的潜伏库会阻碍HIV-1根除工作应该有针对性的。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转化为癌症相关基因。一些挑战阻碍了对HIV-1持续性的研究。首先，HIV-1的罕见性感染的细胞和缺乏可靠的标志物，可以区分细胞含有诱导型HIV-1（约1-10每从含有缺陷型HIV-1的细胞（约100-1000/百万静息CD 4 + T细胞）而未感染的细胞使得HIV-1特异性分析变得困难。第二，大量CD 4 + T细胞的转录组分析来自HIV-1感染个体的转录物主要捕获HIV-1未感染细胞的转录物组。三、方法研究HIV-1整合位点破坏了HIV-1基因组，而研究HIV-1全长序列的方法和复制能力将HIV-1整合位点排除在扩增之外。为此，我们开发了创新的、尖端的HIV-1 RNA SortSeq，它可以识别含有诱导型HIV-1的细胞，细胞RNAseq分析和诱导型HIV-1的整合位点。从病毒感染者的血液样本中抑制的个体，我们鉴定了HIV-1宿主嵌合RNA，其描绘了诱导型HIV-1 RNA， HIV-1感染者与此同时此外，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剪接成HIV-1 RNA，产生编码宿主-HIV-1融合蛋白，和3）HIV-1 RNA剪接到宿主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驱动感染细胞的增殖并促进HIV-1的持续存在交互.我们的目标是检测HIV-1整合到癌症相关基因中是否会导致克隆性的扩增（目标1）和确定HIV-1驱动的增殖机制（目标2）。在目标1中，我们将获得在不同的时间点从病毒抑制个体的血液样品，以确定是否诱导HIV-1 其整合到癌症相关基因中，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单个转录组，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- 宿主RNA相互作用和HIV-1特异性和整合位点特异性刺激后的克隆扩增。总的来说，我们将在单细胞水平上研究HIV-1的持久性和克隆扩增，这将有助于开发一种更有效的HIV-1治疗策略，针对克隆扩张的潜伏库。