HIV transcriptome analysis during viral latency

病毒潜伏期的HIV转录组分析

• 批准号: 9204059
• 负责人: Koh Fujinaga
• 金额: $ 19.81万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-08 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9204059
• 关键词: Acquired Immunodeficiency Syndrome; Antisense RNA; Biological Assay; Biological Markers; CD4 Positive T Lymphocytes; Cataloging; Catalogs; Cells; Color; Detection; Development; Environment; Enzymes; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Goals; HIV; High-Throughput Nucleotide Sequencing; Highly Active Antiretroviral Therapy; Hybrids; Immunologic Surveillance; Individual; Interruption; Life; Maintenance; Messenger RNA; Methods; MicroRNAs; Modeling; Patients; Pattern; Peripheral Blood Mononuclear Cell; Phase; Population; Provirus Integration; Proviruses; RNA; RNA Splicing; Regimen; Reporting; Research Proposals; Rest; Role; Sampling; Signal Transduction; Site; Small Interfering RNA; Sorting - Cell Movement; Staging; Surrogate Markers; Techniques; Technology; Testing; Therapeutic; Variant; Viral; Viral Pathogenesis; Viral Proteins; Virus; Virus Integration; Virus Latency; Withholding Treatment; base; cohort; design; exosome; genome editing; global run on sequencing; immune activation; improved; integration site; knock-down; nanoparticle; next generation sequencing; reactivation from latency; transcriptome; transcriptome sequencing; viral RNA

PROJECT SUMMARY HIV latency is a major hurdle to overcome in the efforts to cure AIDS. Latently infected cells do not express viral proteins and escape immune surveillance during HAART treatment. These cells produce infectious viruses upon cessation of the treatment and immune activation. A challenge to achieving an effective HIV anti- latency therapy (HALT) is that these latently infected cells are very difficult to detect. To this end, it is critical to develop a sensitive method to detect latently infected cells in HIV-infected individuals. Although latently infected cells do not express productive viral mRNAs, defective or non-productive viral RNA are expressed and can be detected, which could serve as an excellent biomarker to detect latently infected cells. Highly sensitive next-generation sequencing techniques have revealed that HIV-infected cells express less characterized HIV RNAs including short RNAs, antisense RNAs, host-viral hybrid RNAs and splice variants from cryptic splicing sites, although the role(s) of these RNA species in the viral latency and pathogenesis are largely unknown. However, RT-qPCR analyses of patient cells would often underestimate these aberrant RNAs because of their instability. It is therefore critical to obtain comprehensive catalogues of viral RNA species expressed in latently infected cells using methods that can also detect unstable and short-lived RNA species. My preliminary results of RNA-seq analysis using well-established Jurkat latent models indicate that these cells exclusively express host-viral hybrid RNAs in unstimulated states. Also, reduction of the RNA exosome components by siRNA increased the level of these hybrid RNAs as well as antisense viral RNA. In the proposed study, exact patterns of viral RNA expression (transcriptome) during the establishment of viral latency and its reactivation will be revealed using ex vivo latency models of primary CD4+ cells as well as HIV-infected cells derived from HAART-suppressed patients. In addition, these analyses will determine the proportion of latently infected cells that express these aberrant RNAs, and whether there is a bias in the correlation between HIV RNA expression and provirus integration sites (orientation, genetic environment). Finally, a highly sensitive assay to detect and quantify HIV RNAs expressed specifically in latently infected cells will be developed. These studies will provide important information for developing an efficient therapeutic approach to improve our current HALT regimen.

项目摘要 艾滋病毒潜伏期是克服治愈艾滋病的主要障碍。潜在感染的细胞不表达 在HAART治疗期间，病毒蛋白并逃避免疫监测。这些细胞产生感染性 停止治疗和免疫激活后的病毒。实现有效的艾滋病毒抗的挑战 潜伏期治疗（停止）是这些潜在感染的细胞很难检测到。为此，至关重要 开发一种灵敏的方法来检测HIV感染的个体中受感染的细胞的潜在感染细胞。虽然潜在 感染的细胞不表达生产性病毒mRNA，缺陷或非生产性病毒RNA，并且表达 可以检测到，可以用作出色的生物标志物来检测潜在感染的细胞。高度敏感 下一代测序技术表明，感染HIV的细胞表达的HIV表现较低 RNA包括短RNA，反义RNA，宿主病毒杂种RNA和剪接变体，来自隐性剪接 尽管这些RNA物种在病毒潜伏期和发病机理中的作用在很大程度上未知。 但是，患者细胞的RT-QPCR分析通常会低估这些异常RNA，因为它们 不稳定。因此，至关重要 使用也可以检测不稳定和短寿命的RNA物种的方法感染细胞。我的初步结果 使用公认的Jurkat潜在模型的RNA-Seq分析表明，这些细胞专门表达 未刺激状态的宿主病毒杂种RNA。另外，siRNA减少RNA外泌体成分 增加了这些混合RNA和反义病毒RNA的水平。在拟议的研究中，精确模式 病毒潜伏期期间病毒RNA表达（转录组）的重新激活将为 使用原代CD4+细胞的离体潜伏期模型以及源自艾滋病毒感染的细胞揭示 哈特抑制的患者。此外，这些分析将确定受到潜在感染细胞的比例 表达这些异常RNA，以及HIV RNA表达之间的相关性是否存在偏差 和原病毒整合位点（方向，遗传环境）。最后，一种高度敏感的检测和 将开发出在潜在感染细胞中特异性表达的HIV RNA。这些研究将提供 开发有效的治疗方法以改善我们当前的停止方案的重要信息。