Mechanism of HIV-1 Latency and Reactivation Kinetics Using Single Cell Analysis

使用单细胞分析研究 HIV-1 潜伏期和再激活动力学的机制

• 批准号: 9298589
• 负责人: GANJAM V KALPANA
• 金额: $ 25.05万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-16 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9298589
• 关键词: Aliquot; Alpha Cell; Biochemical; Biological Assay; CD28 gene; CD3 Antigens; CD4 Positive T Lymphocytes; Cell Count; Cell Culture Techniques; Cell Fraction; Cell Line; Cell Nucleus; Cell membrane; Cell model; Cells; Collaborations; Cytoplasm; DNA analysis; Defective Viruses; Development; Epigenetic Process; Event; Fluorescence; Fluorescent in Situ Hybridization; Future; Genetic Transcription; Goals; Gold; HIV; HIV Infections; HIV-1; Highly Active Antiretroviral Therapy; Host Defense; Immune; Immune system; Immunofluorescence Immunologic; Individual; Kinetics; Lead; Light; Lymphocyte; Maps; Measures; Membrane; Messenger RNA; Methodology; Methods; Microglia; Microscope; Modeling; Molecular; Nature; Nuclear Export; Nuclear RNA; Patients; Pharmaceutical Preparations; Plasma; Procedures; Production; Protein Biosynthesis; Proteins; Proviruses; RNA; RNA Splicing; RNA Stability; RNA primers; Research; Rest; Sampling; Scanning; Shock; Specific qualifier value; System; T-Lymphocyte; Techniques; Technology; Testing; Time; Transcript; Transcription Initiation; Transcriptional Activation; Translations; Viral; Viral Proteins; Viremia; Virion; Virus; Virus Assembly; base; cell type; collaboratory; college; deep sequencing; drug development; gag Gene Products; in vivo; insight; killings; macrophage; memory CD4 T lymphocyte; monocyte; novel; particle; protein expression; reactivation from latency; response; single cell analysis; single molecule; tool; trafficking; viral RNA

The current ART can reduce plasma HIV-1 viremia to undetectable levels, but do not eliminate persistent latent HIV-1 reservoirs. A major form of HIV-1 latency is due to transcriptional silencing of the integrated provirus. Current efforts to eliminating these latent reservoirs include a “wake and kill” strategy, where a Latency Reversing Agents (LRA) is used to transcriptionally activate the virus so that the reactivated cell can be recognized either by host defense system or drugs to kill them. However, the current LRA are ineffective in reversing latency. The mechanistic basis for this inefficient reversal of latency is unknown. It is hypothesized that there could be transcriptional as well as post-transcriptional blocks in these latent cells and that the LRAs may vary in their potential to overcome the post-transcriptional blocks. To facilitate “wake and kill” approach, we will develop strategies to investigate the nature of blocks in latent cells in patient samples by applying a multiplex, single cell RNA-FISH+IF methodologies. This will be done in collaboration with world's expert in RNA-FISH technology, Dr. Robert Singer at Albert Einstein College. We will develop a temporal and subcellular spatial map of reactivation of latent HIV-1 from transcription initiation to virus particle production by investigating expression of three major specifies of mRNA and Gag protein using a combination of RNA-FISH and IF. Using this approach we will be able to determine (i) transcription and expression of three primary transcripts (un-spliced, singly and multiply spliced RNAs); (iii) nuclear export of RNA; (iv) accumulation of viral RNAs in the cytoplasm; (v) p24 protein expression; and (vii) trafficking of viral proteins to plasma membrane. We will apply these techniques to primary T-cell model of latency (Siliciano model) and resting CD4+ memory T-cells from a set of aviremic HIV-infected individuals on suppressive therapy to get a glimpse of different stages of blocks to reactivation in latent cells. These will be done in collaboration with Dr. Siliciano and Dr. Anastos. We will use PerkinElmer Pannoramic 250 Flash II Scanning microscope to detect rare reactivated cells that are positive for RNA-FISH+IF. One of the application for the single cell method is the ability to use this method to simultaneously analyze transcriptional and post-transcriptional events in small amount of sample at single cell level. We will apply this method to test the effect of LRAs not only on their intended use (transcriptional activation) but also on post-transcriptional events in primary latent cell models and in patient derived exVivo latent cells, upon reactivation. It has not been possible to investigate early stages of transcriptional and post-transcriptional mechanisms in reactivated latent cells due to their small numbers and because the current approaches use pooled samples and late time points. We believe that using single cell approach and early time points, a comprehensive account of various blocks in latent cells can be analyzed. These studies are likely to provide novel insight into the nature of blocks to reactivation in latent cells to facilitate future development of effective agents to reverse latency as part of “wake and kill” approach.

目前的抗逆转录病毒疗法可以将血浆HIV-1病毒血症降低到无法检测到的水平，但不能消除 持续潜伏的HIV-1宿主。HIV-1潜伏期的一种主要形式是由于转录沉默 整合的前病毒。目前消除这些潜在油气藏的努力包括一种“唤醒并杀死”策略， 其中潜伏期反转剂(LRA)用于转录激活病毒，从而使重新激活的 细胞既可以被宿主防御系统识别，也可以被药物杀死。然而，目前的上帝抵抗军是 在反转延迟方面无效。这种无效的潜伏期逆转的机制基础尚不清楚。它是 假设在这些潜伏细胞中可能存在转录和转录后阻断 LRA在克服转录后障碍方面的潜力可能有所不同。以促进“唤醒和 方法，我们将制定策略，通过以下方法调查患者样本中潜伏细胞的阻断性质 应用多重、单细胞RNA-FISH+IF方法。这将与世界各地的 阿尔伯特·爱因斯坦学院的罗伯特·辛格博士是RNA-FISH技术的专家。我们将开发一种时态和 潜伏的HIV-1从转录启动到病毒颗粒产生的亚细胞空间图 利用RNA-FISH技术研究GAG蛋白和三种主要基因的表达 如果。使用这种方法，我们将能够确定(I)转录和表达三个主要的 转录本(未剪接的、单个和多重剪接的RNA)；(3)RNA的核输出；(4)病毒的积累 (V)p24蛋白的表达；(Vii)病毒蛋白向质膜的运输。 我们将把这些技术应用于潜伏期的初级T细胞模型(Siliciano模型)和静止的CD4+记忆 一组接受抑制治疗的无氧血症HIV感染者的T细胞获得不同的不同 潜伏细胞从阻断到再激活的各个阶段。这些工作将与Siliciano博士和Dr Siliciano博士合作完成。 阿纳斯托斯。我们将使用PerkinElmer Pannorama 250 Flash II扫描显微镜检测罕见的重新激活 RNA-FISH+IF阳性的细胞。单电池方法的应用之一是能够使用 这种方法可以同时分析少量的转录和转录后事件 单细胞水平的样品。我们将应用这种方法来测试LRA的效果，而不仅仅是对它们的预期用途 (转录激活)，但也对初级潜伏细胞模型和患者的转录后事件 衍生的体外潜伏细胞，在重新激活时。 还不可能研究转录和转录后的早期阶段。 由于潜伏细胞的数量较少，以及目前的方法使用 汇集样本和延迟时间点。我们认为，使用单细胞方法和早期时间点， 综合考虑潜伏细胞中的各个区块可以进行分析。这些研究可能会提供 新洞察阻滞性潜伏细胞重新激活的本质，促进未来发展的有效 代理将扭转延迟作为“唤醒并杀死”方法的一部分。