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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

基本信息

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
项目类别：
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

项目摘要

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ART可以将血浆HIV-1病毒血症降低至不可检测的水平，但并不能消除持续潜伏的 HIV-1 储存库。 HIV-1潜伏期的一个主要形式是由于转录沉默整合原病毒。目前消除这些潜在病毒库的努力包括“唤醒和杀死”策略，其中延迟逆转剂（LRA）用于转录激活病毒，以便重新激活细胞可以被宿主防御系统或药物识别来杀死它们。然而，目前的圣主抵抗军无法有效扭转延迟。这种低效的延迟逆转的机制基础尚不清楚。这是假设这些潜伏细胞中可能存在转录和转录后阻断，并且 LRA 克服转录后阻断的潜力可能有所不同。为了促进“唤醒和杀死”方法，我们将制定策略来研究患者样本中潜伏细胞块的性质应用多重、单细胞 RNA-FISH+IF 方法。这将与世界各地的合作完成 RNA-FISH技术专家，阿尔伯特·爱因斯坦学院的罗伯特·辛格博士。我们将开发一个临时的和潜伏 HIV-1 从转录起始到病毒颗粒产生再激活的亚细胞空间图使用 RNA-FISH 组合研究 mRNA 和 Gag 蛋白的三个主要特性的表达和如果。使用这种方法，我们将能够确定（i）三个主要的转录和表达转录本（未剪接、单剪接和多重剪接 RNA）； (iii) RNA 的核输出； (iv) 病毒积累细胞质中的RNA； (v) p24蛋白表达； (vii) 将病毒蛋白运输至质膜。我们将把这些技术应用于原代 T 细胞潜伏期模型（Siliciano 模型）和静息 CD4+ 记忆来自一组接受抑制治疗的无病毒血症 HIV 感染者的 T 细胞，以了解不同的情况潜伏细胞重新激活的阻断阶段。这些将与 Siliciano 博士和 Dr. Siliciano 博士合作完成。阿纳斯托斯。我们将使用 PerkinElmer Pannoramic 250 Flash II 扫描显微镜来检测罕见的重新激活 RNA-FISH+IF 呈阳性的细胞。单细胞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，而不仅仅是对其预期用途的影响（转录激活）以及原代潜伏细胞模型和患者中的转录后事件重新激活后衍生的离体潜伏细胞。尚不可能研究转录和转录后的早期阶段由于潜伏细胞数量较少并且当前的方法使用重新激活潜伏细胞的机制合并样本和晚期时间点。我们相信，使用单细胞方法和早期时间点，可以对潜在细胞中的各个块进行综合分析。这些研究可能会提供对潜伏细胞重新激活的阻断性质的新颖见解，以促进未来有效的开发代理逆转延迟作为“唤醒和终止”方法的一部分。