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.

当前的艺术可以将血浆HIV-1病毒血症降低到无法检测到的水平，但不会消除持续的潜在HIV-1水库。 HIV-1潜伏期的一种主要形式是由于转录沉默综合病毒。目前消除这些潜在水库的努力包括“唤醒和杀戮”策略，其中使用潜伏期逆转剂（LRA）用于转录激活病毒，以使重新激活可以通过宿主防御系统或药物杀死细胞来识别细胞。但是，当前的LRA是无效逆转延迟。这种无效的潜伏期逆转的机械基础尚不清楚。这是假设这些潜在细胞中可能存在转录和转录后块 LRA可能会在克服转录后块的潜力上有所不同。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在细胞中块的性质如果方法，则应用多重单细胞RNA-FISH+。这将与世界合作完成 RNA-Fish Technology的专家，Albert Einstein学院的Robert Singer博士。我们将发展一个暂时的潜在的HIV-1从转录启动到病毒颗粒产生的潜在HIV-1的亚细胞空间图使用RNA-Fish的组合研究了三个主要指定mRNA和GAG蛋白的表达如果。使用这种方法，我们将能够确定（i）三个主要的转录和表达笔录（未切片，单一和乘剪接的RNA）；（iii）RNA的核出口；（iv）病毒的积累细胞质中的RNA；（v）p24蛋白表达；（vii）将病毒蛋白运输到质膜。我们将将这些技术应用于延迟的主要T细胞模型（硅型模型）和静止的CD4+内存来自一组抑制性疗法的AVIREAMIC HIV感染者的T细胞，以瞥见不同潜在细胞重新激活的块阶段。这些将与Siliciano博士和博士合作完成。阿纳斯托斯。我们将使用Perkinelmer Pannoramic 250 Flash II扫描显微镜检测稀有重新激活 RNA-FISH+IF阳性的细胞。单个单元格方法的应用之一是能够使用的能力这种方法可以同时分析少量的转录和转录后事件样品在单细胞水平上。我们将应用这种方法来测试LRA的影响，不仅对其预期用途（转录激活），但也在原发性潜在细胞模型中的转录后事件和患者中重新激活后，衍生出Exvivo潜在细胞。不可能研究转录和转录后的早期阶段由于其数量少，并且由于当前的方法使用，因此已重新激活潜在细胞的机制汇总样品和较晚的时间点。我们认为，使用单细胞方法和早期时间点可以分析对潜在细胞中各种块的全面说明。这些研究可能会提供对潜在细胞重新激活块的性质的新洞察力，以促进未来的有效发展作为“唤醒和杀死”方法的一部分，代理将潜伏期扭转。