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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.

目前的抗逆转录病毒疗法可以将血浆HIV-1病毒血症降低到检测不到的水平，但不能消除持续潜伏的HIV-1病毒库。HIV-1潜伏的一种主要形式是由于转录沉默，整合前病毒目前消除这些潜在水库的努力包括“唤醒和杀死”战略，其中使用潜伏期逆转剂（LRA）来转录激活病毒，细胞可以被宿主的防御系统识别，也可以被药物杀死。然而，目前的LRA 在逆转延迟方面无效。这种无效的潜伏期逆转的机制基础是未知的。是假设在这些潜伏细胞中可能存在转录和转录后阻滞， LRA可能在克服转录后阻滞的潜力方面有所不同。为了促进“唤醒和我们将制定策略，通过以下方法研究患者样本中潜伏细胞中的块的性质：应用多重、单细胞RNA-FISH+IF方法。这项工作将与世界上 RNA-FISH技术的专家，阿尔伯特爱因斯坦学院的罗伯特·辛格博士。我们将开发一个时间和潜伏HIV-1从转录起始到病毒颗粒产生再活化亚细胞空间图谱使用RNA-FISH组合研究mRNA和Gag蛋白的三种主要特异性的表达如果。使用这种方法，我们将能够确定（i）三个主要的转录和表达，转录物（未剪接的、单剪接的和多剪接的RNA）;（iii）RNA的核输出;（iv）病毒的积累细胞质中的RNA;（v）p24蛋白表达;和（vii）病毒蛋白向质膜的运输。我们将这些技术应用于潜伏期的原代T细胞模型（Siliciano模型）和静息CD 4+记忆来自一组接受抑制治疗的艾滋病病毒感染者的T细胞，以了解不同的从阻滞阶段到潜伏细胞的再激活。这些将与Siliciano博士和Dr. Anaerobic.我们将使用PerkinElmer Pannoramic 250 Flash II扫描显微镜检测罕见的再激活 RNA-FISH+IF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，在单细胞水平上取样。我们将应用这种方法来测试LRA的影响，不仅是其预期用途，（转录激活），而且对原代潜伏细胞模型和患者中的转录后事件衍生的离体潜伏细胞。还不可能研究转录和转录后的早期阶段，由于其数量少，并且由于目前的方法使用合并样本和后期时间点。我们认为，使用单细胞方法和早期时间点，可以分析潜伏细胞中各种块的综合说明。这些研究可能会提供新的见解块的性质，以重新激活潜伏细胞，以促进未来的发展，有效的代理逆转延迟作为“唤醒和杀死”方法的一部分。