Single cell RNA-seq and single molecule RNA-FISH approaches to study stochasticity of latent HIV-1 reactivation

单细胞 RNA-seq 和单分子 RNA-FISH 方法研究潜在 HIV-1 重新激活的随机性

• 批准号: 10082908
• 负责人: GANJAM V KALPANA
• 金额: $ 25.15万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-20 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10082908
• 关键词: Antibodies; Biological Assay; CD4 Positive T Lymphocytes; Cell Culture Techniques; Cells; Computational algorithm; Data; Development; Fishes; Gene Expression Regulation; Genes; Genetic; Genetic Heterogeneity; Genetic Transcription; Goals; HIV; HIV-1; Image; Immunofluorescence Immunologic; Individual; Infection; Lead; Length; Methodology; Methods; Microscopy; Modeling; Molecular; Noise; Pathway interactions; Patients; Procedures; Proteins; Proviruses; Quantitative Reverse Transcriptase PCR; RNA; RNA Probes; Resolution; Rest; Sampling; Scanning; Shock; Site; Slide; Source; Speed; Stimulus; T-Lymphocyte; Techniques; Technology; Transcript; Variant; Viral; Viral Genome; Viral Proteins; Virus Integration; base; cell type; combat; differential expression; epigenetic regulation; in vivo; innovation; integration site; knock-down; multiple omics; new therapeutic target; novel; novel marker; novel therapeutics; reactivation from latency; single cell analysis; single molecule; single-cell RNA sequencing; transcriptome; transcriptome sequencing; viral RNA

The persistent reservoirs are the ultimate hurdle to HIV-1 cure. ‘Shock and Kill’ strategy to eliminate HIV- 1 reservoirs requires reactivation of all latent proviruses with latency reversing agents (LRA), which is currently not possible. Quantitation of patient derived latent reservoirs by QVOA (Quantitative Viral Outgrowth Assay) indicated that only ~1/60th of full-length replication competent latent proviruses are activated by LRA. The reason for this variability is not completely understood but has been attributed to differential epigenetic regulation of provirus (due to different sites of proviral integration), differences in the viral genome (genetic variability), differential expression of cellular factors or different transcriptional or post-transcriptional blocks in the patient- derived latent cells. In the ExVivo and cell culture models of latency, where a single viral clone is used for infection with no genetic heterogeneity, reactivation of only a small percentage of cells and different levels of expression of viral RNA and proteins indicate cell-to-cell variation in reactivation. These studies also revealed that the majority of latent cells are not activated by current approaches. Current strategies do not address cell-to-cell variation in proviral reactivation and understanding this variability in vivo is essential to achieve either full reactivation or full suppression of these reservoirs. The goal of this application is to employ innovative single-cell and multi-omics platforms to investigate mechanisms of HIV persistence at the single-cell level with greater precision and higher resolution than has been achieved previously using traditional techniques. Stellaris-based quantitative single molecule RNA-FISH (smRNA-FISH), that allows the greater resolution and precise quantitation of the number of RNA molecules within single cells, have been used to study stochasticity in eukaryotic gene transcription. We have applied smRNA-FISH combined with Immunofluorescence (IF) termed SMIRA (Single cell and single molecule IF and RNA-FISH based Assay) to study HIV-1 reactivation in latency models and quantitated the single RNA molecules using FISH-quant, a computational algorithm. In addition, we have combined these studies with a high speed and high resolution scanning (HSHRS) microscopy to identify rare positive cell/s in a large pool of negative cells. Using these methods, our goal is to quantitate the cell-to-cell variability in latent cells. Our hypothesis is that cell- to-cell variation in HIV-1 reactivation is due to both ‘intrinsic’ and ‘extrinsic’ factors. By combining single cell and single molecule approaches with single cell RNA-sequencing (scRNA-seq) methodologies we propose to identify the determinants responsible for variation in the HIV-1 reactivation and extend these studies to identify these markers in patient samples. In aim I, we will characterize and determine the genes and pathways involved in the stochastic activation in HIV-1 latent cells using SMIRA, HSHRS and RNA-seq analysis. In aim II, we will validate the genes and pathways identified to influence HIV-1 stochastic activation in models of latency and in ART-suppressed patient samples. Our analyses will identify novel genes and pathways that influence cell-to-cell variability in HIV-1 reactivation and ultimately will lead to the development of novel therapies to combat HIV-1 latency.

持久的病毒库是治愈 HIV-1 的最终障碍。消除艾滋病毒的“休克和杀戮”策略 1 病毒库需要使用潜伏期逆转剂 (LRA) 重新激活所有潜伏原病毒，目前已 不可能。通过 QVOA（定量病毒生长分析）对患者来源的潜伏病毒库进行定量 表明只有大约 1/60 的全长复制能力的潜伏原病毒被 LRA 激活。原因 因为这种变异性尚未完全被理解，但已归因于不同的表观遗传调控 原病毒（由于原病毒整合的不同位点），病毒基因组的差异（遗传变异性）， 患者体内细胞因子或不同转录或转录后模块的差异表达 衍生的潜伏细胞。在 ExVivo 和细胞培养潜伏模型中，使用单个病毒克隆进行感染 无遗传异质性，仅重新激活一小部分细胞和不同水平的表达 病毒RNA和蛋白质的变化表明细胞与细胞之间重新激活的差异。这些研究还表明， 目前的方法并未激活大多数潜伏细胞。目前的策略并没有解决细胞间的问题 前病毒再激活的变异性和理解体内这种变异性对于实现完全 重新激活或完全抑制这些储存库。 该应用程序的目标是采用创新的单细胞和多组学平台来研究 与以往相比，在单细胞水平上具有更高的精度和分辨率来研究艾滋病毒持续存在的机制 以前使用传统技术实现的。基于 Stellaris 的定量单分子 RNA-FISH (smRNA-FISH)，可实现更高的分辨率和精确的 RNA 分子数量定量 在单细胞内，已用于研究真核基因转录的随机性。我们已经申请了 smRNA-FISH 与免疫荧光 (IF) 结合称为 SMIRA（单细胞和单分子 IF 和 基于 RNA-FISH 的检测）用于研究潜伏模型中 HIV-1 的再激活并定量单个 RNA 分子 使用 FISH-quant（一种计算算法）。此外，我们将这些研究与高速结合起来 高分辨率扫描 (HSHRS) 显微镜可识别大量阴性细胞中罕见的阳性细胞。 使用这些方法，我们的目标是量化潜伏细胞中细胞间的变异性。我们的假设是细胞 HIV-1 重新激活的细胞变异是由“内在”和“外在”因素造成的。通过将单细胞和 我们建议采用单细胞 RNA 测序 (scRNA-seq) 方法来识别单分子方法 导致 HIV-1 重新激活变异的决定因素，并扩展这些研究以确定这些因素 患者样本中的标记物。在目标 I 中，我们将表征并确定所涉及的基因和途径 使用 SMIRA、HSHRS 和 RNA-seq 分析研究 HIV-1 潜伏细胞的随机激活。在目标二中， 我们将在模型中验证已确定影响 HIV-1 随机激活的基因和途径 潜伏期和 ART 抑制的患者样本中。我们的分析将确定新的基因和途径 影响 HIV-1 重新激活的细胞间变异，最终将导致新疗法的开发 对抗 HIV-1 潜伏期。