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.

持续的水库是HIV-1治疗的最终障碍。消除艾滋病毒的“震惊和杀戮”策略 1个水库需要用潜伏期逆转剂（LRA）重新激活所有潜在病毒，目前是 不可能。定量QVOA（定量病毒生长测定法）对患者衍生的潜在储层的定量 表明只有〜1/60的全长复制能力的潜在病毒被LRA激活。原因 因为这种变异性尚未完全理解，但已归因于差异表观遗传调节 病毒病毒（由于病毒整合的不同位点），病毒基因组的差异（遗传变异性）， 细胞因子的差异表达或患者中不同的转录或转录后阻滞 派生的潜在细胞。在延迟的Exvivo和细胞培养模型中，其中单个病毒克隆用于感染 没有遗传异质性，只有一小部分细胞的重新激活和不同水平的表达 病毒RNA和蛋白质的重新激活表明细胞间变化。这些研究还表明 大多数潜在细胞不会被当前方法激活。当前的策略无法解决细胞到细胞 病毒重新激活和理解体内这种变异性的变化对于实现完整至关重要 重新激活或完全抑制这些储层。 该应用程序的目的是采用创新的单电池和多派平台来调查 与以前相比 以前使用传统技术实现。基于星式的定量单分子RNA鱼 （smRNA-fish），可以更大的分辨率和精确定量RNA分子数量 在单个细胞中，已用于研究真核基因转录中的随机性。我们已经申请了 SmRNA-Fish与称为Smira的免疫荧光（IF）结合（单细胞和单分子if和 基于RNA的基于RNA的测定）研究潜伏模型中的HIV-1重新激活并定量单个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相结合 以及高分辨率扫描（HSHRS）显微镜，以鉴定大量阴性细胞中的稀有细胞/s。 使用这些方法，我们的目标是量化潜在细胞中细胞对细胞的变异性。我们的假设是细胞 HIV-1重新激活中的电池差异是由于“内在”和“外部”因素引起的。通过组合单细胞和 单分子方法采用单细胞RNA - 序列（SCRNA-SEQ）方法，我们建议识别 负责HIV-1重新激活变化并扩展这些研究以确定这些研究的确定因素 患者样品中的标记。在目标I中，我们将表征并确定所涉及的基因和途径 在AIM II中，使用SMIRA，HSHRS和RNA-SEQ分析的潜在细胞。 我们将验证确定的基因和途径，以影响HIV-1随机激活 潜伏期和抑制艺术的患者样本。我们的分析将确定新的基因和途径 影响HIV-1重新激活中细胞间的细胞变异性，并最终导致新疗法的发展 打击HIV-1潜伏期。