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:
- 金额:$ 25.05万
- 依托单位:
- 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:
- 关键词:AliquotAlpha CellBiochemicalBiological AssayCD28 geneCD3 AntigensCD4 Positive T LymphocytesCell CountCell Culture TechniquesCell FractionCell LineCell NucleusCell membraneCell modelCellsCollaborationsCytoplasmDNA analysisDefective VirusesDevelopmentEpigenetic ProcessEventFluorescenceFluorescent in Situ HybridizationFutureGenetic TranscriptionGoalsGoldHIVHIV InfectionsHIV-1Highly Active Antiretroviral TherapyHost DefenseImmuneImmune systemImmunofluorescence ImmunologicIndividualKineticsLeadLightLymphocyteMapsMeasuresMembraneMessenger RNAMethodologyMethodsMicrogliaMicroscopeModelingMolecularNatureNuclear ExportNuclear RNAPatientsPharmaceutical PreparationsPlasmaProceduresProductionProtein BiosynthesisProteinsProvirusesRNARNA SplicingRNA StabilityRNA primersResearchRestSamplingScanningShockSpecific qualifier valueSystemT-LymphocyteTechniquesTechnologyTestingTimeTranscriptTranscription InitiationTranscriptional ActivationTranslationsViralViral ProteinsViremiaVirionVirusVirus Assemblybasecell typecollaboratorycollegedeep sequencingdrug developmentgag Gene Productsin vivoinsightkillingsmacrophagememory CD4 T lymphocytemonocytenovelparticleprotein expressionreactivation from latencyresponsesingle cell analysissingle moleculetooltraffickingviral 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。
项目成果
期刊论文数量(0)
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GANJAM V KALPANA其他文献
GANJAM V KALPANA的其他文献
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{{ truncateString('GANJAM V KALPANA', 18)}}的其他基金
RNA-mimicry to guide the intra-cellular targeting of host virus protein and viral RNA-protein interactions to inhibit HIV replication.
RNA模拟引导宿主病毒蛋白的细胞内靶向和病毒RNA-蛋白相互作用以抑制HIV复制。
- 批准号:
10554025 - 财政年份:2022
- 资助金额:
$ 25.05万 - 项目类别:
RNA-mimicry to guide the intra-cellular targeting of host virus protein and viral RNA-protein interactions to inhibit HIV replication.
RNA模拟引导宿主病毒蛋白的细胞内靶向和病毒RNA-蛋白相互作用以抑制HIV复制。
- 批准号:
10618961 - 财政年份:2022
- 资助金额:
$ 25.05万 - 项目类别:
Single cell RNA-seq and single molecule RNA-FISH approaches to study stochasticity of latent HIV-1 reactivation
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10082908 - 财政年份:2020
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Structure-based design of stapled peptides to target Gag-Pol and INI1 interaction to block assembly
基于结构的钉合肽设计,以靶向 Gag-Pol 和 INI1 相互作用来阻止组装
- 批准号:
10302316 - 财政年份:2020
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Effect of drugs of abuse on CNS HIV-1 reservoirs and neuropathogenesis
滥用药物对 CNS HIV-1 储存库和神经发病机制的影响
- 批准号:
9532834 - 财政年份:2016
- 资助金额:
$ 25.05万 - 项目类别:
Effect of drugs of abuse on CNS HIV-1 reservoirs and neuropathogenesis
滥用药物对 CNS HIV-1 储存库和神经发病机制的影响
- 批准号:
9333307 - 财政年份:2016
- 资助金额:
$ 25.05万 - 项目类别:
Effect of drugs of abuse on CNS HIV-1 reservoirs and neuropathogenesis
滥用药物对 CNS HIV-1 储存库和神经发病机制的影响
- 批准号:
9977143 - 财政年份:2016
- 资助金额:
$ 25.05万 - 项目类别:
Effect of Drugs of Abuse on CNS HIV-1 Reservoirs and Neuropathogenesis
滥用药物对中枢神经系统 HIV-1 病毒库和神经发病机制的影响
- 批准号:
10419775 - 财政年份:2016
- 资助金额:
$ 25.05万 - 项目类别:
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
- 批准号:
9207956 - 财政年份:2016
- 资助金额:
$ 25.05万 - 项目类别:
Effect of drugs of abuse on CNS HIV-1 reservoirs and neuropathogenesis
滥用药物对 CNS HIV-1 储存库和神经发病机制的影响
- 批准号:
9926378 - 财政年份:2016
- 资助金额:
$ 25.05万 - 项目类别:
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