Molecular Mechanisms of HIV Latency

HIV潜伏期的分子机制

基本信息

批准号：
10200723
负责人：
Eric M. Verdin
金额：
$ 126.08万
依托单位：
BUCK INSTITUTE FOR RESEARCH ON AGING
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：
2016-04-15 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10200723
关键词：
Binding Sites Biological Blood CD4 Positive T Lymphocytes CRISPR interference CRISPR/Cas technology Cell Line Cells Cessation of life Chimeric Proteins Clustered Regularly Interspaced Short Palindromic Repeats Cocaine Collaborations Data Drug user Epigenetic Process Exhibits Experimental Models FRAP1 gene Fluorescence Gene Expression Genes Genetic Epistasis Genetic Transcription Genomics Goals Guide RNA HIV HIV Genome HIV Infections HIV-1 Human In Vitro Individual Infection Label Lymphoid Cell Maintenance Maps Methamphetamine Modeling Molecular Mononuclear Paint Pathway interactions Patients Pharmaceutical Preparations Phenotype Process Proteins RNA Regulation Reporter Repression Resolution Rest Role Shock Systems Biology T-Cell Activation Techniques Testing United States Viral Viral Load result Viral reservoir Virus Virus Integration Virus Latency antiretroviral therapy base cell type crosslink differential expression drug abuser drug of abuse env Gene Products exhaustion experimental study follow-up genome wide screen genome-wide illicit drug use innovation mTOR Inhibitor memory CD4 T lymphocyte methamphetamine effect new technology novel novel therapeutics public health relevance reactivation from latency response small hairpin RNA small molecule small molecule inhibitor stimulant use synergism tool transcription factor transcriptome sequencing

项目摘要

DESCRIPTION: Post-integration viral latency is a major barrier to eradicating HIV-1 infection. The current theoretical paradigm for eliminating the viral reservoir is known as `Shock and Kill', reactivation of latent virus using non-targeted small molecules. Key hurdles to this approach have recently emerged such as inefficient and/or stochastic viral reactivation, avoidance of global T-cell activation, and limited cellular death upon re-activation. It is also becoming apparent that our understanding of the molecular mechanisms of HIV latency are fragmentary and in particular the relevance of drugs of abuse on the initiation and maintenance of HIV latency. Here, we propose to explore on a genomic scale the landscape of cellular factors that regulate HIV latency establishment and maintenance and their relationship to drugs of abuse. Our exploration will be based on validating a recently completed genome scale shRNA screen for genes that control latency maintenance and reactivation. We propose to expand this screen using the novel CRISPR-Cas9 technology based on targeting via a guide RNA fusion proteins that either activate (CRISRa) or inhibit (CRISPRi) gene expression. These screens will focus on the mTOR pathway and on methamphetamine as we have recently uncovered evidence that they may act independently or together to modulate the reactivation of latent HIV. We will validate and mechanistically explore both pathways and other top hits in primary CD4 T cells and in cells from HIV-infected patients. We also propose to further develop and exploit new dual-fluorescence reporter HIV-1 genomes to identify, quantify, and purify latently infected cells in their native state, without inducing viral reactivation. This new latency model will allow us to study the effect of drugs of abuse, particularly methamphetamine, on the establishment and maintenance of latency in primary human lymphoid cells and to study the very earliest mechanisms of HIV-1 latency establishment. By combining the power of our dual-labeled latency model with high-resolution single-cell systems-biology techniques, we are uniquely suited to map out the cellular regulatory networks that control HIV latency and the role of drugs of abuse in this process.

描述：整合后病毒潜伏期是消除 HIV-1 感染的主要障碍，目前消除病毒库的理论范式被称为“休克和杀伤”，即使用非靶向小分子重新激活潜伏病毒。最近出现了这种方法，例如低效和/或随机的病毒再激活、避免全局 T 细胞激活以及再激活时有限的细胞死亡。我们对病毒的分子机制的理解也逐渐加深。 HIV潜伏期是零散的，特别是滥用药物与HIV潜伏期的启动和维持的相关性在这里，我们建议在基因组规模上探索调节HIV潜伏期建立和维持的细胞因素及其与药物的关系。我们的探索将基于验证最近完成的基因组规模 shRNA 筛选，以筛选控制潜伏期维持和重新激活的基因。 (CRISRa) 或抑制(CRISPRi) 基因表达。这些筛选将重点关注 mTOR 途径和甲基苯丙胺，因为我们最近发现的证据表明它们可能独立或共同作用来调节潜在 HIV 的重新激活，我们将验证和机械地探索这两种途径和其他热门产品。我们还建议进一步开发和利用新的双荧光报告基因 HIV-1 基因组来识别、量化和纯化其天然的潜伏感染细胞。状态，而不诱导病毒重新激活，这种新的延迟模型将使我们能够研究滥用药物（特别是甲基苯丙胺）对原代人类淋巴细胞潜伏期建立和维持的影响，并通过将我们的双标记潜伏期模型的力量与高潜伏期相结合来研究 HIV-1 潜伏期建立的最早机制。 -分辨率单细胞系统生物学技术，我们非常适合绘制控制艾滋病毒潜伏期的细胞调控网络以及滥用药物在此过程中的作用。