Molecular Mechanisms of HIV Latency

HIV潜伏期的分子机制

基本信息

批准号：
9547084
负责人：
Eric M. Verdin
金额：
$ 127.88万
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 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9547084
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 Genome 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 Technology 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 genome-wide analysis illicit drug use innovation killings 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 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：融合后病毒潜伏期是根除HIV-1感染的主要障碍。消除病毒储层的当前理论范例被称为“冲击和杀伤”，使用非靶向的小分子重新激活潜在病毒。最近出现了这种方法的关键障碍，例如无效和/或随机病毒重新激活，避免全局T细胞激活以及重新激活后的细胞死亡有限。同样很明显，我们对艾滋病毒潜伏期分子机制的理解是碎片性的，尤其是滥用药物在主动和维持艾滋病毒潜伏期方面的相关性。在这里，我们建议在基因组规模上探索细胞因素的景观，这些细胞因素调节艾滋病毒潜伏期的建立和维持及其与滥用药物的关系。我们的探索将基于验证最近完成的基因组量表shRNA筛选的基因，以控制潜伏期维持和重新激活。我们建议使用新型的CRISPR-CAS9技术扩展此屏幕，该技术基于通过引导RNA融合蛋白进行靶向，该蛋白要么激活（CRISRA）或抑制（CRISPRI）基因表达。这些屏幕将集中在MTOR途径和甲基苯丙胺上，因为我们最近发现了它们可以独立起作用或共同起作用以调节潜伏HIV的重新激活。我们将验证并机械探索原代CD4 T细胞和HIV感染患者细胞中的途径和其他最高命中。我们还建议进一步开发和利用新的双荧光记者HIV-1基因组，以识别，量化和净化其本地感染的潜在感染细胞，而无需诱导病毒重新激活。这种新的延迟模型将使我们能够研究滥用药物，尤其是甲基苯丙胺的药物对原代人淋巴样细胞的潜伏期的建立和维持，并研究HIV-1潜伏期建立的早期机制。通过将双标记潜伏期模型与高分辨率单细胞系统生物学技术相结合，我们非常适合绘制控制艾滋病毒潜伏期和滥用药物在此过程中的作用的细胞调节网络。