Molecular Mechanisms of HIV Latency

HIV潜伏期的分子机制

• 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
• 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

 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根除HIV-1感染的主要障碍。目前消除病毒储存库的理论范例被称为“休克和杀死”，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的关键障碍，例如低效和/或随机的病毒再活化，避免整体T细胞活化，以及再活化后有限的细胞死亡。同样明显的是，我们对艾滋病毒潜伏期的分子机制的理解是零碎的，特别是滥用药物对艾滋病毒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的景观，调节艾滋病毒潜伏期的建立和维护，以及它们的关系，滥用药物的细胞因子。我们的探索将基于验证最近完成的基因组规模的shRNA筛选控制潜伏期维持和再激活的基因。我们建议使用新的CRISPR-Cas9技术来扩展这种筛选，该技术基于通过引导RNA融合蛋白进行靶向，该融合蛋白可以激活（CRISRa）或抑制（CRISPRi）基因表达。这些筛选将集中在mTOR通路和甲基苯丙胺上，因为我们最近发现的证据表明，它们可能独立或共同作用，以调节潜伏的HIV的再激活。我们将验证和机械地探索这两种途径和其他顶级命中在原代CD 4 T细胞和艾滋病毒感染患者的细胞。我们还建议进一步开发和利用新的双荧光报告HIV-1基因组，以识别、量化和纯化天然状态的潜伏感染细胞，而不会诱导病毒重新激活。这个新的延迟模型将允许我们 研究滥用药物，特别是甲基安非他明对人类初级淋巴细胞潜伏期的建立和维持的影响，并研究HIV-1潜伏期建立的最早机制。通过将我们的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HIV潜伏期的细胞调控网络以及滥用药物在此过程中的作用。