Molecular Mechanisms of HIV Latency

HIV潜伏期的分子机制

• 批准号: 10308273
• 负责人: Eric M. Verdin
• 金额: $ 6.89万
• 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 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308273
• 关键词: Award; CD4 Positive T Lymphocytes; CRISPR interference; CRISPR/Cas technology; Cells; Cessation of life; Chimeric Proteins; FRAP1 gene; Fluorescence; Gene Expression; Genes; Genomics; Guide RNA; HIV; HIV Genome; HIV-1; Human; In Vitro; Infection; Label; Lymphoid Cell; Maintenance; Maps; Methamphetamine; Modeling; Molecular; Parents; Pathway interactions; Patients; Process; Reporter; Resolution; Rest; Role; Shock; Systems Biology; T-Cell Activation; Techniques; Viral; Viral reservoir; Virus; Virus Integration; Virus Latency; antiretroviral therapy; base; drug abuser; drug of abuse; genome-wide; memory CD4 T lymphocyte; novel; parent grant; reactivation from latency; small hairpin RNA; small molecule; tool

PROJECT SUMMARY FROM PARENT AWARD 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.

来自上级奖项的项目总结 整合后病毒潜伏期是根除HIV-1感染的主要障碍。当前的理论 消除病毒库的范例被称为“休克和杀死”，即潜伏病毒的重新激活 使用非靶向的小分子。这种方法的关键障碍最近出现了，例如 低效和/或随机的病毒重新激活，避免全球T细胞激活，以及有限的细胞 在重新激活时死亡。同样明显的是，我们对分子的理解 艾滋病毒潜伏期的机制是零散的，特别是与滥用药物对 艾滋病毒潜伏期的启动和维持。 在这里，我们建议在基因组规模上探索调节艾滋病毒的细胞因子的图景 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 基于对最近完成的基因组规模shRNA筛查中控制基因的验证 延迟维护和重新激活。我们建议使用新的CRISPR来扩展这一屏幕- Cas9技术基于通过指南靶向RNA融合蛋白激活(CRISRa)或 抑制(CRISPRi)基因表达。这些屏幕将重点放在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 共同调节潜伏的艾滋病毒的重新激活。我们将验证并机械地探索这两者 在原始的CD4T细胞和HIV感染者的细胞中的通路和其他最高命中率。 我们还建议进一步开发和利用新的双荧光报告HIV-1基因组来 识别、量化和纯化处于天然状态的潜伏感染细胞，而不会引发病毒 重新激活。这一新的潜伏期模型将使我们能够研究滥用药物的影响，特别是 甲基苯丙胺对人初级淋巴组织潜伏期的建立和维持 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-生物学技术的力量， 我们非常适合规划控制HIV潜伏期和 滥用药物在这一过程中的作用。