Molecular Mechanisms of HIV Latency

帕萨特奖项目总结 整合后病毒潜伏期是根除HIV-1感染的主要障碍。当前理论 消除病毒储存库的范例被称为“休克和杀死”，即潜伏病毒的再激活 使用非靶向小分子。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，以及有限的细胞毒性。 重新激活后死亡。我们对分子生物学的理解 艾滋病毒潜伏的机制是零碎的，特别是滥用药物对艾滋病毒感染的影响。 启动和维持HIV潜伏期。 在这里，我们建议在基因组水平上探索调节HIV的细胞因子的景观 潜伏期的建立和维持及其与药物滥用的关系。我们的探索将 基于验证最近完成的基因组规模的shRNA筛选基因， 延迟维护和重新激活。我们建议使用新的CRISPR来扩大这种筛选- Cas9技术基于通过引导RNA融合蛋白的靶向，所述引导RNA融合蛋白激活（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，因为我们最近发现的证据表明，他们可能独立行动， 共同调节潜伏的HIV的再激活。我们将验证和机械地探索两者 在原代CD 4 T细胞和来自HIV感染患者的细胞中， 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， 鉴定、定量和纯化处于天然状态的潜伏感染细胞，而不诱导病毒 重新激活这种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 甲基苯丙胺对人淋巴细胞潜伏期的建立和维持 细胞和研究HIV-1潜伏期建立的最早机制。通过组合 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， 我们非常适合绘制出控制HIV潜伏期的细胞调节网络， 药物滥用在这一过程中的作用。

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.