Cytor lncRNA as a positive regulator of HIV gene expression and viral latency

Cytor lncRNA 作为 HIV 基因表达和病毒潜伏期的正调节因子

• 批准号: 10681321
• 负责人: Koh Fujinaga
• 金额: $ 20.48万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-10 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10681321
• 关键词: Binding; Biochemical; CD4 Positive T Lymphocytes; Cell Separation; Cell physiology; Cells; Complex; Cytoskeleton; Development; Event; Foundations; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; HIV; HIV Infections; HIV tat Protein; Histones; In Vitro; Integration Host Factors; Knowledge; Light; Mediating; Molecular; Monitor; Patients; Phosphotransferases; Physiological; Play; Positive Transcriptional Elongation Factor B; Proteins; Proteomics; Protocols documentation; RNA; RNA Polymerase II; Regulation; Regulator Genes; Rest; Role; Testing; Therapeutic; Transcription Elongation; Transcriptional Elongation Factors; Untranslated RNA; Viral; Viral reservoir; Virus Latency; Virus Replication; antiretroviral therapy; chromatin isolation by RNA purification sequencing; clinically relevant; clinically significant; cofactor; cyclin T1; differential expression; effective therapy; efficacy testing; experimental study; genetic testing; genome-wide; improved; insight; new therapeutic target; promoter; reactivation from latency; recruit; therapeutic RNA; tool; transcriptome; transcriptome sequencing

ABSTRACT To successfully eliminate the HIV reservoir, it is critical to understand the molecular events that control HIV latency. While the key roles that host protein factors play in the regulation of HIV transcription and viral latency have been extensively studied, our understanding of how the non-coding transcriptome, especially long non- coding RNA (lncRNA), contribute to viral latency control is limited. To better understand the regulatory roles lncRNAs play in the control of HIV transcription and viral latency, we employed RNA-Seq analysis and compared the transcriptome in activated versus resting HIV-infected cells. We identified numerous lncRNAs that are differentially expressed, therefore are candidates for HIV latency regulation. Among them, one lncRNA, Cytoskeleton Regulator (Cytor), activates HIV transcription and viral latency. Our results also indicate that the depletion of Cytor suppresses latent HIV reactivation by reducing the occupancy of RNA Polymerase II (Pol II) and the levels of histone activation markers on the viral promoter. Additional biochemical and proteomic analyses showed that Cytor occupies the HIV promoter and associates with Positive Transcription Elongation Factor b (P- TEFb), which is an essential cellular factor for transcription elongation of HIV and cellular genes. In light of these findings, we hypothesize that by recruiting P-TEFb to the HIV promoter, Cytor activates HIV gene expression. To test this hypothesis, we will determine whether Cytor directly binds P-TEFb and recruits the cellular transcription elongation complex to the HIV promoter. Additional preliminary RNA-Seq analysis indicated that Cytor depletion results in broad changes in the host transcriptome. We will therefore identify downstream targets of Cytor and determine their indirect effects on HIV gene expression. Significantly, our results will be further confirmed in a clinically relevant context, as we will manipulate Cytor expression in CD4+ primary cells and determine the magnitude of Cytor’s therapeutic potential for HIV reactivation and latency reversal or, alternatively, to latency induction. Our study will provide new insights into the regulation of HIV transcription and viral latency by lncRNAs. Its successful completion will lay the groundwork for the development of new RNA-based therapies that will be added to current therapeutic protocols to eliminate HIV infection and the persistent viral reservoir.

摘要 为了成功地消除艾滋病病毒库，了解控制艾滋病病毒的分子事件至关重要 延迟。虽然宿主蛋白因子在调节HIV转录和病毒潜伏期中起着关键作用， 已经得到了广泛的研究，我们对非编码转录组，特别是长非编码转录组的理解， 编码RNA（lncRNA），有助于病毒潜伏期控制是有限的。为了更好地理解监管角色， lncRNA在HIV转录和病毒潜伏期的控制中发挥作用，我们采用RNA-Seq分析并比较了 激活的和静止的HIV感染细胞中的转录组。我们发现了大量的lncRNAs， 因此，差异表达的基因是HIV潜伏期调节的候选者。其中，一种lncRNA， 细胞骨架调节因子（Cytor），激活HIV转录和病毒潜伏期。我们的研究结果还表明， Cytor的耗竭通过减少RNA聚合酶II（Pol II）的占据抑制潜伏的HIV再活化 和病毒启动子上组蛋白活化标记物的水平。其他生化和蛋白质组学分析 表明Cytor占据HIV启动子并与阳性转录延伸因子B（P- TEFb），其是HIV和细胞基因转录延伸的必需细胞因子。鉴于这些 根据这些发现，我们假设通过将P-TEFb募集到HIV启动子，Cytor激活HIV基因表达。 为了验证这一假设，我们将确定Cytor是否直接结合P-TEFb并募集细胞内的 转录延伸复合物的HIV启动子。额外的初步RNA-Seq分析表明， 细胞因子耗竭导致宿主转录组的广泛变化。因此，我们将确定下游目标 并确定它们对HIV基因表达的间接影响。值得注意的是，我们的结果将进一步 在临床相关背景下证实，因为我们将操纵CD 4+原代细胞中的Cytor表达， 确定Cytor对HIV再激活和潜伏期逆转的治疗潜力，或者， 到潜伏期诱导我们的研究将为HIV转录和病毒潜伏期的调控提供新的见解 通过lncRNA。它的成功完成将为开发基于RNA的新疗法奠定基础 这将被添加到目前的治疗方案，以消除艾滋病毒感染和持久的病毒库。