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

摘要 为了成功地消除艾滋病毒的蓄积，了解控制艾滋病毒的分子事件是至关重要的 延迟。而宿主蛋白因子在调节HIV转录和病毒潜伏时间中发挥的关键作用 已经进行了广泛的研究，我们对非编码转录组的理解如何，特别是长时间的非编码 编码RNA(LncRNA)，对病毒潜伏期的控制有限。为了更好地理解监管角色 LncRNAs在控制HIV转录和病毒潜伏时间方面发挥作用，我们采用RNA-Seq分析并比较 激活的和静止的HIV感染细胞中的转录组。我们确定了大量的lncRNA是 因此，差异表达的是HIV潜伏期调控的候选基因。其中，一个lncRNA， 细胞骨架调节因子(Cytor)，激活HIV转录和病毒潜伏期。我们的结果还表明， Cytor缺失通过减少RNA聚合酶II(POL II)的占有率抑制潜在的HIV重新激活 以及病毒启动子上的组蛋白激活标志物的水平。额外的生化和蛋白质组学分析 研究表明，Cytor占据HIV启动子，并与阳性转录延伸因子b(P-B)相关。 TEFb)，它是HIV和细胞基因转录延长所必需的细胞因子。鉴于这些， 发现，我们假设通过招募P-TEFb到HIV启动子，Cytor激活HIV基因的表达。 为了验证这一假设，我们将确定Cytor是否直接结合P-TEFb并招募细胞 转录延伸复合体到HIV启动子。另外的初步RNA-Seq分析表明 Cytor耗尽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 在临床相关的背景下得到证实，因为我们将在CD4+原代细胞中操纵Cytor的表达 确定Cytor对HIV重新激活和潜伏期逆转的治疗潜力的大小，或者， 潜伏期诱导。我们的研究将为HIV转录和病毒潜伏的调控提供新的见解 通过InncRNAs。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中，以消除艾滋病毒感染和持久的病毒库。

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.