The interplay between m6A and viral lncRNA during KSHV replication

这一建议将阐明在卡波西肉瘤相关疱疹病毒（KSHV）复制的背景下，多聚腺苷酸化核（PAN）长非编码（lnc）RNA的m6 A表位转录调控。KSHV编码过多的产物来控制其感染的两个阶段，但也复杂地破坏正常的细胞途径，并建立终身感染。在潜伏期，KSHV以休眠状态存在，并且即使已报道PAN lncRNA表达，其功能和潜在的表位转录调控尚未确定。KSHV裂解再活化以高度确定的顺序发生，PAN是最丰富表达的转录物之一。大量研究表明PAN在调节细胞和病毒基因表达以及病毒RNA输出细胞核中起着重要作用，但PAN相互作用组及其调控机制尚未得到深入研究。表位转录组学修饰，其中m6 A是最普遍的，已被证明会影响RNA的稳定性，结构，相互作用，并在许多人类致病病毒的生命周期中发挥关键作用。在这项提案中，我们将探讨PAN RNA生物学中的作用，以及PAN m6 A状态对KSHV基因表达程序和病毒粒子产生的影响。在目标1中：我们将定义KSHV复制期间的PAN m6 A定位和相关甲基化组。我们的初步数据显示，在KSHV裂解再活化过程中，PAN上的m6 A的精确定位，并表明特定的m6 A相关的细胞酶，作为PAN相互作用的伙伴。使用siRNA，我们将敲除这些酶，以验证它们参与PAN修饰。体外甲基转移酶和脱甲基酶测定将提供对该过程的动力学的了解，并且定点诱变将允许描绘促进PAN上的m6 A沉积的RNA结构决定簇In Aim 2：我们将探索m6 A对PAN RNA生物学和KSHV复制的影响。我们已经通过在BCBL-1细胞中进行SHAPE-MaP探测，用siRNA定向敲低调节m6 A的两种主要酶，确定了m6 A对PAN RNA结构的影响。我们还建立了在HEK 293中重建感染性KSHV的细菌人工染色体系统，并提供了一种易于处理的工具，允许直接操作m6 A位点。该系统将用于解决1）通过应用我们实验室中建立的RNA反义纯化和质谱方案，m6 A在介导PAN分子间接触中的作用，2）研究PAN上m6 A的破坏如何影响其稳定性和亚细胞定位，以及4）PAN m6 A状态对KSHV基因表达程序的影响。该提案的结果预计将提供对lncRNA的“表观转录组密码”的更好理解，其可以应用于更大的非编码RNA群体，提供对病毒lncRNA和细胞表观转录组过程之间几乎没有解决的关系的第一次洞察，并确定PAN m6 A修饰对KSHV生命周期的影响。

This proposal will shed light on the m6A epitranscriptomic regulation of polyadenylated nuclear (PAN) long non-coding (lnc) RNA in the context of Kaposi's sarcoma-associated herpesvirus (KSHV) replication. KSHV encodes a plethora of products to govern its two phases of infection, but also to intricately subvert normal cellular pathways, and establish a lifelong infection. During the latency, KSHV exists in a dormant state, and even though PAN lncRNA expression has been reported, its function and potential epitranscriptomic regulation have not been determined. KSHV lytic reactivation occurs in a highly defined order, with PAN being one of the most abundantly expressed transcripts. Numerous studies designate the critical function of PAN in regulating the cellular and viral gene expression, and export of viral RNAs out of the nucleus, yet, PAN interactome and its regulation have not been explored. Epitranscriptomic modifications, with m6A being the most prevalent, have been shown to affect RNA stability, structure, interactions, and play critical roles in the lifecycle of many human pathogenic viruses . In this proposal, we will explore the role of m6A on PAN RNA biology, and the influence of PAN m6A status on the KSHV gene expression program, and virion production. In Aim 1: we will define the PAN m6A localization and related methylome during KSHV replication. Our preliminary data show the precise localization of m6A on PAN during KSHV lytic reactivation and indicate specific m6A-related cellular enzymes, as PAN interacting partners. Using siRNAs, we will knockdown these enzymes to verify their involvement in PAN modification. The in vitro methyltransferase and demethylase assays will provide insight into the kinetics of the process, and the site-directed mutagenesis will allow delineating the RNA structural determinants that facilitate m6A deposition on PAN In Aim 2: we will explore the influence of m6A on PAN RNA biology and KSHV replication. We have identified the impact of m6A on PAN RNA structure by performing the SHAPE-MaP probing in BCBL-1 cells with a siRNA-directed knockdown of two major enzymes that regulate m6A. We have also established the bacterial artificial chromosome system that reconstitutes infectious KSHV in HEK293, and provides a tractable tool, allowing the direct manipulation of m6A sites. This system will be employed to address 1) the role of m6A in mediating PAN intermolecular contacts by applying the established in our lab RNA antisense purification and mass-spectrometry protocols, 2) investigate how the disruption of m6A on PAN affects its stability and subcellular localization, and 4) the influence of PAN m6A status over the KSHV gene expression program. The results of this proposal are expected to provide a better understanding of `epitranscriptomic code' of lncRNA that can apply to a much larger non-coding RNA population, provide the first insight in the scarcely addressed relationship between the viral lncRNAs and cellular epitranscriptomic processes, and identify the influence of PAN m6A modification over KSHV lifecycle.