Regulation of KSHV replication by N6-methyladenosine (m6A) - Diversity Supplement

N6-甲基腺苷(M6A)是Poly(A)RNA中含量最丰富的内部修饰。动态调节 M6A表位转录组参与多种细胞功能。M6A通过影响这些功能来调节这些功能 MRNA翻译、选择性剪接、核输出和降解，以及miRNA的生物发生和结合 通过三组蛋白质：甲基转移酶或“编写者”、去甲基酶或“擦除器”和m6A结合 蛋白质或“读者”。M6A存在于RNA病毒的基因组中，并调节这些病毒的复制 病毒。卡波西肉瘤相关疱疹病毒(KSHV)是卡波西肉瘤(KS)的病原体。 原发性渗出性淋巴瘤(PEL)多见于艾滋病患者。对调控机制的认识 KSHV的潜伏和裂解复制不仅可以为KSHV诱导的癌症的发病机制提供洞察 也是开发新疗法的基础。在目前的资助期内，我们已经做出了 朝着这一目标取得了重大进展。在这次更新中，我们发现KSHV上M6A基因丰富 M6A阅读蛋白YTHDF2在病毒裂解复制过程中起到抗病毒因子的作用。我们 演示KSHV潜伏和裂解复制过程中m6A的动态变化，以及在不同类型的支持 独特的病毒复制程序。尽管有这些工作，M6A在KSHV感染中的作用才刚刚开始 会被揭穿。此应用程序的目标是系统地绘制m6A修改的动态图 KSHV表位转录组中m6A阅读器蛋白的单碱基分辨和结合，并测定其 KSHV在生命周期不同阶段的功能，以及KSHV诱导的肿瘤发生。中心假说是 KSHV m6A修饰是动态调节的，这些修饰调节了不同阶段的 KSHV的生命周期，因此KSHV诱导的肿瘤发生。我们将通过映射m6A标记来检验这一假设 在KSHV转录组中，以单一碱基分辨率，并确定m6A编写器和擦除器蛋白在 KSHV生命周期的不同阶段和KSHV诱导的肿瘤发生(目标1)；确定KSHV的功能 KSHV生命周期不同阶段的M6A阅读器蛋白 检查它们与KSHV转录本的结合(目标2)；以及检查KSHV m6A标记在 使用Crispr-Cas9引导的m6A编写器和擦除器以及通过定点突变进行病毒感染的背景 (目标3)。这是我们的期望，这个项目将提供全面的地图和功能划定 在KSHV生活史的不同阶段，M6A标记和M6A写入器、擦除器和读取器蛋白，以及KSHV- 诱导肿瘤形成。这项工作意义重大，因为它将首次系统地揭示 这些RNA修饰在KSHV感染中的作用，从而为深入研究KSHV的调控机制提供了依据 KSHV的生活史及其致病机制。这项研究还将确定潜在的预后标志和 治疗靶点。拟议的工作具有很高的创新性，因为它将使用新技术来绘制M6A标志 和m6A阅读器蛋白的结合，以及通过以下方式直接操纵关键病毒转录本上的m6A标记 CRISPR-Cas9指导的M6A写入器和橡皮擦、KSHV反向遗传学和创新的肿瘤模型。 此外，生成的数据集、信息和试剂将对科学界有价值。

N6-methyladenosine (m6A) is the most abundant internal modification on poly(A) RNA. Dynamic regulation of the m6A epitranscriptome is involved in diverse cellular functions. m6A mediates these functions by affecting mRNA translation, alternative splicing, nuclear export and degradation, and miRNA biogenesis and binding through three groups of proteins: methyltransferases or “writers”, demethylases or “erasers”, and m6A-binding proteins or “readers”. m6A is present in the genomes of RNA viruses and regulates the replication of these viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) commonly found in AIDS patients. Understanding the mechanism regulating KSHV latent and lytic replication can not only provide insights into the pathogenesis of KSHV-induced cancers but also serve as the basis for developing novel therapy. In the current funding period, we have made significant progresses toward this goal. For this renewal, we have discovered that m6A is abundant on KSHV transcripts, and that m6A reader protein YTHDF2 acts as an antiviral factor during viral lytic replication. We demonstrate m6A dynamics during KSHV latent and lytic replications, and in different cell types that support distinct viral replication programs. Despite these works, the roles of m6A in KSHV infection have just started to be revealed. The Objective of this application is to systematically map the dynamics of m6A modifications at a single base resolution and bindings of m6A reader proteins in KSHV epitranscriptome, and determine their functions in different phases of KSHV life cycle, and KSHV-induced tumorigenesis. The Central Hypothesis is that KSHV m6A modifications are dynamically regulated, and these modifications mediate different phases of KSHV life cycle, and hence KSHV-induced tumorigenesis. We will test this hypothesis by mapping m6A marks in KSHV transcriptome at a single base resolution, and determine the roles of m6A writer and eraser proteins in different phases of KSHV life cycle and KSHV-induced tumorigenesis (Aim 1); determining the functions of m6A reader proteins in different phases of KSHV life cycle by gain- and loss-of-function approaches and by examining their bindings to KSHV transcripts (Aim 2); and examining the functions of KSHV m6A marks in the context of viral infection using Crispr-Cas9-guided m6A writer and eraser, and by site-specific mutagenesis (Aim 3). It is our expectations that this project will provide comprehensive mapping and functional delineation of m6A marks, and m6A writer, eraser and reader proteins in different phases of KSHV life cycle, and KSHV- induced tumorigenesis. This work is highly significant as it will, for the first time, systematically reveal the functions of these RNA modifications in KSHV infection, thus providing insights into the mechanism regulating KSHV life cycle and KSHV-induced pathogenesis. This study will also identify potential prognostic markers and therapeutic targets. The proposed work is highly innovative as it will use new technologies to map m6A marks and bindings of m6A reader proteins, as well as directly manipulate m6A marks on key viral transcripts by Crispr-Cas9-guided m6A writer and eraser, KSHV reverse genetics, and innovative tumor models. Furthermore, the generated datasets, information and reagents will be valuable to the scientific community.