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

N6-甲基腺苷 (m6A) 对 KSHV 复制的调节 - Diversity Supplement

• 批准号: 10533427
• 负责人: Shou-Jiang Gao
• 金额: $ 9.77万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10533427
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Alternative Splicing; Binding; Binding Proteins; Biogenesis; CRISPR/Cas technology; Cell physiology; Communities; Data Set; Etiology; Funding; Genetic Translation; Genome; Goals; Herpesviridae Infections; Human; Human Herpesvirus 8; Kaposi Sarcoma; Life Cycle Stages; Malignant Neoplasms; Maps; Mediating; MicroRNAs; Modeling; Modification; Morbidity - disease rate; Multicentric Angiofollicular Lymphoid Hyperplasia; Nuclear Export; Pathogenesis; Patients; Phase; Poly(A)+ RNA; Prevention; Prognostic Marker; Protein Methyltransferases; Proteins; RNA; RNA Viruses; Reader; Reagent; Regulation; Resolution; Role; Site-Directed Mutagenesis; Societies; Testing; Time; Transcript; Viral; Virus Diseases; Virus Replication; Work; base; cell type; epitranscriptome; expectation; innovation; insight; loss of function; lytic replication; mortality; new technology; novel; novel therapeutics; primary effusion lymphoma; programs; reverse genetics; therapeutic target; transcriptome; tumor; tumorigenesis

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.

N6-甲基腺苷（m6 A）是poly（A）RNA上最丰富的内部修饰。动态调节 m6 A表转录组参与多种细胞功能。m6 A通过影响 mRNA翻译、选择性剪接、核输出和降解以及miRNA生物合成和结合 通过三组蛋白质：甲基转移酶或“作家”，脱甲基酶或“橡皮擦”，和m6 A结合 蛋白质或“读者”。m6 A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）是艾滋病患者常见的恶性淋巴瘤。了解调节机制 KSHV的潜伏性和裂解性复制不仅可以为KSHV诱导的癌症的发病机制提供见解， 也是开发新疗法的基础。在目前的融资期内，我们已 朝着这一目标取得重大进展。对于这种更新，我们发现m6 A在KSHV上丰富 m6 A阅读器蛋白YTHDF 2在病毒裂解复制期间充当抗病毒因子。我们 在KSHV潜伏和裂解复制过程中以及在不同的细胞类型中证明了m6 A的动力学， 不同的病毒复制程序尽管有这些工作，m6 A在KSHV感染中的作用才刚刚开始， 被揭露。本申请的目的是系统地绘制m6 A修饰的动力学， KSHV表观转录组中m6 A阅读器蛋白的单碱基分辨率和结合，并确定其 在KSHV生命周期的不同阶段的功能，以及KSHV诱导的肿瘤发生。中心假设是 KSHV m6 A修饰是动态调节的，这些修饰介导了不同的阶段， KSHV的生命周期，因此KSHV诱导的肿瘤发生。我们将通过绘制m6 A标记来检验这一假设。 在KSHV转录组中以单碱基分辨率，并确定m6 A写入器和擦除器蛋白在KSHV转录组中的作用。 KSHV生命周期的不同阶段和KSHV诱导的肿瘤发生（目的1）;确定 通过获得和丧失功能的方法以及通过 检查它们与KSHV转录本的结合（目的2）;以及检查KSHV m6 A标记在细胞中的功能。 使用Crispr-Cas9引导的m6 A写入器和擦除器，以及通过位点特异性诱变， (Aim 3）。我们期望该项目将提供全面的绘图和功能划分， 的m6 A标记，和m6 A写入器，擦除器和阅读器蛋白在KSHV生命周期的不同阶段，和KSHV- 诱发肿瘤发生。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感染中的功能，从而提供了对调节KSHV感染的机制的见解。 KSHV的生命周期和致病机制。这项研究还将确定潜在的预后标志物， 治疗目标拟议的工作是高度创新的，因为它将使用新技术来映射m6 A标记 和m6 A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物上的m6 A标记： Crispr-Cas9引导的m6 A写入器和擦除器，KSHV反向遗传学和创新的肿瘤模型。 此外，生成的数据集、信息和试剂对科学界也很有价值。