The interplay between m6A and viral lncRNA during KSHV replication

KSHV 复制过程中 m6A 和病毒 lncRNA 之间的相互作用

• 批准号: 10363754
• 负责人: Joanna Sztuba-Solinska
• 金额: $ 22.61万
• 依托单位: AUBURN UNIVERSITY AT AUBURN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-04 至 2022-10-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10363754
• 关键词: Address; Adenine; Affect; Affinity; Antibodies; Antisense RNA; Antiviral Response; Bacterial Artificial Chromosomes; Biological; Biological Assay; Biological Process; Biology; Cell Nucleus; Cells; Chemicals; Code; Data; Deposition; Disease; Enzymes; Exhibits; Gene Expression; Genes; Herpesviridae Infections; Human; Human Herpesvirus 8; In Vitro; Infection; Kinetics; Knowledge; Lead; Life Cycle Stages; Light; Lytic; Lytic Phase; Maps; Mass Spectrum Analysis; Mediating; Methods; Methylation; Methyltransferase; Modification; Molecular; Molecular Conformation; Nuclear; Nuclear Proteins; Nuclear RNA; Nuclear Structure; Oncogenic; Pathway interactions; Phase; Play; Poly A; Population; Positioning Attribute; Process; Production; Proteins; Protocols documentation; RNA; RNA Stability; Reader; Regulation; Reporting; Role; Site; Site-Directed Mutagenesis; Small Interfering RNA; Structure; System; Transcript; Untranslated RNA; Viral; Viral Genes; Virion; Virus Diseases; Virus Replication; Work; antiviral drug development; chromatin remodeling; crosslink; deep sequencing; epitranscriptomics; gammaherpesvirus; insight; intermolecular interaction; knock-down; latent infection; lytic gene expression; lytic replication; methylome; nanopore; pathogen; pathogenic virus; prevent; programs; reconstitution; recruit; tool; transcriptome sequencing; viral RNA; virus host interaction

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.

该提案将揭示卡波西肉瘤相关疱疹病毒 (KSHV) 复制背景下多聚腺苷酸核 (PAN) 长非编码 (lnc) RNA 的 m6A 表观转录组调控。 KSHV 编码大量产物来控制其感染的两个阶段，同时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处于休眠状态，尽管PAN lncRNA表达已被报道，但其功能和潜在的表观转录组调控尚未确定。 KSHV 裂解再激活以高度明确的顺序发生，其中 PAN 是表达最丰富的转录本之一。许多研究表明 PAN 在调节细胞和病毒基因表达以及将病毒 RNA 从细胞核输出方面发挥着关键作用，但 PAN 相互作用组及其调控尚未得到探索。表观转录组修饰（其中 m6A 最为普遍）已被证明会影响 RNA 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。在本提案中，我们将探讨 m6A 在 PAN RNA 生物学中的作用，以及 PAN m6A 状态对 KSHV 基因表达程序和病毒颗粒产生的影响。目标 1：我们将定义 KSHV 复制过程中的 PAN m6A 定位和相关甲基化组。我们的初步数据显示了 KSHV 裂解再激活过程中 m6A 在 PAN 上的精确定位，并表明特定的 m6A 相关细胞酶作为 PAN 相互作用伙伴。使用 siRNA，我们将敲低这些酶以验证它们参与 PAN 修饰。体外甲基转移酶和去甲基化酶测定将深入了解该过程的动力学，定点诱变将允许描绘促进 m6A 在 PAN 上沉积的 RNA 结构决定因素目标 2：我们将探索 m6A 对 PAN RNA 的影响生物学和 KSHV 复制。我们通过在 BCBL-1 细胞中进行 SHAPE-MaP 探测，并通过 siRNA 定向敲低调节 m6A 的两种主要酶，确定了 m6A 对 PAN RNA 结构的影响。我们还建立了细菌人工染色体系统，可在 HEK293 中重建感染性 KSHV，并提供易于处理的工具，允许直接操作 m6A 位点。该系统将用于解决 1) 通过应用我们实验室建立的 RNA 反义纯化和质谱方案，m6A 在介导 PAN 分子间接触中的作用，2) 研究 m6A 对 PAN 的破坏如何影响其稳定性和亚细胞定位、4) PAN m6A 状态对 KSHV 基因表达程序的影响。该提案的结果预计将有助于更好地理解lncRNA的“表观转录组密码”，该密码可应用于更大的非编码RNA群体，为病毒lncRNA与细胞表观转录组过程之间几乎没有解决的关系提供第一个见解，并确定 PAN m6A 修饰对 KSHV 生命周期的影响。