Regulation of EBV Latency by Purine Metabolism and Signaling

通过嘌呤代谢和信号传导调节 EBV 潜伏期

• 批准号: 10407656
• 负责人: PAUL M LIEBERMAN
• 金额: $ 45.76万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-19 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10407656
• 关键词: ATAC-seq; ATRX gene; Adenine; Adenosine; Adenosine Kinase; Anabolism; B Cell Proliferation; B lymphocyte immortalization; B-Lymphocytes; Cells; Cellular biology; ChIP-seq; Chromatin; Chromatin Modeling; Chromosomes; Complex; DAXX gene; DNA Binding; Data; Deaminase; EBV-associated disease; Enzymes; Epigenetic Process; Epstein-Barr Virus Infections; Epstein-Barr Virus Nuclear Antigens; Epstein-Barr Virus latency; Event; G-Protein-Coupled Receptors; Gene Expression; Genes; Genome; Histone H3; Human Herpesvirus 4; Human Herpesvirus 8; Immune; Immune signaling; Inflammatory; Link; Lymphomagenesis; Lytic Virus; Malignant Neoplasms; Mass Spectrum Analysis; Metabolic; Metabolic Control; Metabolism; Molecular Chaperones; Nuclear; Pathway interactions; Play; Primary Infection; Process; Proteins; Purines; Purinoceptor; Regulation; Role; Signal Transduction; Testing; Therapeutic Intervention; Transcriptional Activation; Transcriptional Regulation; Viral; Viral Genes; Viral Physiology; Virus; Virus Diseases; Virus Latency; cell transformation; gammaherpesvirus; helicase; inhibitor; metabolomics; mouse model; new therapeutic target; novel; purine metabolism; selective expression; transcriptional reprogramming; transcriptome sequencing; tumor; tumorigenesis; virus related cancer

Epstein-Barr Virus (EBV) reprograms host cell gene expression and metabolism during the establishment of latency and the immortalization of B-lymphocytes. The regulatory mechanisms coordinating this reprogramming with EBV latency reflect important events in viral oncogenesis, yet remain poorly understood. We have found that key viral regulators of EBV latency, including the major tegument protein BNRF1 and the EBV Nuclear Antigen EBNA1 coordinate key aspects of purine metabolism during establishment of latency and immortalization of primary B-cells. In this R01, we focus on how EBV reprograms purine metabolic gene expression, and how purine metabolites contribute directly to EBV tumorigenesis. One clue to this coordinate regulation is provided by the viral-encoded tegument protein BNRF1 that shares extensive structural similarity to the purine biosynthetic enzyme FGARAT (also called PFAS) and functions in viral chromatin assembly during primary infection. Orthologues of BNRF1 are found in all gamma herpesviruses, including KSHV ORF75, and share the common function of disarming components of the PML-nuclear body (PML-NB) and its anti-viral functions. We have previously shown that BNRF1 interacts with the histone H3.3 chaperone DAXX and displaces its interaction with the ATP-dependent SNF2-like helicase ATRX to enable selective expression of latency-specific viral genes during primary infection. However, it has not yet been shown how the viral FGARAT homology domain is linked to cellular purine biosynthesis and/or signaling. Using metabolomics mass spectrometry, we provide new preliminary data indicating that the purine biosynthetic pathway is among the most significantly perturbed by EBV during B-cell immortalization. Integrating gene expression (RNA-Seq), chromatin accessibility (ATAC-Seq), and EBNA1-DNA binding to host chromosome (ChIP-Seq), we identified cellular metabolic genes, including adenine deaminase (ADA), adenosine kinase 4 (AK4), and purinergic receptors P2RY8 and P2RX5 as direct targets of EBNA1 transcriptional regulation during EBV immortalization. We now propose to investigate the mechanisms by which EBV senses and reprograms purine metabolism and how purinergic signaling regulates establishment of EBV latency and host cell transformation. We will test the central hypothesis that EBV coordinately regulates cellular purine metabolism with viral and cellular gene expression during the B-cell immortalization process, and that purinergic signaling is critical for viral latency and oncogenesis. Specifically, we will investigate how EBV regulates expression of purine metabolic genes during primary infection (Aim 1), elucidate how purine metabolism impacts the establishment of EBV latency (Aim 2), and investigate the role of purine metabolism and signaling in B-cell immortalization, immune signaling, and EBV-induced tumorigenesis (Aim 3). These studies will advance our understanding of basic mechanisms coordinating gene expression with metabolism and identify new targets for therapeutic intervention in viral latency and viral-associated cancers.

Epstein-Barr 病毒 (EBV) 在 B 淋巴细胞潜伏期建立和永生化过程中重新编程宿主细胞基因表达和代谢。协调这种重编程与 EBV 潜伏期的调节机制反映了病毒肿瘤发生中的重要事件，但仍知之甚少。我们发现，EBV 潜伏期的关键病毒调节因子，包括主要皮膜蛋白 BNRF1 和 EBV 核抗原 EBNA1，在原代 B 细胞潜伏期建立和永生化过程中协调嘌呤代谢的关键方面。在本 R01 中，我们重点关注 EBV 如何重新编程嘌呤代谢基因表达，以及嘌呤代谢物如何直接促进 EBV 肿瘤发生。这种协调调节的一条线索是由病毒编码的外皮蛋白 BNRF1 提供的，该蛋白与嘌呤生物合成酶 FGARAT（也称为 PFAS）具有广泛的结构相似性，并且在初次感染期间在病毒染色质组装中发挥作用。 BNRF1 的直向同源物存在于所有 γ 疱疹病毒中，包括 KSHV ORF75，并且具有解除 PML 核体 (PML-NB) 成分及其抗病毒功能的共同功能。我们之前已经证明，BNRF1 与组蛋白 H3.3 伴侣 DAXX 相互作用，并取代其与 ATP 依赖性 SNF2 样解旋酶 ATRX 的相互作用，从而能够在初次感染期间选择性表达潜伏期特异性病毒基因。然而，尚未表明病毒 FGARAT 同源结构域如何与细胞嘌呤生物合成和/或信号传导相关。利用代谢组学质谱分析，我们提供了新的初步数据，表明嘌呤生物合成途径是 B 细胞永生化过程中受 EBV 干扰最显着的途径之一。整合基因表达 (RNA-Seq)、染色质可及性 (ATAC-Seq) 和 EBNA1-DNA 与宿主染色体结合 (ChIP-Seq)，我们鉴定了细胞代谢基因，包括腺嘌呤脱氨酶 (ADA)、腺苷激酶 4 (AK4) ，以及嘌呤能受体 P2RY8 和 P2RX5 作为 EBV 永生化过程中 EBNA1 转录调控的直接靶标。我们现在建议研究 EBV 感知和重新编程嘌呤代谢的机制，以及嘌呤信号如何调节 EBV 潜伏期的建立和宿主细胞转化。我们将检验以下中心假设：EBV 在 B 细胞永生化过程中通过病毒和细胞基因表达协调调节细胞嘌呤代谢，并且嘌呤能信号传导对于病毒潜伏期和肿瘤发生至关重要。具体来说，我们将研究 EBV 在初次感染期间如何调节嘌呤代谢基因的表达（目标 1），阐明嘌呤代谢如何影响 EBV 潜伏期的建立（目标 2），并研究嘌呤代谢和信号传导在 B 细胞永生化中的作用、免疫信号传导和 EBV 诱导的肿瘤发生（目标 3）。这些研究将增进我们对协调基因表达与代谢的基本机制的理解，并确定病毒潜伏期和病毒相关癌症的治疗干预的新靶点。