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.

EB病毒(Epstein-Barr Virus，EBV)在B淋巴细胞潜伏期的建立和永生化过程中，对宿主细胞的基因表达和代谢进行了重新编程。协调这种重编程与EBV潜伏期的调节机制反映了病毒致癌中的重要事件，但仍知之甚少。我们发现，EBV潜伏期的关键病毒调节因子，包括主要的被膜蛋白BNRF1和EBV核抗原EBNA1，在建立潜伏期和原代B细胞永生化的过程中协调嘌呤代谢的关键方面。在这本R01中，我们重点研究EBV如何重新编程嘌呤代谢基因的表达，以及嘌呤代谢产物如何直接促进EBV肿瘤的发生。病毒编码的被膜蛋白BNRF1提供了这种协调调控的线索之一，它在结构上与嘌呤生物合成酶FGARAT(也称为PFAS)有广泛的相似性，并在初次感染期间参与病毒染色质的组装。BNRF1的同源基因存在于包括KSHV ORF75在内的所有伽玛疱疹病毒中，它们具有解除PML核体成分的共同功能(PML-NB)及其抗病毒功能。我们先前已经证明BNRF1与组蛋白H3.3伴侣DAXX相互作用，并取代其与ATP依赖的SNF2样解旋酶ATRX的相互作用，从而使潜伏期特异性病毒基因在初次感染期间能够选择性表达。然而，目前还没有研究表明病毒的FGARAT同源结构域如何与细胞嘌呤的生物合成和/或信号转导相联系。利用代谢组学质谱仪，我们提供了新的初步数据，表明在B细胞永生化过程中，嘌呤生物合成途径是受EBV干扰最明显的途径之一。结合基因表达(RNA-Seq)、染色质可及性(ATAC-Seq)和EBNA1-DNA与宿主染色体的结合(ChIP-Seq)，我们确定了细胞代谢基因，包括腺嘌呤脱氨酶(ADA)、腺苷激酶4(AK4)和嘌呤能受体P2RY8和P2RX5，它们是EBNA1在EBV永生化过程中转录调控的直接靶点。我们现在建议研究EBV感知和重新编程嘌呤代谢的机制，以及嘌呤能信号如何调节EBV潜伏期的建立和宿主细胞的转化。我们将验证中心假设，即EBV在B细胞永生化过程中协调调节细胞嘌呤代谢与病毒和细胞基因表达，以及嘌呤能信号对病毒潜伏期和肿瘤发生至关重要。具体地说，我们将研究EBV如何在初次感染期间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在B细胞永生化、免疫信号和EBV诱导的肿瘤形成中的作用(目标3)。这些研究将促进我们对基因表达与新陈代谢协调的基本机制的理解，并为病毒潜伏和病毒相关癌症的治疗干预确定新的靶点。