Metabolic regulation of Epstein-Barr Virus-infected tonsillar B cells by EBNA-LP

爱泼斯坦-巴尔病毒(EBV)感染全世界90%以上的成年人，最初在口腔中感染， 包括扁桃体B细胞。在感染幼稚B细胞时，EBV表达包括EBV核在内的病毒蛋白 抗原(EBNAs)，转录共激活因子，促进B细胞成熟并建立潜伏感染 记忆B细胞储存库。EB病毒相关的恶性肿瘤包括传染性单核细胞增多症，以及免疫性 宿主受损，肿瘤形成，包括淋巴瘤。最近，人们认识到EBV可以诱导 感染后的新陈代谢变化。我们的实验室发现EBV上调氧化磷酸化 (OXPHOS)，以促进细胞增殖，避免停滞。同样，幼稚的B细胞也需要增加 OXPHOS在被抗原激活后进行生发中心重塑并产生记忆B 细胞。因此，这一提议的最终目的是阐明EBV改变的分子机制。 OXPHOS，这将增强我们对EBV潜伏期要求的理解，以及新陈代谢在 B细胞成熟。有趣的是，病毒蛋白EBNA-Leader蛋白(EBNA-LP)是感染幼稚病毒所必需的 B细胞，而不是记忆B细胞-尽管EBNA-LP的基本作用还没有得到很好的描述。我们的 初步数据表明，病毒蛋白EBNA-Leader蛋白(EBNA-LP)可能在上调表达中起重要作用 OXPHOS通过代谢基因的转录共激活。我们的数据表明EBNA-LP与 调控氧合酶相关基因表达的转录因子包括nrf1、errα和yy1以及 然后招募染色质重塑因子，如P300。这种转录共激活机制模仿了 PGC蛋白家族，它使用富含亮氨酸的基序结合相同的OXPHOS转录因子和 招募改造者。我们的初步数据支持这一模型，因为EBNA-LP含有多种富含亮氨酸的蛋白质 基序，这可能是结合这些转录因子所必需的。此外，EBNA-LP形成核体， 或无膜细胞器，这可能是EBNA-LP调节转录所必需的，包括OXPHOS 基因。我们的初步数据表明，EBNA-LP含有翻译后修饰的羟脯氨酸， 这促进了蛋白质齐聚和胶原蛋白等修饰底物中的高阶结构。 因此，我认为EBNA-LP的羟脯氨酸作用是形成EBNA-LP核小体和共同激活转录所必需的。我的总体假设是EBNA-LP通过使用以下方式诱导OXPHOS基因转录 富含亮氨酸的基序，以模拟细胞PGC蛋白家族，并通过在 羟基催乳素。在目标1中，我将评估富含亮氨酸的基序在介导OXPHOS转录中的作用 扁桃体B细胞中细胞OXPHOS的基因和上调在目标2中，我将确定 EBNA-LP在核体形成中的羟脯氨酸作用及其在感染细胞中的代谢调节这些 研究将阐明病毒蛋白调节转录和B细胞代谢的新机制 并将为理解EBV相关恶性肿瘤提供洞察力。

Epstein-Barr Virus (EBV) infects over 90% of adults worldwide and initially establishes infection in the oral cavity, including tonsillar B cells. Upon infecting naïve B cells, EBV expresses viral proteins including EBV Nuclear Antigens (EBNAs), transcriptional co-activators that promote B cell maturation and establish latently infected memory B cell reservoirs. EBV-associated malignancies include infectious mononucleosis, and, in immune compromised hosts, tumorigenesis including lymphomas. Recently, it has been appreciated that EBV induces metabolic changes upon infection. Our lab has discovered that EBV upregulates oxidative phosphorylation (OXPHOS) in order to promote cell proliferation and avoid arrest. Similarly, naïve B cells also require increased OXPHOS upon activation by antigen in order to undergo germinal center remodeling and produce memory B cells. Therefore, the ultimate goal of this proposal is to elucidate the molecular mechanisms by which EBV alters OXPHOS, which will enhance our understanding of EBV requirements for latency, and the role of metabolism in B cell maturation. Intriguingly, the viral protein EBNA-Leader Protein (EBNA-LP) is required for infection of naïve B cells, but not memory B cells – although the essential role of EBNA-LP is not well characterized. Our preliminary data suggests the viral protein EBNA-Leader Protein (EBNA-LP) may be essential in upregulation OXPHOS through transcriptional co-activation of metabolic genes. Our data suggests that EBNA-LP binds transcription factors that regulate expression of OXPHOS-related genes including NRF1, ERRα, and YY1 and then recruits chromatin remodeling factors such as P300. This mechanism of transcriptional co-activation mimics the PGC family of proteins, which uses leucine-rich motifs to bind the same OXPHOS transcription factors and recruit remodelers. Our preliminary data supports this model in that EBNA-LP contains multiple leucine-rich motifs, which may be required for binding these transcription factors. Additionally, EBNA-LP forms nuclear bodies, or membraneless organelles, that may be essential for EBNA-LP to regulate transcription, including OXPHOS genes. Our preliminary data suggests that EBNA-LP contains a post-translational modification, hydroxyproline, which promotes protein oligomerization and higher order structures in modified substrates such as collagen. Therefore, I propose that hydroxyprolination of EBNA-LP is required to form EBNA-LP nuclear bodies and co-activate transcription. My overall hypothesis is that EBNA-LP induces transcription of OXPHOS genes by using leucine-rich motifs to mimic the cellular PGC family of proteins, and through forming nuclear bodies upon hydroxyprolination. In Aim 1, I will assess the role of leucine-rich motifs in mediating transcription of OXPHOS genes and upregulation of cellular OXPHOS in tonsillar B cells. In Aim 2, I will determine the role of hydroxyprolination of EBNA-LP in formation of nuclear bodies and metabolic regulation in infected cells. These studies will elucidate novel mechanisms by which a viral protein regulates transcription and B cell metabolism and will provide insight towards understanding EBV-related malignancies.