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

EB病毒（EBV）感染全世界超过90%的成年人，最初在口腔中建立感染， 包括扁桃体B细胞。在感染幼稚B细胞后，EBV表达病毒蛋白，包括EBV核蛋白， 抗原（EBNA），促进B细胞成熟并建立潜伏感染的转录共激活因子 记忆B细胞库。EBV相关的恶性肿瘤包括传染性单核细胞增多症， 受损宿主，肿瘤发生，包括淋巴瘤。最近，人们已经认识到EBV诱导了 感染后的代谢变化。我们的实验室发现EBV上调氧化磷酸化 （OXPHOS）以促进细胞增殖并避免停滞。同样，幼稚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，本提案的最终目标是阐明EBV改变的分子机制。 OXPHOS，这将增强我们对EB病毒潜伏期要求的理解，以及代谢在 B细胞成熟有趣的是，病毒蛋白EBNA-Leader蛋白（EBNA-LP）是感染幼稚型 B细胞，但不是记忆B细胞-尽管EBNA-LP的基本作用还没有得到很好的表征。我们 初步数据表明，病毒蛋白EBNA-Leader蛋白（EBNA-LP）可能是上调 OXPHOS通过代谢基因的转录共激活。我们的数据表明EBNA-LP结合 调节OXPHOS相关基因表达的转录因子，包括NRF 1、ERRα和YY 1， 然后招募染色质重塑因子如P300。这种转录共激活机制模拟了 PGC蛋白家族，其使用富含亮氨酸的基序结合相同的OXPHOS转录因子， 招募改造者我们的初步数据支持这一模型，因为EBNA-LP含有多个富含亮氨酸的 基序，其可能是结合这些转录因子所需的。此外，EBNA-LP形成核体， 或无膜细胞器，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，羟脯氨酸， 其促进蛋白质寡聚化和改性基质如胶原中的更高级结构。 因此，我认为EBNA-LP的羟脯氨酸是形成EBNA-LP核体和共激活转录所必需的。我的总体假设是EBNA-LP通过使用 富含亮氨酸的基序来模拟细胞PGC蛋白家族，并通过在 羟丙基在目的1中，我将评估富含亮氨酸的基序在介导OXPHOS转录中的作用 基因和扁桃体B细胞中细胞OXPHOS的上调。在目标2中，我将确定 EBNA-LP的羟脯氨酸在感染细胞中的核体形成和代谢调节中的作用。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相关的恶性肿瘤提供见解。

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.