Effects of EBV Type on Viral Reactivation

项目摘要/摘要 爱泼斯坦-巴尔病毒(EBV)可引起传染性单核细胞增多症，是人类B细胞和 上皮性细胞癌。潜伏感染和裂解感染之间的切换是由两种病毒即刻早期(IE)介导的 蛋白质、BZLF1(Z)和BRLF1(R)。有两种类型的EBV，类型1(T1)和类型2(T2)，但相对较少 已知T2 EB病毒。T2 EB病毒在体外转化B细胞的能力因下降而受损 EB病毒癌蛋白LMP1的表达。然而，我们的初步研究表明，T2 EBV可以诱导 人源化小鼠模型中的B细胞淋巴瘤是高度溶解的，在口腔上皮细胞中也更容易溶解。 因此，我们假设增强型溶血性感染是T2 EBV的主要表型。我们对公众的分析 现有的EBV基因组表明，所有T2 EBV共享相同的病毒启动子(ZP)的变体(ZP-V3) 控制EBV感染在B细胞中是潜伏的还是裂解的，并且还包含Z和R IE的相同变体 蛋白质。T2编码的Z蛋白与T1病毒编码的Z蛋白相比，包含9个氨基酸(AA)差异， 它们都位于245aa蛋白的重要功能区。无论是T1还是T2的功能 也比较了Z启动子的T1型和T2型的活性。两者都是T2形式 和Z蛋白的T2形式，据报道在某些类型中过度表达 EBV感染的癌症与其在非恶性样本中的频率有关，我们最近发现 T1/T2杂交病毒(在其他T1 EBV病毒内包含Z/R IE基因的T2形式)是过度- 在从Burkitt淋巴瘤(BLS)分离的EBV中出现。我们还发现T2，而不是T1， Z启动子的形式增强了抗原刺激的B细胞中EBV的裂解再激活，这是因为它能够 结合NFATC1细胞转录因子，我们的初步结果表明T2 Z/R蛋白也 具有增强的诱导B细胞裂解再激活的能力。我们假设T2 EBV毒株 由于Z启动子、Z和/或R IE蛋白的差异，比T1 EBV株的裂解能力更强，并降低 LMP1。我们还假设，混合T1/T2 EBV毒株(包含Z/R IE基因座的T2形式)具有 与纯T1株相比，进入裂解感染的能力增强，这种差异增强了它们的 可能是恶性的。我们的具体目标是1)使用人源化的小鼠模型来确定EBV基因 T2EBV感染B细胞的增强裂解表型，并确定混合的T1/T2EBV病毒 在人类BLS中发现的类似杂交病毒比纯T1 EBV更具裂解性，或更具转化性；2)比较 未分化和分化的口腔上皮细胞中T1、T2和T1/T2杂合病毒的表型； 3)体外比较T1与T2 Z和R蛋白，T1与T2 Z和R启动子的功能 B细胞和上皮细胞模型，并明确机制(S)有何不同。建议的研究应 扩大我们对T2 EBV的理解，并可能揭示为什么T1/T2杂交EBV毒株在 癌症。

PROJECT SUMMARY / ABSTRACT Epstein-Barr virus (EBV) causes infectious mononucleosis and is an important cause of human B-cell and epithelial cell cancers. The switch between latent and lytic infection is mediated by two viral immediate-early (IE) proteins, BZLF1 (Z) and BRLF1 (R). There are two types of EBV, type 1 (T1) and type 2 (T2), but relatively little is known about T2 EBV. T2 EBV is impaired for the ability to transform B cells in vitro due to decreased expression of an EBV oncoprotein, LMP1. However, our preliminary studies demonstrate that T2 EBV induces B-cell lymphomas in a humanized mouse model that are highly lytic, and is also more lytic in oral epithelial cells. Thus, we hypothesize that enhanced lytic infection is a major phenotype of T2 EBV. Our analysis of publically available EBV genomes indicates that all T2 EBV share the same variant (Zp-V3) of the viral promoter (Zp) that governs whether EBV infection is latent or lytic in B cells, and also contain the same variants of the Z and R IE proteins. The T2-encoded Z protein contains 9 amino acid (aa) differences compared to Z encoded by T1 viruses, all located within functionally important regions of the 245 aa protein. Neither the functions of the T1 versus T2 forms of Z, nor the activities of theT1 versus T2 forms of the Z promoter, have been compared. Both the T2 form of the Z promoter, and the T2 form of the Z protein, have been reported to be over-represented in certain types of EBV-infected cancers relative to their frequency in non-malignant samples, and we have recently discovered that T1/T2 hybrid viruses (containing the T2 form of the Z/R IE locus within otherwise T1 EBV viruses) are over- represented in EBV isolated from Burkitt lymphomas (BLs). We have also discovered that the T2, but not T1, form of the Z promoter confers enhanced lytic EBV reactivation in antigen-stimulated B cells due to its ability to bind the NFATC1 cellular transcription factor, and our preliminary results indicate that the T2 Z/R proteins also have an enhanced ability to induce lytic reactivation in B cells. We hypothesize that T2 EBV strains are much more lytic than T1 EBV strains due to differences in the Z promoter, the Z and/or R IE proteins, and decreased LMP1. We also hypothesize that hybrid T1/T2 EBV strains (containing the T2 form of the Z/R IE locus) have an increased ability to enter lytic infection relative to pure T1 strains, and that this difference enhances their malignant potential. Our Specific Aims are to 1) use a humanized mouse model to define EBV genes contributing to the enhanced lytic phenotype of T2 EBV infection in B cells, and to determine if a hybrid T1/T2 EBV virus resembling hybrid viruses found in human BLs is more lytic, or more transforming, than pure T1 EBV; 2) compare the phenotypes of T1, T2, and T1/T2 hybrid viruses in undifferentiated and differentiated oral epithelial cells, and 3) compare the functions of the T1 versus T2 Z and R proteins, and T1 versus T2 Z and R promoters, in vitro in B cell and epithelial cell models, and define mechanism(s) for any differences. The proposed studies should expand our understanding of T2 EBV, and may reveal why T1/T2 hybrid EBV strains are over-represented in cancers.