Effects of EBV Type on Viral Reactivation

项目总结/摘要 EB病毒（EBV）引起传染性单核细胞增多症，并且是人类B细胞和淋巴细胞增殖的重要原因。 上皮细胞癌潜伏性感染和溶解性感染之间的转换是由两种病毒即早期（IE）介导的。 蛋白质，BZLF 1（Z）和BRLF 1（R）。EBV有两种类型，1型（T1）和2型（T2），但相对较少 已知T2 EBV。T2 EBV在体外转化B细胞的能力受损，这是由于T2 EBV在体外转化B细胞的能力降低。 EBV癌蛋白LMP 1的表达。然而，我们的初步研究表明，T2 EBV诱导 人源化小鼠模型中的B细胞淋巴瘤，其具有高度溶解性，并且在口腔上皮细胞中也更具溶解性。 因此，我们假设增强的溶解性感染是T2 EBV的主要表型。我们的分析表明， 可用的EBV基因组表明所有T2 EBV共享病毒启动子（Zp）的相同变体（Zp-V3）， 控制EBV感染在B细胞中是潜伏的还是溶解的，并且还包含Z和R IE的相同变体 proteins.与T1病毒编码的Z相比，T2编码的Z蛋白含有9个氨基酸（aa）差异， 全部位于245个氨基酸蛋白的功能重要区域内。T1和T2的功能 Z启动子的T1和T2形式的活性也进行了比较。T2型 Z启动子和Z蛋白的T2形式，已经报道在某些类型中过度表达。 EBV感染的癌症相对于其在非恶性样本中的频率，我们最近发现 T1/T2杂合病毒（在其它T1 EBV病毒内含有Z/R IE基因座的T2形式）是过度- 在从伯基特淋巴瘤（BL）分离的EBV中表现。我们还发现，T2，而不是T1， Z启动子的一种形式赋予抗原刺激的B细胞中增强的裂解性EBV再活化，这是由于其能够 结合NFATC 1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细胞中溶解性再活化的能力。我们假设T2 EBV株是多 由于Z启动子、Z和/或R IE蛋白的差异，比T1 EBV株更具裂解性， LMP1。我们还假设，杂交T1/T2 EBV株（含有Z/R IE基因座的T2型）具有一个与T1/T2 EBV株相似的基因型。 相对于纯T1菌株，进入裂解感染的能力增加，并且这种差异增强了它们的 恶性潜能我们的具体目标是：1）使用人源化小鼠模型来定义EBV基因， T2 EBV感染在B细胞中增强的裂解表型，并确定杂交T1/T2 EBV病毒是否 类似于在人BL中发现的杂交病毒，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杂交病毒的表型，和 3)在体外比较T1与T2 Z和R蛋白以及T1与T2 Z和R启动子的功能， B细胞和上皮细胞模型，并定义任何差异的机制。拟议的研究应 扩展了我们对T2 EBV的理解，并可能揭示为什么T1/T2杂交EBV株在 癌的

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.