Functional analysis of EBNA1 protein by using a recombinant EBV with gene replacement

使用基因替换的重组 EBV 进行 EBNA1 蛋白的功能分析

• 批准号: 14570258
• 负责人: KANDA Teru
• 金额: $ 2.56万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570258/
• 关键词: EB virus; latent infection; EBNA1; gene replacement; transcriptional regulation; bacterial artificial chromosome; EBウィルス; ターゲッティング; 大腸菌; 人工染色体; Bリンパ球

EBNA1 protein, a viral protein encoded by Epstein-Barr virus (EBY), is a multi-functional protein. It is well known to be essential for the replication and segregation of EBV episomes in latently-infected cells, and it also regulates viral gene expression as a transcriptional activator. We generated a recombinant EBV, designated HMG1-EBNA1 knock-in EBV, in which the EBNA1 gene is replaced with a HMGI-EBNA1 chimeilc gene. The HMG1-EBNA1 chimeric gene encodes an EBNA1 mutant with greatly-impaired transcriptional activity, while keeping the ability of EBNA1 to episomally riiaintain EBV based plasmids. Akata cells harboring wild-type EBV and HMG1-EBNA1 knock-in virus were established. The mixture of wild-type EBV and HMGI-EBNA1 knock-in EBV Was used to infect: EBV-negative Akata cells and cord blood lymphocytes. We found that HMG1-EBNA1 knock-in virus was maintained as episomes in Akata cells. By contrast, the analyses of BACmids that had been rescued from the established lymphoblastoid cell lines revealed that the HMG1-EBNA1. chimeric gene was frequently replaced with the wild-type EBNA1 gene, presumably due to the recombination between HMGI-EBNA1 knock-in EBY and wild-type EBV. These results strongly suggest that the regulation of viral gene expression by EBNA1 as a transactivator is critical, for EBV-induced B cell growth transformation.

EBNA1蛋白是一种由eb病毒（EBY）编码的病毒蛋白，具有多种功能。众所周知，它对于潜伏感染细胞中EBV发作的复制和分离至关重要，并且它还作为转录激活因子调节病毒基因表达。我们生成了重组EBV，命名为HMG1-EBNA1敲入型EBV，其中EBNA1基因被HMGI-EBNA1嵌合基因取代。HMG1-EBNA1嵌合基因编码了一个转录活性严重受损的EBNA1突变体，同时保持了EBNA1特异性维持基于EBV的质粒的能力。建立了携带野生型EBV和HMG1-EBNA1敲入病毒的Akata细胞。用野生型EBV和HMGI-EBNA1敲入型EBV的混合物感染EBV阴性的Akata细胞和脐带血淋巴细胞。我们发现HMG1-EBNA1敲入病毒在Akata细胞中以发作的形式维持。相比之下，从已建立的淋巴母细胞样细胞系中提取的BACmids的分析显示HMG1-EBNA1。嵌合基因经常被野生型EBNA1基因取代，可能是由于HMGI-EBNA1敲入EBY与野生型EBV之间的重组。这些结果强烈提示EBNA1作为反激活因子调控病毒基因表达对于ebv诱导的B细胞生长转化至关重要。

项目成果

神田 輝: "EBウイルス(基礎・第4章)"診断と治療社. 8 (2003)

Teru Kanda：“EB 病毒（基础/第 4 章）”诊断和治疗出版 8（2003）。

Teru Kanda: "Production of high-titer EBV recombinants derived from Akata cells using a bacterial artificial chromosome system"J.Virol.. (in press). (2004)

Teru Kanda：“使用细菌人工染色体系统从 Akata 细胞中生产高滴度 EBV 重组体”J.Virol..（出版中）。

Yajima, M., Ahsan, N., Tanaka, M., Takada, K.: "Production of high-titer Epstein-Barr virus recombinants derived from Akata cells by using a bacterial artificial chromosome system"J.Virol.. 78(13). 7004-7015 (2004)

Yajima, M.、Ahsan, N.、Tanaka, M.、Takada, K.：“使用细菌人工染色体系统从 Akata 细胞中生产高滴度 Epstein-Barr 病毒重组体”J.Virol.. 78(

Teru Kanda: "Production of high-titer Epstein-Barr virus recombinants derived from Akata cells by using a bacterial artificial chromosome system"Journal of Virology. 78. 7004-7015 (2004)

Teru Kanda：“使用细菌人工染色体系统从 Akata 细胞中生产高滴度 Epstein-Barr 病毒重组体”《病毒学杂志》。