Epstein-Barr病毒潜伏性膜蛋白1 CTAR3缺失突变体的构建与功能分析

Construction and function analysis of the Epstein-Barr Virus-encoded latent membrane protein-1 of CTAR3 region

摘要:

【目的】探讨Epstein-Barr病毒潜伏性膜蛋白1（LMP1）促细胞转化的主要活性部位及其作用机制。【方法】采用PCR方法重组LMP1羧基末端活化域3（aa232-aa351）对应密码子缺失突变体（LMP1△232-351），将突变型LMP1△232-351和野生型LMP1（LMP1WT）分别导入永生化的鼻咽上皮细胞NP69中，比较二者对细胞的转化作用。同时，构建含JAK3启动子序列的荧光素酶表达质粒(pGL-2/JAK3-LUC)，将LMP1△232-351与LMP1WT分别与含有JAK3启动子序列或NF-kB结合序列启动子的荧光酶表达质粒共转染293细胞（用pLNSX质粒作对照），比较二者活化JAK3启动子或转录因子NF-kB的功能。【结果】（1）LMP1△232-351促NP69细胞转化的能力较LMP1WT显著降低（n=3，p<0.01）。（2）LMP1WT能明显呈浓度依赖性活化JAK3启动子，而LMP1△232-351上调能力几乎丧失。【结论】LMP1羧基末端活化域3(aa232-aa351)是LMP1的重要活性部位之一，其促细胞转化的作用与JAK3蛋白表达调节有关。

Abstract:

[Objective] The role of carboxyl terminal activating region of latent membrane protein1 (LMP1) in Epstein-Barr virus infection and oncogenesis is unclear. In this study, we investigated the activating sites and functionary mechanism of LMP1. [Methods] We recombined a deletion mutant type LMP1 (LMP1△232-351), deleted the amino acid residues including 232-351 codons in carboxyl terminal activating region-3 by PCR. Then we compared mutant type LMP1△232-351 with wild type LMP1 (LMP1WT) to alter biological effect in Nasopharyngeal Epithelial Cell line NP69. Moreover, we constructed a Janus Kinase 3 (JKA3) promoter luciferase reporter system (pGL-2/JAK3-LUC). We respectively cotransfected the LMP1△232-351 and LMP1WT with promoter including NF-kB binding sequence or JAK3 promoter luciferase reporter into 293 cells (controlled with pLNSX vector), and compared their actions to activating promoters by results of luciferase activity assay. [Results] (1) The colony forming number (CFN) of NP69-LMP1△232-351 cells significantly decreased to compare with CFN of NP69-LMP1WT (n=3, p<0.01). (2) LMP1WT was able to up-regulate the transcription activity of JAK3 promoter and the level of up-regulation was correlated with its concentration in Human embryonic kidney 293 cell line; while LMP1△232-351 was almost defective ability to activate the promoter. [Conclusion] The carboxyl terminal activating region-3 may be one of the most important function sites of LMP1, which involved in activating the JAK3 promoter and regulating the expression of JAK3 protein.