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

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.

Key words:Epstein-Barr virus; LMP1; deletion mutant; NF-kB; JAK3 promoter