Construction and Function Analysis of a CTAR-2 Region Mutant of the
Epstein-Barr Virus-encoded Latent Membrane Protein-1

HE Zhi-Min*, CHEN Zhu-Chu

( Cancer Research Institute, Xiangya Medical College, Central South University, Changsha 410078, China )

Abstract To investigate the activating sites and functionary mechanism of Epstein-Barr virus (EBV)-latent membrane protein-1 (LMP1), a mutant type LMP1 (mt-LMP1) was made by PCR methods, replacing the amino acid residues YYD with ID in carboxyl terminal activating region-2 (CTAR-2) at position 384-386 codons. The mt-LMP1 and wild type LMP1(wt-LMP1) were cotransfected with transcription factor NF-kB or AP-1 luciferase reporter into 293 cells, respectively, and their actions in activating transcription factors were compared by results of luciferase activity assay. Moreover, mt-LMP1 and wt-LMP1 were transfected into Rat-1 cells to compare their transforming effects by contact inhibition assay. The results showed that (1) Compared with wt-LMP1, mt-LMP1 was 80% defective in NF-kB activation and 100% defective in AP-1 activation. (2) Colony formation number (CFN) of Rat-1 cells expressing mt-LMP1 was significantly decreased compared with CFN of Rat-1 cells expressing wt-LMP1 [(23±3)/well vs. (357±19)/well; (64±8)/well vs. (408±40)/well. n=3, P<0.001]. These results suggest that amino acid residues 384-386 in CTAR-2 may be one of the most important function sites of LMP1 and the activation of NF-kB and AP-1 may be closely related to LMP1-mediated cell transformation and tumorigenesis.

Key words Epstein-Barr virus; LMP1; mutation; nuclear factor (NF-kB); cell transformation

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