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

( 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 carboxy
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-κB
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-κB 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-κB and AP-1 may be closely related to LMP1-mediated cell transformation and tumorigenesis.