[Construction and function analysis of a CTAR-2 region mutant of the Epstein-Barr virus-encoded latent membrane protein-1].
To investigate the activating sites and functional 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-kappa B 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-kappa B 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 suggested 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-kappa B and AP-1 might be closely related to LMP1-mediated cell transformation and tumorigenesis.
He
Zhi-Min
ZM
Cancer Research Institute, Xiangya Medical College, Central South University, Changsha 410078, China. Hezhimin2001@yahoo.com.cn
Chen
Zhu-Chu
ZC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002452:Cell Count; D002460:Cell Line; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000378:metabolism
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; D014763:Viral Matrix Proteins; D008156:Luciferases