Delineation of the cis-acting element mediating EBNA-2 transactivation of latent infection membrane protein expression.
To delineate the cis-acting element through which EBNA-2 transactivates latent membrane protein 1 (LMP1), we assayed the effect of EBNA-2 on the activity of LMP1 promoter upstream deletion mutants in the context of the LMP1 or heterologous promoters controlling chloramphenicol acetyltransferase (CAT) reporter gene expression in Epstein-Barr virus-negative Burkitt lymphoma cells. Assays of progressive 5' deletions of the LMP1 promoter revealed low constitutive and at least eightfold EBNA-2-stimulated activity from -512 to +40 (-512/+40), -334/+40, and -234/+40 LMP1CAT plasmids. More extensive 5'-deleted -205/+40, -155/+40, and -147/+40 LMP1CAT plasmids also had low constitutive activity but were not EBNA-2 responsive. The most 5'-deleted -55/+40 LMP1CAT plasmid had moderate constitutive activity and was not EBNA-2 inducible. Either orientation of the -334/+40 LMP1 sequence conferred EBNA-2 responsiveness when positioned upstream of an enhancerless simian virus 40 or herpes simplex virus thymidine kinase (TK) promoter. EBNA-2 and the cis-acting LMP1 DNA were both required to increase TK promoter-initiated mRNA, indicating that the EBNA-2 effect is at the transcriptional level. Further deletion analysis of the EBNA-2-responsive cis-acting element defined a -234/-92 LMP1 DNA fragment which conveyed EBNA-2 responsiveness to the herpes simplex virus TK promoter. The 5' 30 bp between -234 and -205 were essential for EBNA-2 responsiveness. Thus, these experiments define a 142-bp cis-acting element which is sufficient for conveying EBNA-2 responsiveness and an essential 30-bp component of that element. The role of this element in LMP1 and LMP2B expression and its possible role in LMP2A expression are discussed.
Tsang
S F
SF
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
Wang
F
F
Izumi
K M
KM
Kieff
E
E
D000956:Antigens, Viral / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002472:Cell Transformation, Viral; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D002872:Chromosome Deletion; D019309:Epstein-Barr Virus Nuclear Antigens; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000276:immunology; D006801:Humans; D011401:Promoter Regions, Genetic; D013539:Simian virus 40 / Q000235:genetics; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D015533:Transcriptional Activation; D014162:Transfection; D014763:Viral Matrix Proteins
D000956:Antigens, Viral; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; D014763:Viral Matrix Proteins; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase