Identification of a novel promoter located within the Bam HI Q region of the Epstein-Barr virus genome for the EBNA 1 gene.
EBNA 1 is the only antigen expressed in both Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma cells. Previous studies showed that the mRNA of EBNA 1 in these two tumor tissues was initiated from a promoter located in the Bam HI F fragment (Fp) on the viral genome. Two regulatory elements located in the downstream Bam HI Q region include an EBNA 1 binding site and a positive regulatory region between the Fp and the EBNA1 binding site. This data strongly suggested that a cellular factor(s) may modulate the usage of the Fp. To locate the shortest responsible viral sequence, we constructed a series of luciferase gene and chloramphenicol acetyltransferase (CAT) gene plasmids that contained various portions of the Bam HI F/Q region. Plasmid DNA was then introduced into cells to examine the promoter activity of each construct. By this method, we identified a 186-bp fragment within the Bam HI Q region that possessed the highest activity. This promoter was designated as Qp and found to be orientation-dependent and down-regulated by EBNA 1 in both the type I BL cells and human epithelial cells. Furthermore, RNase protection assay showed that a transcription initiation site was located at nucleotide 62,416 of the EBV genome. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis further confirmed that the transcript was initiated from the Qp, and not the Fp. Therefore, our data suggested that a novel promoter, Qp, located within the Bam HI Q existed for the EBNA 1 expression in the latently infected type 1 BL cells. The biological significance of the selection of the Qp needs further investigation.
Tsai
C N
CN
Graduate Institute of Microbiology and Immunology, National Yang-Ming University, Shih-Pai, ROC.
Liu
S T
ST
Chang
Y S
YS
D000956:Antigens, Viral / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000235:genetics*; D015245:Deoxyribonuclease BamHI; D019309:Epstein-Barr Virus Nuclear Antigens; D017930:Genes, Reporter; D016679:Genome, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic
D000956:Antigens, Viral; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D015245:Deoxyribonuclease BamHI