5-Azacytidine up regulates the expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) through EBNA-6 and latent membrane protein in the Burkitt's lymphoma line rael.
Nonproductive infection of B lymphocytes by Epstein-Barr virus (EBV) is associated with a highly restricted expression of viral genes. In growth-transformed lymphoblastoid cell lines, the products of these genes include a complex of at least six EBV nuclear antigens (EBNAs) (EBNA-1 through EBNA-6) and one membrane protein (latent membrane protein [LMP]). EBV-carrying Burkitt's lymphoma (BL) biopsies and derived cell lines that have retained a representative phenotype (group I BL lines) express only EBNA-1 (M. Rowe, D. T. Rowe, C. D. Gregory, L. S. Young, P. J. Farrell, H. Rupani, and A. B. Rickinson, EMBO J. 6:2743-2751, 1987). We have found that EBNA-2 through EBNA-6 and LMP can be up regulated by treating the group I BL line Rael with the DNA-demethylating agent 5-azacytidine (5-AzaC). The drug acted in a time- and dose-dependent manner. EBNA-2-positive cells were detected by anti-complement immunofluorescence staining just 12 h after addition of 4 microM 5-AzaC and reached a maximum number at 72 h, when up to 75% of the population was positive. EBNA-2, EBNA-3, EBNA-4, EBNA-4, EBNA-6, and LMP were demonstrated immunoblots starting at 48 h. The EBV-encoded early antigens and viral capsid antigens were also induced but at a lower level. EBNA-2 and the lytic cycle-associated antigens appeared with a different time course and in largely nonoverlapping cell subpopulations, as demonstrated by double fluorescence staining. Thus, EBNA-2 expression was not restricted to lytically infected cells, nor was EBNA-2 required for entry into the lytic cycle. The coding and regulatory sequences of EBNA-2 and LMP were found to be highly methylated in Rael cells and were, as expected, demethylated after 5-AzaC treatment. These findings suggest that DNA methylation may participate in the regulation of growth transformation-associated viral genes in BL cells.
Masucci
M G
MG
Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden.
Contreras-Salazar
B
B
Ragnar
E
E
Falk
K
K
Minarovits
J
J
Ernberg
I
I
Klein
G
G
D000956:Antigens, Viral / Q000032:analysis / Q000235:genetics*; D001374:Azacitidine / Q000494:pharmacology*; D002051:Burkitt Lymphoma; D002213:Capsid / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D019309:Epstein-Barr Virus Nuclear Antigens; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects*; D005796:Genes / Q000187:drug effects*; D005814:Genes, Viral / Q000187:drug effects*; D004854:Herpesvirus 4, Human / Q000187:drug effects / Q000235:genetics* / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008745:Methylation; 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; D001374:Azacitidine