Epstein-Barr viral (EBV) latency-associated promotersEpstein-Barr virus (EBV) infection is the cause of infectious mononucleosis and is most closely associated with tumor diseases Burkitt's lymphoma (BL) and nasopharyngeal carcinoma. EBV infection of human B lymphocytes in vitro results in B-cell proliferation and transformation into continuously growing lymphoblastoid cell lines (LCL) (for a review, see reference 42). In latently infected cells, viral genomes are maintained as multiple circular episomal copies which are replicated once per cell cycle (2, 103). Several classes of latency have been described depending on the gene expression pattern (41,77,78). In strict type I latency, represented by BL cells, viral gene expression is restricted to the two RNA polymerase III-transcribed EBER RNA genes and the EBNA1 gene (78) that is transcribed from the Q promoter (Qp) (68). The EBNA1 protein is required for the maintenance of the viral plasmid in dividing cells (45,58). In type III latency, in addition to the EBERs, EBNA-LP, -2, -3A, -3B, -3C, and -1 are expressed from the C promoter (Cp) (6), whereas LMP-1 and -2B are expressed from the bidirectional LMP1 promoter (46), and a larger splice variant of LMP-2, LMP-2A, is expressed from the TP1 promoter (36). Qp generally is supposed to be silent in type III latency (82, 105), although there is also a different view (93). Among the viral proteins expressed in latency type III, EBNA2 plays a central role in switching EBNA transcription from Wp to Cp (W to C switch) (102,104) and in the establishment and maintenance of B-cell transformation (11, 28), as EBNA2 transcriptionally activates the expression of the six nuclear antigens from the C promoter (Cp) and the membrane proteins LMP-1 and -2B from the LMP1 promoter (LMP1p), LMP-2A from the TP1 promoter, and a number of cellular proteins associated with the LCL phenotype (1,12,18,39,44,72,76,90,95,98,99,100,101,102,104,110,111). A crucial mechanism involved in the silencing of Cp and LMP1p in type I latency has been shown to be methylation of CpG dinucleotides (3,15,35,54,60,61,70,73,74,75,84,91,94). In LCL, the EBV genome is mostly free of CpG methylation, whereas in BL cells, EBV genomes are highly methylated. An essential step in understanding the differences between latency types I and III is to elucidate the patterns of methylation and in vivo protein binding of the latency promoters of EBV at nucleotide resolution. Therefore, we decided to examine Qp, Cp, and LMP1p in cells of both latency types.(The contributions of Daniel Salamon to this work were made in partial fulfillment of the requirements for a Ph.D. from Semmelweis University, Budapest, Hungary.)
MATERIALS AND METHODSCell lines and tissue culture. LCL 721 is a B95-8-transformed LCL with type III phenotype (40,52,57). Rael (15,43,61) is a group I BL cell line. Mutu BLI-C1216 is a subclone of the BL line Mutu, representative of latency type I (27). Mutu BLIII-C199 is a subclone of the BL line Mutu, representative of latency type III (27). Raji cells expre...
