FIG 5.

EBNA1 inhibits expression of c-Myc. (A) Levels of c-Myc mRNA quantified by qRT-PCR and normalized to TBP expression at different time points following B-cell infection with wt-EBV or ΔEBNA1-EBV. The levels of c-Myc mRNA were set to 100% for wt-EBV-infected cells (n = 5; error bars show standard error). (B) Expression of c-Myc mRNA in H1299 cells and H1299 cells expressing EBNA1 quantified by qRT-PCR and normalized to GAPDH expression. The level of c-Myc mRNA was set to 100% for cells not expressing EBNA1 (n = 2; error bars show standard error). (C) EBNA1 PWM-predicted binding sites (red and blue sequences) associated with the c-Myc gene. The location of each binding site is given relative to the c-Myc transcriptional start site. (D) An EMSA showing EBNA1 binding to a positive-control oligonucleotide (EBV Rep* site 1; lanes 1 to 3), oligonucleotides that include c-Myc site 1 (lanes 4 to 6) or c-Myc site 2 (lanes 7 to 9), and a negative-control oligonucleotide (a portion of the EBV Raji origin; lanes 10 to 12). For each oligonucleotide, EBNA1 amounts added were none in the left lanes, 1.5 μg EBNA1 in the middle lanes, and 6 μg in the right lanes (n = 3; a representative image is shown). (E) Chromatin immunoprecipitation (ChIP)-qPCR measurement of EBNA1 binding to the sites identified in panel C relative to EBNA1 binding to a negative-control region in the genome (proximal to the rhodopsin gene, where there are no predicted EBNA1-binding sites). Data shown are relative to IgG binding at each of the sites (n = 5; error bars show standard error). *, P < 0.05.