Fig. 5.
LMP2A negatively regulates IL-6 secretion by means of inhibition of NF-κB. (A) EMSA demonstrates that infection of HONE-1 cells with rEBV inhibits NF-κB activity and that this effect is relieved in cells infected with rEBV-2A. Nuclear extracts isolated from HONE-1 cells or HONE-1 cells infected with rEBVs were analyzed for basal NF-κB activity. Tumor necrosis factor α-stimulated HONE-1 parental cells were used as a positive control. The presence of p65 and p50 subunits in the NF-κB complexes was confirmed by using antibodies to these subunits. (B) Blockade of NF-κB activity in rEBV-2A-infected HONE-1 cells with the RAd-IκBαSS/AA virus inhibited IL-6 secretion in a dose-dependent manner as determined by IL-6 ELISA. Data are representative of two separate experiments with triplicate determination, and the mean ± SE were all <5 pg/ml and thus unable to be accurately depicted on the histograms. (C) Transient transfection of the HONE-1 cell panel with an IL-6 reporter plasmid (p-IL-6-luc651) confirms that LMP2A modulates IL-6 expression. Thus, IL-6 promoter acting was similar in parental HONE-1 cells and rEBV-2A-infected HONE-1 cells but significantly reduced in rEBV-infected HONE-1 cells. Data are representative of three experiments and are presented as the mean ± SE of triplicate determinations. (D) NF-κB activity was inhibited in HONE-1 parent cells transiently transfected with a LMP2A-expressing plasmid as determined by EMSA. The pSG5 control vector was used as a negative control. Tumor necrosis factor α-stimulated HONE-1 parent cells served as a positive control for NF-κB activity.