FIG. 3.

NF-κB inhibits KSHV reactivation. (A) Schematic diagram of the dual inducible cell lines. The cassette (for dual induction of IκBα and K-Rta or RelA and K-Rta) was transduced into TREx-BCBL-1 cells by cotransfecting with Flp recombinase expression vector. This cassette integrates at the identical site because of the design of the recombination system. Therefore, the copy number and the transcription levels remain the same. By adding doxycycline, K-Rta triggers reactivation, and the effects of NF-κB on KSHV reactivation can be examined. (B) RelA inhibits K-bZIP expression. After 12 h of induction, total cell protein was prepared and the indicated proteins were probed with specific antibodies. RelA reduced K-bZIP expression. W.B., Western blot. (C) RelA inhibits KSHV gene expression. Total RNA was prepared at 0, 12, and 24 h after induction. Viral gene expression was analyzed by measuring levels of transcripts by real-time qPCR. Cellular actin gene expression was used as an internal control. All values are expressed as the fold increase over the 0-h (uninduced) time point. Triplicate wells were used for each time point, and at least two independent experiments were conducted. (D) RelA inhibits KSHV replication. Viral DNA copy number in culture supernatant, as a measure of virion levels, was analyzed by real-time qPCR.