The motif identified from direct targets is important for transcription from the promoter.

(A) Schematic of plasmid-based promoter activation assay. iSLK-WT or ORF24RAAAG mutant cells reactivated with doxycycline and sodium butyrate were transfected with reporter plasmid carrying the indicated promoter sequence upstream of the firefly luciferase gene and a control renilla luciferase expressing plasmid. 48 h post transfection and reactivation, the cells were assessed for luciferase activity. (B) Reactivated iSLK-WT cells were transfected with the K8.1 promoter reporter plasmid with or without the KSHV left lytic origin of replication cloned in cis and assessed for luciferase activity. (C) Reactivated iSLK-WT cells were transfected with the K8.1 and ORF57 promoter reporter plasmids in the presence or absence of DNA replication inhibitor (PAA) and assessed for luciferase activity. (D) Reactivated iSLK-WT and iSLK- ORF24RAAAG mutant cells were transfected with K8.1 or ORF57 promoter reporter plasmids and assessed for luciferase activity. (E-F) iSLK-WT cells were transfected with plasmids containing the WT K8.1 promoter or the indicated single or double point mutant promoters and assessed for luciferase activity. The data for each mutant was normalized to the WT K8.1 promoter. All data shown are average of 3–5 biological replicates.