The ORF24-34 binding element at the origin is essential for late gene activation.

(A) (Top panel) Schematic of the left lytic origin of replication of the KSHV genome. (Bottom panel) IGV image of the ChIP peak for HA-ORF34 at the origin of replication. (B) iSLK-WT cells were transfected with the K8.1 promoter plasmid containing either the full length Ori (WT), the Ori lacking the ORF24-34 binding element (Delta ORF24-34 BE), or the Ori lacking the minimal Ori-lyt and retaining only the ORF24-34 binding element (ORF24-34 BE). 48 h post transfection and reactivation, the cells were assessed for luciferase activity. (C) iSLK-HAORF34 cells were transfected with a K8.1 promoter containing plasmid containing either the full-length Ori (WT), the Ori lacking the ORF24-34 binding element (Delta ORF24-34 BE), or no Ori. Untagged iSLK-WT cells transfected with K8.1 plasmid served as a control for the IP. The ChIP DNA was quantified using plasmid specific primers (left panel) and genomic K8.1 promoter specific primers (right panel), normalized to the level of input DNA and presented as percent input. Data shown are an average of 3–4 biological replicates.