FIG. 7.

(A) Inhibition of Topo I and Topo II activities with chemical inhibitors abolishes KSHV ori-Lyt-associated DNA replication. BCBL-1 cells were transfected with ori-Lyt-containing plasmid (pOri-A) and RTA expression vector (pCR3.1-ORF50). Transfected cells were cultured in the absence and presence of 0.5 μM of camptothecin (Camp) or 5 μM ellipticine (Ellip). After 48 h of incubation, Hirt DNAs were extracted from the cells and subjected to a viral DNA replication assay as described in Materials and Methods. DpnI-resistant products of DNA replication (Rep'd DNA) were detected by Southern blotting with ³²P-labeled pBluescript plasmid. (B) Effects of chemicals that affect PARP-1 activity on KSHV ori-Lyt-associated DNA replication. Transfected cells were cultured in the absence and presence of the PARP-1 inhibitors 3-AB and NA as well as HU, which raises PARP-1 activity. The DNA replication assay was performed as described above. (C) Effects of the chemicals used in the replication assays on RTA and K8 gene expression. BCBL-1 cells were induced with TPA and incubated in the presence of the chemicals indicated for 48 h. Cells were subjected to Western analyses with monoclonal anti-anti-RTA and K8 antibodies.