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. 2009 Jun 24;83(17):8492–8501. doi: 10.1128/JVI.00922-09

FIG. 2.

FIG. 2.

Effects of LANA on lytic DNA replication. (A) LANA represses oriLyt-dependent DNA replication. Expression of LANA in the cotransfection replication assay suppressed amplification of oriLyt. Vero cells were cotransfected with plasmids that express the required replication proteins along with K-Rta and K-bZIP expression plasmids and a plasmid containing oriLyt. A LANA expression plasmid was either present (+LANA) or absent (−LANA) from the cotransfection mixture. The arrow to the left of the figure shows the presence of amplified (DpnI resistant) oriLyt. In the bottom panel is a Southern blot showing input oriLyt DNA. (B) Suppression of oriLyt amplification by LANA expression is not due to a decrease in protein production. Vero cells were transfected with a LANA expression plasmid and a K-Rta, K-bZIP, or ORF59 expression plasmid. Protein lysates were prepared, and protein expression was analyzed by Western blotting (WB). Blots were reacted with an antibody specific for LANA, K-Rta, K-bZIP, or ORF59. (C) K-bZIP modulates amplification of oriLyt and the TR element. Vero cells were cotransfected with the set of plasmids expressing the required replication proteins plus plasmids containing oriLyt and the TR. Southern blotting of total cellular DNA from cotransfections that contained HHV8 plasmids that encoded the required replication proteins along with oriLyt- and/or TR-containing plasmids hybridized with a pGEM 32P-labeled probe was carried out. Cotransfections also contained a LANA expression plasmid and plasmids containing K-bZIP and K-Rta ORFs, as indicated. All transfections contained 1 μg of each replication plasmid, 10 μg of oriLyt- or TR-containing plasmid, and 1 μg of K-bZIP and K-Rta expression plasmids except where otherwise stated. Lane 1, TR plasmid cleaved with EcoRI; lane 2, oriLyt plasmid cleaved with EcoRI; lane 3, cotransfection of required replication plasmids plus oriLyt; lane 4, cotransfection of required replication plasmids plus oriLyt and TR plasmids; lane 5, cotransfection of required replication plasmids plus LANA and TR plasmids; lanes 6, 7, and 8, cotransfection of required replication plasmids plus increasing amounts of a LANA expression plasmid (1, 2.5, and 5 μg, respectively), oriLyt, and TR; lane 9, cotransfection of required replication plasmids plus LANA, oriLyt, and TR and no K-bZIP expression plasmid; lanes 10 and 11, cotransfection of required replication plasmids plus LANA (5 μg), oriLyt, and TR plasmids and a K-bZIP expression plasmid (5 and 10 μg, respectively); lanes 12 and 13, cotransfection of required replication plasmids plus LANA (5 μg), oriLyt, and TR plasmids and plasmid expressing K-bZIP 101 to 134 (5 and 10 μg, respectively); lane 14, cotransfection of required replication plasmids plus LANA (5 μg) and oriLyt but not ORF59 expression plasmid. Input oriLyt and TR plasmids are shown in the bottom panel.