FIG. 2.

Replacement of the RRE-involved promoter in KSHV ori-Lyt with other promoters abolished ori-Lyt-dependent DNA replication. The ori-Lyt promoter region (nucleotides 24022 to 24252) was replaced by a mouse U6 snRNA promoter, an SV40 early promoter, or an HCMV IE promoter in pOri-A to generate pOri-PmU6, pOri-PSV40, and pOri-PCMV, respectively. (A) The transcription initiated by these promoters was examined by Northern analysis. Total RNAs were isolated from 293 cells that had been transfected with pOri-A and its derivatives, as indicated above each lane. These RNAs were separated on a 1.0% agarose-formaldehyde gel and transferred onto a Nytran membrane. The membrane was probed with a ³²P-labeled GC-rich tandem repeat sequence. (B) The abilities of these constructs to mediate DNA replication were tested in BCBL-1 cells by using a transient replication assay as described in Materials and Methods. pBlue, pBluescript; Rep'd, replicated.