FIG. 3.

Southern blot analysis of intracellular replication mediated by various Rep78 proteins. (A) Representative Southern blot of rAAV-LacZ (pAB-11) replication mediated by mutant and wt Rep78 proteins expressed from the HIV LTR in adenovirus type 5-infected 293 cells. The replication assays shown were performed at 37°C, except as indicated for D40,42,44A (lanes 6 to 8). The pHIV-78 and pAB-11 constructs were transfected at a 3:1 molar ratio as described in Materials and Methods. Following transfection, the cells were incubated for 48 h with adenovirus type 5 (MOI of 5). At 48 h postinfection, Hirt DNA was extracted, digested with DpnI, and analyzed by Southern blotting with a ³²P-labeled probe specific for β-galactosidase sequences. Replicative monomer (M) and dimer (D) DNA forms are indicated. ∗, replicated forms of pAB-11 were detected only after long-term exposure of blots. Rep68 was expressed from pHIV-68, a derivative of the pHIV-78 construct that has the rep gene intron removed as described previously (74). wt Rep is expressed from the pHIV-Rep construct, which expresses all four rep gene products, as described in Materials and Methods. (B) Southern blot analysis of replication of an rAAV-UF5 virus (GFP) mediated by transfected wt pIM45 or mutD40A,D42A,D44A pIM45 Rep expression plasmids. The pIM45 constructs express all four rep gene products (see Materials and Methods). Transfection infections were performed at permissive (32°C) and nonpermissive (39.5°C) temperatures in adenovirus type 5-infected HEK 293 cells. Rep-containing plasmids (wt pIM45 or mutD40,D42,D44A pIM45) were transfected into subconfluent HEK 293 cells and simultaneously coinfected with adenovirus type 5 (MOI of 5) and rAAV-UF5 (MOI of 5). At various time points, Hirt DNA was extracted and analyzed by Southern blotting as described above with a ³²P-labeled probe specific for GFP sequences. No-plasmid lanes, cells were infected with adenovirus type 5 only; pIM45 (wt) lanes, cells were coinfected with adenovirus type 5 and rAAV-UF5 and transfected with wt pIM45; mutD40,42,44A lanes, cells were coinfected with adenovirus type 5 and rAAV-UF5 and transfected with mutD40,D42,D44A,pIM45.