FIG. 4.

WT AAV replication is restricted by the Mre11 complex. (A) WT- or mutant E1b55K-expressing HeLa cells were infected with WT AAV with or without the Ad mutant dl1016 or Ad5 (MOI of 50) for 24 h before being harvested for immunoblot analysis or Hirt DNA extraction and QPCR of viral DNA. QPCR levels were normalized to those in GFP-expressing cells infected with AAV alone. Viral replication and Rep accumulation were greatest in E1b55K cells where the MRN complex was degraded (Ad5 or WT E1b55K and R240A cells with dl1016). (B) AAV replication in E1b55K cell lines by transfection with helper plasmids. HeLa-derived cell lines expressing GFP or E1b55K proteins were transfected with the AAV plasmid pNTC244 in the presence or absence of plasmids expressing E4orf6 and DBP. Infection with Ad5 (MOI of 50) served as a positive control. Viral DNA was extracted from cells after 48 h and analyzed by Southern blot hybridization with a Rep probe. Rfd (replication form dimer), Rfm (replication form monomer), and ssDNA (single-stranded DNA) refer to replicative forms of AAV. (C) NBS cell lines complemented with empty vector (NBS−) or WT Nbs1 (NBS+) were infected with WT AAV in the presence or absence of Ad5 (MOI of 50) or the Ad mutant dl1004 (MOI of 50) for 24 h. Cells were processed for viral DNA and immunoblotting. DNA was subject to Southern blot hybridization with a Rep probe. Rfd, Rfm, and ssDNA refer to replicative forms of AAV as in panel B. Lysates were analyzed by immunoblotting for levels of cellular (MRN) and adenoviral (E1b55K, DBP, and E1A) proteins as shown below.