FIG 4.

68k-ank is required for replication of uncoated DNA. (A) Overview of plasmid replication assay. HeLa cells were transfected with pUC19 and infected with VACV 24 h later. The methylated template plasmids and unmethylated replicated DNA are represented by gray and red circles, respectively. Total DNA was extracted and digested with BamHI to cut DNA into unit-length segments and DpnI to cleave methylated template plasmids. After the digestion, droplet digital PCR (ddPCR) was performed to detect newly synthesized DNA. (B) The single BamHI and multiple DpnI restriction sites in pUC19 are shown. The ddPCR primers (P+ and P−) flanked a region with multiple DpnI sites. (C) The number of copies of replicated plasmid DNA was determined at 6 and 12 h after infection of HeLa cells with 5 PFU/cell of MVA or MVA-Δ186. **, P < 0.01.