FIG. 8.

Involvement of HSV-1 UL12 protein in AAV replication. (A) Colocalization of UL12 with Rep within AAV RCs. HeLaAAVtCR cells were either infected with the indicated HSV-1 strain or transfected with the HSV-1 helper plasmids and pSAKUL12 plasmid and then stained with an anti-UL12 antibody and TO-PRO-3. Bars, 5 μm. (B) Analysis of the effect of UL12 on AAV DNA replication. Total DNA was extracted from HeLaAAVtCR cells either infected with wt HSV-1 or HSVΔUL12 or transfected with HSV-1 helper plasmids and a plasmid encoding either UL12, UL12.5, or an exonuclease-negative UL12 mutant (pUL12exo−) and then quantified by qPCR, using rep-specific primers. Data presented are means with error bars calculated for three independent experiments. (C) The same samples used in panel B were analyzed by Southern blotting, using a DIG-labeled rep probe. mRF, monomer replicative form; dRF, dimer replicative form. (D) Effect of UL12 on rAAV-GFP particle production. HeLa cells transfected with rAAV-GFP and rep-cap-expressing plasmids were either cotransfected with HSV-1 helper plasmids or infected with HSVΔUL12 in the absence or presence of a plasmid encoding UL12. As a control, the cells were cotransfected with adenoviral helper plasmids (pXX6 and pGKE1) or infected with wt HSV-1. Cell lysates were then used to infect HeLa cells in the presence of wt adenovirus, and GFP-positive cells were counted by flow cytometry at 24 h p.i. Mean values with error bars, calculated for at least three independent experiments, are presented.