Zymoxin-Mediated Removal of HCV Infected Cells Figure 6. Expression of mCherry-NS3 activated MazF results in growth inhibition and morphological changes in NS3-expressing cells. 16105 Tet-NS3/activated MazF or Tet-NS3/uncleavable MazF cells were seeded on poly-L-lysine coated cover-slips in a 24 well-plate. 12 h later, cells were supplemented with 10 ng/ml or 1000 ng/ml of tetracycline, or left untreated. 36 h later, cells were fixed. Following nuclear staining by Hoechst 33258 (Blue), slides were examined by fluorescence microscopy. The bar represents 50 mm. doi:10.1371/journal.pone.0032320.g006 uncleavable version of the construct (mCherry-uncleavable MazF). Red-fluorescent comet-like adenovirus-producing foci were ap- parent upon infection of packaging cells with both recombinant viruses (encoding cleavable or uncleavable constructs) (see Figure S1). The production yields for both viruses were ,36108 plaque forming units (PFU)/ml, after two ‘‘cycles’’ of virus amplification (see Text S2). In order to evaluate the ability of adenovirus-mediated delivery of NS3 activated MazF encoding cassette to eradicate NS3 expressing hepatocytes, wild-type Huh7.5 hepatoma cells and our previously described EGFP-full NS3-4A expressing Huh7.5 cells [10] were infected with the NS3-activated or uncleavable MazF encoding viruses. Since our previous unpublished observations and studies of others have indicated that high level of transgene expression and adenoviral infection, per se, may adversely affect cell viability and growth [36,37,38,39]; the cells were infected with a series of multiplicity of infection (MOI) ratios in order to find an optimal MOI that leads to eradication of NS3-expressing cells while maintaining minimal toxicity to na¨ıve cells. As shown in Figure 7A (left panel), infection with the NS3- activated zymoxin resulted in a considerable cytotoxic effect against NS3-expressing hepatocytes, leading to their almost complete eradication at MOI’s$12. However, infection at these ratios also adversely affected the growth of wild-type hepatocytes. In contrast, infection at MOI of ,3 decreased the viability of the NS3-expressing Huh7.5 cells to about 35% of the untreated control without affecting the viability of the wild- type hepatocytes (see also microscopic examination in Figure 7B). Therefore, infection at this MOI was applied during the next experiments. As expected, no substantial enhancement in cytotoxicity against NS3 expressing Huh7.5 cells (relatively to wild-type Huh7.5 cells) was observed, at any MOI, upon infection with the uncleavable MazF encoding viruses (Figure 7A, right panel). PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e32320