Zymoxin-Mediated Removal of HCV Infected Cells Figure 5. Eradication of NS3 expressing cells by mCherry-NS3 activated MazF. Upper panel: Tet-inducible full NS3-4A (No MazF), Tet-NS3/ activated MazF (NS3-activated MazF) or Tet-NS3/uncleavable MazF (uncleavable MazF) cells were seeded in 96 well plates (26104 cells per well). After 24 h, cells were supplemented with 3 fold dilutions of tetracycline starting with concentration of 1000 ng/ml, or left untreated. 72 hours later, the fraction of viable cells (relatively to the untreated controls) was determined using an enzymatic MTT assay. Each bar represents the mean 6SD of a set of data from six wells. Lower panel: 30 ng of total protein from lysates of Tet-NS3/uncleavable MazF cells that were supplemented with 3 fold dilutions of tetracycline for 48 h were analyzed by immunoblotting with mouse anti-GFP (for the detection of EGFP-NS3) and mouse anti-actin antibodies (loading control) followed by HRP-conjugated secondary antibodies and ECL development. doi:10.1371/journal.pone.0032320.g005 media, with around 10 ng/ml as an intermediate concentration for induction of low NS3 expression level. Indeed, strong cytotoxicity was clearly evident when Tet-NS3/activated MazF cells where treated with tetracycline concentrations of down to 4 ng/ml. No cytotoxic effect was detected when the controls Tet- NS3/uncleavable MazF or Tet-inducible full NS3-4A (protease only) expressing cells were similarly treated (Figure 5, upper panel). These findings demonstrate the deleterious effect of the MazF based zymoxin specifically toward NS3 protease expressing cells, as well as its competence to be activated by very low cellular levels of protease. In order to obtain a visual insight at the cell level, Tet-NS3/activated MazF and Tet-NS3/uncleavable MazF cells were supplemented with tetracycline to a final concentration of 10 ng/ml or 1000 ng/ml (for low and high induction levels of NS3 expression, respectively), or left untreated. 36 h later, nuclei were stained and cells were examined under a fluorescence microscope. The results show that both lower and higher induction levels of NS3 protease caused growth inhibition and rounding of cells that constitutively expresses the cleavable MazF. Furthermore, both green and red fluorescence were faint in these cells, probably as a result of the destructive ribonuclease activity of the cleaved toxin toward the NS3 protease and its own mRNA. As expected, none of the above observations was evident when these cells were not supplemented with tetracycline or when NS3 expression was induced to high level in cells that constitutively express the uncleavable toxin (Figure 6). Adenovirus-mediated delivery of mCherry-NS3 activated MazF encoding cassette specifically eradicates NS3 expressing hepatocytes To achieve efficient DNA delivery into mammalian cells, the expression cassette encoding mCherry-NS3 activated MazF was cloned into a ‘‘first generation’’ DE1/DE3 human type 5 adenoviral vector plasmid DNA by homologous recombination in bacteria [35]. Virus particles were propagated in HEK293 packaging cells as described in the ‘‘materials and methods’’ section. In addition, a control adenoviral vector was constructed for the delivery of a similar cassette that encodes for the PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e32320