(only the fluorescent protein) or EGFP- MazF (where MazF is not fused to its inhibitory peptide), respectively. Transfection was carried out using FuGENE 6 reagent (Roche, Germany) according to the manufacturer instructions. After 48 h, transfection efficiency was assessed by fluorescence microscopy and was determined as equal between the plasmids. Transfected cells were then trypsinized, counted and seeded in 3 fold dilutions (starting from 150,000 cells/well) in 6 well plates and were incubated for 10 days in the presence of 1 mg/ml of G418 (to which all the three plasmids confer resistance). Surviving colonies were fixed with 4% formaldehyde in PBS and stained with Giemsa (Sigma, USA). Number of surviving colonies from wells that were seeded with 5556 cells was determined by manual counting. Cell-viability assay The cell-killing activities of NS3-activated MazF and unclea- vable MazF zymoxins were measured by a Thiazolyl Blue Tetrazoliam Bromide (MTT) assay as described below: Cytotoxicity assay of intracellularly expressed mCherry- NS3 activated MazF or mCherry-uncleavable MazF in T- REx HEK293 cells inducibly expressing EGFP-Full NS3- 4A. Tet-inducible full NS3-4A, Tet-NS3/activated MazF or Tet-NS3/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 h later, the media was replaced by fresh media (100 ml per well) containing 1 mg/ml MTT (Thiazolyl Blue Tetrazoliam Bromide (Sigma, Israel) dissolved in PBS) reagent and the cells were incubated for further 30 minutes. MTT-formazan crystals were dissolved by the addition of extraction solution (20% SDS, 50% N, N-Dimethyl Formamide (DMF), pH 4.7) (100 ml per well) and incubation for 16 h at 37uC. Absorbance at 570 nm was recorded on an automated microtiter plate reader. The results were expressed as percentage of living cells relatively to the untreated controls. Cytotoxicity assay of recombinant adenoviruses encoding for mCherry-NS3 activated MazF or mCherry-uncleavable MazF on full NS3-4A expressing Huh7.5 cells. 16104 wild- type or EGFP-full NS3-4A expressing Huh7.5 cells [10] were seeded per well in 96 well plates. After 24 h, growth media were replaced by fresh media containing recombinant adenoviruses encoding for mCherry-NS3 activated MazF or mCherry-uncleavable MazF at indicated multiplicity of infection (MOI) ratios. Control cells remained untreated. Four days post infection, the media was replaced by fresh media (100 ml per well) containing 1 mg/ml MTT (except in representative wells in which cells were fixed and microscopically examined) and the cells were incubated for further 60 minutes. The next steps were identical to theses described above. Cytotoxicity assay of recombinant adenoviruses encoding for mCherry-NS3 activated MazF or mCherry-uncleavable MazF on a mixed culture of uninfected and HCV-infected Huh7.5 cells. Uninfected Huh7.5 cells and mixed culture of HCV infected and uninfected cells at 1:1 ratio (50% infected culture) were seeded in 96-well plates (16104 cells/well). After 24 h, cells were treated with recombinant adenoviruses (at MOI of ,3) encoding for mCherry-NS3 activated MazF or mCherry-uncleavable MazF zymoxins. Control cells remained untreated. 72 h later, the media was replaced by fresh media (100 ml per well) containing 1 mg/ml MTT (except in representative wells in which cells were fixed and microscopically examined) and the cells were incubated for further 60 minutes. The next steps were identical to these described above. For HCV-infection immunofluorescence analysis, 36104 cells from the HCV infected and uninfected mixed culture were seeded per well into 8-well chamber slides (Nalge Nunc, USA). 24 h later, Table S1 study. (DOC) Text S1 ins. (DOC) PLoS ONE | www.plosone.org 15 February 2012 | Volume 7 | Issue 2 | e32320 Zymoxin-Mediated Removal of HCV Infected Cells cells were treated with recombinant adenoviruses (MOI of ,3) encoding for the mCherry-NS3 activated MazF or mCherry- uncleavable MazF zymoxins. Control cells were remained untreated. 72 h post treatment, cells were fixed and permeabilized with 1:1 acetone/methanol mixture and stained with 1:300 diluted mouse monoclonal antibody C7-50 (Affinity BioReagents, USA) specific for the HCV core protein followed by staining with 1:100 diluted FITC-conjugated goat anti-mouse IgG (Jackson Immu- noResearch Laboratories, USA). Nuclei were then stained with DAPI and slides were mounted (SouthernBiotech, USA) and examined using a fluorescence microscope. For each treatment, evaluation of the fraction of the HCV-infected cells from the general cell population was performed by dividing the number of the green, HCV-core positive cells by the general number of cells (DAPI stained) from five representative microscopic fields. [3H]-leucine incorporation assay 16105 Tet-NS3/activated MazF or Tet-NS3/uncleavable MazF cells were seeded per well in 24-wells plate. 24 or 48 h later, cells were supplemented with tetracycline to a final concentration of 1000 ng/ml, or left untreated. 72 h after seeding, cells were supplemented with [3H]-leucine (Perkin Elmer, USA) to a final concentration of 1 mCi/ml and returned to incubation. After 6 h, cells were scraped, washed with PBS and lysed by four freeze/thaw cycles. 7 mg total protein form the lysate of each treatment were then add to a solution containing PBS and a final concentration of 150 mg bovine serum albumin (BSA) in a total volume of 75 ml. The solution was then mixed with an identical volume of ice-cold 10% trichloro acetic acid (TCA). Mixtures were then incubated on ice for 30 minutes and centrifuged for 10 minutes at 20,000 g, 4uC, after which the pellet was washed with ice-cold 5% TCA, followed by washing with ice-cold 80% ethanol. The pellet was then dissolved in 300 ml of 0.1 M NaOH, transferred to a scintillation tube and neutralized with 200 ml 1 M HCl. 4 ml of scintillation liquid was added and radioactivity was counted by a beta-counter device. Supporting Information Figure S1 Fluorescence microscopy analysis of adeno- virus producing foci. 36105 HEK293 cells were seeded per well in 6 wells plate. When reached 90% confluence, cells were infected with 10 fold dilutions of recombinant adenoviruses encoding for mCherry-NS3 activated MazF or mCherry-unclea- vable MazF, starting from 2.56106 PFU per well. After 36 h, cells were fixed and examined under a fluorescence microscope. Red fluorescent adenovirus-producing foci from wells infected with 2.56103 PFU are shown. The bar represents 200 mm. (DOC) Oligonucleotides that have been used in this Amino-acid sequences of MazF based zymox- Text S2 Construction and propagation of recombinant adenoviral vectors. (DOC) Acknowledgments We thank Prof. Matti Sa¨llberg (Karolinska Institute, Sweden) for the plasmid carrying the NS3-4A gene of the 1a genotype. We thank Prof.