Zymoxin-Mediated Removal of HCV Infected Cells Figure 7. Eradication of NS3-expressing Huh7.5 cells by recombinant adenovirus-mediated delivery of mCherry-NS3 activated MazF encoding cassette. 16104 wild-type (W.T) or EGFP-full NS3-4A expressing Huh7.5 cells were seeded per well in 96 well plates. After 24 h, recombinant adenoviruses encoding for mCherry-fused NS3 activated MazF or uncleavable-MazF zymoxins were added at the indicated MOI’s. Control cells remained untreated. (A) MTT viability assay: 4 days post infection, the fraction of viable cells (relatively to uninfected controls) was determined using an enzymatic MTT assay. A representative graph of three independent experiments is shown. Each bar represents the mean 6SD of a set of data determined in triplicates. (B) Microscopic examination: 4 days post infection, wild-type (lower panel) or EGFP-full NS3-4A expressing Huh7.5 cells (upper panel), uninfected or infected with recombinant adenoviruses encoding for mCherry fused NS3-activated MazF zymoxin (at MOI of ,3), were fixed and subjected to microscopic examination. The bar represents 200 mm. doi:10.1371/journal.pone.0032320.g007 Adenovirus-mediated delivery of NS3-activated MazF encoding cassette specifically eradicates HCV infected hepatocytes To test the competence of the MazF based zymoxin to specifically eradicate HCV infected hepatocytes, we utilized the infectious chimeric virus HJ3-5 [40]. This is one of the currently used models to study hepatitis C virus in which recombinant infectious HCV particles are produced in cell culture (HCVcc) (for review, see [41,42]). As the main purpose of zymoxins based treatment is the specific eradication of HCV infected cells from a background of healthy cells, ‘‘mixed culture’’ experiments were carried out. In these experiments, Huh7.5 hepatoma cells were infected with the HCV 1a/2a chimeric virus HJ3-5 (encoding the NS3 protease of genotype 2a strain JFH1 [40]). When infection reached ,50% (about 50% of the cultured cells showed expression of the HCV-core protein, as detected by immuno- staining and fluorescence microscopy), the mixed culture and a culture of uninfected cells were treated with NS3 activated MazF or uncleavable-MazF encoding adenoviruses at MOI of ,3. Control cells remained untreated. 72 h post adenoviral infection; viability assay and microscopic examination that included immunostaining for HCV-core protein were performed. As shown, treating the mixed culture with NS3-activated MazF zymoxin-encoding adenovirus reduced the viable cells popula- tion to about 65% relatively to untreated control; while viability of the uninfected cells (‘‘HCV negative’’) was barely affected by this treatment (Figure 8, upper panel). Microscopic exami- nation of the treated mixed culture revealed two cell populations that differ in their appearance. While one population is characterized by a ‘‘typical’’ Huh7.5 cell morphology (hollow arrows, Figure 8, lower panel), the other is composed of partially detached cells with round, condensed or distorted shape (filled arrows) that are hypothesized to be zymoxin-intoxicated PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e32320