Zymoxin-Mediated Removal of HCV Infected Cells Figure 8. Treatment of HCV infected/uninfected mixed cell culture with recombinant adenovirus-delivered MazF based zymoxin. Uninfected (HCV-negative) Huh7.5 cells and a 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 (MOI of ,3) encoding for the mCherry fused NS3- activated MazF or uncleavable-MazF zymoxins. Control cells remained untreated. Upper panel: MTT viability assay: 72 h post treatment, the fraction of viable cells (relatively to untreated 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. Lower panel: Microscopic examination: 72 h post treatment, the uninfected (HCV-negative) Huh7.5 cells, the mixed culture of HCV infected and uninfected cells and the control untreated cells were fixed and subjected to microscopic examination. Hollow arrows point to cells that are characterized by a ‘‘typical’’ Huh7.5 cell morphology. Filled arrows point to partially detached cells with round, condensed or distorted shape. The bar represents 200 mm. doi:10.1371/journal.pone.0032320.g008 HCV infected cells. In order to validate this assumption, the fraction of HCV infected cells from the general population was evaluated by immunofluorescence analysis using anti-HCV core protein specific antibodies. Indeed, treatment with the NS3- activated zymoxin showed a ‘‘curing effect’’ upon the partially infected culture, considerably reducing the fraction of the HCV infected cells from the general population (Figure 9). As expected, no significant effect upon the HCV infected cell population was observed following treatment with the unclea- vable zymoxin. PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e32320