Zymoxin-Mediated Removal of HCV Infected Cells Figure 1. Schematic representation of the construct ‘‘mCherry-NS3 activated MazF’’ and hypothetical mechanism of activation by NS3 protease. The NS3-activated MazF zymoxin was constructed by fusing 5 elements in the following order (from the N terminus): monomeric red fluorescence protein mCherry, E. coli MazF ribonuclease, HCV P10-P10’ NS3 cleavage sequence derived from 2a genotype (strain JFH1) NS5A/B junction, a short inhibitory peptide corresponding to MazE C-terminal 35 amino-acids (which encompass the 23 amino-acids inhibitory peptide (MazEp) that has been described by Li et al. [25]) and the C-terminal ER membrane anchor of the tyrosine phosphatase PTP1B [28]. After being anchored to the ER membrane, the NS3 cleavage site that is located between the ribonuclease and the inhibitory peptide in the ‘‘mCherry-NS3 activated MazF’’ construct (which is active as a dimer but for convenience is illustrated here in its monomeric form) is cleaved by the HCV- NS3 protease which is also localized to the cytoplasmic side of the ER membrane. The toxic ribonuclease, no longer covalently tethered to its ER-anchored inhibitor, is now free to diffuse to the cytoplasm (which lacks the antidote) and exert its destructive activity. doi:10.1371/journal.pone.0032320.g001 Expression of NS3 activated MazF is well tolerated by na¨ıve cells To verify that expression of ‘‘mCherry-NS3 activated MazF’’ is tolerated by cells that do not express the NS3 protease, a colony formation assay was carried out. In this assay, the toxicity of an expressed transgene can be comparatively and qualitatively assessed by testing its effect on the competency of transfected cells to evolve into colonies under selection. HEK293 T-REx cells where transfected with plasmids encoding either mCherry-NS3 activated MazF, mCherry (only the fluorescent protein) or EGFP- MazF (where MazF is not fused to its inhibitory peptide). 48 hours (h) later, expression of the encoded transgenes was confirmed by fluorescence microscopy (data not shown) and transfected cells were seeded in 3 fold dilutions and were treated with G418 (to which all three plasmids confer resistance). After 10 days of selection, surviving colonies were stained. As shown in Figure 2, similar numbers of surviving colonies were observed when the cells were transfected with the plasmids encoding mCherry-NS3 activated MazF or the red fluorescent protein alone, suggesting that expression of NS3-activable ribonuclease in na¨ıve HEK293 T-REx cells (that do not express NS3) cause minimal toxicity, if any. As expected, growth was severely inhibited when cells were transfected with the EGFP-fused active (uninhibited) toxin. The ER membrane-targeted zymoxin colocalizes with NS3 protease in vivo Previously we described a HEK293 cell line which inducibly expresses (by addition of tetracycline) a fusion between EGFP and the coding sequence of the full length NS3 (including the helicase domain) followed by NS4A from HCV 1a genotype [10]. These cells, denoted ‘‘Tet-inducible full NS3-4A expressing cells’’, were stably transfected with a plasmid encoding the NS3-activated zymoxin ‘‘mCherry-NS3 activated MazF’’ or with the uncleavable control (‘‘mCherry-uncleavable MazF’’) encoding plasmid. Fol- lowing selection, stable clones that constitutively express high level of the cleavable construct (denoted ‘‘Tet-NS3/activated MazF cells’’) or the uncleavable control (denoted ‘‘Tet-NS3/uncleavable MazF cells’’) were isolated. In order to characterize the intracellular distribution of the mCherry fused zymoxins, Tet- NS3/activated or uncleavable MazF cells were subjected to immunofluorescence microscopy analysis. As shown in Figure 3 (A and B), both zymoxins have a similar cellular distribution, colocalizing with the ER marker calnexin at the juxtanuclear region of the ER. Importantly, upon induced expression of NS3- A4 in Tet-NS3/uncleavable MazF cells, a colocalization of the two fluorescent fusion proteins could be observed (Figure 3, C and D).These observations confirm that indeed both the protease and the modified ribonuclease are tethered to a common cellular compartment, presumably the cytoplasmic side of the ER membrane (see scheme in Figure 1). NS3-mediated proteolytic activation of MazF based zymoxin inhibits de-novo cellular protein synthesis Since expression of active MazF was found to inhibit de-novo protein synthesis in mammalian cells [27], we hypothesized that such an effect may be observed also in cells in which MazF based zymoxin is proteolytically activated. In order to validate this assumption, Tet-NS3/activated MazF and Tet-NS3/uncleavable MazF cells were supplemented with tetracycline for 24 or 48 h, or left untreated. Levels of de-novo protein synthesis were than 3 determined by [ H]-leucine incorporation assay. As shown in Figure 4 (upper panel), a complete shutoff in protein synthesis was observed as soon as 24 h post NS3 induction in cells that express the cleavable construct, indicating proteolytic activation of the zymoxin. As expected, protein synthesis was not impaired following NS3 induction in cells that express the uncleavable toxin. PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e32320