a fusion between the binding and translocation domains of Pseudomonas exotoxin A and NS3-activable modified catalytic domains of the bacterial or the plant toxins diphtheria toxin (DTA) or ricin toxin (RTA), respectively. Upon binding and translocation into cells cytoplasm by virtue of the corresponding Pseudomonas exotoxin A domains, activation of these toxins is mediated by HCV-NS3 protease cleavage that separates between the toxic domains and a fused, rationally designed inhibitory peptide. These zymoxins showed a higher level of cytotoxicity when applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. Still, the zymoxins we described had two limitations: first, they required high levels of NS3 protease for efficient activation, and second, they were not totally inactive in the zymogenized state, and their basal cytotoxic activity without proteolytic activation resulted in quite narrow therapeutic windows [10]. Apparently, the zymoxins were only partially inhibited by the rationally designed inhibitory peptides. Toxic proteins that are completely inhibited by a natural inhibitor can be found in bacterial ‘‘toxin-antitoxin’’ systems. Natural bacterial plasmids ensure their survival within the bacterial host by replicating inside their host cell and using mechanisms that function to segregate them prior to cell division [11]. In addition, several low copy number plasmids use a unique system called ‘‘addiction module’’ or ‘‘toxin-antitoxin (TA) system’’ which functions to prevent the proliferation of plasmid- free cells by executing the so called ‘‘post-segregational killing effect’’. The system consists of a pair of genes encoding for a stable toxin and an unstable antitoxin organized in a bicistronic operon that is transcriptionally autoregulated either by the toxin-antitoxin complex itself or by the antitoxin alone. When coexpressed in plasmid harboring cells, the antitoxin component interferes with the lethal action of the toxin. If a cell loses the plasmid, the cellular concentration of the labile antitoxin (that is degraded faster than the more stable toxin) is rapidly diminished, enabling the toxin to exert its action which eventually results in cell death (reviewed by [12,13,14,15,16]). Toxin-antitoxin systems have also been found integrated to the chromosome of various bacteria species where their function has been the subject of considerable speculation [14,17,18,19,20,21]. One of the most studied ‘‘genomic’’ TA modules was found on the chromosome of E. coli as a negatively autoregulated bicistronic operon. The system, which is activated by several stress conditions, was denoted MazEF after its two active protein components: the long lived MazF toxin and the labile MazE antitoxin. MazF induced toxicity is executed by blocking de- novo protein synthesis through its endoribonuclease activity that catalyze the cleavage of single-stranded mRNAs at ACA sequences [22]. When coexpressed with MazF, the MazE antitoxin complexed with the toxin and a catalytically inactive heterohex- amer is formed in which a MazE dimer is sandwiched between two MazF dimers (MazF2-MazE2-MazF2) (reviewed by [21,23]). The crystal structure of the MazE-MazF complex indicates that the interactions between the toxin and the antitoxin are primarily mediated by the acidic C terminus of MazE which wraps around the MazF homodimer crossing the edge of the dimer interface [24]. Later on, Li et al had discovered that a short acidic peptide corresponding to MazE C-terminal 23 amino-acids, which they denoted ‘‘MazEp’’, binds strongly to the homodimer of MazF which possesses two identical active sites. Interestingly, it was found that one inhibitory peptide, occupying a single active site on the MazF homodimer, affects the conformation of both sites that consequently become catalytically inactive. This unique mecha- nism also explains the inhibitory activity of MazE toward MazF at a 1:2 molar ratio [25]. The discovery of the MazF mechanism of action was soon followed by demonstrations that it is also toxic to Zymoxin-Mediated Removal of HCV Infected Cells eukaryotic cells, causing Bak-dependent programmed cell death in mammalian cells, suggesting it may be used as a tool for gene therapy against diseases such as cancer and AIDS [26,27]. Here we describe the design of a different zymoxin than those described in [10], based on NS3-activable MazF ribonuclease that is delivered as a transgene by an adenoviral vector. The delivered transgene encodes for a fusion between MazF, and a potent inhibitory peptide derived from its natural antidote MazE, through an NS3 cleavable linker. We show that the self-inhibited, zymogenized toxin is well tolerated when expressed in na¨ıve cells. In contrast, in NS3 expressing or in HCV infected cells, NS3- mediated cleavage separates between the toxin and its inhibitor which results in inhibition of protein synthesis followed by death of the cells. Finally, we demonstrate that treatment with the MazF based zymoxin has a ‘‘curing effect’’ when applied to mixed culture of healthy and HCV infected cells, leading to specific eradication of the infected cell population. Results The construction of MazF-based zymoxin For the construction of NS3-activated MazF based zymoxin, the MazF coding sequence was fused through its C terminus to the HCV P10-P10’ NS3 cleavage sequence derived from the 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]) was fused, preceded by a short flexible linker, to the C terminus of the MazF-NS3 cleavage site sequence. A flexible linker, followed by the C-terminal ER membrane anchor of the tyrosine phosphatase PTP1B [28], was than fused to the C terminus of the inhibitory peptide and the whole construct was fused through its N terminus to the monomeric red fluorescence protein mCherry [29] (see Figure 1). The rationale behind the design of this construct, which was denoted ‘‘mCherry-NS3 activated MazF’’, was that the coupling between the ribonuclease and its antidote may enable high level of expression of the non-toxic fusion on the ER membrane of uninfected mammalian cells without causing any deleterious effect. In contrast, in HCV infected cells, the fusion protein is expected to colocalize with the ER membrane-bound viral NS3 protease (in infected cells, NS3 is localized to the cytosolic side of the ER membrane and membranes of ER-like modified compartments [30,31,32,33,34]). As a result, the NS3- cleavable linker between the toxin and its inhibitory peptide is expected to be cleaved. The toxic ribonuclease, no longer covalently tethered to its ER membrane-anchored inhibitor, is now free to diffuse into the cytoplasm (which lacks the antidote) and exert its cytotoxic activity. Finally, fusion to the fluorescent protein mCherry makes the whole construct trackable and facilitates the determination of its expression level and intracel- lular localization by fluorescence microscopy. As a control, an uncleavable construct (denoted ‘‘mCherry-uncleavable MazF’’) was constructed in which the NS3 cleavage sequence was replaced by a mutated 14 amino acids cleavage sequence (P10- P4’) from HCV genotype 1a NS5A/B junction in which the P3 valine was substituted by alanine, the P2 cysteine by glycine, the P1 cysteine by glycine and the P4’ tyrosine by alanine. A schematic representation of the NS3-activated MazF-based zymoxin (‘‘mCherry-NS3 activated MazF’’) and the hypothetical mechanism of its cleavage by NS3 protease on the cytoplasmic side of the ER membrane are shown in Figure 1. The amino- acid sequence of the MazF based zymoxins can be found in Text S1. PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e32320