the effector domain and the domain bearing the intracellular signaling moiety which specifies a cleavage site for a predeter- mined protease (e.g. a viral encoded protease). Following the introduction of the sitoxin into the target cell (that expresses the specific protease), cleavage by the predetermined protease separates the toxic effector domain of the sitoxin from the intracellular signaling moiety, resulting either in a longer-lived (and therefore more toxic) effector domain or in an effector domain that moves from a cellular compartment where the domain is nontoxic to a cellular compartment where the domain is able to exert its effect [51]. We developed and successfully evaluated two rationally designed viral-protease-activated chimeric toxins which we named ‘‘zymoxins’’ for ‘‘zymogenized toxins’’ [10]. In contrast to sitoxins, in which the inhibitory activity is mediated by intracellular components which recognize a cleavable signaling polypeptide fused to a constitutively active toxin; the concept of zymoxins is based on the idea of reengineering a toxic enzyme into an inactive zymogen which is specifically activated by a predetermined protease. This general approach was demon- strated by other elegant studies in which several enzymes, including bovine RNase A, Vip2 and the maize ribosome inactivating protein (maize-RIP) were converted into protease activated forms [53,54,55,56,57]. Our previous work focused on the design of protein-delivered toxins that were converted into protease-activated forms by means of fusion to specific, rationally designed inhibitory peptides through HCV-NS3 protease cleavable linker. When tested in-vitro and on NS3-overexpressing or HCV infected cells, a clear NS3 protease cleavage-dependent enhancement in activity/cytotoxicity was observed. However, these constructs had two major drawbacks, as mentioned in the introduction: The first is the incomplete inhibition of the toxic enzymatic activity that is conferred by the rationally designed fused peptide. The second relates to the necessity of relatively high level of expressed viral protease for achieving adequate zymoxin activation inside the cells [10]. In the current study, we turned to evaluate a similar strategy for eradicating HCV-infected cells, namely, by converting a consti- tutively active enzyme into an NS3 activated zymogen. As a toxic moiety, we chose MazF, an endoribonuclease that together with its polypeptidic antidote, MazE, constitute one of the most studied ‘‘genomic’’ toxin-antitoxin systems in E. coli. In order to convert MazF into a zymoxin, a fusion polypeptide was constructed in which an inhibitory peptide derived from the MazE antitoxin was fused to the C terminus of the MazF toxin via an NS3-cleavable linker. Regarding the issue of incomplete inhibition of zymoxin’s activity in uncleaved form, it should be mentioned that in contrast to our previously described constructs, in which a ‘‘rationally designed’’ peptide is appended to provide the inhibition of the toxin’s activity; in the MazF based-zymoxin, a ‘‘natural’’ inhibitory polypeptide was chosen for that purpose. Antidote polypeptides in toxin-antitoxin systems were ‘‘evolutionary shaped’’ to strongly inhibit the destructive activity of their toxic counterparts. Thus, a very efficient inhibition may be achieved when using them as inhibitory peptides in the construction of zymoxins. Indeed, our results show that the fused MazE-derived inhibitory peptide diminishes the toxin’s activity to such an extent that enables its non-lethal overexpression in na¨ıve cells. In order to improve the responsiveness of the zymoxin to the presence of low levels of cellular-expressed viral protease, an ER membrane ‘‘anchoring peptide’’ was fused to the C terminus of the construct, subsequently to the MazE derived inhibitory peptide. By using that design, a colocalization between the ER-bound NS3 Zymoxin-Mediated Removal of HCV Infected Cells protease and its zymoxin substrate might be achieved, resulting in an improved cleavage efficiency. Indeed, such a colocalization could be observed, as shown above (Figure 3). Moreover, activation of the zymoxin was evident also upon expression of very low cellular levels of the viral protease (Figures 5 and 6). It should also be mentioned that while being extremely sensitive to minute amounts of expressed NS3 protease, zymoxin expressing cells can still tolerate some degree of activating proteolysis (as shown above for NS3-activated MazF expressing cells that show a very low, basal NS3 proteolytic activity without tetracycline induction (Figure 4)). This property may, in fact, contribute to the specificity and general safety of zymoxin-based therapeutics, even though ‘‘spontaneous’’, unspecific proteolytic activity toward NS3 substrates could not be detected in na¨ıve cells during our experiments (data not shown). As discussed, tethering the zymoxin to the ER membrane through its inhibitory peptide may also enhance its cleavage-dependent toxicity. This might be achieved by enabling spatial separation between the activated toxin and its antidote following NS3-cleavage and diffusion of the active MazF to the inhibitory peptide-free cytoplasm. Supporting this assump- tion, we found that although cleaved, a cytoplasmic version of the NS3-activated MazF based zymoxin is barely toxic to NS3 protease-expressing cells (data not shown). In contrast to our previous study, in which the toxins were delivered into mammalian cells as purified recombinant proteins; in the current research an adenoviral vector gene-delivery system was used. Widely applied in gene therapy, recombinant viral vaccines and basic science studies, this system enables efficient delivery and high level of transgene expression in a wide variety of human cells. Using this system, we have demonstrated specific eradication of full NS3-4A expressing Huh7.5 cells, sparing ‘‘na¨ıve’’ cells which do not express the protease. Furthermore, specific eradication of HCV infected cells has also been achieved, demonstrating a prominent ‘‘curing effect’’ when tested on mixed cultures of healthy and HCV infected hepatocytes. The use of viral gene delivery for treating HCV infection by eradication of infected cells has been proved successful in previous study on mice with chimeric human livers. In that study, Hsu et al designed a modified proapoptotic BID molecule in which its endogenous cleavage sites were replaced by NS3 recognition site. As BID requires proteolytic processing in order to activate its apoptotic function, infection with adenoviral vector that delivers a transgene encoding the engineered BID molecule was demon- strated to induce activation of apoptosis in cells expressing the HCV NS3 protease. Moreover, a significant reduction in HCV titer in the serum of the HCV infected mice was detected following injection of the engineered adenoviral vector into the jugular vein [58]. Although such an approach had proved successful; it should be taken into account that a delivered transgene which encodes for a modified form of a protein that is naturally expressed in the target cell has the potential to negatively influence normal cellular processes in which its endogenous counterpart plays a role [59,60,61,62,63]. For example, when overexpressed in non- infected cells, an NS3-cleavable apoptotic molecule, such as that described above, may act as a ‘‘dominant negative’’ by competing with the natural, endogenous protein for interactions with activators, regulators or substrates. Delivery of a transgene encoding for a foreign protein which has no endogenous counterpart in target cells, such as MazF, may reduce that risk. Furthermore, it should be noted that MazF possess a ribonuclease activity that is also capable of processing multiple potential cleavage sites in the HCV genome. Although not evaluated in this study, a direct attack on the parasite’s genetic material may PLoS ONE | www.plosone.org 12 February 2012 | Volume 7 | Issue 2 | e32320