represent an additional mode of anti-viral activity that works in parallel with host-cell protein synthesis shutoff. Of note, the applicability of the described zymoxin may be extended by replacing the protease cleavage site that separates between the toxic moiety and the inhibitory peptide. This could facilitate eradication of cells that are infected with other protease expressing viruses other than HCV (a partial list of protease expressing viruses is given at the beginning of the discussion) or different strains of HCV that differ in their NS3 protease cleavage specificity. In addition, one may replace the C terminal ER anchoring peptide with sequence that tethers the construct to a different intracellular location in which the viral protease resides. In that way, a colocalization between the zymoxin and the predetermined viral protease may be achieved. In conclusion, the presented anti-viral agent was designed following our previously described ‘‘zymoxins’’ concept in which a constitutively active toxin is converted into a ‘‘zymogenized’’, viral-protease activated from. The MazF-based zymoxin, that is introduced to target cells by means of adenovirus mediated gene delivery, shows very low toxicity to na¨ıve cells and enhanced responsiveness to low viral-protease expression level, when compared to our previously presented constructs. As evident from our results, the MazF based zymoxin eradicates NS3-expressing model cells and HCV infected cells with remarkable efficiency and specificity, providing further proof to the concept of zymoxins and a potential new means of fighting viral diseases. Obviously, further optimizations and pharmacological assessments in animal models may be required in order to determine the safety and efficiency of zymoxins treatment in the context of the whole organism. Materials and Methods Bacterial strains The following Escherichia coli (E. coli) strains were used: XL-1 Blue and DH5a (Stratagene, USA) for plasmid propagation and BJ5183 (Stratagene, USA) for the generation of recombinant adenovirus plasmid DNA. Recombinant DNA techniques and vectors Recombinant DNA techniques were carried out according to standard protocols or as recommended by the suppliers. Nucleotide sequences were determined using the PRISM 3100 Genetic Analyzer (Applied Biosystems, USA) according to the supplier’s recommendations. The eukaryotic CMV promoter- based GFP-fusion expression vector pEGFP C2, which was used for expression of mCherry, MazF and MazF-based zymoxins, was from Clontech (USA). The AdEasy plasmid system (pShuttle and pAdEasy-1) [35], that was used for generation of recombinant human type 5 adenoviral vectors for gene delivery of the zymoxins expression cassettes, was a generous gift from Prof. Nadir Arber, Integrated Cancer Prevention Center, Tel Aviv Sourasky Medical Center, Israel. All plasmid and DNA fragment purifications were carried out with High-Speed Plasmid Mini Kit and Gel/PCR DNA fragments Extraction Kit (Geneaid Biotech Ltd., Taiwan) unless mentioned otherwise. T4 DNA ligase and restriction enzymes were purchased from New England Biolabs (USA). DNA ligations were carried out at 16uC overnight. Molecular cloning Oligonucleotides. All the oligonucleotides that were used in this study were purchased from Hylabs, Israel. Oligonucleotides that were used in this study are listed in Table S1. Construction of the vector encoding for ‘‘mCherry-NS3 activated MazF’’. A polymerase chain reaction (PCR) was Zymoxin-Mediated Removal of HCV Infected Cells carried out using a single colony of Escherichia coli strain XL-1 as a template, the forward primer: 40- clvmazf and the reverse primers: 41-clvmazf, 42-clvmazf, 43-clvmazf, 44-clvmazf, 45-clvmazf, 46- clvmazf and 47-clvmazf. The PCR product, encoding for a fusion polypeptide composed of (from the N terminus) MazF, HCV P10- P10’ NS3 cleavage sequence derived from 2a genotype (strain JFH1) NS5A/B junction, a short flexible linker, 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]), a flexible linker and the C- terminal ER membrane anchor of the tyrosine phosphatase PTP1B [28] was digested with XhoI and EcoRI and was cloned between the corresponding sites in the plasmid pEGFP-C2, generating plasmid ‘‘pEGFP-NS3 activated MazF’’. Next, the sequence of the red fluorescent protein mCherry [29] was amplified by PCR from an expression cassette (kindly provided by Prof. Adi Avni, Department of Molecular Biology and Ecology of Plants, Tel-Aviv university, Israel) using the forward primer: 48- clvmazf and the reverse primer: 49- clvmazf. The PCR product was digested with NheI and XhoI and was cloned between the corresponding sites of the plasmid ‘‘pEGFP-NS3 activated MazF’’ (replacing the EGFP coding sequence), generating plasmid: ‘‘pmCherry-NS3 activated MazF’’. The amino-acid sequence of the encoded cleavable zymoxin can be found in Text S1. Construction of the vector encoding for ‘‘mCherry- uncleavable MazF’’. A PCR was carried out using DNA of plasmid ‘‘pmCherry-NS3 activated MazF’’ as a template, the forward primer: 40- clvmazf and the reverse primers: 50-unclmazf and 51-unclmazf. The PCR product was digested with EcoRV and NruI, and the digestion product of 233 bp was cloned between the corresponding sites of the same plasmid that has been used as a template, generating the plasmid: ‘‘pmCherry–uncleavable MazF’’. This plasmid encodes for an uncleavable construct 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 P3 valine was substituted by alanine, P2 cysteine by glycine, P1 cysteine by glycine and P4’ tyrosine by alanine. The amino-acid sequence of the encoded uncleavable zymoxin can be found in Text S1. Construction of the vector encoding for ‘‘EGFP- MazF’’. This is a mutated variant of an ‘‘intermediate’’ vector used in the construction process of the ‘‘mCherry-NS3 activated MazF’’ encoding vector. In this variant, a nonsense mutation was inserted instead of the tyrosine in the SMSY sequence of the NS3 recognition site, generating the plasmid ‘‘pEGFP-MazF’’ that encodes for a truncated EGFP-MazF fusion protein that lacks the MazE derived inhibitory peptide and the ER anchor. Construction of the vector encoding for mCherry. The sequence of the red fluorescent protein mCherry [29] was amplified by PCR from an expression cassette (see construction of the vector encoding for ‘‘mCherry-NS3 activated MazF’’) using the forward primer: 48- clvmazf and the reverse primer: 49- clvmazf. The PCR product was digested with NheI and BglII and was cloned between the corresponding sites of the plasmid pEGFP C2 (replacing the EGFP coding sequence), generating the plasmid ‘‘pmCherry’’. Construction and propagation of recombinant adenoviral vectors. Construction and propagation of recombinant human type 5 adenoviral vectors for gene delivery of the mCherry-NS3 activated MazF and mCherry-uncleavable MazF expression cassettes was carried out using the AdEasy system essentially as described in [35,64]. A more detailed description of the procedure is provided in Text S2. PLoS ONE | www.plosone.org 13 February 2012 | Volume 7 | Issue 2 | e32320