Removal of Hepatitis C Virus-Infected Cells by a Zymogenized Bacterial Toxin Assaf Shapira1,2, Shiran Shapira3,4, Meital Gal-Tanamy 2 , Romy Zemel2, Ran Tur-Kaspa2,5, Itai Benhar1* 1 Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, Israel, 2 Molecular Hepatology Research Laboratory, Sackler School of Medicine, Felsenstein Medical Research Center, Tel-Aviv University, Petah Tikva, Israel, 3 The Integrated Cancer Prevention Center, Tel Aviv Medical Center, Tel-Aviv, Israel, 4 Sackler School of Medicine, Tel-Aviv University, Ramat Aviv, Israel, 5 Department of Medicine D and Liver Institute, Rabin Medical Center, Beilinson Campus, Petah Tikva, Israel Abstract Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and has become a global health threat. No HCV vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. Moreover, there is no preventive therapy for recurrent hepatitis C post liver transplantation. The NS3 serine protease is necessary for HCV replication and represents a prime target for developing anti HCV therapies. Recently we described a therapeutic approach for eradication of HCV infected cells that is based on protein delivery of two NS3 protease-activatable recombinant toxins we named ‘‘zymoxins’’. These toxins were inactivated by fusion to rationally designed inhibitory peptides via NS3-cleavable linkers. Once delivered to cells where NS3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. The zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. Here, we present a solution that overcame the major limitations of the ‘‘first generation zymoxins’’ by converting MazF ribonuclease, the toxic component of the E. coli chromosomal MazEF toxin-antitoxin system, into an NS3-activated zymoxin that is introduced to cells by means of gene delivery. We constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, MazE, linked via an NS3-cleavable linker. While covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in na¨ıve, healthy cells. In contrast, activating proteolysis that is induced by even low levels of NS3, results in an eradication of NS3 expressing model cells and HCV infected cells. Zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases. Citation: Shapira A, Shapira S, Gal-Tanamy M, Zemel R, Tur-Kaspa R, et al. (2012) Removal of Hepatitis C Virus-Infected Cells by a Zymogenized Bacterial Toxin. PLoS ONE 7(2): e32320. doi:10.1371/journal.pone.0032320 Editor: Sung Key Jang, Pohang University of Science and Technology, Korea Received November 2, 2011; Accepted January 26, 2012; Published February 16, 2012 Copyright: 2012 Shapira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the Israel Science Foundation (ISF) (grant no. 397/08) (http://www.isf.org.il) and grant no. 3-3981 from the Israel Ministry of Health (http://www.health.gov.il/pages/default.asp?maincat = 14). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: benhar@post.tau.ac.il Introduction Hepatitis C virus (HCV) is a small, enveloped RNA virus belonging to the Hepacivirus genus of the Flaviviridae family. HCV has been recognized as a major cause of chronic liver disease and affects approximately 200 million people worldwide at the present time. Persistent infection is associated with the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. A protective vaccine for HCV is not yet available and even the most recent combination of antiviral therapy is often poorly tolerated [1]. The HCV genome encodes one large open reading frame that is translated as a polyprotein which is proteolytically processed to yield the viral structural and nonstructural (NS) proteins. The non-structural proteins include the p7 ion channel, the NS2-3 protease, the NS3 serine protease/RNA helicase and its co-factor NS4A, the NS4B and NS5A proteins and the NS5B RNA-dependent RNA polymerase (RdRp) [2,3]. Two virally encoded proteases participate in polyprotein processing, the NS2-3 autoprotease (which cleaves in cis at the NS2-3 junction) and the NS3-4A serine protease (which cleaves at four downstream NS protein junctions). NS3 is an extensively studied HCV protein that possesses multiple enzymatic activities that are essential for HCV replication, making NS3-4A the most attractive target for anti HCV drug development [4]. The N-terminus of NS3, in complex with its endoplasmic reticulum (ER) membrane-anchored co- factor NS4A, primarily functions as a serine protease, which cleaves the viral polyprotein precursor downstream to NS3. The remaining 2/3 of the protein has a helicase and NTPase activities, both of which are essential for HCV replication [5]. Zymogens are inactive enzyme precursors that are converted to their active form following a biochemical modification, such as proteolytic processing. Among the known and important groups of enzymes that are proteolytically activated are secreted digestive enzymes like pepsin and trypsin [6,7], the cysteine aspartic acid proteases (caspases) which play an essential role at various stages of the apoptotic process [8]; and blood coagulating factors [9]. Recently we described a proof of concept for potential new anti- viral agents that can specifically eradicate virally infected cells (thus limiting virus production and spread). These agents, which we named ‘‘zymoxins’’ (for ‘‘zymogenized toxins’’), were composed of PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e32320