Zymoxin-Mediated Removal of HCV Infected Cells Figure 9. Eradication of HCV-infected hepatocytes by recombinant-adenovirus delivered MazF based zymoxin. 36104 cells from mixed HCV infected and uninfected culture (at 1:1 ratio) were seeded per well into 8-well chamber slides. 24 h later, cells were treated with recombinant adenoviruses (MOI of ,3) encoding for the mCherry fused NS3-activated MazF or uncleavable-MazF zymoxins. Control cells remained untreated. 72 h post treatment, cells were fixed, permeabilized and immunostained with mouse anti-HCV core and FITC-conjugated goat anti mouse antibodies for visualization of infected cells (green). Cell nuclei were then stained with DAPI (cyan) and slides were visualized by fluorescence microscopy. The bar represents 100 mm (upper panel). The fraction (given in percentage) of HCV-infected cells from the general cell population was evaluated, for each treatment, by dividing the number of the green, HCV-core positive cells by the general number of cells (DAPI stained) (lower panel). Each bar represents the mean 6SD of a set of data collected from five representative microscopic fields. Numbers in brackets represent the percentage of the HCV-infected cells in each treatment relatively to their percentage in the untreated culture. doi:10.1371/journal.pone.0032320.g009 Discussion Fight against viral infections is considered as one of the most challenging areas in modern medicine. While vaccination is considered to be the most efficient method for fighting viral infections; for some viral pathogens which cause world-wide health problem like human immunodeficiency virus (HIV) and hepatitis C virus (HCV), no efficient vaccine has yet been developed. Over the last years, efforts have been focused on the discovery and development of anti-viral agents that target crucial steps in the viral replication cycle which includes viral entry, RNA translation and post-translational processing, reverse transcription, genome integration, viral assembly and release [43,44,45]. An essential step in the replication cycle of many viruses is the processing of a polyprotein precursor by a viral-encoded protease. A partial list of human diseases associated viruses encoding protease(s) in their genome include flaviviruses such as hepatitis C virus (HCV), West Nile virus (WNV), dengue fever virus (DFV) and yellow fever virus (YFV); retroviruses such as HIV-1; picornaviruses such as coxsackievirus, poliovirus and hepatitis A virus; nidoviruses such as coronaviruses (CoV), including the severe acute respiratory syndrome (SARS) causative SARS-CoV and herpesviruses such as varicella-zoster virus (VZV) and Epstein- Bar virus (EBV) [46,47]. Therefore, a large part of antiviral drug discovery is focused on inhibiting viral proteases [46]. Our group too has published several studies where HCV replication was inhibited by intracellular expression of antibodies and peptide aptamers [48,49,50]. The opposite ‘‘side of the coin’’ – taking advantage of the activity of a viral protease per-se as an antiviral approach has been rather neglected. Recently we described a study that was based on the concept of ‘‘sitoxins’’ which are anti-viral agents that are designed to eradicate viral-infected cells by taking advantage of a specific viral activity instead of inhibiting it [51,52]. specifically, a sitoxin is comprised of an effector domain (e.g. a toxin); a domain bearing an intracellular signaling moiety (e.g. a degradation or an intracellular localization signal); and a domain located between PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e32320