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Engineered toxins "zymoxins" are activated by the HCV NS3 protease by removal of an inhibitory protein domain.

Shapira A, Gal-Tanamy M, Nahary L, Litvak-Greenfeld D, Zemel R, Tur-Kaspa R, Benhar I - PLoS ONE (2011)

Bottom Line: Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window.The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells.This suggests that the "zymoxin" approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, Israel.

ABSTRACT
The synthesis of inactive enzyme precursors, also known as "zymogens," serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated "zymogenized" chimeric toxins (which we denote "zymoxins"). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the "zymoxin" approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

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Tetracycline induced expression of active EGFP-scNS3 and EGFP- full NS3-4A in T-Rex 293 cells.Upper panel: immunofluorescence analysis of inducible NS3 expressing cells: 105 cells inducibly expressing EGFP-scNS3 or EGFP-full NS3-4A were seeded on cover-slips in a 24 well-plate. After 12 hours, the cells were treated with 1µg/ml of tetracycline for 24 hours and then were fixed and permeabilized. Following immunostaining with rabbit anti-calnexin and Cy3-conjugated goat anti rabbit for ER visualization (red) and nuclei staining by Hoechst 33258 (Blue), slides were examined by confocal fluorescence microscopy. (A). Uninduced cells. (B). Induced scNS3 expressing cells. (C). Induced full NS3-4A expressing cells. Lower panel: in-vivo substrate cleavage by NS3: cells inducibly expressing EGFP-scNS3 were transfected with 2µg of the plasmid pCMV/MBP-EGFP-NS5AB-CBD (“cleavable substrate”- see scheme in D). After 24 hours, cells were treated with 1µg/ml of tetracycline or left untreated. 24 hours later, cells were lysed and 20µg protein from total cell extract where analyzed by immunoblotting with rabbit anti-EGFP antibodies (for detection of EGFP-scNS3) and mouse-serum anti-MBP (which is more sensitive than anti-EGFP in detecting the cleavage products of the substrate) followed by HRP-conjugated secondary antibodies and ECL development (E). Bands corresponding to the full length substrate, cleaved substrate and EGFP-scNS3 are indicated by arrows. Similar results were obtained also in cells inducibly expressing EGFP-full NS3-A4.
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pone-0015916-g001: Tetracycline induced expression of active EGFP-scNS3 and EGFP- full NS3-4A in T-Rex 293 cells.Upper panel: immunofluorescence analysis of inducible NS3 expressing cells: 105 cells inducibly expressing EGFP-scNS3 or EGFP-full NS3-4A were seeded on cover-slips in a 24 well-plate. After 12 hours, the cells were treated with 1µg/ml of tetracycline for 24 hours and then were fixed and permeabilized. Following immunostaining with rabbit anti-calnexin and Cy3-conjugated goat anti rabbit for ER visualization (red) and nuclei staining by Hoechst 33258 (Blue), slides were examined by confocal fluorescence microscopy. (A). Uninduced cells. (B). Induced scNS3 expressing cells. (C). Induced full NS3-4A expressing cells. Lower panel: in-vivo substrate cleavage by NS3: cells inducibly expressing EGFP-scNS3 were transfected with 2µg of the plasmid pCMV/MBP-EGFP-NS5AB-CBD (“cleavable substrate”- see scheme in D). After 24 hours, cells were treated with 1µg/ml of tetracycline or left untreated. 24 hours later, cells were lysed and 20µg protein from total cell extract where analyzed by immunoblotting with rabbit anti-EGFP antibodies (for detection of EGFP-scNS3) and mouse-serum anti-MBP (which is more sensitive than anti-EGFP in detecting the cleavage products of the substrate) followed by HRP-conjugated secondary antibodies and ECL development (E). Bands corresponding to the full length substrate, cleaved substrate and EGFP-scNS3 are indicated by arrows. Similar results were obtained also in cells inducibly expressing EGFP-full NS3-A4.

Mentions: In order to monitor specific NS3 proteolytic activity in Tet induced cells, we constructed a substrate-encoding plasmid that encodes for a modification of our previously described polypeptide which serves as a substrate for proteolysis by the NS3 protease [33]. This plasmid, denoted pCMV/MBP-EGFP-NS5AB-CBD, encodes for a fusion of maltose binding protein (MBP), enhanced green fluorescence protein (EGFP), the 10 amino acid minimal NS3 cleavage sequence (P6-P4′) from HCV NS5A/B site derived from HCV genotype 1b/1a ( for both 1b and 1a genotypes this sequence is identical) [41] and cellulose binding domain (CBD). As shown in Fig. 1, addition of Tet to selected stable clones of the inducible system results in EGFP-scNS3 and EGFP- full NS3-4A expression, as assayed by fluorescence confocal microscopy (Fig. 1B and 1C, respectively) and immunoblotting (Fig. 1E, lower panel). Regarding the intracellular localization of the proteases, EGFP-full NS3-4A was only partially colocalized with calnexin (ER) in these cells, but apparently was absent from the nucleus and has less diffuse distribution pattern in comparison to the nucleocytoplamic EGFP-scNS3, as was expected. A strong colocalization, however, was observed between EGFP-full NS3-4A and a chimeric fluorescent protein which was fused to the C-terminal ER membrane anchor of the tyrosine phosphatase PTP1B [42] (data not shown). Immunoblot analysis also shows efficient cleavage of MBP-EGFP-NS5AB-CBD (referred to as the “cleavable substrate” (Fig. 1D)) in cells transfected with pCMV/MBP-EGFP-NS5AB-CBD and induced for NS3 expression (Fig. 1E). A faint band, corresponding to the cleavage product, was also detected in a sample from uninduced cells. This is probably due to some degree of “leakiness” of the Tet inducible system, allowing a very low basal transcription of the protease in the absence of externally added Tet. In support to this assumption, only a single band corresponding to the full length substrate was detected when “naïve” HEK293 T-Rex cells were transfected with the substrate encoding vector (data not shown).


Engineered toxins "zymoxins" are activated by the HCV NS3 protease by removal of an inhibitory protein domain.

Shapira A, Gal-Tanamy M, Nahary L, Litvak-Greenfeld D, Zemel R, Tur-Kaspa R, Benhar I - PLoS ONE (2011)

Tetracycline induced expression of active EGFP-scNS3 and EGFP- full NS3-4A in T-Rex 293 cells.Upper panel: immunofluorescence analysis of inducible NS3 expressing cells: 105 cells inducibly expressing EGFP-scNS3 or EGFP-full NS3-4A were seeded on cover-slips in a 24 well-plate. After 12 hours, the cells were treated with 1µg/ml of tetracycline for 24 hours and then were fixed and permeabilized. Following immunostaining with rabbit anti-calnexin and Cy3-conjugated goat anti rabbit for ER visualization (red) and nuclei staining by Hoechst 33258 (Blue), slides were examined by confocal fluorescence microscopy. (A). Uninduced cells. (B). Induced scNS3 expressing cells. (C). Induced full NS3-4A expressing cells. Lower panel: in-vivo substrate cleavage by NS3: cells inducibly expressing EGFP-scNS3 were transfected with 2µg of the plasmid pCMV/MBP-EGFP-NS5AB-CBD (“cleavable substrate”- see scheme in D). After 24 hours, cells were treated with 1µg/ml of tetracycline or left untreated. 24 hours later, cells were lysed and 20µg protein from total cell extract where analyzed by immunoblotting with rabbit anti-EGFP antibodies (for detection of EGFP-scNS3) and mouse-serum anti-MBP (which is more sensitive than anti-EGFP in detecting the cleavage products of the substrate) followed by HRP-conjugated secondary antibodies and ECL development (E). Bands corresponding to the full length substrate, cleaved substrate and EGFP-scNS3 are indicated by arrows. Similar results were obtained also in cells inducibly expressing EGFP-full NS3-A4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3021518&req=5

pone-0015916-g001: Tetracycline induced expression of active EGFP-scNS3 and EGFP- full NS3-4A in T-Rex 293 cells.Upper panel: immunofluorescence analysis of inducible NS3 expressing cells: 105 cells inducibly expressing EGFP-scNS3 or EGFP-full NS3-4A were seeded on cover-slips in a 24 well-plate. After 12 hours, the cells were treated with 1µg/ml of tetracycline for 24 hours and then were fixed and permeabilized. Following immunostaining with rabbit anti-calnexin and Cy3-conjugated goat anti rabbit for ER visualization (red) and nuclei staining by Hoechst 33258 (Blue), slides were examined by confocal fluorescence microscopy. (A). Uninduced cells. (B). Induced scNS3 expressing cells. (C). Induced full NS3-4A expressing cells. Lower panel: in-vivo substrate cleavage by NS3: cells inducibly expressing EGFP-scNS3 were transfected with 2µg of the plasmid pCMV/MBP-EGFP-NS5AB-CBD (“cleavable substrate”- see scheme in D). After 24 hours, cells were treated with 1µg/ml of tetracycline or left untreated. 24 hours later, cells were lysed and 20µg protein from total cell extract where analyzed by immunoblotting with rabbit anti-EGFP antibodies (for detection of EGFP-scNS3) and mouse-serum anti-MBP (which is more sensitive than anti-EGFP in detecting the cleavage products of the substrate) followed by HRP-conjugated secondary antibodies and ECL development (E). Bands corresponding to the full length substrate, cleaved substrate and EGFP-scNS3 are indicated by arrows. Similar results were obtained also in cells inducibly expressing EGFP-full NS3-A4.
Mentions: In order to monitor specific NS3 proteolytic activity in Tet induced cells, we constructed a substrate-encoding plasmid that encodes for a modification of our previously described polypeptide which serves as a substrate for proteolysis by the NS3 protease [33]. This plasmid, denoted pCMV/MBP-EGFP-NS5AB-CBD, encodes for a fusion of maltose binding protein (MBP), enhanced green fluorescence protein (EGFP), the 10 amino acid minimal NS3 cleavage sequence (P6-P4′) from HCV NS5A/B site derived from HCV genotype 1b/1a ( for both 1b and 1a genotypes this sequence is identical) [41] and cellulose binding domain (CBD). As shown in Fig. 1, addition of Tet to selected stable clones of the inducible system results in EGFP-scNS3 and EGFP- full NS3-4A expression, as assayed by fluorescence confocal microscopy (Fig. 1B and 1C, respectively) and immunoblotting (Fig. 1E, lower panel). Regarding the intracellular localization of the proteases, EGFP-full NS3-4A was only partially colocalized with calnexin (ER) in these cells, but apparently was absent from the nucleus and has less diffuse distribution pattern in comparison to the nucleocytoplamic EGFP-scNS3, as was expected. A strong colocalization, however, was observed between EGFP-full NS3-4A and a chimeric fluorescent protein which was fused to the C-terminal ER membrane anchor of the tyrosine phosphatase PTP1B [42] (data not shown). Immunoblot analysis also shows efficient cleavage of MBP-EGFP-NS5AB-CBD (referred to as the “cleavable substrate” (Fig. 1D)) in cells transfected with pCMV/MBP-EGFP-NS5AB-CBD and induced for NS3 expression (Fig. 1E). A faint band, corresponding to the cleavage product, was also detected in a sample from uninduced cells. This is probably due to some degree of “leakiness” of the Tet inducible system, allowing a very low basal transcription of the protease in the absence of externally added Tet. In support to this assumption, only a single band corresponding to the full length substrate was detected when “naïve” HEK293 T-Rex cells were transfected with the substrate encoding vector (data not shown).

Bottom Line: Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window.The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells.This suggests that the "zymoxin" approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, Israel.

ABSTRACT
The synthesis of inactive enzyme precursors, also known as "zymogens," serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated "zymogenized" chimeric toxins (which we denote "zymoxins"). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the "zymoxin" approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

Show MeSH
Related in: MedlinePlus