Hepatitis C virus (HCV) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer. The high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for HCV. The HCV NS3 is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. Here, we report the development of a novel bacterial genetic screen for inhibitors of NS3 catalysis and its application for the isolation of single-chain antibody-inhibitors. Our screen is based on the concerted co-expression of a reporter gene, of recombinant NS3 protease and of fusion-stabilized single-chain antibodies (scFvs) in Escherichia coli. The reporter system had been constructed by inserting a short peptide corresponding to the NS5A/B cleavage site of NS3 into a permissive site of the enzyme β-galactosidase. The resulting engineered lacZ gene, coding for an NS3-cleavable β-galactosidase, is carried on a low copy plasmid that also carried the NS3 protease-coding sequence. The resultant β-galactosidase enzyme is active, conferring a Lac+ phenotype (blue colonies on indicator 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-gal) plates), while induction of NS3 expression results in loss of β-galactosidase activity (transparent colonies on X-gal plates). The identification of inhibitors, as shown here by isolating NS3-inhibiting single-chain antibodies, expressed from a compatible high copy number plasmid, is based on the appearance of blue colonies (NS3 inhibited) on the background of colorless colonies (NS3 active). Our source of inhibitory scFvs was an scFv library that we prepared from spleens of NS3-immunized mice and subjected to limited affinity selection. Once isolated, the inhibitors were validated as genuine and specific NS3 binders by an enzyme-linked immunosorbent assay and as bone fide NS3 serine protease inhibitors by an in vitro catalysis assay. We further show that upon expression as cytoplasmic intracellular antibodies (intrabodies) in NS3-expressing mammalian cells, three of the scFvs inhibit NS3-mediated cell proliferation. Although applied here for the isolation of antibody-based inhibitors, our genetic screen should be applicable for the identification of candidate inhibitors from other sources.
Bibliographical noteFunding Information:
This work was supported by research grants from the United States Israel Binational Science Foundation (BSF) and from the Israel Cancer Research Fund (ICRF). M.G.-T. was supported by a travel fellowship from the Joan and Jaime Constantiner Institute for Molecular Genetics.
- Genetic screen
- NS3 protease