Abstract
Genetic suppressor elements (GSEs) are biomolecules derived from a gene or genome of interest that act as transdominant inhibitors of biological functions presumably by disruption of critical biological interfaces. We exploited a cell death reporter cell line for hepatitis C virus (HCV) infection, n4mBid, to develop an iterative selection/enrichment strategy for the identification of anti-HCV GSEs. Using this approach, a library of fragments of an HCV genome was screened for sequences that suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 derives from a single-base frameshift of the enhanced green fluorescent protein (eGFP) which was used as a filler during fragment cloning. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa) ratio of 1.5. We show that B1 expression specifically inhibits HCV replication. In addition, five highly positively charged B1 fragments produced from progressive truncation at the C-terminus all retain the ability to inhibit HCV, suggesting that a high positive charge, rather than a particular motif in B1, likely accounts for B1’s anti-HCV activity. Another supercharged protein, +36GFP, was also found to strongly inhibit HCV replication when added to cells at the time of infection. This study reports a new methodology for HCV inhibitor screening and points to the anti-HCV potential of positively charged proteins/peptides.
Highlights
Hepatitis C virus (HCV) is a single-stranded, enveloped, positive-sense RNA virus of the Flaviviridae family [1]
DNA fragments sized in the range 100– 200 bp were obtained by DNaseI digestion of a plasmid encoding full-length Jc1 HCV [30]
HCV replicates in complexes associated with the lipid raft membrane [34,35] and HCV nonstructural (NS) proteins have been detected around lipid droplets in most HCV-permissive cells [36]
Summary
Hepatitis C virus (HCV) is a single-stranded, enveloped, positive-sense RNA virus of the Flaviviridae family [1]. HCV RNA polymerase exhibits a high mutation rate, causing the virus to exist as a quasispecies in a single patient [2]. Until recently the only approved HCV therapy involved a 24 or 48 week regimen of combination therapy using pegylated interferon alpha and ribavirin [8,9]. Interferon a-ribavirin therapy yields a sustained virological response (SVR) in only 50% of treated patients infected with the most common genotype [12]. Recent pharmacological advances have led to the development and approval of two new drugs, boceprevir and telaprevir, which greatly improve the treatment response to up to 79% of the patients [13,14]. Molecules that target specific viral proteins, including boceprevir, telaprevir and most of those in advanced clinical development, tend to foster drug-resistant variants [15,16]
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