title: A screen for genetic suppressor elements of hepatitis C virus identifies a supercharged protein inhibitor of viral replication.
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.
authors.affiliation: Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Rudo L
authors.initials: RL
authors.lastname: Simeon
authors.affiliation: Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, Texas, United States of America ; Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, Texas, United States of America.
authors.forename: Zhilei
authors.initials: Z
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology*; D016923:Cell Death; D016368:Frameshift Mutation / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000494:pharmacology*; D016174:Hepacivirus / Q000235:genetics / Q000472:pathogenicity*; D006526:Hepatitis C / Q000378:metabolism / Q000517:prevention & control* / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D013489:Suppression, Genetic / Q000502:physiology*; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D019151:Peptide Library; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins