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.
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