Open AccessDevelopment and Validation of a High-Throughput Screening Assay for the Hepatitis C Virus p7 Viroporin
Author(s) -
Christian Gervais,
Florence Dô,
Ariane Cantin,
George Kukolj,
Peter W. White,
Annick Gauthier,
Frédéric H. Vaillancourt
Publication year - 2011
Publication title -
slas discovery
Language(s) - English
Resource type - Journals
eISSN - 2472-5560
pISSN - 2472-5552
DOI - 10.1177/1087057110396215
Subject(s) - high throughput screening , liposome , recombinant dna , melittin , escherichia coli , chromatography , biology , biochemistry , small interfering rna , microbiology and biotechnology , chemistry , peptide , rna , virology , gene
The HCV p7 protein is not involved in viral RNA replication but is essential for production of infectious virus. Based on its putative ion channel activity, p7 belongs to a family of viral proteins known as viroporins that oligomerize after insertion into a lipid membrane. To screen for compounds capable of interfering with p7 channel function, a low-throughput liposome-based fluorescent dye permeability assay was modified and converted to a robust high-throughput screening assay. Escherichia coli expressing recombinant p7 were grown in high-density fed-batch fermentation followed by a detergent-free purification using a combination of affinity and reversed-phase chromatography. The phospholipid composition of the liposomes was optimized for both p7 recognition and long-term stability. A counterscreen was developed using the melittin channel-forming peptide to eliminate nonspecific screening hits. The p7 liposome-based assay displayed robust statistics (Z' > 0.75), and sensitivity to inhibition was confirmed using known inhibitors.