Journal
JOURNAL OF BIOMOLECULAR SCREENING
Volume 16, Issue 3, Pages 363-369Publisher
SAGE PUBLICATIONS INC
DOI: 10.1177/1087057110396215
Keywords
hepatitis C virus; p7 viroporin; liposome leakage; high-throughput screening
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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. (Journal of Biomolecular Screening 2011;16:363-369)
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