Selection and target-site mapping of peptides inhibiting HCV NS5B polymerase using phage display.
A series of peptides binding to the HCV NS5B polymerase was selected from phage display peptide libraries. A conserved motif of Ser-Arg-X-Arg/Leu was identified among the selected peptides, and Pep2 (Trp-Ser-Arg-Pro-Arg- Ser-Leu) was chosen for further characterization. The binding of Pep2 to HCV NS5B in vivo was shown by a yeast twohybrid assay and by subcellular colocalization analysis using immunofluorescence confocal microscopy. The in vitro interaction was also confirmed by GST pulldown assay. The replication of the HCV 1b subgenomic replicon was efficiently inhibited by the presence of the peptide. By using a subtractive biopanning against Pep2, the binding site of the peptide was mapped at the pocket of Pro388 to Pro391 in the thumb subdomain of the polymerase. A yeast two-hybrid analysis using Pro388Ala and Pro391Ala mutants of NS5B confirmed the binding.
Kim
Min Soo
MS
Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin 449-791, Korea.
Park
Chanhee
C
Lee
Jong-Ho
JH
Myung
Heejoon
H
D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D016174:Hepacivirus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008647:Mesocricetus; D019151:Peptide Library; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D019151:Peptide Library; D010455:Peptides; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase