Japanese encephalitis virus NS2B-NS3 protease binding to phage-displayed human brain proteins with the domain of trypsin inhibitor and basic region leucine zipper

Virus Res. 2006 Mar;116(1-2):106-13. doi: 10.1016/j.virusres.2005.09.002. Epub 2005 Nov 10.

Abstract

Flavivirus NS2B-NS3 proteases are associated with neurovirulence, becoming an important target for insight into the virus-induced pathogenesis. In this study, a phage-displayed human brain cDNA library was used to detect possible interaction between brain proteins and the Japanese encephalitis virus (JEV) NS2B-NS3 protease. After six rounds of biopanning, eight high-affinity NS2B-NS3 protease-interacting phages were identified. Identified NS2B-NS3 protease-interacting brain proteins contained several repeats of the consensus motifs E(R/K)(R/K)K and G(R/K)(R/K) with the dibasic residues, being similar to the conserved cleavage sites among flavivirus proteases. In addition, three identified brain proteins (phage-24, 34, and 44) were predicted as the domain of trypsin inhibitor and basic region leucine zipper (bZIP) using the SMART genome search. Immunoprecipitation and cleavage of two brain fusion proteins (phage-24 and phage-46) by the NS2B-NS3 protease confirmed the specific interaction between identified brain proteins and the JEV NS2B-NS3 protease. Fluorogenic peptide substrate assays revealed dose-manner inhibitory effects of these two brain fusion proteins on the trans-cleavage activity of NS2B-NS3 protease. Moreover, in vitro signaling pathway assay revealed that the JEV NS2B-NS3 protease significantly inhibited the signaling pathway of activator protein 1(AP1), a member of the bZIP family. Our results provide an insight into the protein interaction network of the JEV NS2B-NS3 protease in human brain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Chlorocebus aethiops
  • Gene Library
  • Humans
  • Immunoprecipitation
  • Leucine Zippers* / genetics
  • Molecular Sequence Data
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / metabolism*
  • Peptide Library
  • Protein Binding
  • RNA Helicases / metabolism
  • Serine Endopeptidases / metabolism
  • Signal Transduction
  • Transcription Factor AP-1 / antagonists & inhibitors
  • Trypsin Inhibitors / chemistry*
  • Vero Cells
  • Viral Nonstructural Proteins / metabolism*

Substances

  • NS3 protein, flavivirus
  • Nerve Tissue Proteins
  • Peptide Library
  • Transcription Factor AP-1
  • Trypsin Inhibitors
  • Viral Nonstructural Proteins
  • Serine Endopeptidases
  • RNA Helicases