Japanese encephalitis virus NS2B-NS3 protease binding to phage-displayed human brain proteins with the domain of trypsin inhibitor and basic region leucine zipper.
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.
Lin
Cheng-Wen
CW
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan, Taiwan, ROC. cwlin@mail.cmu.edu.tw
Lin
Kuan-Hsun
KH
Lyu
Ping-Chiang
PC
Chen
Wei-June
WJ
D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D015723:Gene Library; D006801:Humans; D047468:Immunoprecipitation; D016350:Leucine Zippers / Q000235:genetics; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D019151:Peptide Library; D011485:Protein Binding; D020365:RNA Helicases / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors; D014361:Trypsin Inhibitors / Q000737:chemistry*; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D009419:Nerve Tissue Proteins; D019151:Peptide Library; D020365:RNA Helicases; D012697:Serine Endopeptidases; D018808:Transcription Factor AP-1; D014361:Trypsin Inhibitors; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases