title: Phage display technique identifies the interaction of severe acute respiratory syndrome coronavirus open reading frame 6 protein with nuclear pore complex interacting protein NPIPB3 in modulating Type I interferon antagonism.
abstract: OBJECTIVE
  Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins including ORF6 inhibit Type I interferon (IFN) signaling.
  
  
  METHODS
  This study identified SARS-CoV ORF6-interacting proteins using the phage displayed human lung cDNA libraries, and examined the association of ORF6-host factor interaction with Type I IFN antagonism. After the fifth round of biopanning with Escherichia coli-synthesized ORF6-His tagged protein, the relative binding affinity of phage clones to ORF6 was determined using direct enzyme-linked immunosorbent assay.
  
  
  RESULTS
  The highest affinity clone to ORF6 displayed the C-terminal domain of NPIPB3 (nuclear pore complex interacting protein family, member B3; also named as phosphatidylinositol-3-kinase-related kinase SMG-1 isoform 1 homolog). The coimmunoprecipitation assay demonstrated the direct binding of ORF6 to the C-terminal domain of NPIPB3 in vitro. Confocal imaging revealed a close colocalization of SARS-CoV ORF6 protein with NPIPB3 in human promonocytes. The dual luciferase reporter assay showed that the C-terminal domain of NPIPB3 attenuated the antagonistic activity of SARS-CoV ORF6 on IFN-β-induced ISRE (IFN stimulated response element)-responsive firefly luciferase activity. In addition, confocal imaging and Western blotting assays revealed that the increases in STAT-1 nuclear translocation and phosphorylation occurred in the transfected cells expressing both genes of ORF6 and NPIPB3, but not in the ORF6-expressing cells in response to IFN-β.
  
  
  CONCLUSIONS
  The overexpression of NPIPB3 restored the IFN-β responses in SARS-CoV ORF6 expressing cells, indicating that the interaction of SARS CoV ORF6 and NPIPB3 reduced Type I IFN antagonism by SARS-CoV ORF6.
authors: Department of Biotechnology, Asia University, Wufeng, Taichung, Taiwan.
authors: Su-Hua
authors: SH
authors: Huang
authors: Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan.
authors: Tzu-Ying
authors: TY
authors: Lee
authors: Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung, Taiwan.
authors: Ying-Ju
authors: YJ
authors: Lin
authors: Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung, Taiwan.
authors: Lei
authors: L
authors: Wan
authors: School of Medicine, Department of Microbiology, China Medical University, Taichung, Taiwan.
authors: Chih-Ho
authors: CH
authors: Lai
authors: Department of Biotechnology, Asia University, Wufeng, Taichung, Taiwan; Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan. Electronic address: cwlin@mail.cmu.edu.tw.
authors: Cheng-Wen
authors: CW
authors: Lin
mesh_terms: D061505:Cell Surface Display Techniques; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D054884:Host-Pathogen Interactions; D006801:Humans; D047468:Immunoprecipitation; D007370:Interferon Type I / Q000037:antagonists & inhibitors*; D018613:Microscopy, Confocal; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D045473:Severe acute respiratory syndrome-related coronavirus / Q000502:physiology*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D007370:Interferon Type I; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D014764:Viral Proteins; C514704:SMG1 protein, human
keywords: IFN antagonism; NPIPB3; ORF6; SARS-CoV; phage display