Dataset Information
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.
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ABSTRACT: Background/purpose
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.Conclusion
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.
SUBMITTER: Huang SH
PROVIDER: S-EPMC7105049 | biostudies-literature | 2017 Jun
REPOSITORIES: biostudies-literature
Publications
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.
Journal of microbiology, immunology, and infection = Wei mian yu gan ran za zhi 20150731 3
<h4>Background/purpose</h4>Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins including ORF6 inhibit Type I interferon (IFN) signaling.<h4>Methods</h4>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 c ...[more]
