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

Su Hua Huang, Tzu Ying Lee, Ying Ju Lin, Lei Wan, Chih Ho Lai, Cheng Wen Lin*

*此作品的通信作者

研究成果: 期刊稿件文章同行評審

18 引文 斯高帕斯(Scopus)

摘要

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.

原文英語
頁(從 - 到)277-285
頁數9
期刊Journal of Microbiology, Immunology and Infection
50
發行號3
DOIs
出版狀態已出版 - 06 2017
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© 2015

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