Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jun;50(3):277-285.
doi: 10.1016/j.jmii.2015.07.002. Epub 2015 Jul 31.

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  1 , Tzu-Ying Lee  2 , Ying-Ju Lin  3 , Lei Wan  3 , Chih-Ho Lai  4 , Cheng-Wen Lin  5
Affiliations

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 et al. J Microbiol Immunol Infect. 2017 Jun.

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.

Keywords: IFN antagonism; NPIPB3; ORF6; SARS-CoV; phage display.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Expression and purification of Escherichia coli-synthesized SARS-CoV ORF6 protein. The ORF6 gene was amplified using PCR and cloned into pTriEx-4 Neo vector (A). ORF6-His tagged proteins were synthesized in transformed E. coli BL21 (DE3), purified using immobilized-metal affinity chromatography, separated using SDS-PAGE, and then examined using Western blotting with anti-His tag (B). ORF = open reading frame; PCR = polymerase chain reaction; SARS-CoV = severe acute respiratory syndrome coronavirus; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Figure 2
Figure 2
Biopanning of phage display a human lung cDNA library with ORF6-His tagged protein. After the fifth round of biopanning with ORF6, each phage clone was randomly picked up from individual plaques, amplified in Escherichia coli, and performed using direct binding ELISA with ORF6-coated plates and antiphage antibodies. * p < 0.05, by Scheffe's test. ** p < 0.01, by Scheffe's test. *** p < 0.001, by Scheffe's test. ELISA = enzyme-linked immunosorbent assay; ORF = open reading frame.
Figure 3
Figure 3
Direct binding ELISA of high-affinity phage clones to ORF6-His tagged protein. High-affinity phage clone was amplified in Escherichia coli, quantitated using plaque assay, and then performed by direct binding ELISA with ORF6-coated plates and antiphage antibodies. * p < 0.05, by Scheffe's test. ** p < 0.01, by Scheffe's test. *** p < 0.001, by Scheffe's test. ELISA = enzyme-linked immunosorbent assay; ORF = open reading frame.
Figure 4
Figure 4
Coimmunoprecipitation of ORF6-His tagged protein with Trx-NPIPB3 fusion protein. Purified ORF6-His tagged protein mixed with Trx-NPIPB3 fusion protein was incubated with anti-Trx antibodies at 4°C overnight, followed by incubation with protein A-Sepharose beads for a further 2 hours. After centrifugation, the pellet was washed with NET buffer, samples were analyzed by SDS-PAGE, Western blotting, and immunoanalysis using rabbit anti-Trx and mouse anti-His tag antibodies. NPIPB3 = nuclear pore complex interacting protein family, member B3; ORF = open reading frame; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Trx = thioredoxin.
Figure 5
Figure 5
Colocalization analysis of SARS-CoV ORF6 and C-terminal domain of NPIPB3 in human promonocytes using confocal microscopy. Transfected cells with single or both of pTriEx-ORF6 and pDsRed-NPIPB3 were fixed, and stained with anti-His tag, followed by FITC-conjugated antimouse immunoglobulin G antibodies, and then analyzed by confocal microscopy. FITC = fluorescein isothiocyanate; NPIPB3 = nuclear pore complex interacting protein family, member B3; ORF = open reading frame; SARS-CoV = severe acute respiratory syndrome coronavirus; Trx = thioredoxin.
Figure 6
Figure 6
Effect ofNPIPB3 overexpression on the activity of the ISRE-based cis reporter. Cells were transiently cotransfected with single or both of pTriEx-ORF6 and pDsRed-NPIPB3, plus control and ISRE luciferase reporters. Firefly and renilla luciferase enzymes were measured 4 hours after IFN-β treatment. According to the dual Luciferase Reporter Assay System, the relative firefly luciferase activity was normalized by renilla luciferase. * p < 0.05, by Scheffe's test. ** p < 0.01, by Scheffe's test. *** p < 0.001, by Scheffe's test. IFN = interferon; ISRE = IFN stimulated response element; NPIPB3 = nuclear pore complex interacting protein family, member B3; ORF = open reading frame.
Figure 7
Figure 7
Effect of NPIPB3 overexpression on IFN-β-induced nuclear translocation of STAT1 in vector control and ORF6-expressing cells. (A) Mock and (B) IFN-β-treated cells transfected with single or both of pTriEx-ORF6 and pDsRed-NPIPB3 were fixed, and reacted with anti-STAT1 and FITC-conjugated antimouse immunoglobulin G antibodies. Finally, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 minutes, imaging analyzed by confocal microscopy. FITC = fluorescein isothiocyanate; IFN = interferon; NPIPB3 = nuclear pore complex interacting protein family, member B3; ORF = open reading frame; STAT1 = signal transducer and activator of transcription.
Figure 8
Figure 8
Effect of NPIPB3 overexpression on IFN-β-induced STAT1 phosphorylation at Tyr701 in vector control and ORF6-expressing cells using Western blotting. Cells were transiently transfected with single or both of pTriEx-ORF6 and pDsRed-NPIPB3, and then treated with IFN-β. After 0 minutes, 15 minutes, and 30 minutes of treatment, the lysate was analyzed by Western blotting with antiphosphotyrosine STAT1 (Tyr701), and anti-β-actin antibodies. The immune complexes were visualized using horseradish peroxidase-conjugated goat antimouse immunoglobulin G antibodies and enhanced chemiluminescence. IFN = interferon; NPIPB3 = nuclear pore complex interacting protein family, member B3; ORF = open reading frame; STAT1 = signal transducer and activator of transcription.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Drosten C., Günther S., Preiser W., van der Werf S., Brodt H.R., Becker S. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med. 2003;348:1967–1976. - PubMed
    1. Marra M.A., Jones S.J., Astell C.R., Holt R.A., Brooks-Wilson A., Butterfield Y.S. The Genome sequence of the SARS-associated coronavirus. Science. 2003;300:1399–1404. - PubMed
    1. Tan Y.J., Lim S.G., Hong W. Characterization of viral proteins encoded by the SARS-coronavirus genome. Antiviral Res. 2005;65:69–78. - PMC - PubMed
    1. Narayanan K., Huang C., Makino S. SARS coronavirus accessory proteins. Virus Res. 2008;133:113–121. - PMC - PubMed
    1. Liu D.X., Fung T.S., Chong K.K., Shukla A., Hilgenfeld R. Accessory proteins of SARS-CoV and other coronaviruses. Antiviral Res. 2014;109:97–109. - PMC - PubMed

MeSH terms