Movatterモバイル変換

[0]ホーム
URL:

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

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

BioMed Central full text link BioMed Central Free PMC article
Full text links

Actions

Share

doi: 10.1186/1743-422X-8-25.

Analysis of the PDZ binding specificities of Influenza A virus NS1 proteins

Affiliations

Analysis of the PDZ binding specificities of Influenza A virus NS1 proteins

Miranda Thomas et al. Virol J..

Abstract

The Influenza A virus non-structural protein 1 (NS1) is a multifunctional virulence factor with several protein-protein interaction domains, involved in preventing apoptosis of the infected cell and in evading the interferon response. In addition, the majority of influenza A virus NS1 proteins have a class I PDZ-binding motif at the C-terminus, and this itself has been shown to be a virulence determinant.In the majority of human influenza NS1 proteins the consensus motif is RSxV: in avian NS1 it is ESxV. Of the few human strains that have the avian motif, all were from very high mortality outbreaks of the disease. Previous work has shown that minor differences in PDZ-binding motifs can have major effects on the spectrum of cellular proteins targeted. In this study we analyse the effect of these differences upon the binding of Influenza A virus NS1 protein to a range of cellular proteins involved in polarity and signal transduction.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Human and avian type NS1 protens differ in PDZ-binding activity. A GST-pulldown assay was performed using bacterially expressed GST and GST-Dlg, with GST-p53 as a non-PDZ-containing control. These were incubated with in vitro translated radiolabelled Avian type (A) or Human type (H) NS1 proteins. After extensive washing the bound proteins were eluted and analysed by SDS-PAGE and autoradiography (upper panel). The lower panel shows the Coomassie-stained gel; the GST fusion proteins are arrowed.
Figure 2
Figure 2
The NS1 binding to Dlg is PDZ-dependent.A. The cartoon shows the last 11 amino acid residues of Avian (A) and Human (H) NS1, together with the non-PDZ-binding mutant of Avian NS1 (Aa).B. GST-pulldown assay, using the NS1 proteins shown in panel A.C. Histogram showing the collated results of at least 3 such assays.
Figure 3
Figure 3
Mapping the site of NS1 binding on Dlg.A. A cartoon showing the GST-Dlg wild type and mutant fusion proteins used in this assay.B. GST pulldown assay, as before, using the mutants shown in 3A.C. Histogram showing the collated results of at least three such assays.
Figure 4
Figure 4
The non-canonical residues of the PBM determine NS1 binding affinity for Dlg.Upper panel. The cartoon shows the last 11 amino acid residues of Avian (A) and Human (H) NS1, together with the non-PDZ-binding mutant of Avian NS1 (Aa), plus the avian human-like (Ah) and the human avian-like (Ha) mutants.Lower panel. GST pulldown assay using these NS1 proteins.
Figure 5
Figure 5
The avian PBM is not sufficient for binding all class 1 PDZ domains.Upper panel. The cartoon shows the wild type MAGI-1c and the GST fusion proteins M1P1 and M1P5.Lower Panel. A GST pulldown assay was performed using these fusion proteins together with the NS1 proteins described in Figure 4.
Figure 6
Figure 6
Different PDZ domains have different binding characteristics for the same ligand.Upper panel. The cartoon shows the GST-NTMAGI-1 and GST-CTMAGI-1 fusion proteins. Also shown is the GST-Scrib4PDZ fusion protein, which comprises all four of the PDZ domains of Scribble, aligned with the full-length protein for reference.Lower panel. A GST pulldown assay was performed with these fusion proteins plus the GST alone and GST-DLG as negative and positive controls, respectively. They were incubated as before with thein vitrotranslated Avian, Human and non-PDZ binding mutant NS1 proteins. Human papillomavirus type 18 E6 was included for comparison.
Figure 7
Figure 7
Expression of wild type avian NS1 reduces Interferon-induced STAT1 activation.Upper panel. Western blot analysis of 293 cells transfected with plasmids expressing wild type avian NS1 (A), wild type human NS1 (H), non-PDZ binding avian NS1 mutant (Aa) or vector alone (C). Cells were treated for 5 h with 1 × 104U/ml Hplc-purified Interferon-α prior to harvesting. The blot was probed with anti phospho-STAT-1 antibodies to detect activated STAT1.Lower panel. The blot was reprobed with anti-α-actinin antibody to control for cellular protein input.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Zhirnov OP, Konakova TE, Wolff T, Klenk TD. NS1 protein of influenza A virus down-regulates apoptosis. J Virol. 2002;76:1617–1625. doi: 10.1128/JVI.76.4.1617-1625.2002. - DOI - PMC - PubMed
    1. Ehrhardt C, Wolff T, Pleschka S, Planz O, Beermann W, Bode JG, Schmolke M, Ludwig S. Influenza A virus NS1 protein activates the PI3K/Akt pathway to mediate antiapoptotic signaling responses. J Virol. 2007;81:3058–3067. doi: 10.1128/JVI.02082-06. - DOI - PMC - PubMed
    1. Shin YK, Li Y, Liu Q, Anderson DH, Babiuk LA, Zhou Y. SH3 binding motif 1 in influenza A virus NS1 protein is essential for PI3K/Akt signaling pathway activation. J Virol. 2007;81:12730–12739. doi: 10.1128/JVI.01427-07. - DOI - PMC - PubMed
    1. Hale BG, Randall RE. PI3K signalling during influenza A virus infections. Biochem Soc Trans. 2007;35:186–187. doi: 10.1042/BST0350186. - DOI - PubMed
    1. Obenauer JC, Denson J, Mehta PK, Su X, Mukatira S, Finkelstein DB, Xu X, Wang J, Ma J, Fan Y, Rakestraw KM, Webster RG, Hoffmann E, Krauss S, Zheng J, Zhang Z, Naeve CW. Large-scale sequence analysis of avian influenza isolates. Science. 2006;311:1576–1580. doi: 10.1126/science.1121586. - DOI - PubMed

MeSH terms

Substances

Related information

LinkOut - more resources

Full text links
BioMed Central full text link BioMed Central Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp