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.2011 Oct;85(20):10639-48.
doi: 10.1128/JVI.05070-11. Epub 2011 Aug 17.

The avian influenza virus NS1 ESEV PDZ binding motif associates with Dlg1 and Scribble to disrupt cellular tight junctions

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The avian influenza virus NS1 ESEV PDZ binding motif associates with Dlg1 and Scribble to disrupt cellular tight junctions

Lisa Golebiewski et al. J Virol.2011 Oct.

Abstract

The influenza A virus NS1 protein contains a conserved 4-amino-acid-residue PDZ-ligand binding motif (PBM) at the carboxyl terminus that can function as a virulence determinant by targeting cellular PDZ proteins. The NS1 proteins from avian and human viral isolates have consensus PBM sequences ESEV and RSKV, respectively. Currently circulating highly pathogenic H5N1 viruses contain the ESEV PBM which specifically associates with the PDZ proteins Scribble, Dlg1, MAGI-1, MAGI-2, and MAGI-3. In this study, we found NS1 proteins from viral isolates that contain the PBM sequence RSKV, KSEV, or EPEV are unable to associate with these PDZ proteins. Other results showed that the ESEV PBM mediates an indirect association with PDZ protein, Lin7C, via an interaction with Dlg1. Infection with a virus that expresses a NS1 protein with the ESEV PBM results in colocalization of NS1, Scribble, and Dlg1 within perinuclear puncta and mislocalization of plasma membrane-associated Lin7C to the cytoplasm. Infection of polarized MDCK cells with the ESEV virus additionally results in functional disruption of the tight junction (TJ) as measured by altered localization of TJ markers ZO-1 and Occludin, decreased transepithelial electrical resistance, and increased fluorescein isothiocyanate (FITC)-inulin diffusion across the polarized cell monolayer. A similar effect on the TJ was observed in MDCK cells depleted for either Scribble or Dlg1 by small interfering RNA (siRNA). These findings indicate that ESEV PBM-mediated binding of NS1 to Scribble and Dlg1 functions to disrupt the cellular TJ and that this effect likely contributes to the severe disease associated with highly pathogenic H5N1 influenza A viruses.

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Figures

Fig. 1.
Fig. 1.
Requirements of Dlg1 and Lin7C for binding to the avian influenza NS1 proteinin vitro. (A) Domain structure of Dlg1 is shown: three PDZ domains, a SRC homology domain 3 (SH3), an I3 domain, and an inactive guanylate kinase (GK) domain. Deletion mutants of the indicated PDZ domains are shown. (B) The indicated GFP-tagged Dlg1 deletion mutants were transfected into 293T cells, cell extracts were prepared at 24 h posttransfection, and binding assays performed with the GST-NS1 ED containing the ESEV PBM. Products of binding reactions were evaluated by immunoblots using GFP antisera; binding reaction products were also evaluated by Coomassie blue staining shown in the bottom panel. Lanes indicated by “−” represent input in binding reactions; lanes indicated by “+” are products of the binding reactions. (C) 293T cells were transfected with siRNAs targeting Scribble or Dlg1 for 48 h; cultures were then transfected with full-length FLAG-tagged NS1 protein (H6N6) with indicated PBM or were mock transfected. Lysates were prepared at 24 h posttransfection and used in coimmunoprecipitations. Products of immunoprecipitations were analyzed in immunoblots.
Fig. 2.
Fig. 2.
In vitro binding assays with NS1 PBMs from a variety of viral isolates. (A) 293T cells were transfected with expression plasmids for the indicated epitope-tagged PDZ proteins, cell extracts were prepared at 48 h posttransfection, and binding assays were performed with GST-NS1 EDs containing the indicated PBM sequences. Products of binding reactions were examined in immunoblots. Coomassie blue stains of the products of binding reactions confirmed that equivalent amounts of GST-NS1 fusion proteins were used in binding assays (not shown). (B) H5N1 and H6N6 GST-NS1 ED fusion proteins were used in binding assays with extracts from 293T cells transfected with the PDZ protein expression plasmids indicated in panel A. The products of binding assays were resolved by SDS-PAGE and examined by immunoblotting. Gels were also stained with Coomassie blue to demonstrate that equivalent levels of GST-NS1 ED proteins were used in binding assays (not shown). (C) 293T cells were transfected with full-length H6N6 or H3N2 FLAG-tagged NS1 protein with the indicated PBMs. Cell extracts were prepared 24 h posttransfection and used in coimmunoprecipitations. Products of immunoprecipitations were examined by immunoblotting.
Fig. 3.
Fig. 3.
Avian NS1 with ESEV PBM colocalizes with Scribble and Dlg1 in cytoplasmic puncta while disrupting Lin7C and tight junction markers. (A) A549 cells were infected at an MOI of 3 with the indicated influenza viruses. Cells were fixed at 10 h postinfection for immunofluorescence analysis with Dlg1, Scribble, and NS1 antisera. (B) Enlargement of indicated infected cell. Puncta with Scribble, Dlg1, and NS1 are displayed as white; puncta with only NS1 and Dlg1 are displayed as cyan; puncta with Scribble and Dlg1 are displayed as yellow; puncta with NS1 and Scribble are displayed as violet. (C) A549 cells were infected at an MOI of 3 with the indicated influenza viruses, and cells were fixed at 10 h postinfection for immunofluorescence analysis with Lin7C and nucleoprotein antisera. (D) A549 cells were infected at an MOI of 3 with the indicated influenza virus, and cells were fixed at 10 h postinfection for immunofluorescence analysis with Lin7C, Scribble, and Dlg1 antisera. (E) MDCK cells were infected at an MOI of 3 with the indicated viruses. Cells were fixed at 10 h postinfection for immunofluorescence analysis with nucleoprotein, ZO-1, or Occludin antisera.
Fig. 4.
Fig. 4.
ESEV PBM of NS1 disrupts tight junctions and increases leakage of infected monolayers. (A) Polarized MDCK cells grown on transwell filter inserts were infected in the apical chamber with an MOI of 1 with the indicated virus or were mock infected. TER was measured at 0, 3, and 6 h postinfection. Readings are expressed as a percentage of mock-infected monolayer TER. Error bars indicate standard deviations from three independent experiments (*,P < 0.05 by Student'st test) with at least four transwell inserts per infection condition. Monolayers were processed for immunofluorescence and stained for influenza virus NP and Scribble (data not shown). Five random fields from two monolayers were then counted for the presence of nucleoprotein. ESEV monolayers were 36.1% infected and ESEA monolayers were 35.8% infected. (B) MDCK cells were reverse transfected with siRNA targeting canine Dlg1, canine Scribble, or negative-control siRNA and grown on transwell filter inserts. TER was measured on days 3 and 4 posttransfection. Readings are expressed as a percentage of negative-control siRNA-treated monolayer TER. Data shown are from one representative experiment that was repeated in triplicate. Error bars indicate standard deviations with four transwell inserts per experimental condition (*,P < 0.05 by Student'st test). (C) MDCK inserts from panel C were fixed on day 4 posttransfection for immunofluorescence analysis with Dlg1, Scribble, ZO-1, and Occludin antisera. Scribble is displayed as blue, Dlg1 as red, and ZO-1 or Occludin as green. Images are X-Z representations from a Z-stack. (D) Polarized monolayers were infected with indicated viruses at an MOI of 1. After 1 h, the apical inoculum was replaced with medium containing FITC-inulin (2 mg/ml). Samples were removed from the basolateral chamber at 3 and 6 h postinfection and quantified by fluorescence spectrometry. Paracellular permeability was calculated as a percentage of mock permeability. Error bars indicate standard deviations from three independent experiments (*,P < 0.05 by Student'st test) with at least four transwell inserts per infection condition.
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