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. 2010 Nov;84(21):11164-74.
doi: 10.1128/JVI.01278-10. Epub 2010 Aug 11.

The ESEV PDZ-binding motif of the avian influenza A virus NS1 protein protects infected cells from apoptosis by directly targeting Scribble

Hongbing Liu  1 , Lisa Golebiewski, Eugene C Dow, Robert M Krug, Ronald T Javier, Andrew P Rice
Affiliations

The ESEV PDZ-binding motif of the avian influenza A virus NS1 protein protects infected cells from apoptosis by directly targeting Scribble

Hongbing Liu et al. J Virol. 2010 Nov.

Abstract

The NS1 protein from influenza A viruses contains a four-amino-acid sequence at its carboxyl terminus that is termed the PDZ-binding motif (PBM). The NS1 PBM is predicted to bind to cellular PDZ proteins and functions as a virulence determinant in infected mice. ESEV is the consensus PBM sequence of avian influenza viruses, while RSKV is the consensus sequence of human viruses. Currently circulating highly pathogenic H5N1 influenza viruses encode an NS1 protein with the ESEV PBM. We identified cellular targets of the avian ESEV PBM and identified molecular mechanisms involved in its function. Using glutathione S-transferase (GST) pull-down assays, we found that the ESEV PBM enables NS1 to associate with the PDZ proteins Scribble, Dlg1, MAGI-1, MAGI-2, and MAGI-3. Because Scribble possesses a proapoptotic activity, we investigated the interaction between NS1 and Scribble. The association between NS1 and Scribble is direct and requires the ESEV PBM and two Scribble PDZ domains. We constructed recombinant H3N2 viruses that encode an H6N6 avian virus NS1 protein with either an ESEV or mutant ESEA PBM, allowing an analysis of the ESEV PBM in infections in mammalian cells. The ESEV PBM enhanced viral replication up to 4-fold. In infected cells, NS1 with the ESEV PBM relocalized Scribble into cytoplasmic puncta concentrated in perinuclear regions and also protected cells from apoptosis. In addition, the latter effect was eliminated by small interfering RNA (siRNA)-mediated Scribble depletion. This study shows that one function of the avian ESEV PBM is to reduce apoptosis during infection through disruption of Scribble's proapoptotic function.

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Figures

FIG. 1.
FIG. 1.
GST-NS1 in vitro binding assays. (A) The indicated GST fusion proteins (NS1 ED + PBM) attached to glutathione-Sepharose beads were incubated with 293T cell extracts. Bead complexes were washed and then incubated with [γ-32P]ATP under kinase reaction conditions. Products of kinase reactions were analyzed on a 9% SDS-polyacrylamide gel. A Colloidal blue stain (showing GST-NS1 proteins used in the assay) and autoradiograph of the dried gel are shown. Major substrates of the kinase reaction with the GST-avNS1 protein are indicated by asterisks. (B and C) Cultures of 293T cells were transfected with expression plasmids for the indicated epitope-tagged PDZ proteins. Cell extracts were prepared and incubated with the indicated GST proteins (NS1 ED + PBM) attached to glutathione-Sepharose beads. Proteins bound to GST fusion proteins were examined in immunoblots using the appropriate antisera to detect epitope tags. The amounts of input GST-NS1 proteins used in binding assays are shown at the bottom (Coomassie-stained gel). (D) Cultures of 293T cells were transfected with expression plasmids for epitope-tagged Scribble or Dlg1. Cell extracts were prepared and incubated with the indicated GST proteins containing the H6N6 or H3N2 ED plus the indicated PBM. Scribble or Dlg1 bound to GST fusion proteins was examined in immunoblots. The amounts of input GST-NS1 proteins used in binding assays are shown at the bottom (Coomassie-stained gel).
FIG. 2.
FIG. 2.
Requirements of Scribble for binding in vitro to the avian influenza virus NS1 protein. (A) The domain structure of Scribble is shown: 16 leucine-rich repeats (LRRs), a LAPS domain (LAPSD), and four PDZ domains. Portions of Scribble expressed from Myc-tagged vectors are shown; the vectors 1F2R-1A and 1F2R-4A contain one and four alanine substitutions in PDZ domain two. The binding activities of Scribble proteins for avian influenza virus NS1 are summarized. (B) The indicated Myc-tagged Scribble vectors were transfected into 293T cells, extracts were prepared, and binding assays were performed with indicated GST proteins. Products of binding assays were evaluated in immunoblots.
FIG. 3.
FIG. 3.
Direct binding of Scribble to avian influenza virus NS1. (A) The GST-avNS1 and GST-avNS1-ESEA proteins bound to glutathione-Sepharose beads were incubated with purified His-Scribble-1F2R (see Fig. 4A) and washed, and proteins eluted with 2× SDS sample buffer. Eluates were analyzed on a 10% SDS-polyacrylamide gel. A Coomassie blue stain of the gel is shown. Immunoblotting with anti-His antiserum was performed on eluates to detect His-Scribble. (B) His-Scribble-1F2R was bound to Ni beads and incubated with purified GST-NS1 and GST-NS1-ESVA proteins. After binding, bead complexes were washed, and proteins were then eluted with 2× SDS sample buffer. Eluates were analyzed on a 10% SDS-polyacrylamide gel. A Coomassie blue stain of the gel is shown. Immunoblotting with anti-GST antiserum was performed on eluates to detect GST-NS1 proteins.
FIG. 4.
FIG. 4.
ESEV PBM enhances viral replication in MDCK cells. Duplicate cultures of confluent MDCK cells were infected at an MOI of 0.01 with recombinant H3N2 viruses encoding the H6N6 NS1 protein with ESEV or mutant ESEA PBM. Viral replication at 24 and 48 h postinfection was measured by plaque assays in MDCK cells, with triplicate culture dishes used to quantify plaques for each time point of the duplicate infections. Viral yields at 24 h postinfection (p.i.) were 3.6 × 106 and 1.4 × 106 for the ESEV and ESEA viruses, respectively. Viral yields at 48 h p.i. were 8.1 × 106 and 2.0 × 106 for the ESEV and ESEA viruses, respectively. Asterisk indicates P value of 0.005 according to Student's t test; error bars are standard deviations.
FIG. 5.
FIG. 5.
The avian NS1 protein colocalizes with Scribble in cytoplasmic puncta. A549 cells were infected at an MOI of 1 with the indicated influenza viruses, and cells were fixed at 10 h postinfection for immunofluorescence analysis with NS1 and Scribble antisera. DAPI stains were performed to visualize cell nuclei. Scale bar, 10 μm.
FIG. 6.
FIG. 6.
Avian NS1 sequesters Scribble in insoluble complexes. Cultures of 293T or A549 cells were infected with the indicated influenza viruses at an MOI of ∼2, and whole-cell extracts were prepared at 8 h postinfection. Cell lysates were fractionated into soluble and insoluble fractions. Amounts of soluble (S) and insoluble (I) cell fractions that represented equal numbers of cells were loaded on an 8% SDS-polyacrylamide gel. The indicated proteins were examined by immunoblotting.
FIG. 7.
FIG. 7.
ESEV PBM of NS1 reduces apoptosis during infection. (A) HeLa cells were infected with the indicated influenza viruses at an MOI of 5, and cells were fixed at 12 and 24 h postinfection. The percentages of apoptotic cells in two fields of at least 250 cells were quantified by a TUNEL assay. Error bars indicate standard deviations from four independent experiments (*, P < 0.05 by Student's t test). (). Cultures of HeLa cells were infected with the ESEV or ESEA virus at an MOI of 5, and cell extracts were prepared at 6 h postinfection. Cleavage of PARP-1 was evaluated in an immunoblot, and β-actin was used as a loading control. Quantitation of the immunoblot indicated that approximately 19% or 31% of full-length PARP-1 was cleaved in infection with the ESEV or ESEA virus, respectively. (C) HeLa cells were transfected with the indicated siRNAs and, 48 h later, infected with the indicated viruses at an MOI of 5. The percentages of apoptotic cells in random fields of at least 250 cells were quantified by a TUNEL assay. Error bars indicate standard deviations from four independent experiments (**, P < 0.05 by Student's t test). An immunoblot verified that the siRNAs were effective in Scribble depletions.
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