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. 2010 Nov 10;5(11):e13927.
doi: 10.1371/journal.pone.0013927.

Influenza virus non-structural protein 1 (NS1) disrupts interferon signaling

Danlin Jia  1 , Ramtin Rahbar, Renee W Y Chan, Suki M Y Lee, Michael C W Chan, Ben Xuhao Wang, Darren P Baker, Bing Sun, J S Malik Peiris, John M Nicholls, Eleanor N Fish
Affiliations

Influenza virus non-structural protein 1 (NS1) disrupts interferon signaling

Danlin Jia et al. PLoS One. .

Abstract

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections.

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Conflict of interest statement

Competing Interests: Darren P. Baker is employed by Biogen Idec. Biogen Idec did not provide funds for these studies, nor was Biogen Idec involved in the study design, data analysis or any decisions relating to manuscript preparation or submission. Biogen Idec provided interferon-beta for these studies at no cost. Accordingly, the relationship with Darren P. Baker (and therefore Biogen Idec) in no way affects adherence to all of the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. H5N1 NS1 expression inhibits IFN-inducible STAT phosphorylation.
A) HeLa cells transfected with vector alone (formula image) or HA-tagged NS1 plasmid (▪) were left untreated (−) or treated (+) with IFN-β (1×103 U/mL) for 15 mins, 24 hr post-transfection. Cells were harvested, lysates were resolved by SDS-PAGE and immunoblotted with the indicated anti-phospho-STAT1, anti-phospho-STAT2, anti-phospho-STAT3 and anti-HA(NS1) antibodies. Membranes were stripped and reprobed with anti-STAT1, anti-STAT2, anti-STAT3 and anti-β-tubulin antibodies as loading controls. Relative fold induction of phosphorylated STAT proteins was calculated using signal intensity of phospho-STATs over total STATs and normalized with untreated, vector transfected cells. The data plots are representative of three independent experiments. B) HeLa cells transfected with HA-tagged NS1 plasmid were treated with IFN-β (1×103 U/mL) for 15 mins. Cells were then fixed and stained for HA (red) and phospho-STAT2 (blue), and analyzed by confocal microscopy as described in Materials and Methods. The white arrow identifies the nuclear predominance of NS1and the broken line in the middle panel defines the nucleus showing reduced phospho-STAT2. Data are representative of two independent experiments.
Figure 2
Figure 2. H5N1 NS1 expression inhibits IFN-inducible STAT:SIE complex formation.
A) HeLa cells transfected with either vector alone or HA-tagged NS1 plasmid were left untreated (−) or treated (+) with IFN-β (1×103 U/mL) for 15 mins, 24 hr post-transfection. Nuclear extracts were isolated and equal amounts of protein were incubated with 32P-labeled SIE probe. Complexes were resolved by native gel electrophoresis and visualized by autoradiography. Data are representative of two independent experiments. HeLa cells were treated with IFN-β (1×103 U/mL) for 15 mins. Nuclear extracts were isolated and equal amount of protein were incubated with 32P-labeled SIE probe in the presence or absence of 2 µg of anti-STAT1 and anti-STAT3 antibodies, as indicated. Complexes were resolved by native gel electrophoresis and visualized by autoradiography.
Figure 3
Figure 3. H5N1 NS1 reduces surface IFNAR1 but not IFNAR2 expression.
A) HeLa cells were transfected with either GFP vector alone (green) or GFP vector containing HA-tagged NS1 (red), then 24 hr post-transfection, GFP-positive cells were FACS sorted and stained for IFNAR1 or IFNAR2 and analyzed by FACS. Data are representative of three independent experiments. B) HeLa cells were transfected with HA-tagged NS1 plasmid and 24 hr post-transfection were fixed and stained for HA (green) and either IFNAR1 (red; upper panel) or IFNAR2 (red; lower panel), and analyzed by confocal microscopy. Data are representative of two independent experiments. C) HeLa cells were transfected with either GFP vector alone (formula image) or GFP vector containing HA-tagged NS1 (▪). 24 hr post-transfection, GFP positive cells were FACS sorted, RNA extracted and cDNA synthesized. Ifnar1, ifnar2 and β-actin gene expression were analyzed by RT-PCR. Gene expression was calculated relative to β-actin gene expression and normalized to cells transfected with GFP vector alone. Data are representative of two independent experiments. Significant differences (asterisk) were determined by Student's t-test (p<0.05).
Figure 4
Figure 4. Influenza virus infection reduces ifnar1 and ifnar2 expression at 24 hours post-infection in human monocyte-derived macrophages and ex vivo lung tissues.
Human monocyte-derived macrophages were infected with A/HK/483/97 H5N1 or A/HK/54/98 H1N1 virus (Multiplicity of Infection (MOI)  = 2). RNA was extracted from the cells at A) 3 hr, B) 6 hr, and C) 24 hr post-infection. Following cDNA synthesis, ifnar1 (▪) and ifnar2 (formula image) expression was assayed by real-time PCR. Data shown are fold induction of gene expression relative to mock-infected control after normalizing to β-actin in each sample. Representative data of duplicate experiments with means of triplicate assays are shown; D) Human lung explant tissue was either mock-infected (PBS) or infected with A/HK/483/97 H5N1 or A/HK/54/98 H1N1 influenza A viruses, as described in Materials & Methods. 18 hr post-infection tissue was processed to extract RNA. cDNA was synthesized and expression of ifnar1, ifnar2 and β-actin gene expression was measured by RT-PCR analysis. Gene expression was calculated relative to β-actin gene expression and normalized to mock infected tissues. Data are representative of two independent experiments. Significant differences (asterisk) were determined by Student's t-test (p<0.05).
Figure 5
Figure 5. NS1 regulates SOCS1 and SOCS3 expression.
A) HeLa cells were transfected with vector alone or HA-tagged NS1 plasmid, then 24 hr post-transfection cells were harvested and lysates were resolved by SDS-PAGE and immunoblotted with the indicated anti-SOCS1 and anti-SOCS3 antibodies. Membranes were probed with anti-HA antibody to confirm expression of NS1, and anti-tubulin antibody was applied as a loading control. Relative fold induction of proteins was calculated using signal intensity over loading and normalized against vector transfected cells (SOCS1 formula image, SOCS3 ▪). Data are representative of two independent experiments. B) HeLa cells were transfected with either GFP vector alone (formula image) or GFP vector containing HA-tagged NS1 (▪). 24 hr post-transfection, GFP+ cells were sorted, RNA extracted and cDNA synthesized. Gene expression of socs1 (formula image), socs3 (▪) and β-actin gene expression were analyzed by RT-PCR. Gene expression was calculated relative to β-actin gene expression and normalized to cells transfected with GFP vector alone. Data are representative of two independent experiments. C) RNA from human lung tissue either mock-infected (PBS) or infected with A/HK/54/98 H1N1 or H5N1 influenza A viruses was collected 18 hr post-infection. cDNA was synthesized and expression of socs1(▪), socs3(▪) and β-actin gene expression was measured by RT-PCR analysis. Gene expression was calculated relative to β-actin gene expression and normalized to mock infected cells. Data are representative of two independent experiments. Significant differences (asterisk) were determined by Student's t-test (p<0.05).
Figure 6
Figure 6. Treatment with IFN alfacon-1 inhibits H5N1 and H1N1 replication in primary human lung cells and upregulates ISG expression.
Different human surgical lung tissue explants (1–6) were either left untreated (♦) or treated with IFN alfacon-1 (1.2×104 U/mL) (○) for 16 hr. Tissues were then infected with H5N1 (1–4) or H1N1 influenza A viruses (5, 6), as indicated. At different time points post infection, RNA from cells was collected and cDNA synthesized. Gene expression for A) influenza A m gene, was measured by RT-PCR. Gene expression for B) pkr (formula image), isg15 (▪), 2′5′-oas (formula image), and β-actin, was measured by RT-PCR analysis at 18 hr post-infection with H5N1 or A/HK/54/98 H1N1 for explant 5. Data are representative of two independent experiments and normalized to mock infected controls.
Figure 7
Figure 7. Treatment with IFN alfacon-1 inhibits pandemic H1N1 2009 virus replication in human lung tissue.
Three different human surgical lung tissue explants were infected with pandemic H1N1 2009 virus for 24 hr, then either left untreated (▪) or treated with IFN alfacon-1 (1.2×104 U/mL) (formula image) for a further 48 hr. At the indicated times A) Viral titers (TCID50) and B) Influenza A m gene expression were measured; Significant differences were determined by Student t-test: * ∼ p<0.05; ** ∼ p<0.01. C) Thin sections of infected human lung explants, either untreated (i) or IFN-treated (ii) were stained for influenza A nucleoprotein (pink).
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