A Conserved Residue, Tyrosine (Y) 84, in H5N1 Influenza A Virus NS1 Regulates IFN Signaling Responses to Enhance Viral Infection

Abstract

The non-structural protein, NS1, is a virulence factor encoded by influenza A viruses (IAVs). In this report, we provide evidence that the conserved residue, tyrosine (Y) 84, in a conserved putative SH2-binding domain in A/Duck/Hubei/2004/L-1 [H5N1] NS1 is critical for limiting an interferon (IFN) response to infection. A phenylalanine (F) substitution of this Y84 residue abolishes NS1-mediated downregulation of IFN-inducible STAT phosphorylation, and surface IFNAR1 expression. Recombinant IAV (rIAV) [H1N1] expressing A/Grey Heron/Hong Kong/837/2004 [H5N1] NS1-Y84F (rWSN-GH-NS1-Y84F) replicates to lower titers in human lung epithelial cells and is more susceptible to the antiviral effects of IFN-β treatment compared with rIAV expressing the intact H5N1 NS1 (rWSN-GH-NS1-wt). Cells infected with rWSN-GH-NS1-Y84F express higher levels of IFN stimulated genes (ISGs) associated with an antiviral response compared with cells infected with rWSN-GH-NS1-wt. In mice, intranasal infection with rWSN-GH-NS1-Y84F resulted in a delay in onset of weight loss, reduced lung pathology, lower lung viral titers and higher ISG expression, compared with mice infected with rWSN-GH-NS1-wt. IFN-β treatment of mice infected with rWSN-GH-NS1-Y84F reduced lung viral titers and increased lung ISG expression, but did not alter viral titers and ISG expression in mice infected with rWSN-GH-NS1-wt. Viewed altogether, these data suggest that the virulence associated with this conserved Y84 residue in NS1 is, in part, due to its role in regulating the host IFN response.

Keywords: influenza A viruses; interferon signaling; interferon-stimulated genes; interferon-β; non-structural protein 1.

Figure 1

In silico modeling of the Y84F (tyrosine to phenylalanine) mutation in the putative Src homology 2 (SH2)-binding domain of A/Duck/Hubei/L-1/2004 [H5N1] non-structural protein 1 (NS1). (A) Ribbon diagrams of the A/Vietnam/1203/2004 [H5N1] H5N1 NS1 (PDB: 3F5T) and p85β complex, based on a crystallized structure of the H1N1 NS1 and p85β internal SH2 (i-SH2) domain complex (PDB: 3L4Q). (B) Ribbon diagrams showing the effect of the Y84F mutation on NS1-p85β binding.

Figure 2

H5N1 NS1-Y84F is unable to upregulate protein kinase B (AKT) phosphorylation. HeLa cells were transfected with vector alone, vector carrying the NS1-wt complementary DNA (cDNA) (□), or NS1-Y84F cDNA (■). 24 hours (h) post-transfection, cells were lysed and lysates were resolved by SDS-PAGE and immunoblotted with an anti-phospho (p)-AKT (Ser473) antibody. The blot was then stripped and re-probed with an antibody against AKT. A separate aliquot of the same cell lysate was resolved by SDS-PAGE and immunoblotted with antibodies against HA (NS1) and α-tubulin. Band intensities were quantitated and the relative induction in p-AKT was determined, normalizing to AKT. Data are presented as the mean +/− standard error (SE) and are representative of three independent experiments. ** p < 0.01.

Figure 3

The Y84F mutation abrogates H5N1 NS1-mediated inhibition of interferon (IFN)-inducible signal transducer and activator of transcription (STAT) phosphorylation. HeLa cells were transfected with vector alone, or vector carrying the NS1-wt cDNA (□), or NS1-Y84F cDNA (■). 24 h post-transfection, cells were either left untreated or treated with 1000 U/mL of IFN-β for 15 minutes (min). Cells were lysed, lysates resolved by SDS-PAGE and immunoblotted with antibodies against p-STAT (Tyr701), p-STAT2 (Tyr690), or HA (NS1). Blots were then stripped and re-probed with antibodies against STAT1 or STAT2. Band intensities were quantitated and the relative induction in p-STAT1 and p-STAT2 was determined, normalizing to STAT1 and STAT2, respectively. Data are presented as the mean +/− SE and are representative of three independent experiments.

Figure 4

NS1-Y84F does not affect IFN-α/β receptor subunit (IFNAR) 1 expression. HeLa cells were transfected with vector alone (▬, black), vector carrying the NS1-wt cDNA (▬, grey), or NS1-Y84F cDNA (---). 24 h later, cells were stained with antibodies against IFNAR1 or IFNAR2. Transfected cells stained with the Alexa Fluor 647 secondary antibody alone served as the isotype control (▬, light grey fill). Untransfected HeLa cells (no line, dark gray fill) were also stained. Cells were analyzed using a FACSCalibur, gating on green fluorescent protein (GFP)+ cells, then analyzing for IFNAR1 or IFNAR2 expression. Data are representative of two independent experiments.

Figure 5

The Y84F mutation inhibits recombinant IAV (rIAV) [H1N1] replication and enhances IFN-β production in human A549 lung epithelial cells. (A) A549 cells were infected with rWSN-GH-NS1-wt (○) or rWSN-GH-NS1-Y84F (●) at a multiplicity of infection (MOI) of 0.01. Culture supernatants from rWSN-GH-NS1-wt (□) or rWSN-GH-NS1-Y84F (■) infected cells were collected at 6, 12, 24, 36, and 48 h post-infection and viral titers were determined by plaque assay in Madin-Darby canine kidney (MDCK) cells. (B) IFN-β levels were measured in the culture supernatants collected at 12 and 24 h post-infection by enzyme-linked immunosorbent assay (ELISA). Data are presented as the mean +/− SE and are representative of three (titration) and two (ELISA) independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 6

The Y84F mutation confers greater sensitivity to the antiviral effects of IFN-β. A549 cells were left uninfected (■) or infected with rWSN-GH-NS1-wt (□) or rWSN-GH-NS1-Y84F (■) at a MOI of 0.01. At 12 h post-infection, cells were either left untreated, or treated with 50, 100, or 1000 U/mL of IFN-β. RNA was purified from cells at 24 h post-infection (i.e., 12 h post-IFN-β treatment) and cDNA was synthesized. Quantitative polymerase chain reaction (qPCR) was performed to determine the relative expression of IFN-inducible (A) EIF2AK2 and (B) MxA, normalized to HPRT1. Data are presented as the fold-induction of each IFN stimulated gene (ISG) relative to infected or uninfected A549 cells that were not treated with IFN-β. (C) IAV M gene expression was determined by qPCR at 48 h post-infection (36 h post-IFN-β treatment). (D) Viral titers in culture supernatants collected at 48 h post-infection (36 h post-IFN-β treatment) were determined by plaque assay in MDCK cells. Data are presented as the mean +/− SE and are representative of two independent experiments.

Figure 7

IFN-β pre-treatment inhibits rIAV replication. A549 cells were either left untreated or treated with 50, 100, or 1000 U/mL of IFN-β for 16 h and then infected with rWSN-GH-NS1-wt (□) or rWSN-GH-NS1-Y84F (■) at a MOI of 0.01. Culture supernatants were collected at (A) 24 and (B) 48 h post-infection (40 and 64 h post-IFN-β treatment, respectively) and viral titers were determined by plaque assay in MDCK cells. Data are presented as the mean +/− SE and are representative of two independent experiments. ND ~ not detected.

Figure 8

rWSN-GH-NS1-wt and rWSN-GH-NS1-Y84F replicate to higher titers in STAT1^−/− mouse embryonic fibroblasts (MEFs) in comparison to STAT1^+/+ MEFs. STAT1^+/+ and STAT1^−/− MEFs were infected with rWSN-GH-NS1-wt (○) or rWSN-GH-NS1-Y84F (●) at a MOI of 0.01. Culture supernatants were collected at 6, 12, 24, 36 and 48 h post-infection and viral titers were determined by plaque assay in MDCK cells. Data are presented as the mean +/− SE and are representative of two independent experiments.

Figure 9

Mice infected with rIAV expressing the Y84F mutant NS1 experience delayed weight-loss and lower lung viral titers on day 1 and day 3 post-infection. C57BL/6 mice were infected with 1 × 10⁵ plaque-forming units (PFU) of rWSN-GH-NS1-wt (○) or rWSN-GH-NS1-Y84F (●) by intranasal inoculation. (A) Weight-loss was monitored up to day 3 post-infection. Mice were euthanized on days 1 and 3 post-infection and (B) lung viral titers were determined by plaque assay in MDCK cells and (C) IFN-β production in the lungs was determined by ELISA. (D) RNA was purified from the lung tissues of rIAV-infected mice on days 1 and 3 post-infection and cDNA was synthesized. qPCR was performed to determine the relative expression of murine ISG15, EIF2AK2, OAS1, IFNA4, and IFNB1, normalized to HPRT1 and lung viral titer. Data are representative of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 10

Mice infected with rIAV expressing the Y84F mutant NS1 experience reduced lung pathology and neutrophil infiltration early in infection. C57BL/6 mice were infected with 1 × 10⁵ PFU of rWSN-GH-NS1-wt (○) or rWSN-GH-NS1-Y84F (●) by intranasal inoculation. (A) Lungs were harvested on days 1 and 3 post-infection and processed to prepare thin tissue sections (5 μm) for H&E staining. Black bar = 200 μm. (B) Lungs were harvested on day 1 post-infection and perfused with PBS. Lung tissues were mashed mechanically and ammonium-chloride-potassium (ACK) lysis was performed to remove red blood cells. Neutrophils were quantified by flow cytometry with antibodies targeting CD45, CD11b, and Ly6G. (C) Lungs were harvested on day 1 post-infection, RNA was extracted, and cDNA synthesized. qPCR was performed to determine the relative expression of murine CXCL1 and CXCL2, normalized to HPRT1. Data are representative of two independent experiments. ** p < 0.01.

Figure 11

Mice infected with rIAV expressing the Y84F mutant NS1 are more sensitive to the antiviral effects of IFN-β. C57BL/6 mice were infected with 1 × 10⁵ PFU of rWSN-GH-NS1-wt (○) or rWSN-GH-NS1-Y84F (●) by intranasal inoculation and injected by the intraperitoneal route with PBS or 1 × 10⁵ U of murine IFN-β1 at 8 h post-infection. (A) Lung viral titers were determined on day 1 and 3 post-infection by plaque assay in MDCK cells. On day 1 post-infection, one of the five mice in the Y84F-infected and IFN-treated cohort, did not have a detectable lung viral titer (titer < 10¹ PFU/mL). (B) RNA was purified from the lung tissues of rIAV infected and PBS or murine IFN-β1 treated mice on days 1 and 3 post-infection. cDNA was synthesized and qPCR was performed to determine the relative expression of murine EIF2AK2, OAS1, IFNA4, and IFNB1, normalized to HPRT1 and lung viral titer. Data are representative of two independent experiments. ** p < 0.01, and *** p < 0.001.