Identification of two residues within the NS1 of H7N9 influenza A virus that critically affect the protein stability and function

Abstract

The emerging avian-origin H7N9 influenza A virus, which causes mild to lethal human respiratory disease, continues to circulate in China, posing a great threat to public health. Influenza NS1 protein plays a key role in counteracting host innate immune responses, allowing the virus to efficiently replicate in the host. In this study, we compared NS1 amino acid sequences of H7N9 influenza A virus with those of other strains, and determined NS1 protein variability within the H7N9 virus and then evaluated the impact of amino acid substitutions on ability of the NS1 proteins to inhibit host innate immunity. Interestingly, the amino acid residue S212 was identified to have a profound effect on the primary function of NS1, since S212P substitution disabled H7N9 NS1 in suppressing the host RIG-I-dependent interferon response, as well as the ability to promote the virus replication. In addition, we identified another amino acid residue, I178, serving as a key site to keep NS1 protein high steady-state levels. When the isoleucine was replaced by valine at 178 site (I178V mutation), NS1 of H7N9 underwent rapid degradation through proteasome pathway. Furthermore, we observed that P212S and V178I mutation in NS1 of PR8 virus enhanced virulence and promoted the virus replication in vivo. Together, these results indicate that residues I178 and S212 within H7N9 NS1 protein are critical for stability and functioning of the NS1 protein respectively, and may contribute to the enhanced pathogenicity of H7N9 influenza virus.

Figure 1

H7N9 NS1 protein impairs host innate immune response. A 293T cells transfected with plasmids expressing H7N9 NS1 or empty vector (EV) were infected with WSN virus (MOI = 1) for 12 h, followed by quantitative real-time PCR to detect the mRNA levels of the indicated genes. In each experiment, the value observed in mock-infected cells was normalized to 1. Plotted are the average levels from three independent experiments. The error bars represent the s.e.m. **P < 0.01. B–D 293T cells transfected with plasmids expressing H7N9 NS1 or EV were infected with WSN virus as described in (A). Then the cells were harvested and cell extracts were prepared for Western blotting using indicated antibodies. Shown are representative data of three independent experiments with similar results. E 293T cells transfected with plasmids expressing H7N9 NS1 or EV were treated as described in (A), and viral titers in the supernatants of the cells were examined by plaque assay.

Figure 2

H7N9 NS1-S212P mutation reduces its capacity to inhibit WSN and PR8 delNS1 virus-induced host antiviral response. A 293T cells were transfected with plasmids expressing H7N9 NS1-WT (WT), NS1 mutants (R55E, H63Q, E70K, P87S, S114P, A143T, I178V, S212P) or EV for 24 h, then the cells were infected with WSN virus (MOI = 1) for 12 h, followed by quantitative real-time PCR to detect IFN-β mRNA expression levels. B–G After 293T cells were transfected with plasmids expressing H7N9 NS1-WT (WT), NS1-S212P (S212P) or EV for 24 h, the cells were then infected with WSN or PR8 delNS1 virus (MOI = 1) for 12 h, followed by quantitative real-time PCR to detect IFN-α (B), IFN-β (C), IL-28 (D), OASL (E), MxA (F) and IL-6 (G) mRNA levels. In each experiment, the value observed in mock-infected cells was normalized to 1. Plotted are the average levels from three independent experiments. The error bars represent the s.e.m. ^*P < 0.05, ^**P < 0.01.

Figure 3

H7N9 NS1 S212P mutation causes a decrease of its ability to suppress RIG-I expression and STAT1 activation. A, B 293T cells transfected with plasmids expressing H7N9 NS1-WT (WT), NS1-S212P (S212P) or EV were infected with WSN (A) or PR8 delNS1 virus (B) (MOI = 1) for 12 h, followed by quantitative real-time PCR to detect RIG-I mRNA levels. In each experiment, the RIG-I level in mock-infected cells was normalized to 1. Plotted are the average levels from three independent experiments. The error bars represent the s.e.m. (C, D) 293T cells transfected with plasmids expressing H7N9 NS1-WT (WT), NS1-S212P (S212P) or EV were infected with WSN (C) or PR8 delNS1 virus (D) as described in (A, B), then cells were harvested and cell extracts were prepared for Western blotting using indicated antibodies. The results are representative of three independent experiments. ^*P < 0.05, ^**P < 0.01.

Figure 4

S212P mutation of H7N9 NS1 results in a reduction in the viral replication. A 293T cells transfected with plasmids expressing H7N9 NS1-WT (WT), NS1-S212P (S212P) or EV were infected with WSN (MOI = 1) for 12 h, followed by Western blotting to detect viral NP expression. B NP levels in (A) were quantitated by densitometry, and normalized to β-actin levels. In each experiment, the NP level in NS1-WT transfected cells is 100. C 293T cells transfected with plasmids expressing H7N9 NS1-WT (WT), NS1-S212P (S212P) or EV were infected with PR8 delNS1 virus (MOI = 1) for 12 h, followed by Western blotting to detect viral NP expression. D NP levels in (C) were quantitated by densitometry, and normalized to β-actin levels. In each experiment, the NP level in NS1-WT transfected cells is 100. E, F 293T cells transfected with plasmids expressing H7N9 NS1-WT (WT), NS1-S212P (S212P) or EV were infected with WSN (E) or PR8 delNS1 virus (F) (MOI = 1) for 12 h, and viral titers in the supernatants of the cells were examined by plaque assay. ^**P < 0.01.

Figure 5

I178V mutation decreases stability of H7N9 NS1 protein. A 293T cells were transfected with plasmids expressing H7N9 NS1-WT (WT) or NS1 mutants (R55E, H63Q, E70K, P87S, S114P, A143T, I178V, S212P) for 24 h, followed by Western blotting to detect the NS1 protein levels. The results are representative of three independent experiments. B 293T cells were transfected with plasmids expressing H7N9 NS1-WT (WT) or NS1-I178V (I178V) as described in (A). Then the mRNA levels of NS1 were detected by RT-PCR. C Half-lives of NS1-WT (WT) and NS1-I178V (I178V) were examined. 293T cells were transfected with plasmids expressing H7N9 NS1-WT (WT) or NS1-I178V (I178V) as described in (A), then the cells were treated with CHX (100 μg/mL). At the indicated times after treatment, cells were harvested and cell extracts were prepared for Western blotting to analyze NS1 protein levels. D NS1 levels in (C) were quantitated by densitometry, and normalized to β-actin levels. Plotted are the average levels from three independent experiments. ^*P < 0.05, ^**P < 0.01.

Figure 6

I178V mutation induces proteasome degradation of NS1 protein. A–C 293T cells transfected with plasmids expressing H7N9 NS1-WT (WT) or NS1-I178V (I178V) were treated with CHX (100 μg/mL), then were either mock treated or treated with MG132 (10 μM) (A), NH4Cl (20 mM) (B) or chloroquine (50 μM) (C). At the indicated times, cells were harvested and analyzed by Western blotting with the indicated antibodies. Shown are representative data of three independent experiments with similar results.

Figure 7

P212S and V178I mutation in NS1 of PR8 virus enhances its virulence in vivo. A Shown is the body weight change of mice mock infected or infected with PR8-WT, PR8-S212 or PR8-I178 (1 × 10⁴ PFU/mouse). Body weight was measured daily. The results are shown as mean percentage weight changes from three independent experiments. B Survival rate of mice infected with PR8-WT, PR8-S212 or PR8-I178 (n = 10 mice per group). Mice were monitored for up to 14 days. During this period, mice were sacrificed when they displayed severe unrelieved distress, hind limb paralysis or excessive weight loss (25% weight loss from initial body weight). C Shown is the viral load measured on day 5 post-infection in the lungs of mice infected with PR8-WT, PR8-S212 or PR8-I178. D Mice were mock infected or infected with PR8-WT, PR8-S212 or PR8-I178 for 5 days. Shown are representative micrographs of mouse lungs stained with hematoxylin and eosin (HE). Bars, 200 μm. E, F Three groups of mice (five mice per group) were mock infected or infected with PR8-WT and PR8-S212 for 5 days. Then the mRNA expression level of RIG-I (E) and IFN-β (F) was detected by quantitative real-time PCR. In each experiment, the value observed in mock-infected cells was normalized to 1. The error bars represent the s.e.m. G A549 cells were infected with PR8-WT and PR8-I178 for 16 h, then the cells were treated with CHX (100 μg/mL) for indicated times, followed by Western blotting to detect the NS1 protein levels. The results are representative of three independent experiments. **P < 0.01.