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. 2021 Feb 24;95(6):e02001-20.
doi: 10.1128/JVI.02001-20. Print 2021 Feb 24.

Protein Tyrosine Phosphatase SHP2 Suppresses Host Innate Immunity against Influenza A Virus by Regulating EGFR-Mediated Signaling

Qingsen Wang #  1 , Wenliang Pan #  1 , Song Wang  1 , Chen Pan  1 , Hongya Ning  1 , Shile Huang  2   3 , Shih-Hsin Chiu #  4 , Ji-Long Chen #  4   5
Affiliations

Protein Tyrosine Phosphatase SHP2 Suppresses Host Innate Immunity against Influenza A Virus by Regulating EGFR-Mediated Signaling

Qingsen Wang et al. J Virol. .

Abstract

Influenza A virus (IAV) is a highly contagious pathogen, causing acute respiratory illnesses in human beings and animals and frequently giving rise to epidemic outbreaks. Evasion by IAV of host immunity facilitates viral replication and spread, which can be initiated through various mechanisms, including epidermal growth factor receptor (EGFR) activation. However, how EGFR mediates the suppression of antiviral systems remains unclear. Here, we examined host innate immune responses and their relevant signaling to EGFR upon IAV infection. IAV was found to induce the phosphorylation of EGFR and extracellular signal-regulated kinase (ERK) at an early stage of infection. Inhibition of EGFR or ERK suppressed the viral replication but increased the expression of type I and type III interferons (IFNs) and interferon-stimulated genes (ISGs), supporting the idea that IAV escapes from antiviral innate immunity by activating EGFR/ERK signaling. Meanwhile, IAV infection also induced the activation of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2). Pharmacological inhibition or small interfering RNA (siRNA)-based silencing of SHP2 enhanced the IFN-dependent antiviral activity and reduced virion production. Furthermore, knockdown of SHP2 attenuated the EGFR-mediated ERK phosphorylation triggered by viral infection or EGF stimulation. Conversely, ectopic expression of constitutively active SHP2 noticeably promoted ERK activation and viral replication, concomitant with diminished immune function. Altogether, the results indicate that SHP2 is crucial for IAV-induced activation of the EGFR/ERK pathway to suppress host antiviral responses.IMPORTANCE Viral immune evasion is the most important strategy whereby viruses evolve for their survival. This work shows that influenza A virus (IAV) suppressed the antiviral innate immunity through downregulation of IFNs and ISGs by activating EGFR/ERK signaling. Meanwhile, IAV also induced the activation of protein tyrosine phosphatase SHP2, which was found to be responsible for modulating the EGFR-mediated ERK activity and subsequent antiviral effectiveness both in vitro and in vivo The results suggest that SHP2 is a key signal transducer between EGFR and ERK and plays a crucial role in suppressing host innate immunity during IAV infection. The finding enhances our understanding of influenza immune evasion and provides a new therapeutic approach to viral infection.

Keywords: EGFR; SHP2; influenza A virus; innate immunity; interferon.

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Figures

FIG 1
FIG 1
IAV infection induces activation of the EGFR/ERK pathway. (A) A549 cells were infected with WSN or PR8 (MOI = 1) and collected at 0, 5, 10, 15, 30, 60, 90, and 120 min. (B and C) A549 cells were infected with WSN or PR8 (MOI = 1) without (Live) or with treatment at 56°C or 65°C, and then the cells were further cultured for 0, 5, 10, 15, 30, and 60 min. The cell lysates were harvested and subjected to Western blotting with the indicated antibodies.
FIG 2
FIG 2
Inhibition of the EGFR/ERK pathway reduces IAV replication. (A and B) A549 cells were pretreated with 1 μM afatinib (Afa), 10 μM U0126, or DMSO as a vehicle control for 12 h, followed by WSN, PR8, or CA04 infection (MOI = 1) for 30 min, and the protein samples were harvested. The expression levels of p-EGFR and p-ERK were detected by Western blotting. (C and D) Culture supernatants were harvested at 15 h postinfection and subjected to plaque assay to determine the virus titer. (E) A549 cells were transfected with siRNA corresponding to EGFR (siEGFR) or scrambled control siRNA (siCtrl) for 24 h, and the knockdown efficiency of siEGFR was determined by Western blotting. (F) After transfection with siEGFR or siCtrl at 80 nM for 24 h, the cells were exposed to WSN (MOI = 1) for 15 h, and then the culture supernatants were collected for plaque assay. Data are means and standard deviations (SD). *, P < 0.05.
FIG 3
FIG 3
Inhibition of IAV-induced EGFR/ERK signaling upregulates the expression of innate immunity-related genes. A549 cells were pretreated with 1 μM afatinib (Afa) for 12 h, followed by infection with WSN (A and C), PR8 (E), or CA04 (F) (MOI = 1). (B and D) After U0126 treatment at a concentration of 10 μM for 12 h, A549 cells were infected with WSN (MOI = 1). Total RNA was extracted at 8 h postinfection, and the mRNA levels of IFNs and ISGs were determined by qRT-PCR. (G and H) A549 cells were treated with 1 μM Afa or 10 μM U0126 prior to WSN infection (MOI = 1). IFN-β and IL-29 levels in the culture supernatants collected at 15 h postinfection were measured by ELISA. Data are means and SD. *, P < 0.05; **, P < 0.01.
FIG 4
FIG 4
Knockdown of EGFR upregulates the expression of innate immunity-related genes during IAV infection. A549 cells were transfected with siEGFR or scrambled control siRNA (siCtrl) at 80 nM for 24 h, followed by infection with WSN (MOI = 1). At 8 h postinfection, total RNA was extracted, and the mRNA levels of IFNs (A) and ISGs (B) were determined by qRT-PCR. (C) IFN-β and IL-29 levels in the culture supernatants collected at 15 h postinfection were measured by ELISA. Data are means and SD. **, P < 0.01.
FIG 5
FIG 5
Activation of SHP2 mediates IAV replication upon infection. (A) A549 cells were incubated with WSN, PR8, or CA04 (MOI = 1) for 0, 5, 10, 15, 30, 60, 90, and 120 min prior to protein extraction and analysis using Western blotting with the indicated antibodies. (B and C) To examine the efficacy of SHP2 inhibitor and siRNA, A549 cells were treated with the SHP2 inhibitor SHP099 (SHP) for 12 h prior to infection with WSN (MOI = 1) for 30 min or transfected with siRNA corresponding to SHP2 (siSHP2) or scrambled control siRNA (siCtrl) for 24 h. The cell lysates were analyzed by Western blotting with the indicated antibodies. The titer of WSN was determined by plaque assay at 8, 16, and 24 h postinfection (MOI = 1) after pretreatment of cells with 10 μM SHP099 for 12 h (D) or 120 nM siSHP2 for 24 h (E). A549 cells were pretreated with 10 μM SHP099 for 12 h, followed by PR8 (F) or CA04 (G) infection (MOI = 1) for 15 h, and then the culture supernatants were subjected to plaque assay to determine the virus titer. In mouse experiments, BALB/c mice were treated with SHP099 for 3 days prior to infection with WSN. At 24 h postinfection, lung homogenates of the mice (n = 4 per group; three independent experiments) were collected for assessing the phosphorylation status of SHP2 (H), the IAV NP mRNA level (I), and the virus titer (J). Data are means and SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 6
FIG 6
Inhibition of IAV-induced SHP2 activation upregulates the expression of innate immunity-related genes. A549 cells were treated with 10 μM SHP099 (SHP) for 12 h, followed by infection with WSN (A and B), PR8 (C), or CA04 (D) (MOI = 1). A549 cells were transfected with siRNA corresponding to SHP2 (siSHP2) or scrambled control siRNA (siCtrl) at 120 nM for 24 h prior to WSN infection (MOI = 1) (F and G). After infection for 8 h, total RNA samples were harvested and subjected to qRT-PCR for assessing the levels of IFN and ISG mRNA. Following inhibition (E) and knockdown (H) of SHP2, IFN-β and IL-29 levels in the culture supernatants collected at 15 h after WSN infection were measured by ELISA. Data are means and SD. *, P < 0.05; **, P < 0.01.
FIG 7
FIG 7
SHP2 is involved in EGFR-mediated activation of ERK. (A) A549 cells were serum starved for 12 h and then treated with 10 ng/ml EGF for 0, 5, 10, 15, 30, and 60 min. The cell lysates were harvested and subjected to Western blotting with the indicated antibodies. (B and C) After transfection with siRNA corresponding to SHP2 (siSHP2) or scrambled control siRNA (siCtrl) at 120 nM for 24 h, A549 cells were infected with WSN (MOI = 1) or treated with 10 ng/ml EGF for 30 min, and the protein extracts were analyzed by Western blotting with the indicated antibodies. (D and E) A549 cells were pretreated with 1 μM afatinib (Afa), 10 μM SHP099 (SHP), or 10 μM U0126 for 12 h and then infected with WSN (MOI = 1) or stimulated with 10 ng/ml EGF for 30 min. After incubation, the cell lysates were harvested and subjected to Western blotting with the indicated antibodies.
FIG 8
FIG 8
Activated SHP2 is required for ERK activation and promotes IAV replication. (A) A549 cells were transiently transfected with empty vector (EV), wild-type SHP2 (WT), or a constitutively active SHP2 mutant (EK) for 24 h, followed by protein extraction and analysis using Western blotting with the indicated antibodies. (B) A549 cells harboring EV, WT, or EK were pretreated with or without 1 μM afatinib (Afa) for 12 h and then infected with WSN or PR8 (MOI = 1) for 30 min. After infection, the cell lysates were harvested and subjected to Western blotting with the indicated antibodies. (C) A549 cells harboring EV, WT, or EK were infected with WSN (MOI = 1). The culture supernatants were collected at 15 h postinfection, and the virus titer was determined by plaque assay. (D) A549 cells harboring EV, WT, or EK were infected with WSN (MOI = 1) for 8 h. Total RNA was extracted after infection, and the mRNA levels of IFNs and ISGs were assessed by qRT-PCR. Data are means and SD. *, P < 0.05; **, P < 0.01.
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