Enterovirus 71 disrupts interferon signaling by reducing the level of interferon receptor 1

Abstract

The recent outbreak of enterovirus 71 (EV71) infected millions of children and caused over 1,000 deaths. To date, neither an effective vaccine nor antiviral treatment is available for EV71 infection. Interferons (IFNs) have been successfully applied to treat patients with hepatitis B and C viral infections for decades but have failed to treat EV71 infections. Here, we provide the evidence that EV71 antagonizes type I IFN signaling by reducing the level of interferon receptor 1 (IFNAR1). We show that the host cells could sense EV71 infection and stimulate IFN-β production. However, the induction of downstream IFN-stimulated genes is inhibited by EV71. Also, only a slight interferon response and antiviral effects could be detected in cells treated with recombinant type I IFNs after EV71 infection. Further studies reveal that EV71 blocks the IFN-mediated phosphorylation of STAT1, STAT2, Jak1, and Tyk2 by reducing IFNAR1. Finally, we identified the 2A protease encoded by EV71 as an antagonist of IFNs and show that the protease activity is required for reducing IFNAR1 levels. Taken together, our study for the first time uncovers a mechanism used by EV71 to antagonize type I IFN signaling and provides new targets for future antiviral strategies.

Fig 1

EV71 infection activated type I IFN production but inhibited ISG activation. RD cells were mock infected or infected with EV71 at an MOI of 1 (A and D) or 10 (B and E). The expression levels of IFN-β (A and B) and ISGs (D and E) were measured by qRT-PCR. (C) RD cells were cultured in DMEM with 2% FBS for 24 h, and then the culture medium was replaced with the EV71-conditioned medium. After incubation for 2 h, the cells were harvested to check the mRNA levels of ISGs. IFN-β-specific antibody was used for the neutralization. Con, treated with control medium; VM, treated with EV71-conditioned medium; VM+Ab, treated with EV71-conditioned medium and IFN-β antibody. Data are shown as mean ± SD of three independent experiments; each was done in triplicate. *, P < 0.05.

Fig 2

EV71 antagonized the antiviral effect of IFN-α treatment. (A) RD cells were infected with EV71 at an MOI of 1. IFN-α treatment (100 U/ml, frames c to g; or 1,000 U/ml, frames h to l) was performed before or after viral infection as indicated on the figure. Cells uninfected or infected without IFN treatment are shown in frames a or b, respectively. Photomicrographs were taken at 20 h postinfection (original magnification, ×100). (B) Cellular viral loads were quantified by qRT-PCR at 20 h p.i. The cells infected with EV71 but without IFN treatment were set as controls. (C) RD cells were infected with EV71 at an MOI of 10. At 9 h after infection, cells were stimulated with IFN-α2b for another 2 h (Virus+IFN). Cellular RNA was extracted, and expression levels of relative ISGs were measured by qRT-PCR. The expression level of each gene was calculated relative to GAPDH gene expression and normalized to mock-treated cells. Data are shown as mean ± SD of three independent experiments; each was done in triplicate. *, P < 0.05, versus the control; **, P < 0.001, versus the control; #, P < 0.001, Virus+IFN versus Con+IFN.

Fig 3

EV71 inhibited phosphorylation of STAT1/STAT2. RD cells (A and C) or HeLa cells (B and D) were infected with EV71 at an MOI of 1 or 10 for 9 h, and cells were left untreated (−) or treated (+) with IFN-α2b (A and B) or IFN-β (C and D) for another 30 min. Western blotting was performed by detecting STAT1, STAT2, phosphorylated STAT1 (p-STAT1), phosphorylated STAT2 (p-STAT2), and viral structural protein VP1 with relative antibodies. GAPDH was also detected as a loading control.

Fig 4

EV71 inhibited type I IFN-mediated Jak/STAT signaling by reducing IFNAR1 protein levels. (A) RD cells were infected with EV71 at an MOI of 10 for 9 h, and cells were left untreated (−) or treated (+) with IFN-α2b for another 10 min. The phosphorylated Jak1 (p-Jak1), Tyk2 (p-Tyk2), and VP1 were detected with specific antibodies. GAPDH was also detected as a loading control. (B) RD cells were mock infected or infected with EV71 at an MOI of 10. Cells were treated with IFN-α2b for another 30 min at 0, 3, 6, and 9 h after infection. The mock-infected cells without IFN treatment were set as negative controls. The expression levels of IFNAR1, STAT1, VP1, and p-STAT1 were measured with related antibodies.

Fig 5

Identification 2A^pro as an antagonist of IFN signaling. HEK293T cells were cotransfected with pAAV-EGFP and plasmids expressing individual EV71 proteins. The null vector was used as a control. At 24 h after transfection, cells were mock treated (A) or stimulated with IFN-α2b (B) for another 2 h. Then, total cellular RNA was extracted, and the mRNAs of MxA and EGFP as well as of each viral protein were quantified by qRT-PCR. The mRNA level of MxA was calculated relative to that of each viral protein and normalized to that of the control (set as 1). Data are shown as mean ± SD of three independent experiments; each was done in triplicate. *, P < 0.05, versus the control; **, P < 0.001, versus the control.

Fig 6

2A^pro inhibited Jak/STAT signaling by reducing IFNAR1 levels. HEK293T cells were transfected with a plasmid expressing 2A^pro or a control vector. At 24 h after transfection, cells were treated with or without IFN-α2b. Western blotting was performed to detect IFNAR1, p-Jak1, p-Tyk2, STAT1, STAT2, p-STAT1, p-STAT2, and eIF4G with related antibodies. The arrow indicates the cleaved eIF4G in HEK293T cells. GAPDH was detected as a loading control.

Fig 7

Protease activity is required for 2A^pro-mediated inhibition of IFN signaling. (A) HEK293T cells were transfected with a plasmid expressing 2A^pro or the mutant 2A^C110A or with a control vector. At 24 h after transfection, cells were treated without or with IFN-α2b (100 U/ml) for 30 min. Western blotting was performed to detect IFNAR1, STAT1, STAT2, p-STAT1, p-STAT2, and eIF4G with related antibodies. The arrow indicates the cleaved eIF4G. GAPDH was detected as a loading control. (B) A model for EV71 to evade surveillance of type I IFN. During the EV71 replication cycle, the virus encoded two proteases, 2A^pro and 3C^pro. 3C^pro interacts with RIG-1 and TRIF to suppress the production of IFN-β, while 2A^pro reduces the IFN receptor IFNAR1 and then blocks the downstream signaling pathway. As a consequence, IFN-induced antiviral effectors like MxA, OAS1, ISG15, and ISG56 are all suppressed during the whole infection process, and high efficient replication and persistent infection could finally be achieved.