Enterovirus 71 Antagonizes Antiviral Effects of Type III Interferon and Evades the Clearance of Intestinal Intraepithelial Lymphocytes

FIGURE 1. EV71 can replicate in human intestinal epithelial cells. (A) HT29 cells were inoculated with EV71 at an MOI of 1, and the images were obtained at the indicated time points. Representative images were shown. (B) The kinetic curve of EV71 replication in HT29 cells. HT29 cells and culture supernatants were harvested at indicated time points (MOI = 1). Total RNAs were extracted for reverse transcription (cDNA) to determine the mRNA level of EV71-VP1 using VP1 specific primers by qRT-PCR, and dates were calculated relative to GAPDH gene expression. (C) The cell culture supernatants were collected for virus titer detection by plaque assay. (D) HT29 cells were fixed in 4% paraformaldehyde at the indicated infection time points, then incubated with anti-EV71 VP1 antibody, followed by staining with Alexa Fluor 488-conjugated secondary antibody and DAPI to visualize the EV71 and nuclei. Olympus confocal laser scanning microscope was used for imaging. Mock was cells without infection. The results were presented from three independent experiments as mean ± SD.

FIGURE 2. IFN-λ effectively inhibits EV71 infection in intestinal epithelial cells. (A) HT29 and Jurkat T cells were uninfected or infected with EV71 at an MOI of 1, qRT-PCR was used to detect transcriptional upregulation of both IFN-β, IFN-λ1, and IFN-λ2 at 48 hpi. (B) At the same infection conditions, the production and secretion of IFN-β, IFN-λ1, and IFN-λ2 proteins in the supernatant were detected by ELISA. Mock was cells without infection. (C) HT29 and Jurkat T cells were stimulated without or with 10 ng/ml recombinant protein IFN-β or IFN-λ1 for 12 h. Then the expression levels of ISG54, ISG15, PKR, and OAS were measured by qRT-PCR. Calculated the expression level of each gene relative to the expression of GAPDH and normalized it to mock-treated cells. Control was cells without treatment. (D,E) HT29 cells were pretreated with 10 ng/ml IFN-λ or 10 ng/ml IFN-β for 6 h, respectively, and then infected with EV71 at MOI of 1 for 24 h. The virus replication was monitored by qRT-PCR and plaque experiments. Control was cells with infected but without IFN treatment. Dates were presented as mean ± SD of three replicates (n = 3 independent experiments, **P < 0.01, ***P < 0.001, *⁣*⁣**P < 0.0001…

FIGURE 3. The antiviral function of IFN-λ is antagonized by EV71. (A) HT29 cells were infected with EV71 at an MOI of 1. The expression levels of IFN-β, IFN-λ1, and IFN-λ2 were detected at 0, 12, 24, 48, and 72 hpi by qRT-PCR. (B) At the same infection conditions, ISG54, ISG15, PKR, and OAS expression levels were detected by qRT-PCR. (C) Recombinant protein IFN-λ treatment (10 ng/ml) was performed 6 h before infection or 6 h after infection (MOI = 1). (D) After 24 hpi, the cellular RNA was extracted, and ISG54, ISG15, PKR, and OAS expression levels were measured by qRT-PCR. HT29 cells were mock-infected and were not treated by IFN-λ as “control (Ctrl)”. HT29 cells were infected with EV71 as “EV71”. The “IFN-λ + EV71” meant that HT29 was treated with IFN-λ 6 h before infection, and “EV71 + IFN-λ” meant that HT29 was treated with IFN-λ 6 h after infection. (E) The viral titers in the culture supernatant were monitored by plaque experiments at 24 hpi (F). The intracellular viral protein (VP1) load was measured by confocal fluorescence microscopy. Dates were presented as mean ± SD (n = 3 independent experiments, *P < 0.05, ***P < 0.001, *⁣*⁣**P < 0.0001 and ns, not significant).

FIGURE 4. The 2Apro and 3Cpro of EV71 antagonize the antiviral function of IFN-λ and inhibit the expression level of IFN-λ induced by poly(I:C). HT29 cells were transfected with pcDNA3.1-2A (control plasmid: pcDNA3.1) or p-EGFP-3C (control plasmid: p-EGFP). After transfection 24 h, cells were treated with IFN-λ (10 ng/ml) 6 h before infection. Then cells were infected with EV71 at an MOI of 1. Cells and culture mediums were collected at 24 hpi. (A) The protein expression level of VP1 was determined by incubating with anti-EV71 VP1 antibody, followed by staining with Alexa Fluor 555-conjugated secondary antibody. (B) The mRNA expression levels of VP1 were determined by qRT-PCR. (C) Cell culture medium was prepared for virus titer analysis. Control was cells with infected only. IFN-λ group was cells infected and using IFN-λ pretreatment. (D,E) HT29 cells were transfected with pcDNA3.1-2A (pcDNA3.1) or p-EGFP-3C (p-EGFP), and after transfection 24 h, cells were treated with poly(I:C) (4 μg/ml). Cells and culture mediums were collected 24 h after stimulation. The protein expression levels of IFN-λ1 and IFN-λ2 were determined by ELISA, and the mRNA expression levels were determined by qRT-PCR.…

FIGURE 5. The expression level of IFN-λR on intestinal epithelial cells is inhibited by EV71, and its 2Apro and 3Cpro play an important inhibitory effect. (A) The expression levels of IFNAR1, IFNAR2, (B) IL-28Rα, and IL-10Rβ on HT29 or Jurkat T were measured by FACS. (C) HT29 cells were infected with EV71 at an MOI of 1. The membrane expression level of IL-28Rα was detected at 48 hpi by FACS. MFI, mean fluorescence intensity. (D) At 48 hpi, cells were fixed and stained with anti-EV71 (green) antibodies, anti-IL-28Rα (red) antibodies, and DAPI (blue) and examined by a confocal microscope. Mock was cells without infection. Merged images of the different channels were shown. (E–G) HT29 cells were transfected with pcDNA3.1-2A (pcDNA3.1) or p-EGFP-3C (p-EGFP). After 24 h transfection, the cells were treated with PIC (4 μg/ml) for another 24 h. Then cells were collected to detect the expression level of IL-28Rα by FACS and IF, and the mRNA expression level was determined by qRT-PCR. Control was cells without any treatment. PIC group was cells stimulated only by poly(I:C). Dates were presented as mean ± SD (n = 3 independent experiments, *P < 0.05, **P < 0.01 and *⁣*⁣**P < 0.0001).

FIGURE 6. The 2Apro and 3Cpro of EV71 inhibit the production of antiviral proteins downstream of the IFN-λ pathway. HT29 cells were transfected with pcDNA3.1-2A (pcDNA3.1) or p-EGFP-3C (p-EGFP). After transfection 24 h, cells were stimulated by IFN-λ (10 ng/ml). The cells were collected 24 h later, and the mRNA expression levels of (A) ISG54, (B) ISG15, (C) PKR, and (D) OAS were measured by qRT-PCR. Control was cells without any treatment. Dates were presented as mean ± SD (n = 3 independent experiments, *P < 0.05, **P < 0.01 and *⁣*⁣**P < 0.0001).

FIGURE 7. EV71+IECs express lower levels of MICA/B and higher levels of PD-L1 and are less susceptible to iNK and CD3+iIELs lysis. (A) The intestinal intraepithelial lymphocytes from the C57BL/6 mouse were separated and extracted, and the ratio of CD3+iIELs and NK1.1+iNK cells was analyzed by flow cytometry. (B) HT29 cells were infected with EV71 at an MOI of 1 for 48 h. LDH was applied to analyze the susceptibility of EV71–HT29 and EV71+HT29 to NK-92 cell lysis at 6 h at the indicated E:T ratios. (C,E) The intestinal intraepithelial lymphocytes were prepared to enrich iNK or CD3+iIELs using the MojoSort™ Mouse NK Cell Isolation Kit or the MojoSort™ Mouse CD3 T Cell Isolation Kit by MACS. Cells were stained with anti-NK1.1 antibody or anti-CD3 antibody. (D,F) The h-SCARB2-MC38 cells were infected with EV71 at an MOI of 1 for 48 h. LDH was applied to analyze the susceptibility of EV71–h-SCARB2-MC38 and EV71+h-SCARB2-MC38 to iNK or CD3+iIELs cell lysis on 10 h at the indicated E:T ratios. (G,H) HT29 cells were infected with EV71 at an MOI of 1. The membrane expression levels of MICA/B or PD-L1 were detected at 48 hpi by FACS. (I,J) At 48 hpi, cells were fixed and stained with anti-EV71…

FIGURE 8. The 2Apro and 3Cpro reduce the susceptibility of IECs to NK lysis by suppressing NKG2DL expression and improving PD-L1 expression on IECs. (A) Analyze the susceptibility of HT29 cells transfected with the pcDNA3.1-2A (pcDNA3.1) or p-EGFP-3C (p-EGFP) plasmids to NK-92 cell lysis on 6 h at the indicated E:T ratios by LDH. (B,D) HT29 cells were transfected with the four kinds of plasmids. After 24 h transfection, the cells were collected to detect the expression levels of PD-L1 and MICA/B by FACS. (C,E,F) The mRNA expression levels of PD-L1, MICA/B, and ULBP1∼6 were determined by qRT-PCR. Control was cells without any treatment. Dates were presented as mean ± SD (n = 3 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001, *⁣*⁣**P < 0.0001 and ns, not significant).

FIGURE 9. The 2Apro and 3Cpro of enterovirus 71 inhibit the IFN-λ production and IFN-λ receptor expression and further decrease the response of IECs to IFN-λ. Also, 2Apro and 3Cpro reduce the susceptibility of IECs to iNK and CD3+iIELs lysis, possibly by down-regulation of NKG2DL and up-regulation of PD-L1.