Enhancing MyD88 oligomerization is one important mechanism by which IBDV VP2 induces inflammatory response

Abstract

The inflammatory response is an essential component of innate immunity to defense against pathogens. Infectious bursal disease (IBD) is the most important immunosuppressive disease in chickens and is caused by the infectious bursal disease virus (IBDV). Acute inflammation is a typical pathogenic process for IBD, however, the underlying mechanism is not clear. Here, we report that IBDV induces obvious inflammatory response in vivo and in vitro. Furthermore, viral VP2 is identified as an important inflammatory stimulus. It is observed that IBDV VP2 can activate NF-κB signaling pathway and then increase IL-1β production. In detail, IBDV VP2 interacts with myeloid differentiation primary response gene 88 (MyD88), potentiates the oligomerization of MyD88 and assembly of MyD88 complex, which is one important element leading to NF-κB signaling pathway activation and IL-1β production increase. More meaningfully, residues 253/284 of viral VP2 are significantly involved in IBDV-induced inflammatory response through modulating the interaction strength between VP2 and MyD88 and the following MyD88-NF-κB-IL-1β signaling pathway. This study reveals one molecular mechanism that trigger inflammation during IBDV infection, which is of great significance for a deeper understanding of the pathogenic mechanisms of IBDV.

Fig 1. IBDV infection induces inflammatory response in vivo and in vitro.

(A) Macroscopic lesions of the bursa of vvIBDV-infected chicken at 3 dpi. (B, C) Protein abundance of IL-1β (B) and TNF-α (C) in serums at indicated times post-infection were detected by ELISA. (D) Representative H&E stained analysis of the bursa sections of mock and vvIBDV infected chickens at 3 dpi. The lymphoid follicles which infected with vvIBDV were severely atrophied with hemorrhage, lymphocytes depletion, and inflammatory cells infiltration including macrophages (green arrow). (E) The vvIBDV growth dynamic in HD11 cells was analysed by RT-qPCR. (F) The vvIBDV subcellular localization. HD11 cells were incubated with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) for 2 h. The vvIBDVs were stained as green color and the engulfed vvIBDVs by cell pseudopodia were highlighted with the white arrows and white boxes. (G, H) Effect of vvIBDV infection on IL-1β or TNF-α gene expression in HD11 cells. HD11 cells were infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells), and the mRNA levels of IL-1β (G) or TNF-α (H) were assessed by RT-qPCR at 0, 6, 12, 24 hpi. (I, J) Effect of vvIBDV infection on IL-1β production in HD11 cells. In the HD11 infection experiments, the IL-1β in the cell supernatants at indicated times post infection were detected by ELISA, and the pro-IL-1β in the cell protein extracts were measured by immunoblot analysis (I). In addition, different infection doses of vvIBDV on IL-1β production in HD11 cells at 6 h were detected by ELISA and immunoblot analysis (J). HD11 cells stimulated with LPS (1 μg/mL) was measured at 6 h after stimulating as a control. All data are representative of at least three independent experiments. Graphs show mean ± SD, n = 3, * P < 0.05, *** P < 0.001.

Fig 2. IBDV capsid protein VP2 can activate the inflammatory response.

(A, B) Effect of viral VP1, VP2, VP3, VP4, and VP5 on IL-1β or TNF-α gene expression. HD11 cells were transfected with the recombinant plasmid expressing viral protein fusing HA-tag (3 μg/1×10⁶ cells) for 24 h, and levels of IL-1β (A) or TNF-α (B) mRNA were assessed by RT-qPCR. (C, D) Effect of viral VP1, VP2, VP3, VP4, and VP5 on IL-1β production. HD11 cells were transfected with the recombinant plasmid expressing viral protein fusing HA-tag (3 μg/1×10⁶ cells) for 24 h, and IL-1β in the cell supernatant were measured by ELISA (C), and pro-IL-1β in the total cell protein extracts were resolved by immunoblot analysis (D). (E) VLP of VP2 is engulfed by HD11 cells. HD11 cells were incubated with VLP of VP2 (100 μg/mL) for 2 h. VLP staining using IBDV VP2 monoclonal antibody was showed as green color and the engulfed VLP by cell pseudopodia was highlighted with the white arrows. (F, G) Effect of VLP incubation on IL-1β or TNF-α gene expression. HD11 cells were incubated with VLP (50 μg/mL or 100 μg/mL) for 0, 6, 12 h, and the IL-1β (F) or TNF-α (G) mRNA were assessed by RT-qPCR. (H) Effect of VLP incubation on IL-1β production. HD11 cells were incubated with VLP (50 μg/mL and 100 μg/mL) for 6 h or stimulated with LPS (1 μg/mL) for 6 h, the pro-IL-1β in the total cell protein extracts were detected by immunoblot analysis with specific antibodies shown on the left, and IL-1β in the cell supernatants were measured by ELISA. (I, J) Effect of viral VP2 on IL-1β or TNF-α gene expression. HD11 cells were transfected with the plasmid expressing HA-VP2 (3 μg/1×10⁶ cells) for 0, 6, 12 and 24 h, and levels of IL-1β (I) or TNF-α (J) mRNA were assessed by RT-qPCR. (K, L) Effect of VP2 on IL-1β production. HD11 cells were transfected with the plasmid expressing HA-VP2 (1.5 μg/1×10⁶ cells and 3 μg/1×10⁶ cells) for 6, 12 and 24 h, and pro-IL-1β in the total cell protein extracts were resolved by immunoblot analysis (K), and IL-1β in the cell supernatant were measured by ELISA (L). HD11 cells stimulated with LPS (1 μg/mL) was used as a control. All data are representative of at least three independent experiments. Graphs show mean ± SD, n = 3, *** P < 0.001.

Fig 3. IBDV VP2 promotes the NF-

κB signaling pathway activation. (A, B) Effect of IBDV or viral VP2 on NF-κB mediated gene expression. HD11 cells were infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) (A) or transfected with the plasmid expressing HA-VP2 (3 μg/1×10⁶ cells) (B) for 0, 6, 12 and 24 h, and levels of p65 mRNA were assessed by RT-qPCR. (C, D) Effect of IBDV or viral VP2 on the phosphorylation and nuclear translocation of p65. HD11 cells were infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) (C) or transfected with the plasmid expressing HA-VP2 (3 μg/1×10⁶ cells) (D) for 0, 6, 12, 24 h or stimulated with LPS (1 μg/mL) (C) for 6 h, and p65 in the total cell protein extracts were detected by immunoblot analysis. (E) Effect of IBDV on the nuclear translocation of p65. HD11 cells were infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) or stimulated with LPS (1 μg/mL) for 12 h, and p65 was stained using p65 monoclonal antibody. The nuclear translocation of p65 is highlighted with the white arrows. (F, G) Effect of viral VP2 on the phosphorylation and nuclear translocation of p65. HD11 cells were transfected with the plasmid expressing Flag-p65 (2 μg/1×10⁶ cells) or HA-VP2 (2 μg/1×10⁶ cells) respectively or simultaneously for 24 h, and p65 was stained using p65 monoclonal antibody. The nuclear translocation of p65 is highlighted with the white arrows (F). The cytoplasmic and nuclear extracts were detected by immunoblot analysis (G). (H) Effect of NF-κB inhibitor on IL-1β production induced by IBDV. HD11 cells mock treated or pre-treated with BAY 11-7082 (20 μM) for 6 h were mock infected, or infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) in the presence or absence of drug pre-treated for 6 h and then harvested at 6 hpi, or stimulated with LPS (1 μg/mL) in the presence or absence of drug pre-treated for 6 h and then harvested at 6 hpi. The total cell protein extracts were detected by immunoblot analysis, and IL-1β in cell supernatants were measured by ELISA. (I) Effect of NF-κB inhibitor on IL-1β production induced by viral VP2. HD11 cells mock treated or pre-treated with BAY 11-7082 (20 μM) for 6 h were mock transfected, or transfected with the plasmid expressing HA-VP2 (3 μg/1×10⁶ cells) in the presence or absence of drug pre-treated for 6 h and then harvested at 24 h. Total cell protein extracts were detected by immunoblot analysis, and the protein production of IL-1β were measured by ELISA. (J, K) RT-qPCR (J) and immunoblot (K) analysis of p65 in HD11 cells untreated or treated with indicated shRNA (1 μg/1×10⁵ cells). Twenty-four hours later, cells were infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) for 6 h. (L) Effect of p65 interference on IL-1β production induced by IBDV. HD11 cells mock treated or infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) in the presence or absence of shRNA-mediated specific gene silence pre-treated for 24 h and then harvested at 6 hpi, or stimulated with LPS (1 μg/mL) in the presence or absence of shRNA-mediated specific gene silence pre-treated for 24 h and then harvested at 6 h. Total cell protein extracts were detected by immunoblot analysis, and the protein production of IL-1β were measured by ELISA. (M) Effect of p65 interference on IL-1β production induced by viral VP2. HD11 cells mock treated or transfected with the plasmid expressing HA-VP2 (3 μg/1×10⁶ cells) in the presence or absence of shRNA-mediated specific gene silence pre-treated for 24 h and harvested at 24 h post-transfection. Total cell protein extracts were detected by immunoblot analysis, and the protein production of IL-1β were measured by ELISA. HD11 cells stimulated with LPS (1 μg/mL) was used as a control. All data are representative of at least three independent experiments. Graphs show mean ± SD, n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig 4. MyD88 is associated with NF-

κB-dependent inflammatory response activated by IBDV. (A-C) Effect of IBDV or VLP or viral VP2 on TIRAP, MyD88, and TRAF6 gene expression. HD11 cells were infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) (A) or incubated with VLP (100 μg/mL) (B) or transfected with the recombinant plasmid expressing HA-VP2 (3 μg/1×10⁶ cells) (C), and levels of TIRAP, MyD88, and TRAF6 mRNA were assessed by RT-qPCR. (D, E) RT-qPCR (D) and immunoblot (E) analysis of TIRAP in HD11 cells untreated or treated with indicated shRNA (1 μg/1×10⁵ cells). Twenty-four hours later, cells were incubated with LPS (1 μg/mL) for 6 h. (F) Effect of TIRAP interference on IL-1β production. HD11 cells mock treated or infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) in the presence or absence of shRNA-mediated specific gene silence pre-treated for 24 h and then harvested at 6 hpi, or stimulated with LPS (1 μg/mL) or VLP (100 μg/mL) in the presence or absence of shRNA-mediated specific gene silence pre-treated for 24 h and then harvested at 6 h. Total cell protein extracts were detected by immunoblot analysis, and protein production of IL-1β were measured by ELISA. (G, H) RT-qPCR (G) and immunoblot (H) analysis of MyD88 in HD11 cells untreated or treated with indicated shRNA (1 μg/1×10⁵ cells). Twenty-four hours later, cells were incubated with LPS (1 μg/mL) for 6 h. (I) Effect of MyD88 interference on IL-1β production. HD11 cells mock treated or infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) in the presence or absence of shRNA-mediated specific gene silence pre-treated for 24 h were harvested at 6 hpi, or stimulated with LPS (1 μg/mL) or VLP (100 μg/mL) in the presence or absence of shRNA-mediated specific gene silence pre-treated for 24 h and then harvested at 6 h post stimulation. Total cell protein extracts were detected by immunoblot analysis, and IL-1β in the cell supernatants were measured by ELISA. (J) Effect of MyD88 inhibitor on IL-1β production. HD11 cells mock treated or pre-treated with ST2825 (15 μM) for 6 h were mock infected, or infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) in the presence or absence of drug pre-treated for 6 h and then harvested at 6 hpi, or stimulated with LPS (1 μg/mL) or VLP (100 μg/mL) in the presence or absence of drug pre-treated for 6 h and then harvested at 6 h post stimulation. Total cell protein extracts were detected by immunoblot analysis, and protein production of IL-1β were measured by ELISA. (K, L) RT-qPCR (K) and immunoblot (L) analysis of TRAF6 in HD11 cells untreated or treated with indicated shRNA (1 μg/1×10⁵ cells). Twenty-four hours later, cells were incubated with LPS (1 μg/mL) for 6 h. (M) Effect of TRAF6 interference on IL-1β production. HD11 cells mock treated or infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) in the presence or absence of shRNA-mediated specific gene silence pre-treated for 24 h were harvested at 6 hpi, or stimulated with LPS (1 μg/mL) or VLP (100 μg/mL) in the presence or absence of shRNA-mediated specific gene silence pre-treated for 24 h and then harvested at 6 h post stimulation. Total cell protein extracts were detected by immunoblot analysis, and IL-1β in the cell supernatants were measured by ELISA. (N) Luciferase assay of NF-κB promoter. HD11 cells co-transfected with NF-κB reporter plasmid (1 μg/1×10⁶ cells) and HA-TIRAP (1 μg/1×10⁶ cells), or HA-MyD88 (1 μg/1×10⁶ cells), or HA-TRAF6 (1 μg/1×10⁶ cells), and then harvested at 24 h post transfection. The protein production of Luciferase was measured by ELISA. HD11 cells stimulated with LPS (1 μg/mL) as a control. All data are representative of at least three independent experiments. Graphs show mean ± SD, n = 3, ^* P < 0.05, ** P < 0.01, *** P < 0.001.

Fig 5. IBDV VP2 interacts with MyD88.

(A) Luciferase assay of NF-κB promoter. DF-1 cells co-transfected with NF-κB reporter plasmid (1 μg/1×10⁶ cells) and HA-VP2 (1 μg/1×10⁶ cells) in the presence or absence of Flag-MyD88 (1 μg/1×10⁶ cells) and then harvested at 24 h post-transfection. The protein production of Luciferase was measured by ELISA. (B, C) Co-localization of MyD88 with VP2. HD11 cells transfected with the plasmid expressing Flag-MyD88 (2 μg/1×10⁶ cells) and HA-VP2 (2 μg/1×10⁶ cells) respectively or simultaneously for 24 h, MyD88 and VP2 were stained using the Flag Tag and HA Tag, respectively. The aggregation of MyD88 was highlighted with the white arrows (B). The cytoplasmic and nuclear extracts were detected by immunoblot analysis (C). (D) The interaction of VP2 and MyD88 detected by Co-IP. HEK293T cells were transfected with indicated plasmid (2 μg/1×10⁶ cells) and harvested at 30 h post-transfection. Cell lysates were immunoprecipitated using Flag Tag and analysed using the HA Tag and Flag Tag. (E) The interaction of VP2 and MyD88 detected by GST pull-down assay. Purified GST-MyD88 or GST was incubated with cell lysates of the plasmid expressing HA-VP2 (2 μg/1×10⁶ cells) in HEK293T cells. Cell extracts were incubated with GST agarose beads for 12 h. Mixtures were analysed by immunoblot using the HA Tag or GST Tag. (F) Schematic of the plasmids expressing the full-length MyD88 and its truncations (DD or TIR). (G, H) VP2 interacts with MyD88 truncations. HEK293T cells were transfected with indicated plasmid (2 μg/1×10⁶ cells) and harvested at 30 h post-transfection. Cell lysates were immunoprecipitated using the Flag Tag and analyzed using the HA and Flag Tag. All data are representative of at least three independent experiments. Graphs show mean ± SD, n = 3, ** P < 0.01, *** P < 0.001.

Fig 6. IBDV VP2 enhances MyD88 complex assembly and MyD88 oligomerization.

(A) VP2 promotes the associations of TIRAP-MyD88-TRAF6. HD11 cells were co-transfected with indicated plasmids (1 μg/1×10⁶ cells) for 24 h. Cell lysates were immunoprecipitated using the HA Tag and analysed using the HA, Flag, and Myc Tag. (B) VP2 promotes the association of endogenous TIRAP-MyD88-TRAF6. HD11 cells were transfected with the plasmid expressing HA-VP2 (3 μg/1×10⁶ cells) for 12 h. Cell lysates were immunoprecipitated using the HA Tag and analysed using TIRAP, MyD88, TRAF6 monoclonal antibody, and HA Tag. (C) Effect of IBDV on MyD88 distribution. HD11 cells were infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) or stimulated with LPS (1 μg/mL) for 12 h, and MyD88 was stained using MyD88 monoclonal antibody. The aggregation of MyD88 was highlighted with the white arrows. (D) VP2 enhances the oligomerization of MyD88. HD11 cells were transfected with plasmids expressing Flag-MyD88 (1.5 μg/1×10⁶ cells), HA-MyD88 (1.5 μg/1×10⁶ cells), and increasing doses of Myc-VP2 (0, 0.7, 1.5 μg) for 24 h. Cell lysates were immunoprecipitated using the Flag Tag and analysed using the HA, Flag, and Myc Tag. (E) VP2 enhances the oligomerization of endogenous MyD88. HD11 cells were transfected with plasmid expressing HA-VP2 (3 μg/1×10⁶ cells) for 12 h. Cell lysates were immunoprecipitated using HA Tag and analysed using MyD88 and HA Tag antibodies. (F) IBDV enhances the oligomerization of endogenous MyD88. HD11 cells were infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) for 24 h. Cell lysates were immunoprecipitated and analysed using anti-MyD88 antibody. (G-H) VP2 enhances oligomerization of MyD88 in the natural condition in HD11 cells. HD11 cells were transfected with the plasmid expressing HA-VP2 (3 μg/1×10⁶ cells) for 12 h. Cell lysates were separated by native or SDS PAGE and analysed by immunoblot with the indicated antibodies. (I) IBDV enhances oligomerization of MyD88 in the natural condition in HD11 cells. HD11 cells were infected with vvIBDV (1×10¹⁰ copies/1×10⁶ cells) for 12 and 24 h. Cell lysates were separated by native or SDS PAGE and analysed by immunoblot with the indicated antibodies. HD11 cells stimulated with LPS (1 μg/mL) as a control. All data are representative of at least three independent experiments.

Fig 7. Residues 253 and 284 of VP2 are involved in the difference of inflammatory response induced by different IBDV strains.

(A) Residues 253/284 mutations downregulate the interaction of VP2 and MyD88. HEK293T cells were transfected with indicated plasmids (2 μg/1×10⁶ cells) for 30 h. Cell lysates were immunoprecipitated using Flag Tag and analysed using the HA Tag and Flag Tag. (B) The growth dynamics of HLJ0504 or HT strain of IBDV in HD11 cells were analysed by RT-qPCR. (C, D) Effect of HLJ0504 or HT strain on MyD88 gene expression and production. HD11 cells were infected with HLJ0504 or HT strain (1×10¹⁰ copies/1×10⁶ cells), and levels of MyD88 mRNA were assessed by RT-qPCR at 6 and 12 hpi (C), and total cell protein extracts were analysed by native or SDS PAGE and probed with specific antibodies shown on the left (D). (E, F) Effect of HLJ0504 or HT strains on NF-κB mediated gene expression and production. HD11 cells were infected with HLJ0504 or HT (1×10¹⁰ copies/1×10⁶ cells) for 6 and 12 hpi, and levels of p65 mRNA were assessed by RT-qPCR (E), and total cell protein extracts were analysed by immunoblot analysis, and probed with specific antibodies shown on the left (F). (G-I) Effect of HLJ0504 or HT strain on the expression of IL-1β, TNF-α, and production of IL-1β. HD11 cells were infected with HLJ0504 (1×10¹⁰ copies/1×10⁶ cells) or HT strain (1×10¹⁰ copies/1×10⁶ cells) for 6 and 12 hpi, and levels of IL-1β (G) and TNF-α (H) mRNA were assessed by RT-qPCR. The pro-IL-1β in the cell lysates were analysed by immunoblot analysis, and IL-1β in the cell supernatants were measured by ELISA (I). All data are representative of at least three independent experiments. Graphs show mean ± SD, n = 3, *** P < 0.001.