Binding of the pathogen receptor HSP90AA1 to avibirnavirus VP2 induces autophagy by inactivating the AKT-MTOR pathway

Abstract

Autophagy is an essential component of host innate and adaptive immunity. Viruses have developed diverse strategies for evading or utilizing autophagy for survival. The response of the autophagy pathways to virus invasion is poorly documented. Here, we report on the induction of autophagy initiated by the pathogen receptor HSP90AA1 (heat shock protein 90 kDa α [cytosolic], class A member 1) via the AKT-MTOR (mechanistic target of rapamycin)-dependent pathway. Transmission electron microscopy and confocal microscopy revealed that intracellular autolysosomes packaged avibirnavirus particles. Autophagy detection showed that early avibirnavirus infection not only increased the amount of light chain 3 (LC3)-II, but also upregulated AKT-MTOR dephosphorylation. HSP90AA1-AKT-MTOR knockdown by RNA interference resulted in inhibition of autophagy during avibirnavirus infection. Virus titer assays further verified that autophagy inhibition, but not induction, enhanced avibirnavirus replication. Subsequently, we found that HSP90AA1 binding to the viral protein VP2 resulted in induction of autophagy and AKT-MTOR pathway inactivation. Collectively, our findings suggest that the cell surface protein HSP90AA1, an avibirnavirus-binding receptor, induces autophagy through the HSP90AA1-AKT-MTOR pathway in early infection. We reveal that upon viral recognition, a direct connection between HSP90AA1 and the AKT-MTOR pathway trigger autophagy, a critical step for controlling infection.

Keywords: AKT-MTOR pathway; ANOVA, analysis of variance; ATG5, autophagy-related 5; BCA, bicinchoninic acid; BECN1, Beclin 1, autophagy-related; CoIP, coimmunoprecipitation; DMEM, Dulbecco's modified Eagle's medium; EBSS, Earle's balanced salt solution; EIF2AK2, eukaryotic translation initiation factor 2-alpha kinase 2; EIF2S1, eukaryotic translation initiation factor 2, subunit 1 alpha; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GOPC, golgi-associated PDZ and coiled-coil motif containing; GST, glutathione S-transferase; Gg, Gallus gallus (chicken); HE-IBDV, heat-inactivated IBDV; HSP90AA1; HSP90AA1, heat shock protein 90 kDa alpha (cytosolic), class A member 1; HSV-1, herpes simplex virus 1; Hs, Homo sapiens (human); IBDV, infectious bursal disease virus; IgG, immunoglobulin G; LPS, lipopolysaccharide; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MOI, multiplicity of infection; MTOR, mechanistic target of rapamycin (serine/threonine kinase); Ni-NTA, nickel-nitrilotriacetic acid; PAMP, pathogen-associated molecular patterns; PBS, phosphate-buffered saline; PI3K, phosphoinositide 3-kinase; PRR, pattern recognition receptors; RNAi, RNA interference; SDS, sodium dodecyl sulfate; SQSTM1, sequestosome 1; SVP, subviral particle; TCID50, 50% tissue culture infectious doses; TLR, toll-like receptors; TSC, tuberous sclerosis complex; VP, viral protein; autophagy; avibirnavirus; cDNA, complementary DNA; dsRNA, double-stranded RNA; eGFP, enhanced green fluorescent protein; hpi, hours post-infection; mAb, monoclonal antibody; shRNA, short hairpin RNA; siRNA, small interfering RNA; viral protein VP2.

Figure 1.

IBDV infection induces autophagosome accumulation in DF-1 cells. (A) Autophagic vacuoles in infected cells observed by transmission electron microscopy. Autophagic vacuole engulfs IBDV particle (black arrows) and organelle (asterisk) in cytoplasm of DF-1 cell infected with IBDV (MOI = 10) at 2 hpi. (B) DF-1 cells transfected with peGFP-LC3 for 48 h, and then infected with IBDV (MOI = 10) or treated with HE-IBDV. At 2 hpi or 4 hpi, cells were incubated with LysoTracker Red (50 nM) for 30 min; intracellular autophagic vacuoles were observed under confocal microscopy. Statistical analysis of the number of cells with >3 autophagic vacuoles. At 2 hpi or 4 hpi, autopgagic vacuoles were counted in IBDV-infected cells. Error bars: Mean ± SD of 3 independent tests. Two-way analysis of variance (ANOVA); ***P < 0.001 compared to control. (C) DF-1 cells transfected with peGFP-LC3 for 48 h, and then infected with IBDV (MOI = 10) or treated with HE-IBDV. At 2 hpi or 4 hpi, cells were incubated with LysoTracker Red (50 nM) for 30 min; intracellular autophagic vacuoles were observed under confocal microscopy. Scale bars: 10 10 μm.mu;m. (D) DF-1 cells transfected with peGFP-LC3 and infected with IBDV at 9 h post-transfection. The cells were fixed, immunostained with anti-VP2 mAb, and autophagosomes were observed under confocal microscopy. Scale bar: 10 μ.

Figure 2.

Characterization of IBDV-triggered autophagosome accumulation. (A) IBDV promotes increase of LC3-II and decrease of SQSTM1 within 1 hpi but inhibits it from 4 hpi. DF-1 cells were infected with IBDV (MOI = 10). (B) Increase of LC3-II and decrease of SQSTM1 was constantly promoted in HE-IBDV-treated cells. (C) LC3 amount and SQSTM1 accumulation in mock-infected cells were unaffected. Cells were harvested and analyzed by immunoblotting using anti-LC3, anti-SQSTM1, anti-VP2, or anti-GAPDH antibody. (D) DF-1 cells were infected with the purified infectious IBDV virions or treated with the purified heat-inactivated IBDV virions (MOI = 10), respectively. The purified infectious IBDV increases the amount of LC3-II within 1 hpi but inhibits it from 4 hpi. However, levels of LC3-II increase constantly in the purified HE-IBDV-treated cells. M, mock infected. HI, purified heat-inactivated IBDV virions. PI, purified infectious IBDV virions. (E, F) The ratio of LC3-II or SQSTM1 to GAPDH was normalized to control conditions in (A to C). (G) The ratio of LC3-II to GAPDH was normalized to control conditions in (D). (H) LC3-II increases and SQSTM1 decreases in VP2-transfected cells. 293T cells were transfected with the vector pFlag-VP2 for 48 h, harvested and analyzed by immunoblotting using anti-LC3, anti-SQSTM1, anti-VP2, or anti-GAPDH antibody. Error bars: Mean ± SD of 3 independent tests. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001 compared to control.

Figure 3.

Effect of autophagy on IBDV replication. (A, B) BECN1 knockdown increases the accumulation of IBDV-encoded VP2 in 293T or DF-1 cells. DF-1 cell and 293T cell monolayers were transfected with negative control (Con.), Scrambled shRNA (Scra.) or Gallus gallus (chicken) BECN1 shRNA (GgBECN1 shRNA) or Homo sapiens (human) BECN1 (HsBECN1 shRNA) for RNA knockdown, respectively, and were infected with IBDV (MOI = 10). At 8 hpi, cells were harvested and analyzed by western blotting with anti-LC3, anti-VP2, and anti-GAPDH antibodies. The ratio of VP2 or BECN1 to GAPDH was normalized to control conditions. (C, D) DF-1 or 293T cells transfected with Scra. or HsBECN1 shRNA, or GgBECN1 shRNA were infected with IBDV (MOI = 0.01) for 24 h. Progeny virus yields in DF-1 cells were determined by TCID₅₀ assay. (E, F) Induction of autophagy with rapamycin (Rapa.) reduced IBDV replication in DF-1 and 293T cells. DF-1 and 293T cells were pretreated in DMEM containing 1 1 μM mu;M or 2 2 μM mu;M rapamycin for 4 h and then infected with IBDV (MOI = 10) for 1 h. After 1 hpi, cells were incubated in DMEM containing 1 1 μM mu;M or 2 2 μM mu;M rapamycin for 7 h, then harvested, lysed, and processed for western blotting with anti-LC3, anti-VP2, and anti-GAPDH antibodies. The ratio of LC3-II or VP2 to GAPDH was normalized to control conditions. (G, H) DF-1 or 293T cells were infected with IBDV (MOI = 0.01) and cultured in DMEM containing 2 2 μM mu;M rapamycin for 24 h to yield progeny virus; virus titers in DF-1 cells were determined by TCID₅₀ assay. (I) Induction of autophagy with starvation reduced IBDV replication in DF-1cells. DF-1 cells were pretreated in EBSS for 2 h. Then the cells were infected with IBDV at MOI of 10 and incubated in EBSS. After cultured for 8 h, cells were harvested and detected by western blotting with anti-LC3, anti-VP2, and anti-GAPDH antibodies. The ratio of LC3-II or VP2 to GAPDH was normalized to control conditions. (J) DF-1 cells pretreated with EBSS for 2 h were infected with IBDV (MOI = 0.01) for 1 h and cultured in EBSS for 24 h to yield progeny virus; virus titers in DF-1 cells were determined by TCID₅₀ assay. Error bars: Mean ± SD of 3 independent tests. Two-way ANOVA; ***P < 0.001 compared to control.

Figure 4.

MTOR and AKT were inactivated at the early stage of IBDV infection. (A) DF-1 cells infected with IBDV (MOI = 10). Cells were harvested at 1, 2, 4, 6, and 8 h, followed by immunoblotting with anti-p-MTOR, anti-MTOR (total protein), anti-p-AKT, anti-AKT1, or anti-GAPDH antibody. (B) Cells incubated with HE-IBDV for 1, 2, 4, 6, and 8 h followed by immunoblotting as in (A). (C) Cells mock-infected and analyzed by immunoblotting as in (A). (D, E) The ratio of p-MTOR to MTOR or p-AKT to AKT was normalized to mock infection and set at 1.0. (F) Cells were infected with purified IBDV or treated with purified HE-IBDV for 1, 2, and 4 h, and analyzed by immunoblotting using the antibody in (A). (G) DF-1 cells pretreated with insulin (500 nM) for 1 h, infected with IBDV and cultured for 2 h, and processed by immunoblotting using the corresponding antibody in (A). (H) DF-1 cells pretreated with insulin (500 nM) for 1 h, incubated with HE-IBDV for 2 h, and processed for immunoblotting using the antibody in (A). The ratio of LC3 to GAPDH, p-MTOR to MTOR, or p-AKT to AKT was normalized to control conditions and set at 1.0. Error bars: Mean ± SD of 3 independent tests. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001 compared to control.

Figure 5.

IBDV VP2 was sufficient for inducing autophagy via AKT and MTOR dephosphorylation. (A) His-tagged VP2 or VP3 expressed in E. coli BL21 and purified in Ni-NTA columns. The purified products were separated using SDS-PAGE and stained with Coomassie brilliant blue. (B) The subviral particles of the purified His-VP2 protein expressed in E.coli. Scale bar: 50 nm. (C) DF-1 cells transfected with eGFP-LC3 for 24 h and incubated with His-VP2 (100 ng/mL), His-VP3 (100 ng/mL), or His-GST (100 ng/mL) for 2 h and observed under confocal microscopy. The ratio of cells with >3 autophagic vacuoles was determined. Scale bars: 10 10 μm.mu;m. Error bars: Mean ± SD of 3 independent tests. (D, E) DF-1 cells incubated in DMEM containing His-GST, His-VP2, or His-VP3 for 4 h, were analyzed by western blotting with anti-LC3, anti-SQSTM1, anti-GAPDH, anti-p-MTOR, anti-MTOR, anti-p-AKT, and anti-AKT1 antibodies. The ratio of LC3 or SQSTM1 to GAPDH, p-MTOR to MTOR, and p-AKT to AKT were normalized to control conditions. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001 compared to the control.

Figure 6.

HSP90AA1 binding to VP2 triggers autophagy via AKT-MTOR dephosphorylation. (A) DF-1 cells transfected with pFlag-VP2 for 24 h. Whole cell lysates (WCL) were used for CoIP and western blotting with anti-Flag or anti-HSP90AA1 antibody (anti-AA1) and irrelevant IgG (Control). (B) DF-1cells cotransfected with Myc-GgHSP90AA1 (Myc-GgAA1) and Flag-GgAKT1 for 48 h. Transfected cells were infected with IBDV for 1 or 2 h, or were incubated in DMEM containing His-VP2(100 ng/ml), His-VP3(100 ng/ml) or His-GST(100 ng/ml). Whole cell lysates of each sample were used for CoIP with anti-MYC antibody and western blotting with anti-Flag or anti-MYC antibody. (C) Western blotting performed using anti-LC3 antibody and anti-SQSTM1 mAb on lysates from DF-1 cells cultured in uncoated plates or in coated plates with anti-HSP90AA1 or irrelevant isotype control IgG for 4 h. The ratio of SQSTM1 or LC3-II to GAPDH was normalized to control conditions. (D) DF-1 cells transfected with peGFP-LC3 for 24 h and cultured in plates coated with negative control, IgG, or anti-HSP90AA1 for 4 h. Autophagic vacuoles were analyzed under confocal microscopy. The ratio of cells containing >3 ring-like GFP structures was determined. Scale bars: 10 10 μm.mu;m. Error bars: Mean ± SD of 3 independent tests. (E) DF-1 cells were incubated respectively with the His-GST, mixture of His-GST and HSP90AA1 (His-GST:HSP90AA1 = 1:2.5), mixture of SVP and HSP90AA1 (SVP:HSP90AA1 = 1:2.5) or SVP for 2 h, and analyzed by immunoblotting with anti-LC3, anti-p-MTOR, anti-MTOR, anti-p-AKT, anti-AKT1, or anti-GAPDH antibody. (F) DF-1 cells incubated with His-GST (100 ng/ml), SVP (100 ng/ml), or purified IBDV (MOI = 10 ) for 2 h at 37°C or 4°C. Cells were analyzed by immunoblotting using the antibody in (E). The ratio of p-MTOR to MTOR, p-AKT to AKT or LC3-II to GAPDH was normalized to mock infection and set at 1.0. Two-way ANOVA; ***P < 0.001 compared to control. C, His-GST; PtdIns, purified IBDV; SVP, His-VP2 subviral particle.

Figure 7.

HSP90AA1, AKT, and MTOR are critical for autophagic induction at the early stage of IBDV infection. DF-1 cells transfected with Con. (Control), Scra. (Scrambled shRNA), GgHSP90 (GgHSP90AA1 shRNA), GgAKT1 (GgAKT1 shRNA), and GgMTOR (GgMTOR shRNA) for RNA knockdown were infected with IBDV (A), and pretreated with purified His-VP2 subviral particles (B) for 2 h and analyzed by immunoblotting with anti-LC3, anti-SQSTM1 or anti-GAPDH antibodies. The purified His-GST was as a control. (C) DF-1 cells were pretreated by specific inhibitors 17-AAG (HSP90AA1), LY294002 (AKT) and rapamycin (MTOR) for 4 h, and incubated with His-GST (100 ng/ml), SVP (100 ng/ml) or purified IBDV (MOI = 10) for 2 h. The cells were harvested and analyzed by immunoblotting with anti-LC3, anti-SQSTM1 or anti-GAPDH antibody. The ratio of LC3 or SQSTM1 to GAPDH was normalized to control conditions. Two-way ANOVA; ***P < 0.001 compared to control. C, His-GST; PI, purified IBDV; SVP, His-VP2 subviral particle.

Figure 8.

Proposed model of IBDV-induced autophagy via the HSP90AA1-AKT-MTOR pathway. IBDV-encoded VP2 binding to cell surface HSP90AA1 and leads to disassociation of phosphorylated AKT from HSP90AA1. The disassociated AKT then loses phosphorylation and results in dephosphorylation of MTOR. The dephosphorylated MTOR then activates autophagosome formation. The autophagosome engulfs IBDV virions, delivering them to lysosomes for final degradation.