ALV-J inhibits autophagy through the GADD45β/MEKK4/P38MAPK signaling pathway and mediates apoptosis following autophagy

Abstract

Autophagy and apoptosis, which are important processes for host immunity, are commonly exploited by viruses to facilitate their survival. However, to the best of our knowledge, very few studies have researched the mechanisms of action of the autophagic and apoptotic signaling pathways following viral infection. Thus, the present study aimed to investigate the mechanisms of action of growth arrest and DNA-damage-inducible β (GADD45β), an important resistance gene involved in the host resistance to ALV-J. Both ALV-J infection and the overexpression of GADD45β inhibited autophagy during the early stages, which prevented the autophagosomes from binding to the lysosomes and resulted in an incomplete autophagic flux. Notably, GADD45β was discovered to interact with MEKK4 in DF-1 cells. The genetic knockdown of GADD45β and MEKK4 using small interfering RNA-affected ALV-J infection, which suggested that ALV-J may promote the binding of GADD45β to MEKK4 to activate the p38MAPK signaling pathway, which subsequently inhibits autophagy. Furthermore, ALV-J was revealed to affect the autophagic pathway prior to affecting the apoptotic pathway. In conclusion, to the best of our knowledge, the present study was the first to investigate the combined effects of ALV-J infection on autophagy and apoptosis, and to suggest that ALV-J inhibits autophagy via the GADD45β/MEKK4/p38MAPK signaling pathway.

Fig. 1. GADD45β expression levels increase in DF-1 cells following ALV-J infection.

a DF-1 cells were infected with ALV-J for 24, 48, or 72 h and the expression levels of GADD45β were analyzed. Statistical differences were determined using two-way ANOVA. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. b Following 24, 48, or 72 h of ALV-J infection, total protein was extracted from DF-1 cells or CEF cells, the expression levels of GADD45β were analyzed using western blotting. The chart (b′) shows the quantification of GADD45β/β-actin in (b). c Cells were infected with ALV-J for 24 h and immunostained with rabbit anti-gp37 antibody (green). The expression levels of GADD45β (red) were analyzed using confocal microscopy. DAPI (blue) was used to stain the nuclear DNA. Scale bar, 10 μm. The chart (c′) shows the fluorescence intensity of endogenous GADD45β in (c). All experiments were repeated in triplicate.

Fig. 2. ALV-J infection and the overexpression of GADD45β inhibits autophagy.

a DF-1 cells or CEF cells were pretreated with 1 μM Rapa for 2 h prior to being infected with ALV-J for 24 h. Expression levels of gp37, LC3B, SQSTM1, and Atg5 were analyzed using western blotting. The chart (a′) shows the quantification of LC3II/β-actin, SQSTM1/β-actin and Atg5/β-actin in (a). b DF-1 cells or CEF cells were transfected with a GADD45β overexpression plasmid and pretreated with Rapa. The expression levels of LC3, SQSTM1, and Atg5 were analyzed using western blotting. The chart (b′) shows the quantification of LC3II/β-actin, SQSTM1/β-actin and Atg5/β-actin in (b). c DF-1 cells were transfected with a GFP-LC3B plasmid for 18 h and either infected with ALV-J or transfected with pRK5-flag-GADD45β for 24 h separately. The number of GFP-LC3B-labeled autophagosomes (green) was observed using confocal microscopy in the presence or absence of Rapa stimulation. The chart (c′) shows the GFP-LC3 fluorescence aggregation point of per cell in (c). Data are presented as the mean ± SD of three independent experimental repeats.

Fig. 3. Autophagosomes are unable to fuse with lysosomes following ALV-J infection or the overexpression of GADD45β.

a DF-1 cells or CEF cells were marked with Lyso Tracker Red (50 nM) for 2 h and GFP-LC3B-labeled autophagosomes (green) were visualized using confocal microscopy to colocalize red-stained acidified vesicles (red). b Cells were stained with anti-LAMP1 antibody (red) and anti-gp37 antibody (blue), and visualized using confocal microscopy to visualize the fusion between the autophagosomes and lysosomes. c Cells were infected with the mCherry-GFP-LC3B recombinant adenovirus to analyze the smoothness of autophagosome formation. Scale bar, 10 μm. The chart (c′) shows the quantification of GFP-mCherry-LC3 tandem reporter in (c).

Fig. 4. GADD45β interacts with MEKK4 in response to ALV-J infection-induced activation of the p38MAPK signaling pathway.

a DF-1 cells or CEF cells were transfected with siRNA targeting GADD45β for 24 h and subsequently infected with ALV-J for 24 h. The infected cells were then collected for western blotting analysis of the expression levels of gp37, p-p38, p38, p-JNK, JNK, p-ERK, ERK, LC3B, SQSTM1, and Atg5. b DF-1 cells were co-transfected with Flag-GADD45β and HA-MEKK4 for 24 h and the colocalization of GADD45β (red) with MEKK4 (green) was detected using the corresponding antibodies under confocal microscopy. Scale bar, 10 μm. c DF-1 cells were co-transfected with Flag-GADD45β and HA-MEKK4 for 48 h, and the interaction between GADD45β and MEKK4 was determined using the indicated antibodies. d ALV-J infection was performed following the transfection with siRNA targeting MEKK4 in DF-1 cells or CEF cells for 24 h. Expression levels gp37, p-p38, p38, p-JNK, JNK, LC3B, SQSTM1, and Atg5 were analyzed using western blotting. Data are presented as the mean ± SD of three independent experimental repeats.

Fig. 5. ALV-J inhibits autophagy through the GADD45β/MEKK4/p38MAPK signaling pathway.

a, b DF-1 cells or CEF cells were treated with SB203580 and SP600125, inhibitors of the p38MAPK and JNK signaling pathways, respectively, and infected with ALV-J for 24 h. Expression levels of gp37, LC3B, SQSTM1, Atg5, p-p38, p38, p-JNK, and JNK were analyzed using western blotting.

Fig. 6. Autophagy precedes apoptosis following ALV-J infection or the overexpression of GADD45β.

a, b Autophagy, but not apoptosis, was detected within 4 h of ALV-J infection or GADD45β overexpression. The expression levels of relevant important autophagy proteins and signaling pathway proteins (gp37, LC3B, SQSTM1, Atg5, p38, JNK, p38, and p-JNK) were detected using western blotting following ALV-J infection or GADD45β overexpression for 2, 4, 6, 8, 12, and 24 h. c, d Caspase-3 and caspase-8 activity assay kits were used to analyze caspase-3 and caspase-8 enzyme activities in DF-1 cells or CEF cells following ALV-J infection or GADD45β overexpression for 2, 4, 6, 8, 12, and 24 h. e As discovered through TEM analysis, autophagic compartments (autophagosomes and autolysosomes), but not apoptotic features, were present 4 h after ALV-J infection or GADD45β overexpression. However, after ALV-J infection or GADD45β overexpression for 12 h, not only were autophagic compartments present, but apoptotic features were also observed in some DF-1 cells. Red arrow indicates an autophagosome or autolysosome. Yellow arrows denote the abnormal morphology of the nuclear membrane.