Fig 1.
Golgi apparatus morphology after AIV infection.
(A) Collapse of the Golgi apparatus induced by AIV infection at different time points. A549 cells were infected with AIV at 1 MOI, fixed on slides at 12, 24, 36, and 48 h post-infection, and incubated with antibodies against IAV-NP, GM130, and DAPI. Monensin (10 μM) treatment and BFA (10 μM) treatment served as different positive controls. (B) Collapse of the Golgi apparatus induced by AIV infection at different infection doses. A549 cells were infected with AIV at 0, 0.01, 0.1, 1, and 10 MOI, fixed on slides at 24 h post-infection, and incubated with antibodies against IAV-NP, GM130, and subjected to DAPI staining. (C) ER stress does not influence the collapse of the Golgi apparatus following AIV infection. A549 cells were infected with AIV at 1 MOI and supplemented with 4-phenylbutyric acid (5 μM), fixed at 36 h post-infection on slides, and incubated with antibodies against IAV-NP, GM130, and subjected to DAPI staining. The cells were visualized using a Zeiss confocal fluorescence microscope LSM880. Scale bar = 10 μm.
Fig 2.
Activation of Golgi apparatus stress (GAS) response pathways following AIV infection.
(A) mRNA levels of GAS-related proteins in response to AIV infection. A549 cells were infected with AIV, and total RNA was extracted at 12, 24, 36, and 48 h post-infection for qRT-PCR analysis. (B) mRNA levels of members of the GAS response pathway. A549 cells were infected with AIV at 1 MOI, and total RNA was extracted at 12, 24, 36, and 48 h post-infection for qRT-PCR analysis. (C) Expression of proteins related to GAS response pathways following AIV infection. A549 cells were infected with AIV at 1 MOI, cell lysates were prepared at 12, 24, 36, and 48 hpi, and incubated with antibodies against IAV-NP, GM130, CREB3, TFE3, HSP47, and ARF4 at 4°C overnight. β-actin served as an internal standard. Bands were visualized using a chemiluminescence imaging analysis system after incubation with peroxidase-conjugated secondary antibodies. (D) Impact of infection doses on the expression of proteins related to GAS response pathways. A549 cells were infected with AIV at 0, 0.01, 0.1, 1, and 10 MOI for 24 h, and cell lysates were prepared and incubated with antibodies against IAV-NP, GM130, CREB3, TFE3, HSP47, and ARF4 at 4°C overnight. β-actin served as an internal standard. Bands were visualized using a chemiluminescence imaging analysis system after incubation with peroxidase-conjugated secondary antibodies. (E) AIV infection-induced nuclear translocation of TFE3. A549 cells were infected with AIV at 1 MOI, fixed at 12, 24, 36, and 48 h post-infection on slides, and incubated with antibodies against IAV-NP and TFE3, followed by DAPI staining. Cells were visualized using a Zeiss confocal fluorescence microscope LSM880 (IAV-NP, red; TFE3, green; DAPI, blue). Scale bar = 10 μm. Error bars represent SD of the means from three independent experiments (*p<0.05, **p<0.01).
Fig 3.
Effect of Golgi apparatus stress (GAS) response on AIV replication.
Effect of knockdown or overexpression of GAS-related genes on AIV replication in A549 cells. Prior to AIV infection, cells were transfected with siCREB3 or FLAG-CREB3 (A), siHSP47 or FLAG-HSP47 (B) and siTFE3 (A) or FLAG-TFE3 (C). Cell lysates were then incubated with antibodies against ARF4, HSP47, and TFE3 to confirm RNA interference, followed by incubation with antibodies against NP. β-actin served as an internal standard. Cell supernatants with siCREB3, siHSP47, and siTFE3 (D) or FLAG-CREB3, FLAG-HSP47, or FLAG-TFE3 (E) were subjected to TCID50 determination at 12, 24, 36, and 48 h post-infection for viral growth curve analysis. Error bars represent SD of the mean from three independent experiments (*p<0.05, **p<0.01).
Fig 4.
Effect of mouse TFE3 knockout on AIV replication and virulence.
(A) Confirmation of TFE3 knockout in mice using western blot analysis. Lungs from 6-week-old BALB/cTFE3+/+ and BALB/cTFE3-/- mice were collected, and their proteins were extracted. Western blotting was performed on protein samples using an antibody against TFE3, with β-actin serving as an internal standard. Bands were visualized using a chemiluminescence imaging analysis system after incubation with peroxidase-conjugated secondary antibodies. (B) Six-week-old BALB/cTFE3+/+ and BALB/cTFE3-/- mice were intranasally infected with 106 EID50 of virus in 50 μL PBS. Mice were monitored daily for weight loss and signs of disease over a 14-day period. The mean body weight change (%±SD) and death rate were presented. A similar experimental set of mice was prepared, and lungs were collected on day 5 post-infection. mRNA levels of virus gene (C) and inflammatory cytokines (D) were detected using qRT-PCR. Error bars represent SD of the means of data from three independent mice (*p< 0.05, **p<0.01).
Fig 5.
Effect of TFE3 on expression of glycosylation enzymes and AIV replication.
(A) AIV infection activated transcription of N-glycosylation enzymes. A549 cells were infected with AIV at 1 MOI. At 12, 24, 36, and 48 h post-infection, the cells were harvested, and total RNA was extracted for RT-qPCR analyses. Effects of TFE3 silencing (B) or overexpression (C) on the regulation of N-glycosylation enzymes following AIV infection. A549 cells were transfected with siTFE3 or FLAG-TFE3 before infection with AIV. At 24 and 48 h post-infection, total RNA was extracted for RT-qPCR analyses. (D) Function of N-glycosylation enzymes on the replication of AIV. A549 cells were transfected with FLAG-B4GALT1, FLAG-MGAT2, FLAG-ST3GAL6, FLAG-ST3GAL1, and FLAG-TFE3 before AIV infection. At 24 h post-infection, cell lysates were subjected to western blotting, and the membranes were incubated with an anti-FLAG antibody (to confirm the expression of transfected constructs) and further incubated with antibodies against NP. β-actin was used as an internal standard. Protein bands were quantified using ImageJ. (E) Role of N-glycosylation enzymes in AIV replication after TFE3 knockdown. A549 cells were transfected with FLAG-B4GALT1, FLAG-MGAT2, FLAG-ST3GAL6, FLAG-ST3GAL1, and FLAG-mutTFE3 before AIV infection. At 24 h post-infection, cell lysates were incubated with antibodies against FLAG (for expression confirmation) and TFE3 (for interference confirmation), followed by incubation with antibodies against NP. β-actin was used as an internal standard. Error bars represent SD of the means from three independent experiments (*p<0.05, **p<0.01).
Fig 6.
Effect of TFE3 on endosome acidification and AIV replication.
(A) mRNA, vRNA, and cRNA levels of the NP gene after TFE3 knockdown. A549 cells were transfected with siTFE3 before AIV infection. mRNA, vRNA, and cRNA were reverse-transcribed and quantified using RT-qPCR. (B) Binding or (C) Endocytosis capacity of AIV after TFE3 knockdown. A549 cells were transfected with siTFE3 before AIV infection. Expression of NP was quantified using RT-qPCR. (D) NP nuclear translocation after TFE3 knockdown. A549 cells were transfected with siTFE3 before AIV infection. A549 cells were infected with AIV at 1 MOI, fixed at 6 h post-infection on slides, and incubated with antibodies against IAV-NP and TFE3 and subjected to DAPI staining. Cells were visualized using a Zeiss confocal fluorescence microscope LSM880 (IAV-NP, red; TFE3, green; DAPI, blue). Scale bar = 10 μm. (E) Expression of proteins related to the endosome. A549 cells were infected with AIV at 1 MOI, cell lysates were then prepared at 6 h post-infection and incubated with antibodies against IAV-NP, RAB5, RAB7, and ATP6V1A at 4°C overnight. β-actin was used as an internal standard. Bands were visualized using a chemiluminescence imaging analysis system after incubation with peroxidase-conjugated secondary antibodies. (F) AIV infection activated transcription of endosome-related genes. A549 cells were infected with AIV at 1 MOI. At 12, 24, 36, and 48 h post-infection, the cells were harvested, and total RNA was extracted for RT-qPCR analyses. (G) Effects of TFE3 knockdown or (H) TFE3 overexpression on the regulation of endosome-related genes following AIV infection. A549 cells were transfected with siTFE3 or FLAG-TFE3 before infection with AIV. At 24 and 48 h post-infection, total RNA was extracted for RT-qPCR analyses. (I) M1 distribution after TFE3 knockdown. A549 cells were transfected with siTFE3 before AIV infection. A549 cells were infected with AIV at 10 MOI, fixed at 1 h, 2.5 h, and 4.5 h post-infection on slides, and incubated with antibodies against IAV-M1 and TFE3 and subjected to DAPI staining. Cells were visualized using a Zeiss confocal fluorescence microscope LSM880 (IAV-M1, red; TFE3, green; DAPI, blue). Scale bar = 10 μm. Error bars represent SD of the means of data from three independent experiments (*p<0.05, **p<0.01).
Fig 7.
Schematic of Golgi apparatus stress response pathway activated by AIV infection.
AIV infection resulted in Golgi apparatus stress (GAS), characterized by swelling and fragmentation of the Golgi apparatus. During GAS response, the TFE3 pathway exhibited robust activation. The activation of the TFE3 pathway within the GAS response promoted endosome acidification and increased the transcription of glycosylation enzymes, thereby facilitating AIV replication.