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ATG7/GAPLINC/IRF3 axis plays a critical role in regulating pathogenesis of influenza A virus [1]

['Biao Chen', 'Key Laboratory Of Animal Pathogen Infection', 'Immunology Of Fujian Province', 'College Of Animal Sciences', 'Fujian Agriculture', 'Forestry University', 'Fuzhou', 'People S Republic Of China', 'Cas Key Laboratory Of Pathogenic Microbiology', 'Immunology']

Date: 2024-02

Autophagy-related protein 7 (ATG7) is an essential autophagy effector enzyme. Although it is well known that autophagy plays crucial roles in the infections with various viruses including influenza A virus (IAV), function and underlying mechanism of ATG7 in infection and pathogenesis of IAV remain poorly understood. Here, in vitro studies showed that ATG7 had profound effects on replication of IAV. Depletion of ATG7 markedly attenuated the replication of IAV, whereas overexpression of ATG7 facilitated the viral replication. ATG7 conditional knockout mice were further employed and exhibited significantly resistant to viral infections, as evidenced by a lower degree of tissue injury, slower body weight loss, and better survival, than the wild type animals challenged with either IAV (RNA virus) or pseudorabies virus (DNA virus). Interestingly, we found that ATG7 promoted the replication of IAV in autophagy-dependent and -independent manners, as inhibition of autophagy failed to completely block the upregulation of IAV replication by ATG7. To determine the autophagy-independent mechanism, transcriptome analysis was utilized and demonstrated that ATG7 restrained the production of interferons (IFNs). Loss of ATG7 obviously enhanced the expression of type I and III IFNs in ATG7-depleted cells and mice, whereas overexpression of ATG7 impaired the interferon response to IAV infection. Consistently, our experiments demonstrated that ATG7 significantly suppressed IRF3 activation during the IAV infection. Furthermore, we identified long noncoding RNA (lncRNA) GAPLINC as a critical regulator involved in the promotion of IAV replication by ATG7. Importantly, both inactivation of IRF3 and inhibition of IFN response caused by ATG7 were mediated through control over GAPLINC expression, suggesting that GAPLINC contributes to the suppression of antiviral immunity by ATG7. Together, these results uncover an autophagy-independent mechanism by which ATG7 suppresses host innate immunity and establish a critical role for ATG7/GAPLINC/IRF3 axis in regulating IAV infection and pathogenesis.

Influenza A virus (IAV) causes acute respiratory diseases in human and animals, posing a great threat to public health. Autophagy plays crucial roles in viral infections including IAV, but mechanisms underlying interaction between autophagy and IAV remain ambiguous. Particularly, function and underlying mechanisms of ATG7, an essential autophagy effector enzyme, in viral infections are largely unexplored, and little information is available about relationship between ATG7 and IAV pathogenesis. Here, we used in vitro and in vivo models to address ATG7 function in the IAV infection and pathogenesis. We found that forced expression of ATG7 facilitates the viral replication, while depletion of ATG7 attenuates viral replication and renders mice more resistant to IAV or pseudorabies virus (PRV) infection. Importantly, we identify that ATG7 suppresses IRF3 activation and interferon production via lncRNA GAPLINC, revealing an autophagy-independent mechanism whereby ATG7 restrains host innate immunity and unveiling a critical role of ATG7/GAPLINC/IRF3 axis in regulating IAV pathogenesis. Moreover, our observations suggest that ATG7 may positively regulate the expression of GAPLINC via suppression of NF-κB activation during IAV infection. Together, these results reveal that ATG7 has multiple biological roles beyond autophagy, and provide an important insight into the complicated interplay between host and IAV.

Funding: This work was supported by National Natural Science Foundation of China (32030110 to JLC, U23A20235 to JLC) and National Key Research and Development Program of China (2021YFD1800205 to JLC).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we used in vitro and in vivo models to address ATG7 function in the viral pathogenesis. The results show that forced expression of ATG7 facilitates the viral replication, while depletion of ATG7 attenuates viral replication and renders mice more resistant to IAV or pseudorabies virus (PRV) infection. Mechanistically, ATG7 promotes the viral replication in autophagy-dependent and -independent manners. Importantly, we identify that ATG7 enhances the expression of lncRNA GAPLINC, resulting in the suppression of IRF3 activation and IFN production, thereby restraining host antiviral immunity. These findings reveal that ATG7 regulates viral infection and pathogenesis via multiple mechanisms, including an autophagy-independent mechanism involving the ATG7/GAPLINC/IRF3 axis.

Notably, it has been shown that autophagy-related proteins (ATG) have extensive biological importance beyond autophagic elimination [ 24 ]. For instance, the ATG5-ATG12/ATG16L1 complex and ATG7, but not the degradative activity of autophagy, are required for the antiviral activity of IFN-γ against murine norovirus (MNV) infection in macrophages through involvement in IFN-γ-mediated inhibition of MNV replication complex formation [ 25 ]. Additionally, ATG9a could co-localize with stimulator of IFN genes (STING), an essential signal transducer required for dsDNA-triggered innate immune responses [ 26 ]. Depletion of ATG9a enhances the assembly of STING and TANK-binding kinase 1 (TBK1) after dsDNA stimulation, leading to aberrant activation of innate immune responses [ 26 ], suggesting a role of ATG9a in the regulation of innate signaling which is independent from its implication in autophagy. Besides, ATG7, an essential autophagy effector enzyme, interacts with p53 and inhibits the expression of pro-apoptotic factors such as Noxa, Puma and Bax, which is independent on its E1-like enzymatic activity [ 27 ]. Accordingly, ATG7-null mouse embryonic fibroblasts displayed augmented DNA damage [ 27 ]. In another study, ATG7 has been shown to directly interact with caspase-9 and suppress the pro-apoptotic activity of caspase-9, which is not related to its function in the autophagic process [ 28 ]. Importantly, a direct association between ATG7 dysfunction and disease was recently established in patients with biallelic ATG7 variants [ 29 ]. However, the functional repertoire and underlying mechanisms of ATG7 in viral infection and pathogenesis are largely unexplored. Particularly, little information is available about relationship between ATG7 and IAV pathogenesis.

Influenza A virus (IAV) is an important member of the Orthomyxoviridae family, which causes acute respiratory diseases in human and a lot of animals, posing a great threat to the health of humans and animals. It has been shown that the PB1 protein of IAV is associated with the selective autophagic receptor neighbor of BRCA1 (NBR1), and the latter recognizes ubiquitinated MAVS and targets it for autophagic degradation, consequently restraining the RIG-I-MAVS-mediated innate immune signaling and facilitating viral infection [ 22 ]. Similarly, the NP protein of IAV is recently reported as a critical regulator of mitophagy, and NP-mediated mitophagy leads to the degradation of MAVS, thereby blocking MAVS-mediated antiviral signaling and promoting viral replication [ 23 ].

On the other hand, emerging data suggest that some viruses have evolved several strategies to hijack and manipulate host autophagy for their own survival and proliferation in host cells [ 16 ]. For instance, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral protein ORF3a interacts with VPS39, and impedes the association of the homotypic fusion and protein sorting (HOPS) complex with STX17 or RAB7, thereby blocking the fusion of autophagosomes with lysosomes and eventually inhibiting the autophagy activity [ 17 , 18 ]. This may be a mechanism employed by SARS-CoV-2 to escape host lysosome degradation. In addition, autophagy has been reported to enhance the replication of some viruses such as coxsackievirus B3 (CVB3), HCV, and poliovirus, as genetic or pharmacological inhibition of autophagy decreases viral yields [ 19 – 21 ], implying that some viruses can take advantage of host autophagy for their infection and replication.

Autophagy is an evolutionarily-conserved cellular degradative pathway, which mediates the degradation of encapsulated cytoplasmic material via the endolysosomal system [ 1 , 2 ]. Autophagy is involved in a variety of biological processes such as the maintenance of cellular homeostasis, cell differentiation, and host defense against invading pathogens [ 3 – 5 ]. Increasing evidence suggests that autophagy plays important roles in viral pathogenesis, although the effects of autophagy on viral replication and the outcome of viral infection differ depending on the viruses and the host cells [ 6 , 7 ]. On the one hand, hosts could utilize their own autophagy to prevent viral infection and pathogenesis [ 8 – 10 ]. For instance, IFN-β-induced endoplasmic reticulum protein SCOTIN interacts with hepatitis C virus (HCV) non-structural protein 5A (NS5A), and targets NS5A to autophagosomes for degradation, hence restricting HCV replication [ 11 ]. In addition, autophagy has been reported to prevent viral invasion through activating innate immune responses [ 12 , 13 ]. A recent study has found that TRIM14 could recruit the deubiquitinase USP14 to remove K48-linked ubiquitin chains of cyclic GMP-AMP synthase (cGAS), an essential DNA virus sensor that triggers type I interferon (IFN) signaling, which leads to the inhibition of p62-mediated autophagic degradation of cGAS, therefore promoting type I IFN response [ 14 ]. Besides, autophagy is also involved in coordinating adaptive immunity by promoting antigen presentation, which is essential for the elimination of invading viruses. Deletion of the key autophagy gene ATG5 in dendritic cells (DCs) of mice results in a significant impairment of CD4 + T cell priming after herpes simplex virus (HSV) infection, thereby accelerating the pathogenesis of HSV in the animal [ 15 ]. These investigations suggest that autophagy may function through different mechanisms to regulate viral infection and pathogenesis.

Results

Altering ATG7 expression has profound effects on replication of IAV Numerous studies have shown that autophagy plays important roles in viral infections. ATG7 is an essential autophagy effector enzyme, but its functional involvement in the IAV pathogenesis is largely unknown. To define the role of ATG7 in IAV infections, we generated A549 cells stably expressing specific shRNAs targeting ATG7 (sh1-ATG7 and sh2-ATG7) or luciferase control (sh-Luc) using lentiviral vectors. The protein levels of ATG7 were dramatically reduced in A549 cells expressing ATG7 shRNAs compared with that in cells expressing control shRNA (S1A Fig). Then, ATG7 knockdown and control cells were infected with influenza virus A/WSN/33 (H1N1), and viral loads were determined by hemagglutination (HA) and plaque forming assays. Notably, ATG7 knockdown significantly decreased IAV titers in the cells compared to the control (Fig 1A and 1B). In line with this, the levels of viral nucleoprotein (NP) protein were dramatically reduced in ATG7 knockdown cells (Fig 1C). These data indicated that depletion of ATG7 impeded the replication of IAV in A549 cells. Furthermore, we infected ATG7 knockdown and control A549 cells with other strains of IAV including A/PR8/34 (H1N1) and H9N2 subtype virus, and similar results were observed in these experiments (Fig 1D–1G). Moreover, Sendai virus (SeV) was also employed, and consistently, ATG7 knockdown hindered the replication of the virus (S1B and S1C Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Altering ATG7 expression has profound effects on the replication of IAV. (A and B) Control and ATG7 knockdown A549 cells were infected with influenza virus A/WSN/33 (MOI = 0.5), and the supernatants were collected for hemagglutination (HA) assay (A) and plaque forming assay (B). (C) Control and ATG7 knockdown A549 cells were infected with influenza virus A/WSN/33 (MOI = 0.5) for the indicated time, and viral NP protein levels in the cells were examined by Western blotting. (D and E) Control and ATG7 knockdown A549 cells were infected with PR8 (MOI = 0.2), and the supernatants were collected at 16 hpi for plaque forming assay (D). Western blotting was performed to detect viral NP protein levels in the cells (E). (F and G) Control and ATG7 knockdown A549 cells were infected with H9N2 influenza virus (MOI = 1). The supernatants were collected at 16 hpi for plaque forming assay (F), and Western blotting was performed to detect viral NP protein levels in the cells (G). (H and I) Control and ATG7 overexpressing A549 cells were infected with WSN (MOI = 0.5) for the indicated time. Viral NP RNA levels in the cells were examined by RT-PCR (H), and the supernatants were collected for HA assay (I). (J and K) Control and ATG7 overexpressing A549 cells were infected with PR8 (MOI = 0.2) for the indicated time. RT-PCR was performed to test viral NP RNA levels in the cells (J), and the supernatants were collected for HA assay (K). (L) Control and ATG7 overexpressing A549 cells were infected with H9N2 influenza virus (MOI = 1) for 16 h. The supernatants were collected for plaque forming assay. RT-PCR and Western blotting data were repeated independently three times with similar results. Shown are representative data of three biologically independent experiments. Data are presented as means ± SD from three independent experiments, **p < 0.01. https://doi.org/10.1371/journal.ppat.1011958.g001 On the other hand, we evaluated the effect of ATG7 overexpression on the viral replication. A549 cell lines stably expressing ATG7 or empty vector (EV) were generated by using lentiviral vectors. These cells were then infected with influenza virus (H1N1 and H9N2) or SeV, and replication of the viruses was examined. As expected, ATG7 overexpression markedly increased IAV titers and elevated viral NP mRNA levels in A549 cells compared to control, in response to infection with H1N1 IAVs including WSN (Figs 1H, 1I and S1D) and PR8 (Figs 1J, 1K and S1E). Similarly, overexpression of ATG7 also resulted in enhanced H9N2 IAV replication (Fig 1L), and a significant increase in SeV NP RNA levels in cells after the viral infection (S1F and S1G Fig). Together, these observations indicate that altering ATG7 expression has profound effects on the replication of IAV, as well as SeV.

In vivo deficiency of ATG7 significantly impairs virulence of IAV in mice To further substantiate the function of ATG7 in IAV infection and pathogenesis under a more elaborate and physiological circumstance, we employed ATG7 conditional knockout (CKO) mice, as the germ line knockout of ATG7 is died within 1 day after birth [30]. ATG7flox/flox mice were crossed with transgenic UBC-CreERT2 mice. ATG7flox/flox/UBC-CreERT2 mice were treated with tamoxifen to induce the Cre recombinase that mediates the knockout of ATG7. We observed that ATG7 protein levels were profoundly decreased in the liver, spleen, lung, kidney, and thymus of ATG7flox/flox/UBC-CreERT2 mice treated with tamoxifen (Fig 2A). The mice were then infected with influenza virus A/PR8/34 (H1N1), and the viral replication was examined. ATG7 CKO mice displayed slower body weight loss and better survival than control mice during IAV infection (Fig 2B and 2C). In addition, ATG7 CKO mice exhibited a lower degree of lung injury caused by IAV infection than control mice (Fig 2D and 2E), suggesting that deficiency of ATG7 attenuated IAV replication in mice. Consistent with these observations, IAV titers were significantly decreased and the viral NP protein levels were clearly reduced in lung tissues from ATG7 CKO mice compared with those in the tissues of control mice (Fig 2F and 2G). These results reveal that depletion of ATG7 renders mice more resistant to the IAV infection. PPT PowerPoint slide

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TIFF original image Download: Fig 2. In vivo deficiency of ATG7 significantly impairs virulence of IAV and PRV in mice. (A) ATG7 protein levels in indicated tissues of WT and ATG7 conditional knockout (CKO) mice were examined by Western blotting. (B-G) WT and ATG7 CKO mice were injected intranasally with 5×104 plaque-forming units (PFU) of PR8. The body weight loss (B), and the survival rate (C) of mice were monitored. The histopathologic changes in the lungs from WT and ATG7 CKO mice at 2 dpi were determined by HE staining (D), and scored for disease severity (E). Scale bar, 200 μm. The viral titers and NP protein levels in the lungs from WT and ATG7 CKO mice were determined by plaque forming assay (F) and Western blotting (G) respectively. (H-K) WT and ATG7 CKO mice were injected intramuscularly with 1×106 PFU of PRV. The body weight loss (H), and survival rate (I) of mice were monitored. Viral gE mRNA and protein levels in the brains from WT and ATG7 CKO mice at 2 dpi were determined by RT-PCR and Western blotting respectively (J). Viral gE mRNA and protein levels in the lungs of mice at 2 dpi were detected by RT-PCR and Western blotting respectively (K). RT-PCR and Western blotting data were repeated independently three times with similar results. Shown are representative data of three biologically independent experiments. Data are presented as means ± SD from three independent experiments, **p < 0.01. https://doi.org/10.1371/journal.ppat.1011958.g002 Since mouse is an ideal animal model for infection with PRV, a DNA virus, we also determined whether ATG7 was involved in the pathogenesis of PRV. To this end, control and ATG7 CKO mice were infected with PRV, and the effect of ATG7 deficiency on PRV replication was evaluated. A time course study showed that ATG7 CKO mice had slower body weight loss and an increased survival rate than control mice challenged with PRV (Fig 2H and 2I). Accordingly, lower viral gE RNA and protein levels were detected in the lung, brain, and liver derived from ATG7 CKO mice than those in the tissues of control mice after PRV infection (Figs 2J, 2K and S2A–S2D). Together, the results suggest that ATG7 facilitates pathogenesis of both IAV and PRV in mice, implying that ATG7 might function as a pro-viral host factor for in vivo pathogenesis of a broad spectrum of viruses.

ATG7 inhibits IAV-induced expression of IFNs in vitro and in vivo Next, we sought to dissect the molecular mechanisms by which ATG7 promotes the viral replication. For this, we performed RNA sequencing (RNA-Seq) to analyze differentially expressed mRNAs between control and ATG7 knockdown A549 cells infected with influenza virus A/PR8/34 (H1N1). Notably, our RNA-Seq analysis revealed that the expression levels of type I and III IFNs, which play central roles in restricting viral infections, were significantly increased in ATG7 knockdown A549 cells as compared to the control cells upon IAV infection (Figs 4A and S4A). This finding was further confirmed by analysis of real-time PCR (Figs 4B–4D and S4B and S4C). Consistently, it was shown that ATG7 knockdown significantly elevated protein levels of IFN-β in the cells infected with IAV (Fig 4E). PPT PowerPoint slide

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TIFF original image Download: Fig 4. ATG7 inhibits IAV-induced expression of IFNs in vitro and in vivo. (A) Transcriptome RNA sequencing analysis of control and ATG7 knockdown A549 cells infected with PR8 (MOI = 0.5) for 16 h. (B-E) Control and ATG7 knockdown A549 cells were infected with WSN (MOI = 0.5) for 0, 12, 15, and 18 h. The RNA levels of IFN-β (B), IL-28 (C), or IL-29 (D) were examined by qRT-PCR. GAPDH was chosen as a reference gene for internal standardization. The IFN-β protein levels in the supernatants were examined by ELISA (E). (F-G) Control and ATG7 knockdown A549 cells were transfected with poly(I:C) for 4 h. The RNA levels of IFN-β were examined by RT-PCR (F) and qRT-PCR (G) respectively. (H-K) Control and ATG7 overexpressing A549 cells were infected with WSN (MOI = 0.5) for 0, 12, 15, and 18 h. The RNA levels of IFN-β (H), IL-28 (I), or IL-29 (J) were examined by qRT-PCR, and the IFN-β protein levels in the supernatants were examined by ELISA (K). (L-N) WT and ATG7 CKO mice were infected with 5×104 PFU of PR8 for 0, 24, and 48 h. The RNA levels of IFN-β (L), and IL-28 (M) in the lungs of mice were determined by qRT-PCR, and the IFN-β protein levels were examined by ELISA (N). Data are presented as means ± SD from three independent experiments, *p < 0.05, **p < 0.01. https://doi.org/10.1371/journal.ppat.1011958.g004 Next, we asked whether ATG7 regulated the IFN expression via autophagy-dependent or -independent mechanism induced by IAV. For this, we used poly(I:C), a dsRNA mimic to induce IFNs expression without induction of autophagy, to examine its influence on the levels of IFN-β in control and ATG7 knockdown A549 cells. Interestingly, disruption of ATG7 expression also led to a significant increase in the expression of IFN-β induced by poly(I:C) (Fig 4F and 4G). These results suggest that ATG7-mediated suppression of IFN response involves an autophagy-independent mechanism. Moreover, SeV was employed, and similarly, the virus-induced expression of IFN-β, IL-28 and IL-29 was obviously upregulated in the absence of ATG7 (S4D–S4G Fig). Additionally, we measured the levels of IFNs in ATG7 overexpressing A549 cells and the control challenged with IAV. As expected, overexpression of ATG7 resulted in a significant decrease in mRNA expression of IFN-β, IL-28 and IL-29 (Figs 4H–4J and S4H), and in protein levels of IFN-β upon IAV infection (Fig 4K). For in vivo analysis, the levels of IFNs were examined in the lungs of ATG7 CKO or WT mice challenged with IAV infection. Consistent with the data obtained from in vitro experiments, in vivo studies showed that ATG7 CKO mice had a significantly increased production of IFN-β and IL-28 in the lungs compared with the control mice infected with IAV (Figs 4L–4N and S4I). Together, both in vitro and in vivo data reveal that ATG7 inhibits the production of IFNs during the IAV infection.

ATG7 suppresses IRF3 activation in an autophagy-independent manner during the IAV infection Next, we further investigated the mechanisms by which ATG7 restrains the IFN response. Since RLR-dependent pathway is a main innate immune signaling activated by IAV infection to trigger the production of interferons, we explored whether ATG7 could suppress this signaling. To this end, IFN-β luciferase reporter system was employed, and ATG7 overexpressing cells and controls were transfected with the reporter and either RIG-I, MAVS, TBK1, WT IRF3 or its 5D mutant expression plasmid. The results showed that ATG7 significantly inhibited RIG-I-, MAVS-, TBK1-, and WT IRF3-mediated IFN-β luciferase activation, as ATG7 overexpressing cells exhibited lower IFN-β luciferase activity than control cells, whereas ATG7 had no significant effect on IFN-β luciferase activity induced by the 5D mutant, an active form of IRF3 (Fig 5A), implying that ATG7 restrains RLR signaling by suppressing IRF3 activation. PPT PowerPoint slide

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TIFF original image Download: Fig 5. ATG7 suppresses IRF3 activation in an autophagy-independent manner during the IAV infection. (A) Control and ATG7 overexpressing 293T cells were transfected with the IFN-β luciferase reporter and either RIG-I, MAVS, TBK1, IRF3 (WT), or IRF3(5D) expressing vector. 24 hours post transfection, the cells were harvested for luciferase assay. (B and C) Control and ATG7 knockdown A549 cells were transfected with poly(I:C) for 4 h (B), or infected with PR8 (MOI = 0.2) for 16 h (C). The levels of IRF3 phosphorylation (p-IRF3) in the cells were examined by Western blotting. The p-IRF3 levels were quantitated by densitometry and normalized to IRF3 levels. Shown was quantification analysis of p-IRF3 levels from three independent experiments. (D and E) Control and ATG7 overexpressing A549 cells were transfected with poly(I:C) for 4 h (D), or infected with PR8 (MOI = 0.2) for 16 h (E). The p-IRF3 levels in the cells were examined by Western blotting. The p-IRF3 levels were quantitated by densitometry and normalized to IRF3 levels. Shown was quantification analysis of p-IRF3 levels from three independent experiments. (F) Control and A549 cells overexpressing WT, or mutants of ATG7 (ATG7C572S, ATG7FAPtoDDD) were infected with PR8 (MOI = 0.2) for 16 h. The levels of p-IRF3 in the cells were examined by Western blotting. (G) Control and ATG7 knockdown A549 cells re-expressing ATG7 WT or its mutants (ATG7C572S, ATG7FAPtoDDD) were infected with PR8 (MOI = 0.2) for 16 h. The levels of p-IRF3 in the cells were examined by Western blotting. Western blotting data were repeated independently three times with similar results. Shown are representative data of three biologically independent experiments. Data are presented as means ± SD from three independent experiments, *p < 0.05, **p < 0.01. https://doi.org/10.1371/journal.ppat.1011958.g005 IRF3 is a key transcriptional factor implicated in the innate immune response to viral infections. This drove us to further evaluate effect of ATG7 on the activation of IRF3 upon viral infection. First, control and ATG7 knockdown A549 cells were transfected with poly(I:C), a dsRNA mimic to activate IRF3 without induction of autophagy. It was observed that ATG7 knockdown led to a significant increase in the phosphorylation levels of IRF3 induced by poly(I:C) (Fig 5B). Then, we examined the phosphorylation of IRF3 in control and ATG7 knockdown A549 cells infected with IAV. As shown in Fig 5C, higher levels of phosphorylated IRF3 were found in ATG7 knockdown cells than those in control cells after IAV infection. Consistently, the nuclear translocation of IRF3 was enhanced in ATG7 knockdown A549 cells compared with control cells following viral infection (S5A Fig). In contrast, knockdown of BECN1 or ATG3 had no significant effect on the phosphorylation of IRF3 compared with control cells after IAV infection (S5B and S5C Fig). These results revealed that depletion of ATG7 enhanced the activation of IRF3 during the viral infection. Moreover, we detected the phosphorylation status of IRF3 in control and ATG7 overexpressing A549 cells after poly(I:C) transfection or IAV infection. Indeed, overexpression of ATG7 resulted in a significant decrease in the phosphorylation of IRF3 induced by either poly(I:C) or IAV (Fig 5D and 5E). In addition, we generated ATG7 knockdown A549 cells re-expressing an shRNA-resistant form of ATG7, followed by transfection with poly(I:C) (S5D and S5E Fig). ATG7 knockdown led to a significant increase in the phosphorylation levels of IRF3 induced by poly(I:C), while re-introduction of ATG7 reversed the enhanced IRF3 phosphorylation caused by loss of ATG7 in the cells treated with poly(I:C) (S5D and S5E Fig). These observations indicate that ATG7 facilitates IAV replication likely through suppression of IRF3 activation and subsequent IFNs expression. Remarkably, the fact that ATG7 can impair the poly(I:C)-induced IRF3 phosphorylation suggests that suppression of IRF3 by ATG7 may be autophagy-independent. Thus, we further asked whether ATG7 regulated the activation of IRF3 via modulating the autophagy process. For this, we employed two ATG7 mutants lacking the ability to regulate autophagy, including the active-site mutant (ATG7C572S) [35], and the mutant defective in the formation of the E2-substrate intermediate of ATG3 and LC3 (ATG7FAPtoDDD) [36]. We generated A549 cells expressing either WT ATG7, each ATG7 mutant, or EV control, followed by infection with IAV. Overexpression of WT ATG7 caused a diminished phosphorylation of IRF3 compared to EV control (Fig 5F). Of note, comparable phosphorylation levels of IRF3 were observed between cells overexpressing WT and ATG7 mutants, although the autophagy was dampened in cells expressing ATG7 mutants (Fig 5F). We also generated ATG7 knockdown A549 cells re-expressing either WT or mutants of ATG7 followed by IAV infection, and the activation of IRF3 was evaluated (Fig 5G). In response to IAV infection, silencing of ATG7 resulted in a significant increase of IRF3 phosphorylation compared with the control, while re-introduction of either WT or mutants of ATG7 reversed the enhanced IRF3 phosphorylation caused by loss of ATG7 in the knockdown cells (Fig 5G). Together, these data imply that there exists an autophagy-independent mechanism underlying ATG7-mediated inactivation of IRF3 during the IAV infection.

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