Influenza A virus use of BinCARD1 to facilitate the binding of viral NP to importin α7 is counteracted by TBK1-p62 axis-mediated autophagy

IAV employs BinCARD1 for efficient replication

BinCARD was identified to be a potential proviral host factor for the replication of IAV in a previous whole-genome siRNA library screen performed with a replication-competent Venus-expressing H5N1 virus [43]. To determine whether BinCARD affects the replication of IAV, A549 cells were transfected with siRNA. For technical reasons, we designed only two siRNAs targeting the CCD region of the two BinCARD isoforms, as well as two siRNAs targeting BinCARD2. At 36 h p.t., the siRNA knockdown efficiency was verified by RT‒qPCR (Fig. 1A, B). Knocking down both BinCARD isoforms or only BinCARD2 with different siRNAs exerted no major effect on cell viability, as measured by a luminescent cell viability assay (Supplementary Fig. 1). The effect of siRNA knockdown of BinCARD on IAV replication was then evaluated after WSN (H1N1) virus infection. We found that when both BinCARD isoforms were simultaneously knocked down in A549 cells, the growth titers of WSN (H1N1) virus were reduced by 15- and 12-fold at 24 h post-infection (p.i.) and 8- and 4-fold at 48 h p.i. However, no significant changes in virus titers were observed between the A549 cells treated with siRNAs targeting BinCARD2 and those treated with scrambled siRNA (Fig. 1C, D). These results indicate that BinCARD1, but not BinCARD2, was required for the efficient replication of IAV. We also selected BinCARD siRNA1, whose downregulating effect on the expression of BinCARD/BinCARD1 at the protein level was confirmed by both confocal microscopy and western blotting (Supplementary Fig. 2A, B). We then examined the effect of reduced BinCARD/BinCARD1 protein levels on the levels of vRNA, mRNA, and cRNA of the NP gene in A549 cells infected with WSN (H1N1) virus. We found that the levels of all three species of viral RNA in the BinCARD siRNA1-treated cells were significantly lower than those in the scrambled siRNA-treated cells at both 3 h p.i. and 6 h p.i. (Supplementary Fig. 3A, B), thereby confirming that BinCARD1 was important for the efficient replication of WSN (H1N1) virus. In addition, the role played by BinCARD1 in the replication of IAV was validated by analyses performed with AH05 (H5N1) and AH13 (H7N9) viruses. We found that treatment with BinCARD siRNA1 in A549 cells led to AH05 (H5N1) and AH13 (H7N9) virus titer reductions of 23- and 13-fold at 24 h p.i. and 21- and 10-fold at 48 h p.i., respectively (Fig. 1E, F). These data demonstrate that BinCARD1 is required by a wide range of IAVs for efficient replication.

Fig. 1figure 1

BinCARD1 promotes IAV replication. SiRNA knockdown of BinCARD and BinCARD2 in A549 cells. A549 cells were transfected with siRNA targeting BinCARD (A) or BinCARD2 (B) or with scrambled siRNA. At 36 h p.t., the knockdown efficiency was determined by RT‒qPCR. ***P < 0.001. Viral replication in siRNA-treated A549 cells. SiRNA-treated A549 cells are the same as those shown in A, B and were infected with WSN (H1N1) (MOI = 0.01) (C, D), AH05 (H5N1) (MOI = 0.1) (E), or AH13 (H7N9) (MOI = 0.1) (F) virus. Supernatants were collected at the indicated timepoints, and virus titers were determined by plaque assays in MDCK cells. ***P < 0.001; ns, not significant. G Knockout of BinCARD in BinCARD_KO A549 cells was confirmed by confocal microscopy with a rabbit anti-BinCARD pAb. Viral replication in BinCARD_KO A549 cells. BinCARD_KO A549 cells or A549 control cells were infected with WSN (H1N1) (MOI = 0.01) (H), AH05 (H5N1) (MOI = 0.1) (I), or AH13 (H7N9) (MOI = 0.1) (J) virus. Supernatants were collected at the indicated timepoints, and virus titers were determined by plaque assays in MDCK cells. ***P < 0.001. K Establishment of A549 cell lines stably overexpressing two isoforms of BinCARD. The stable lentivirus-mediated expression of BinCARD1 and BinCARD2 was confirmed by western blotting with a rabbit anti-BinCARD pAb. L Viral replication in BinCARD1- and BinCARD2-overexpressing A549 cells. BinCARD1- and BinCARD2-overexpressing A549 cells and A549 control cells are the same as those presented in K and were infected with WSN (H1N1) (MOI = 0.01) virus. Supernatants were collected at the indicated timepoints, and virus titers were determined by plaque assays in MDCK cells. ***P < 0.001. The data represent the mean ± SD of three independent experiments (AF, HJ, L)

We next generated the BinCARD-knockout (BinCARD_KO) A549 cell line with the CRISPR/Cas9 system to confirm the positive regulatory effect of BinCARD1 on the replication of IAV. BinCARD/BinCARD1 knockout in BinCARD_KO A549 cells compared with that in control cells was confirmed by confocal microscopy and western blot analysis (Fig. 1G and Supplementary Fig. 4A). BinCARD knockout led to no major effect on cell viability (Supplementary Fig. 4B). The titers of WSN (H1N1) virus produced by BinCARD_KO A549 cells were 26- and 20-fold lower than those of control cells at 24 and 48 h p.i., respectively (Fig. 1H). Consistent with these data, 34-/24-fold and 27-/21-fold reductions in AH05 (H5N1) and AH13 (H7N9) virus titers were observed at 24/48 h p.i., respectively, in BinCARD_KO A549 cells compared with control cells (Fig. 1I, J).

To verify that BinCARD1 was solely responsible for the observed effect of BinCARD on IAV replication, we established BinCARD1-A549 and BinCARD2-A549 cell lines stably overexpressing BinCARD1 and BinCARD2, respectively, using a lentiviral system (Fig. 1K). We found that the titers of WSN (H1N1) virus in the BinCARD1-A549 cells were 5- and 4-fold higher at 24 and 48 h p.i. than those of the control cells. In contrast, overexpression of BinCARD2 in the BinCARD2-A549 cells did not affect the replication of WSN (H1N1) virus (Fig. 1L). Taken together, these data indicate that BinCARD1 positively regulated the replication of IAV.

To evaluate the biological significance of BinCARD1 in the replication cycle of IAV, we determined the expression profile of BinCARD1 in the course of IAV infection. A549 cells were infected with WSN (H1N1) virus (MOI = 0.01), and the level of BinCARD1 was measured by western blotting with a mouse anti-BinCARD1 mAb. We found that the level of BinCARD1 was gradually increased by infection with WSN (H1N1) virus. The level of BinCARD1 peaked at 24–36 h p.i. and then clearly decreased at 48 h p.i., suggesting that BinCARD1 function was important in the replication cycle of IAV (Supplementary Fig. 5).

BinCARD1 positively regulates the nuclear import of the vRNP complex and newly synthesized NP

To define the specific stage of the IAV replication cycle in which BinCARD1 is engaged, we first examined whether BinCARD1 plays a role in viral internalization. BinCARD siRNA1- or scrambled siRNA-treated A549 cells were infected with WSN (H1N1) virus (MOI = 5) for 1 h on ice at 4 °C, and then, the temperature was changed to 37 °C for 30 min to allow viral internalization. After removing virions retained on the cell surface by washing with PBS (pH = 1.3), the amount of internalized viral NP protein was evaluated by western blotting. The amount of NP inside the BinCARD siRNA1-treated A549 cells was similar to that in the scrambled siRNA-treated cells (Fig. 2A), indicating that knocking down BinCARD1 expression had no adverse effect on the viral endocytic process. Next, we asked whether BinCARD1 affects the uncoating process of virus upon entry into cells. SiRNA-treated A549 cells were infected with a high dose of WSN (H1N1) virus (MOI = 50) in the presence of cycloheximide (CHX, 50 μg/mL) to exclude the influence of newly synthesized proteins. The cells were fixed at 1.5 h p.i., stained with an anti-M1 pAb to reveal the uncoating process, and visualized with confocal microscopy. We found that M1 was distributed as puncta in cells treated with siRNA against vATPase (the positive control), indicating that virions were not uncoated (Fig. 2B). In contrast, M1 was diffusely distributed in the cytoplasm of A549 cells treated with BinCARD siRNA1 or scrambled siRNA, indicating completion of the uncoating process; no difference in M1 distribution was observed between the cells treated with BinCARD siRNA1 or scrambled siRNA. Hence, knocking down BinCARD1 did not affect virion uncoating. Together, these results clearly indicate that BinCARD1 was neither involved in the internalization nor the uncoating process in the early stage of the IAV replication cycle.

Fig. 2figure 2

BinCARD1 positively regulates the nuclear import of the vRNP complex and newly synthesized NP. A The effect of BinCARD siRNA1 treatment on the internalization of IAV was analyzed. BinCARD siRNA1- or scrambled siRNA-treated A549 cells were infected with WSN (H1N1) virus (MOI = 5) on ice at 4 °C for 1 h, incubated at 37 °C for 30 min to allow viral internalization, and washed with ice-cold PBS (pH = 1.3) to remove uninternalized viral particles. Cell lysates were subjected to western blotting with a mouse anti-NP mAb to detect the amount of internalized virus particles. B The effect of BinCARD siRNA1 treatment on the uncoating process of IAV was analyzed. BinCARD siRNA1-, scrambled siRNA- or vATPase siRNA-treated A549 cells were treated with CHX to inhibit protein synthesis and were then infected with WSN (H1N1) (MOI = 50) virus. At 1.5 h p.i., the infected cells were stained with a rabbit anti-M1 pAb and Alexa Fluor 633 goat anti-rabbit IgG (H + L) (red) and visualized by confocal microscopy. C The effect of BinCARD siRNA1 treatment on the cellular localization of NP during IAV infection was determined by confocal microscopy. BinCARD siRNA1- or scrambled siRNA-treated A549 cells were infected with WSN (H1N1) (MOI = 5) virus. At 2, 3, 4 and 5 h p.i., the infected cells were fixed and stained with a mouse anti-NP mAb, followed by incubation with Alexa Fluor 633 goat anti-mouse IgG (H + L) (red). The nuclei were stained with DAPI. D Quantitative analysis of NP localization in virus-infected BinCARD siRNA1-treated cells. On the basis of the confocal microscopy images presented in C, the localization of NP (indicative of vRNP localization) upon the appearance of its nuclear localization was divided into the following categories: weak nuclear localization, strong nuclear localization, simultaneous localization at the boundary between the nucleus and the cytoplasm, or predominant cytoplasmic localization. The data shown are derived from 100 cells visualized by confocal microscopy with a 40X objective lens. E The effect of BinCARD1 knockout on the nuclear import of the vRNP complex was determined through a cell fractionation experiment. BinCARD_KO A549 cells and A549 control cells were infected with WSN (H1N1) (MOI = 5) virus. At 3 h p.i., the cells were separated into nuclear (N) and cytoplasmic (C) fractions. The proteins in each fraction were subjected to western blotting with a mouse anti-NP mAb. Densitometric analysis of the western blots was performed with ImageJ software. Lamin B1 and GAPDH were used as the loading controls for the nuclear and cytoplasmic fractions, respectively. F The effect of BinCARD1 knockout on the nuclear import of NP was assessed by confocal microscopy. BinCARD_KO A549 cells and A549 control cells were transfected with a vector to express WSNNP. At 20 h p.t., the cells were stained with a mouse anti-NP mAb and a rabbit anti-BinCARD pAb, incubated with Alexa Fluor 633 goat anti-mouse IgG (H + L) (red) and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (green), and visualized by confocal microscopy

To determine whether BinCARD1 modulates the nuclear import of the vRNP complex, we monitored the cellular localization of NP during the course of IAV infection in siRNA-treated A549 cells. A549 cells were treated with BinCARD siRNA1 or scrambled siRNA and infected with WSN (H1N1) virus (MOI = 5). At 2, 3, 4, and 5 h p.i., the cellular distribution of NP was visualized by confocal microscopy. NP clearly accumulated in the nucleus of approximately 35% and 88% of the scrambled siRNA-treated cells at 2 and 3 h p.i., respectively. However, the ratio of cells with NP accumulation in the nucleus of BinCARD siRNA1-treated cells was 2% and 8%, respectively, at the same timepoints. These results indicate that the vRNP complex is detained in the cytoplasm at 2 and 3 h p.i. in BinCARD siRNA1-treated cells. Due to the defect in the nuclear import of the vRNP complex, the replication cycle of IAV was delayed in the BinCARD siRNA1-treated cells, as indicated by the localization of NP at 4 and 5 h p.i. (Fig. 2C, D). The role played by BinCARD1 in the nuclear import of the vRNP complex after IAV infection was also examined in BinCARD_KO A549 cells. We found that the nuclear import of the vRNP complex was suppressed and that the overall viral replication cycle was delayed in the BinCARD_KO A549 cells compared with the A549 control cells (Supplementary Fig. 6A, B).

We next validated the inhibitory effect of BinCARD1 knockout on the nuclear import of the vRNP complex by performing a cell fractionation experiment. BinCARD_KO and control A549 cells were infected with WSN (H1N1) virus (MOI = 5). At 3 h p.i., the infected cells were lysed, and the cytoplasmic and nuclear fractions were separated and subjected to western blotting. The marker proteins GAPDH and LaminB1 were detected only in the cytoplasm and nucleus, respectively (Fig. 2E). Notably, the amount of NP detected in the nucleus of the BinCARD_KO A549 cells was much lower than that of the control cells (Fig. 2E). These results demonstrate that knocking out BinCARD1 expression suppressed the nuclear accumulation of the vRNP complex, thereby inhibiting viral life cycle progression.

We further investigated the effect of BinCARD1 on the nuclear import of NP protein. BinCARD_KO A549 cells and A549 control cells were transfected with a WSNNP-expressing construct, and the cellular localization of NP was visualized at 20 h p.t. NP accumulated in the nucleus of the A549 control cells but was predominantly retained in the cytoplasm of the BinCARD_KO A549 cells (Fig. 2F).

Collectively, these data demonstrate that BinCARD1 promoted the nuclear import of both the vRNP complex and newly synthesized NP.

BinCARD1 increases the vRNP complex activity of IAV

The vRNP complex is critical for the transcription and replication of the IAV genome [7]. Given the role played by BinCARD1 in the nuclear import of the vRNP complex, we speculated that BinCARD1 might increase vRNP complex activity. To test this hypothesis, we transfected HEK293T cells with BinCARD siRNA1 or scrambled siRNA. A RT‒qPCR analysis showed that BinCARD-specific siRNA treatment indeed downregulated the expression of BinCARD at 36 h p.t. (Fig. 3A). SiRNA-treated cells were then transfected with protein expression constructs for the PB2, PB1, PA, and NP of WSN (H1N1) virus, along with a reporter plasmid for the generation of a vRNA-like luciferase gene [20, 44, 45]. Thirty-six hours later, the luciferase activity in the cell lysates was measured to determine vRNP complex activity. We found that the vRNP activity was decreased by 33% in cells treated with BinCARD siRNA1 compared with cells treated with scrambled siRNA (Fig. 3B). The level of each vRNP complex protein remained unchanged between the two types of siRNA-treated cells, indicating that BinCARD1 did not exert an effect on vRNP complex activity by changing the expression levels of vRNP complex proteins. To confirm the result observed in siRNA-treated cells, the vRNP complex activity of WSN (H1N1) virus was assessed in the presence of exogenously expressed BinCARD1. We found that BinCARD1 overexpression led to dose-dependent increases in vRNP complex activity without affecting the expression level of any vRNP complex protein (Fig. 3C). These data demonstrate that BinCARD1 positively regulated the vRNP complex activity of IAV.

Fig. 3figure 3

BinCARD1 increases the vRNP complex activity of IAV. A SiRNA knockdown of BinCARD in HEK293T cells. HEK293T cells were transfected with BinCARD siRNA1 or scrambled siRNA, and at 36 h p.t., the cells were subjected to RT‒qPCR analysis to determine the effect of BinCARD knockdown. ***P < 0.001. B The vRNP complex activity of IAV in BinCARD siRNA1-treated HEK293T cells. HEK293T cells treated with BinCARD siRNA1 or scrambled siRNA as described in A were further transfected with the set of plasmids for the evaluation of vRNP complex activity, including the four RNP protein expression constructs (PB2, PB1, PA, and NP) derived from WSN (H1N1) virus, pHH21-SC09NS F-Luc, and pRL-TK. Thirty-six hours later, a dual-luciferase assay was performed in which the relative firefly luciferase activity was normalized to the luciferase activity of the Renilla, the internal control. The expression of vRNP complex proteins was measured by western blotting with a mouse anti-PB2, PB1, PA, or NP mAb. ***P < 0.001. C The vRNP complex activity of IAV in HEK293T cells was increased with progressively increased BinCARD1 expression. HEK293T cells were transfected with the set of plasmids for the evaluation of vRNP complex activity as described in B, together with an empty vector or gradually increasing amounts of BinCARD1-expressing constructs. Thirty-six hours later, a dual-luciferase assay was performed as described in B. The expression of the BinCARD1 and vRNP complex proteins was measured by western blotting with a mouse anti-BinCARD1, PB2, PB1, PA, or NP mAb. **P < 0.01; ***P < 0.001. The data represent the mean ± SD of three independent experiments (AC)

BinCARD1 interacts with IAV NP

The nuclear import of the vRNP complex after IAV infection is mediated by the interaction between NP and IMP α isoforms. Since BinCARD1 positively regulated the nuclear import of the vRNP complex and NP, we investigated whether BinCARD1 binds to NP by performing co-IP experiments. HEK293T cells were transfected with V5-tagged WSNNP and Flag-tagged BinCARD1 individually or in combination, and cell lysate proteins were immunoprecipitated with a mouse anti-NP mAb or anti-Flag mAb, and then, western blotting with a rabbit anti-NP pAb or anti-Flag pAb was performed. A clear interaction between NP and BinCARD1 was observed (Fig. 4A, B). Given that the RNA-binding activity of NP is important for its function in the life cycle of IAV, we performed a co-IP experiment with cell lysates that had been first treated with RNase A/T1 to determine whether the interaction between NP and BinCARD1 depends on the RNA-binding activity of NP. We found that the interaction between BinCARD1 and NP was not affected by the removal of the RNA components in the lysates of the transfected cells (Fig. 4C), indicating that the BinCARD1 and NP interaction did not rely on the RNA-binding activity of NP. We also examined the interaction between NP and BinCARD1 in infected cells. HEK293T cells transfected with plasmids expressing Flag-BinCARD1 were infected with WSN (H1N1) virus and then subjected to co-IP. We found that BinCARD1 interacted with NP during the course of IAV infection (Fig. 4D). Furthermore, we examined the interaction between NP and BinCARD1 via confocal microscopy and found that WSNNP and Flag-BinCARD1 colocalized in the nucleus and cytoplasm of transiently transfected A549 cells (Fig. 4E).

Fig. 4figure 4

BinCARD1 interacts with NP. A, B A co-IP assay was performed to examine the interaction between NP and BinCARD1. HEK293T cells were transfected with plasmids expressing V5-WSNNP and Flag-BinCARD1 individually or in combination. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-NP mAb (A) or a mouse anti-Flag mAb (B) and subjected to western blotting with a rabbit anti-NP pAb or a rabbit anti-Flag pAb. C The BinCARD1-NP interaction did not rely on the RNA-binding activity of NP. HEK293T cells were transfected as described in A, B. Cell lysates treated with RNase A/T1 or left untreated were immunoprecipitated with a mouse anti-Flag mAb, and the proteins were subjected to western blotting with a rabbit anti-NP pAb or a rabbit anti-Flag pAb. D A co-IP assay was performed to examine the interaction between NP and BinCARD1 in virus-infected cells. HEK293T cells were transfected with vectors to express Flag-BinCARD1, and at 36 h p.t., the cells were infected with WSN (H1N1) virus (MOI = 5) for 12 h. Cell lysate proteins were immunoprecipitated with a mouse anti-Flag mAb and then subjected to western blotting with a rabbit anti-NP pAb or a rabbit anti-Flag pAb. E Colocalization of NP and BinCARD1 was detected by confocal microscopy. A549 cells were transfected with plasmids expressing WSNNP and Flag-BinCARD1 individually or in combination. At 20 h p.t., the cells were stained with a mouse anti-NP mAb and a rabbit anti-Flag pAb, incubated with Alexa Fluor 633 goat anti-mouse IgG (H + L) (red) and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (green), and visualized by confocal microscopy. F, G Co-IP assays were performed to examine the interaction between truncation mutants of NP and BinCARD1. HEK293T cells were transfected with the indicated constructs. At 36 h p.t., cell lysates were pulled down with glutathione magnetic beads. The bound proteins were eluted and subjected to western blotting with a rabbit anti-NP pAb, a rabbit anti-Flag pAb, or  a rabbit anti-GST pAb

Next, we investigated the key regions within NP and BinCARD1 that are critical for their interaction. Five previously generated NP truncation constructs were used to express peptides with only the amino acid regions of 1–80, 1–162, 1–271, 1–351, and 268–498 [20]. Similarly, we generated two truncation constructs of BinCARD1, one expressed the N-terminal 1–100 amino acids and the other expressing the C-terminal 101–228 amino acids. A GST tag was fused to each truncated NP and BinCARD1 construct at the N-terminus. We then examined the interaction between BinCARD1 and GST-NP truncation mutants as well as the interaction between NP and GST-BinCARD1 truncation mutants by performing a GST pull-down assay. The C-terminal 352–498 amino acid region of NP was found to mediate the interaction of NP with BinCARD1 (Fig. 4F), and the C-terminal 101–228 amino acid region of BinCARD1 was found to be critical for BinCARD1 binding with NP (Fig. 4G).

BinCARD1 facilitates the binding of NP with IMP α7

It had previously been reported that the nuclear import of NP was mediated by its association with IMP α1, α3, α5, or α7 and the subsequent binding of IMP β1 [16, 17]. Given that BinCARD1 interacts with NP and facilitates the nuclear import of the vRNP complex and newly synthesized NP, we hypothesized that BinCARD1 might affect the interaction between NP and IMP α isoforms or the subsequent recruitment of IMP β1. To test this hypothesis, we determined whether BinCARD1 interacts with IMP α isoforms or IMP β1 by performing co-IP assays. HEK293T cells were transfected with plasmids expressing Myc-tagged BinCARD1 and Flag-tagged IMP α1, α3, α5, α7, or β1 individually or in combination. Cell lysate proteins were immunoprecipitated with a mouse anti-Myc or anti-Flag mAb, and then, western blotting was performed with a rabbit anti-Myc or anti-Flag pAb. The data revealed that BinCARD1 did not interact with IMP α1, α3, α5, or β1 (Fig. 5A–D). In contrast, we discovered that BinCARD1 interacts with IMP α7 (Fig. 5E, F). The interaction between BinCARD1 and IMP α7 was also observed during the course of IAV infection, as determined with a co-IP assay performed with HEK293T cells transfected with plasmids expressing Flag-BinCARD1 and subsequently infected with WSN (H1N1) virus (Fig. 5G).

Fig. 5figure 5

BinCARD1 facilitates the binding of NP with IMP α7. AD Co-IP assays were performed to examine the interaction between BinCARD1 and IMP α1, α3, α5, and β1. HEK293T cells were transfected with plasmids expressing Myc-tagged BinCARD1 and Flag-tagged IMP α1 (A), α3 (B), α5 (C), or β1 (D) individually or in combination. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-Myc mAb or a mouse anti-Flag mAb and subjected to western blotting with a rabbit anti-Myc pAb or a rabbit anti-Flag pAb. E, F A co-IP assay was performed to examine the interaction between BinCARD1 and IMP α7. HEK293T cells were transfected with plasmids expressing Myc-tagged BinCARD1 and Flag-tagged IMP α7 individually or in combination. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-Flag mAb (E) or a mouse anti-Myc mAb (F) and subjected to western blotting with a rabbit anti-Myc pAb or a rabbit anti-Flag pAb. G A co-IP assay was performed to examine the interaction between BinCARD1 and endogenous IMP α7 during IAV infection. HEK293T cells were transfected with vectors to express Flag-tagged BinCARD1, and at 36 h p.t., the cells were infected with WSN (H1N1) virus (MOI = 5) for 12 h. Cell lysate proteins were immunoprecipitated with a mouse anti-Flag mAb and subjected to western blotting with a rabbit anti-IMP α7 pAb or a rabbit anti-Flag pAb. H Western blotting analysis to determine the effect of gradually increasing BinCARD1 expression on the expression of IMP α7. HEK293T cells were transfected with vectors to express Flag-tagged IMP α7 and gradually increasing levels of BinCARD1. At 36 h p.t., cell lysate proteins were western blotted with a rabbit anti-Flag pAb and a mouse anti-BinCARD1 mAb. I Co-IP assays were performed to examine the relationship among NP, IMP α7, and BinCARD1. HEK293T cells were transfected with plasmids expressing WSNNP, Flag-tagged IMP α7, and gradually increasing amounts of Myc-tagged BinCARD1 individually or in combination. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-Flag mAb and subjected to western blotting with a rabbit anti-NP pAb, a rabbit anti-Flag pAb, or a rabbit anti-Myc pAb

We next investigated the biological effect of the interaction between BinCARD1 and IMP α7. We found that increasing the amount of BinCARD1 in HEK293T cells exerted no effect on the expression level of IMP α7 (Fig. 5H). We further examined whether the interaction between BinCARD1 and IMP α7 promotes complex formation between NP and IMP α7, thus facilitating the nuclear import of NP through the classical nuclear import pathway. HEK293T cells were transfected with plasmids expressing WSNNP and Flag-tagged IMP α7, together with gradually increasing amounts of the Myc-tagged BinCARD1 construct. The co-IP assay showed that the amount of NP that coimmunoprecipitated with IMP α7 was significantly increased in a dose-dependent manner when BinCARD1 was coexpressed (Fig. 5I). This result indicates that the presence of BinCARD1 promoted the formation of a complex between NP and IMP α7, thereby facilitating the nuclear import of the vRNP complex.

BinCARD1 activates RIG-I innate immune signaling by promoting the K63-linked ubiquitination of TRAF3

When host factors promote the replication of IAV, it is inevitable that mechanisms of host antagonism are activated. Type I IFN plays an important role in host antiviral immunity. Given that BinCARD2 can induce MAVS oligomerization to activate the RIG-I innate immune pathway [39], we hypothesized that BinCARD1 may also play a role in RIG-I innate immune signaling. To test this hypothesis, HEK293T cells were transfected with an ISRE luciferase reporter gene and a BinCARD1 expression construct or an empty vector and were then left untreated or treated with inducers of type I IFN activity [i.e., SeV or poly (I:C)]. By measuring the luciferase activity, we found that cells with overexpressed BinCARD1 showed dramatically enhanced SeV- or poly (I:C)-induced expression of the ISRE luciferase reporter gene compared with cells that did not overexpress BinCARD1 (Fig. 6A, B). Consistent with these data, BinCARD1 knockdown in A549 cells, realized through BinCARD siRNA1 treatment, led to a reduction in IRF3 phosphorylation after SeV stimulation compared with the level in scrambled siRNA-treated cells (Supplementary Fig. 7A). In addition, HEK293T cells were transfected with plasmid expressing  BinCARD1 or an empty vector and then stimulated with SeV or poly (I:C) for 6 h. A RT‒qPCR analysis showed that the expression levels of ISG15 and OAS1, two of the most upregulated type I IFN responsive genes, were much higher in the BinCARD1-overexpressing cells than in the control cells (Supplementary Fig. 7B–E). These data indicate that BinCARD1 was involved in the activation of RIG-I signaling.

Fig. 6figure 6

BinCARD1 activates RIG-I signaling by promoting the K63-linked ubiquitination of TRAF3. A, B An ISRE luciferase reporter assay was performed to determine the effect of BinCARD1 on the activation of RIG-I signaling. HEK293T cells were transfected with an ISRE-Luc reporter plasmid, a pRL-TK control plasmid, and a Flag-BinCARD1-expressing plasmid or an empty pCAGGS vector. At 36 h p.t., the cells were left untreated or stimulated with SeV (A) or poly (I:C) (B) for 12 h. The overexpression of BinCARD1 was confirmed by western blotting with a rabbit anti-Flag pAb. The luciferase activity of the cell lysates was analyzed with a dual-luciferase reporter assay in which firefly luciferase activity was normalized to the luciferase activity of Renilla, which had been coexpressed. The fold-change in the firefly luciferase activity of BinCARD1-overexpressing cells compared with cells transfected with empty vector is shown. ***P < 0.001. C Co-IP assays were performed to examine the interaction between BinCARD1 and RIG-I signaling pathway proteins. HEK293T cells were transfected with plasmids expressing Myc-tagged BinCARD1 and Flag-tagged RIG-I, MAVS, TRAF3, TBK1, or IRF3. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-Myc mAb and subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-Myc pAb. D HEK293T cells were transfected with plasmids expressing Flag-tagged TRAF3 and gradually increasing amounts of Myc-tagged BinCARD1. At 36 h p.t., cell lysate proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-Myc pAb. E TRAF3 impaired the binding between BinCARD1 and IAV NP. HEK293T cells were transfected with combinations of plasmids expressing BinCARD1, V5-WSNNP, and TRAF3-Flag. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-BinCARD1 mAb, and the bound proteins were eluted and subjected to western blotting with a mouse anti-BinCARD1 mAb, a rabbit anti-Flag pAb, or a rabbit anti-NP pAb. F A co-IP assay was performed to assess TRAF3 ubiquitination. HEK293T cells were transfected with plasmids expressing Flag-tagged TRAF3 (C68A/H70A) and HA-tagged ubiquitin, with or without Myc-tagged BinCARD1. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-TRAF3 mAb and then western blotted with a rabbit anti-Flag pAb or a rabbit anti-HA mAb. G A co-IP assay was performed to determine the type of TRAF3 ubiquitination linkage mediated by BinCARD1. HEK293T cells were transfected with plasmids expressing Flag-tagged TRAF3 (C68A/H70A) and HA-tagged ubiquitin (K63) or ubiquitin (K63R), with or without BinCARD1. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-TRAF3 mAb and subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-HA mAb. H K63-linked polyubiquitination of TRAF3 was induced by BinCARD1 in infected cells. RAW264.7 cells were transfected with an empty vector or a BinCARD1-expressing plasmid, and at 48 h p.t., the cells were infected with SeV. At 0 and 6 h p.i., cell lysate proteins were immunoprecipitated with a mouse anti-TRAF3 mAb and subjected to western blotting with a rabbit anti-ubiquitin (linkage-specific K63) mAb, a mouse anti-ubiquitin mAb, a mouse anti-TRAF3 mAb, or a mouse anti-BinCARD1 mAb. I A co-IP assay was performed to determine the effect of BinCARD1 on the interaction between MAVS and IKKε. HEK293T cells were transfected with plasmids expressing Flag-tagged IKKε and Myc-tagged MAVS, with or without BinCARD1. At 36 h p.t., cell lysate proteins were immunoprecipitated with a mouse anti-Myc mAb and subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-MAVS mAb. J The effect of BinCARD1 on the phosphorylation of IRF3 was assessed. HEK293T cells were transfected with plasmids expressing HA-tagged TRAF3, Flag-tagged IRF3, and BinCARD1 individually or in various combinations. At 36 h p.t., cell lysate proteins were subjected to western blotting with a rabbit anti-HA mAb, a rabbit anti-Flag pAb, a rabbit anti-IRF3-p mAb, or a mouse anti-BinCARD1 mAb

To investigate the elements of the RIG-I pathway affected by BinCARD1 function, we performed co-IP experiments with HEK293T cells to examine whether BinCARD1 associates with key adaptors in the RIG-I pathway. BinCARD1 clearly interacted with TRAF3 but not RIG-I, MAVS, TBK1, or IRF3 (Fig. 6C). We found that increasing amounts of BinCARD1 in HEK293T cells exerted no effect on the expression level of TRAF3 (Fig. 6D). Interestingly, we did not detect BinCARD1 in the lysates of cells in which Myc-tagged BinCARD1 and Flag-tagged TBK1 were coexpressed (Fig. 6C). We therefore speculated that BinCARD1 might be degraded by TBK1.

Because BinCARD1 interacts with both IAV NP and TRAF3, we explored whether TRAF3 competes with NP for binding to BinCARD1. A co-IP assay showed that the amount of NP coimmunoprecipitated with BinCARD1 was dramatically reduced when TRAF3 was also expressed (Fig. 6E), indicating that the binding between NP and BinCARD1 was inhibited by the presence of TRAF3.

In the RIG-I pathway, K63-linked ubiquitination of TRAF3 is required for the induction of type I IFNs [46, 47]. We therefore determined whether BinCARD1 activates TRAF3. A TRAF3 mutant deficient in E3 ubiquitin ligase activity (C68A/H70A) was assessed because this mutant is unable to mediate the ubiquitination of substrates, including itself through autoubiquitination [48]. BinCARD1-mediated polyubiquitination of TRAF3 was readily detected in HEK293T cells expressing Flag-tagged TRAF3 (C68A/H70A), HA-tagged ubiquitin, and Myc-tagged BinCARD1 (Fig. 6F). CARD domain-containing proteins can bind unanchored K63 ubiquitin chains, thereby activating target proteins [49, 50]. Therefore, to determine whether the expression of BinCARD1 leads to the K63-linked polyubiquitination of TRAF3, we cotransfected HEK293T cells with different combinations of plasmids to express Flag-tagged TRAF3 (C68A/H70A), BinCARD1, and ubiquitin mutant in which either all lysine residues except K63 were replaced with arginine residues (Ub-K63) or only the K63 residue was replaced with an arginine residue (Ub-K63R). Co-IP analyses showed that BinCARD1 promoted the ubiquitination of TRAF3 (C68A/H70A) in the presence of Ub-K63 but not Ub-K63R (Fig. 6G), indicating that BinCARD1 promoted the K63-linked ubiquitination of TRAF3. Consistent with these data, the SeV-induced K63-linked polyubiquitination of endogenous TRAF3 was clearly enhanced in BinCARD1-overexpressing RAW264.7 cells compared with control cells (Fig. 6H). We further examined the effect of BinCARD1-mediated K63-linked ubiquitination of TRAF3 on downstream signaling in the RIG-I pathway. The interaction between MAVS and IKKε was markedly increased when BinCARD1 was expressed (Fig. 6I). We also measured the phosphorylation rate of IRF3 in the absence or presence of BinCARD1 overexpression. HEK293T cells were transfected with vectors to express TRAF3 and IRF3 in the presence or absence of BinCARD1. We found that when coexpressed with BinCARD1, IRF3 was significantly phosphorylated (Fig. 6J). These results indicate that the K63-linked polyubiquitination of TRAF3, catalyzed by BinCARD1, was important for the recruitment of the TBK1-IKKε kinase complex and activation of RIG-I signaling.

TBK1 mediates BinCARD1 degradation through the autophagy pathway

TBK1 is an important regulator of not only the innate immune system but also autophagy [36, 51, 52]. It has also been documented that TBK1 functions as an E3 ubiquitin ligase to degrade target proteins [53]. When coexpressed with TBK1, BinCARD1 was undetectable (Fig. 6C). In addition, with increases in the abundance of exogenous TBK1-Flag, the amount of Myc-BinCARD1 gradually decreased (Fig. 7A). These results prompted us to hypothesize that BinCARD1 might be degraded by TBK1 via the ubiquitin proteasome pathway or autophagy pathway. Therefore, we used the proteasome inhibitor MG132 and the autophagy inhibitors 3-MA and Baf A1 to identify the pathway through which BinCARD1 is degraded by TBK1. We found that the degradation of BinCARD1 was abolished in the presence of 3-MA or Baf A1 but not MG132 (Fig. 7B, C, Supplementary Fig. 8), indicating that the autophagy activation was required for the degradation of BinCARD1 by TBK1. To confirm this result, we treated HEK293T cells that had been transfected with vectors to overexpress BinCARD1 with the autophagy inducers rapamycin and EBSS. We found that the addition of these autophagy inducers dramatically reduced the level of BinCARD1 (Fig. 7D), indicating that the degradation of BinCARD1 was indeed mediated by TBK1 via the autophagy system. To map the region of TBK1 involved in the degradation of BinCARD1, we generated four TBK1 truncation constructs tagged with Flag at the C-terminus and assessed their effects on the degradation of BinCARD1 in HEK293T cells. BinCARD1 was degraded in the presence of wild-type (WT) TBK1 and the truncated mutants TBK1-KD + ULD and TBK1-ULD + CC (Supplementary Fig. 9), indicating that the ULD domain in TBK1 was important for the degradation of BinCARD1.

Fig. 7figure 7

TBK1 mediates the degradation of BinCARD1 through the autophagy pathway. A TBK1-mediated degradation of BinCARD1 was analyzed by western blotting. HEK293T cells were transfected with plasmids expressing Myc-tagged BinCARD1 and gradually increasing amounts of Flag-tagged TBK1 individually or in combination. At 36 h p.t., cell lysate proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-Myc pAb. B, C Western blotting was performed to identify the pathway through which TBK1 degrades BinCARD1. HEK293T cells were transfected with plasmids expressing BinCARD1, and at a 12-hour interval, the cells were transfected with an empty vector or a TBK1-expressing plasmid. Twenty-four hours later, the cells were treated for 8 h with H2O or 3-MA (B) and DMSO or MG132 (C). Cell lysate proteins were subjected to western blotting with a mouse anti-BinCARD1 mAb or a rabbit anti-TBK1 mAb. D The effect of autophagy inducers on the level of BinCARD1 was assessed. HEK293T cells were transfected with plasmids expressing BinCARD1. At 24 h p.t., cells were treated with rapamycin for 24 h or EBSS for 2 h. Cell lysate proteins were subjected to western blotting with a mouse anti-BinCARD1 mAb

TBK1 promotes the autophagic degradation of BinCARD1 by activating p62 phosphorylation

TBK1 can regulate autophagy by phosphorylating the autophagy receptors OPTN and p62 [

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