A double-edged sword in antiviral defence: ATG7 binding dicer to promote virus replication

Figures

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  Fig. 1. Autophagy and RNAi are involved in AIV-H9N2 replication. A. DF-1 cells were infected with different MOIs (0.01, 0.1, and 1) of AIV-H9N2 for 48 h, and then, immunofluorescence was used to detect AIV-H9N2-HA protein expression. The blue fluorescence represents DAPI-stained nuclei, whereas the red fluorescence represents the AIV-H9N2-HA protein. The magnification is 10×. The right image shows the fluorescence intensity analysis. B. DF-1 cells were infected with AIV-H9N2 at different MOIs (0.01, 0.1, and 1) for 48 h, and the supernatant was collected and incubated with MDCK cells. The release of virus particles was detected through a plaque assay after 72 h, and statistical analysis of the number of speckles was performed. C-D. Western blotting detection of autophagy- and RNAi-related protein expression levels in DF-1 cells infected with different MOIs (0.01, 0.1, and 1) of AIV-H9N2 (C) and grayscale analysis of the data in Fig. 1C (D). GAPDH was used as an internal reference protein, and the expression level of the blank group was set to “1”, while the expression levels of the other groups were multiples of that of the blank group. E-G. RT-qPCR (E) and Western blotting (F) detection…

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  Fig. 2. RNAi is an important antiviral pathway, and autophagy is beneficial for AIV-H9N2 replication. A. DF-1 cells were transfected with the Dicer overexpression plasmid PC-Dicer for 48 h, and then Western blotting was used to detect the expression of the Dicer protein. GAPDH was used as an internal reference protein, and the expression level of the blank group was set to “1”, while the expression levels of the other groups were multiples of that of the blank group. The PC group was used as the control. B. DF-1 cells were transfected with Dicer knockdown plasmids (PS-Dicer-01, PS-Dicer-02, and PS-Dicer-03), and Western blotting was used to detect Dicer protein expression to determine the silencing efficiency. The PS group was used as the control. C-F. DF-1 cells were transfected with the PC-Dicer plasmid or PS-Dicer plasmid for 24 h, and then 0.1 MOI AIV-H9N2 was used for infection for 48 h. The supernatant was collected and incubated with MDCK cells. The release of virus particles was detected through a plaque assay after 72 h, and statistical analysis of the number of speckles was performed (C-D). For 48 h, Western blotting (E) and gray value analysis (F) were performed to detect the…

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  Fig. 3. AIV-H9N2 infection promotes the upregulation of autophagy and inhibits the Dicer-dependent antiviral RNA response via ATG7. A-B. DF-1 cells were treated with 100 nM RAPA or 100 µM CQ for the indicated time points (12 h, 24 h, and 48 h), after which the protein expression levels of Dicer, P62, LC3I, and LC3II were detected via Western blotting (A). The right panel shows the gray value analysis for Fig. 3A (B). C. DF-1 cells were treated with 100 nM RAPA or 100 µM CQ for 24 h, after which the mRNA expression levels of autophagy-related genes were detected via RT-qPCR. D. DF-1 cells were infected with 0.1 MOI AIV-H9N2 for the indicated time points (6 h, 12 h, 24 h, 36 h, and 48 h), after which the protein expression levels of NDP52, HIF1A, Beclin1, and ATG7 were detected via Western blotting. E. DF-1 cells were transfected with the overexpression plasmids PC-Beclin1, PC-HIF1A, PC-ATG7, and PC-NDP52 and then subjected to protein expression detection by immunofluorescence, with the blank group as a negative control and the PC group (containing the mRFP gene) as a positive control. The magnification is 10×. F-I. DF-1 cells were transfected with PC-NDP52 (F), PC-HIF1A (G), PC-Beclin1…

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  Fig. 4. ATG7 protein promotes AIV-H9N2 replication through interaction with dicer. A. 293T cells were transfected with PC-Dicer, PC-NDP52, PC-HIF1A, PC-Beclin1, or PC-ATG7 for 48 h and then pulled down with the Dicer antibody for the CO-IP experiment. The lysates were subjected to detection of NDP52, HIF1A, Beclin1, ATG7, and dicer protein expression via Western blotting. In addition, the internal control and IgG were used as inputs for the negative control. B. Supplement to Fig. 3A. DF-1 cells were transfected with PC-Flag or PC-ATG-Flag for 48 h and subjected to co-IP. In addition, the Flag antibody was used for pulldown on the left, and the Dicer antibody was used on the right. β-actin was added as the control. C. DF-1 cells were transfected with ATG7-knockdown plasmids (PS-ATG7-01, PS-ATG7-02, and PS-ATG7-03) for 48 h, and the silencing effect of ATG7 was detected via Western blotting. The image below the strip is used for gray value analysis. D-F. DF-1 cells were transfected with PC-ATG7 or PS-ATG7 for 24 h and infected with 0.1 MOI AIV-H9N2 for 48 h. The supernatant was collected and incubated with MDCK cells. The release of virus particles was detected through a plaque assay after…

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  Fig. 5. D684 is the main site at which ATG7 interacts with dicer to facilitate AIV-H9N2 replication. A-B. Two-dimensional representations of the ATG7 protein and Dicer protein binding interface (A). Three-dimensional docking of ATG7 and Dicer (green ribbon represents the chain of ATG7; sky blue represents the chain of dicer) (B). C. The CDS region of the Gallus-ATG7 protein and the interaction sites with the Dicer protein, mainly S399, N484, R451, D673, and D684. D. DF-1 cells were transfected with PC, PC-ATG7, and ATG7 mutant plasmids (S399, N484, R451, K673, and D684) for 48 h, and Western blotting was used to detect ATG7 and Dicer protein expression. In addition, PC and PC-ATG7 were used as controls. E. Gray value analysis of Fig. 5D. The data are shown as the means ± SDs, n = 3. The differences were not significant at P ≥ 0.05 (ns) or significant at P < 0.05 (*), P < 0.01 (**)

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  Fig. 6. The ATG7 protein affects the dicer’s ability to cleave and produce small RNAs. A. PC-ATG7 and PS-ATG7 plasmids were transfected into DF-1 cells, and RNA samples were collected for transcriptome sequencing analysis. Statistics of the quantity of 21–23 nt miRNA siRNAs and vsiRNAs. B-C. The volcano plot displays the differences in miRNAs among the different groups (B). KEGG analysis of the impact pathways in each group (C). D. After the DF-1 cells were transfected with PC-ATG7 or PS-ATG7 for 24 h, 0.1 MOI AIV-H9N2 was used for infection. Small RNAs were aligned with the AIV-H9N2 genome, and the abundance and distribution of 21–23 nt vsiRNAs were determined via IGV software

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  Fig. 7. ATG7 suppresses Dicer expression during infection with other RNA viruses. A-B. DF-1 cells were infected with 1 MOI IBDV (A) or 5 MOI IBV (B) for the indicated time points (12 h, 24 h, 48 h, and 72 h), after which Western blotting was used to detect the expression of autophagy-related proteins and viral proteins. The blank group was used as the control, and the gray value analysis was performed on the right. C-D. DF-1 cells were transfected with PC-ATG7 for 24 h and then infected with 1 MOI IBDV (C) or 5 MOI IBV (D) for 24 h. Western blotting was used to detect dicer and viral protein expression. The PC group was used as the control, and the images on the right were subjected to gray analysis. E. Single-cell analysis of the distribution of bursal B cells and FACS validation of the changes in the subtypes of bursary B cells after IBDV infection. t-SNE visualization of the major nine cell types characterized by different colors. The encircled light-green cells represent the total fibroblast population. F. t-SNE of Dicer and ATG7 in the fibroblast population. G. KEGG and GO analyses of the IBDV-infected 2-week-old SPF chickens and the blank control chickens. The data are shown as the…

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  Fig. 8. Pattern diagram of RNA viruses that use the ATG7 protein to inhibit the Dicer protein. The RNA virus pattern diagram shows the inhibition of the RNAi pathway by the ATG7 protein. RNA viruses such as AIV-H9N2 can upregulate the autophagy process in DF-1 cells, hence facilitating the conversion of LC3I to LC3II and increasing the level of the ATG7 protein. Furthermore, the substantial overexpression of the ATG7 protein caused by the virus encouraged ATG7 to bind to the Dicer protein via the D684 site in the RNAi pathway, which prevented the Dicer protein from recognizing and cleaving the viral dsRNA