Tripartite motif-containing protein 46 accelerates influenza A H7N9 virus infection by promoting K48-linked ubiquitination of TBK1

Abstract

Background: Avian influenza A H7N9 emerged in 2013, threatening public health and causing acute respiratory distress syndrome, and even death, in the human population. However, the underlying mechanism by which H7N9 virus causes human infection remains elusive.

Methods: Herein, we infected A549 cells with H7N9 virus for different times and assessed tripartite motif-containing protein 46 (TRIM46) expression. To determine the role of TRIM46 in H7N9 infection, we applied lentivirus-based TRIM46 short hairpin RNA sequences and overexpression plasmids to explore virus replication, and changes in type I interferons and interferon regulatory factor 3 (IRF3) phosphorylation levels in response to silencing and overexpression of TRIM46. Finally, we used Co-immunoprecipitation and ubiquitination assays to examine the mechanism by which TRIM46 mediated the activity of TANK-binding kinase 1 (TBK1).

Results: Type I interferons play an important role in defending virus infection. Here, we found that TRIM46 levels were significantly increased during H7N9 virus infection. Furthermore, TRIM46 knockdown inhibited H7N9 virus replication compared to that in the control group, while the production of type I interferons increased. Meanwhile, overexpression of TRIM46 promoted H7N9 virus replication and decrease the production of type I interferons. In addition, the level of phosphorylated IRF3, an important interferon regulatory factor, was increased in TRIM46-silenced cells, but decreased in TRIM46 overexpressing cells. Mechanistically, we observed that TRIM46 could interact with TBK1 to induce its K48-linked ubiquitination, which promoted H7N9 virus infection.

Conclusion: Our results suggest that TRIM46 negatively regulates the human innate immune response against H7N9 virus infection.

Keywords: Influenza A H7N9; Interferons; TBK1; TRIM46.

Fig. 1

H7N9/ZJU-1 increases TRIM46 expression. A549 cells were seeded in 6-well plate and infected with H7N9/ZJU-1 for indicated times, cells were harvested. The protein expression levels of TRIM46, H7N9/ZJU-1 NP were measured by western blotting and GAPDH was used as an internal control (A), and the relative mRNA of TRIM46 at indicated times were analyzed by RT-qPCR (B)

Fig. 2

Knockdown of TRIM46 inhibits H7N9/ZJU-1 replication. A549 cells were transfected with negative control sequences and TRIM46 knockdown sequences, TRIM46#1 and TRIM46#2, for 72 h, cells were harvested, the protein levels of TRIM46 were measured by western blotting (A) and the mRNA levels were measured by RT-qPCR (B). C. A549 cells were transfected with TRIM46 shRNA sequence or negative control sequences for 72 h, infected with H7N9/ZJU-1 for 12 h, the cells were lysed and subjected to western blotting with indicated antibodies. TRIM46 Knockdown cells and negative control cells were infected with H7N9/ZJU-1 for 12 h, the relative levels of NP mRNA, cRNA and vRNA were measured by RT-qPCR (D). The supernatant of TRIM46 knockdown cells and negative control cells was collected and viral titers were determined by TCID₅₀ method (E)

Fig. 3

Overexpression of TRIM46 promotes H7N9/ZJU-1 infection. A. A549 cells were transfected with lentivirus-mediated TRIM46-Myc plasmids or empty vector plasmids, after 72 h transfection, the cells were harvested and subjected to western blotting for TRIM46 overexpression analysis, GAPDH was used as an internal control. B. A549 cells transfected with lentivirus-mediated TRIM46-Myc plasmids or empty vector plasmids for 72 h, A549 cells were harvested and the relative levels of TRIM46 mRNA were analyzed by RT-qPCR. C. A549 cells were transfected with empty vector plasmids or TRIM46-Myc plasmids, after 72 h, A549 cells were infected with H7N9 /ZJU-1 (MOI = 1) or mock-treated for 12 h, the cells lysates were collected and subjected to western blotting with indicated antibodies. D. TRIM46-Myc overexpression A549 cells or empty vector-transfected A549 cells were infected with H7N9/ZJU-1 (MOI = 1) for 12 h, the relative levels of NP mRNA, cRNA and vRNA were analyzed by RT-qPCR. E. A549 cells were transfected with lentivirus-mediated TRIM46-Myc plasmids or empty vector plasmids, after 72 h transfection, the cells were infected with H7N9/ZJU-1 for 12 h, the supernatant was collected and the viral titers were determined by TCID₅₀ method. The analysis results were presented with mean ± SD, in all situations, a p value < 0.05 was considered statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

A–C Knockdown of TRIM46 accelerates H7N9/ZJU-1-induced type I interferon expression. A549 cells were transfected with lentivirus-mediated TRIM46 knockdown sequences or negative control sequences, after 72 h, the cells were infected with H7N9/ZJU-1 or mock-treated for 12 h, the cells were harvested and subjected to RT-qPCR for analysis of IFNA mRNA (A) and IFNB1 mRNA (B). A549 cells were transfected with lentivirus-mediated TRIM46 shRNA sequence for 72 h, infected with H7N9/ZJU-1 (MOI = 1) for another 12 h. The cells were lysed and pho-IRF3, IRF3 and GAPDH were measured by western blotting (C). D–F Overexpression of TRIM46 inhibits H7N9/ZJU-1-induced type I interferon expression. A549 cells were transfected with TRIM46-Myc endogenous plasmids or empty vector plasmids for 72 h, cells were infected with H7N9/ZJU-1 or mock-infected for 12 h, cells were collected and subjected to RT-qPCR for analyzing relative levels of IFNA mRNA (D) and IFNB1 mRNA (E). A549 cells were transfected with TRIM46-Myc expression plasmids or empty vector plasmids for 72 h, thereafter, infected with H7N9/ZJU-1 (MOI = 1). Cells lysates were collected and subjected to western blotting with anti-pho-IRF3, IRF3 and GAPDH antibodies (F). The results were shown as mean ± SD, **p < 0.01, ***p < 0.001

Fig. 5

TRIM46 interacts with TBK1. A. HEK293T cells were transfected with TRIM46-Myc plasmids with or not with TBK1-FLAG plasmids. After 24 h transfection, the cells were lysed and immuno-precipitated with anti-Myc antibody and subjected to western blotting with anti-Myc and anti-FLAG antibodies. The input cell lysates were analyzed by western blotting with anti-Myc and anti-FLAG antibodies, and GAPDH was used as an internal control. B. HEK293T cells were transfected with empty vector plasmids or TRIM46-Myc expression plasmids for 24 h, the cells were lysed and immuno-precipitated with anti-Flag antibody and subjected to western blotting with anti-Myc or anti-Flag antibodies. Whole cell lysates were subjected to western blotting with anti-Myc and anti-Flag antibodies and GAPDH was used as an internal control. All data were repeated for three independent experiments and the data shown as one representative of the triplicate experiments

Fig. 6

TRIM46 promotes K48-linked ubiquitination of TBK1. HEK293T cells were transfected with indicated plasmids for 24 h, after transfection, the cells were lysed and immune-precipitated with anti-Flag antibody and subjected to western blotting for detection of WT-linked (A), K48-linked (B), K63-linked (C) ubiquitination. Anti-Myc, anti-Flag and GAPDH were used for the input

Fig. 7

Schematic model of TRIM46-mediated K48-linked ubiquitination of TBK1. During avian influenza H7N9 virus infection, TRIM46 increased and interacted with TBK1. Then, TRIM46 mediated K48-linked ubiquitination of TBK1 and degraded TBK1, subsequently, decreased the phosphorylation of IRF3 and type I IFNs expression