TRIM21 Promotes Oxidative Stress and Ferroptosis through the SQSTM1-NRF2-KEAP1 Axis to Increase the Titers of H5N1 Highly Pathogenic Avian Influenza Virus

Abstract

Tripartite motif-containing protein 21 (TRIM21) is involved in signal transduction and antiviral responses through the ubiquitination of protein targets. TRIM21 was reported to be related to the imbalance of host cell homeostasis caused by viral infection. Our studies indicated that H5N1 highly pathogenic avian influenza virus (HPAIV) infection up-regulated TRIM21 expression in A549 cells. Western blot and qPCR results showed that knockdown of TRIM21 alleviated oxidative stress and ferroptosis induced by H5N1 HPAIV and promoted the activation of antioxidant pathways. Co-IP results showed that TRIM21 promoted oxidative stress and ferroptosis by regulating the SQSTM1-NRF2-KEAP1 axis by increasing SQSTM1 K63-linked polyubiquitination under the condition of HPAIV infection. In addition, TRIM21 attenuated the inhibitory effect of antioxidant NAC on HPAIV titers and enhanced the promoting effect of ferroptosis agonist Erastin on HPAIV titers. Our findings provide new insight into the role of TRIM21 in oxidative stress and ferroptosis induced by viral infection.

Keywords: NRF2; TRIM21; ferroptosis; influenza virus; oxidative stress.

Figure 1

DK/212 AIV infection induced oxidative stress and ferroptosis in A549 cells. (A) The ROS level was detected by DCFDA at 12, 24, and 36 h. (B) MMP were detected using flow cytometry by JC-1 staining, CCCP as a positive control. (C) The level of ferrous irons in cell lysate was detected at 24 h post infection, A549 cells treated by 5 μM Erastin as a positive control. (D) The level of MDA was detected at 24 h post infection. A549 cells were treated by 5 μM Erastin or 20 nM liproxstatin-1. (E) The level of GSH was detected at 24 h post infection. A549 cells were treated by 20 nM liproxstatin-1 after infection. (F) The cell viability was detected at 24 h post infection. A549 cells were treated by 5 μM Erastin or 20 nM liproxstatin-1. All data were expressed as means ± SD; the Student’s t-test was used to analyze the differences (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns p > 0.05).

Figure 2

DK/212 AIV infection increased the mRNA and protein level of TRIM21, and reduced mRNA and protein level of related antioxidant genes. (A,B) A549 cells were infected with DK/212 at an MOI of 0.1. The mRNA and protein level of TRIM21 was detected at 12, 24, and 36 h (C–J) A549 cells were infected with DK/212 at an MOI of 0.1. The mRNA level of NRF2, SLC7A11, GPX4, NQO1, HO-1, GCLC, and GCLM genes was detected at 24 h. (K) A549 cells were infected with DK/212 at an MOI of 0.1. The protein level of NRF2, SLC7A11, and GPX4 was detected at 12, 24, and 36 h. All data were expressed as means ± SD; the Student’s t-test was used to analyze the differences (* p < 0.05, ** p <0.01, *** p < 0.001 ns p > 0.05).

Figure 3

Knockdown of TRIM21 alleviated oxidative stress and ferroptosis induced by DK/212 AIV infection. (A–C) A549 cells were transfected with TRIM21 si RNA1, si RNA2, and si RNA3. The mRNA and protein level of TRIM21 was detected. (D) A549 cells were transfected with TRIM21 si RNA or si NC. The level of ROS was detected at 12, 24, and 36 h after virus infection. (E) A549 cells were transfected with TRIM21 si RNA or si NC. The level of MDA was detected at 24 h after virus infection. (F) A549 cells were transfected with TRIM21 si RNA or si NC. The level of GSH was detected at 24 h after virus infection. (G) The cell viability was detected by cck8 after knockdown TRIM21. All data were expressed as means ± SD; the Student’s t-test was used to analyze the differences (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns p > 0.05).

Figure 4

TRIM21 inhibited antioxidant genes and promoted ferroptosis by down-regulating NRF2. (A–F) A549 cells were transfected with TRIM21 Si RNA. The mRNA level of antioxidant genes was detected at 24 h after DK/212 infection. (G) A549 cells were transfected with TRIM21 Si RNA. The protein level of NRF2, SLC7A11, and GPX4 was detected at 24 h after DK/212 infection. (H) A549 cells were transfected with FLAG-NRF2 plasmid. The protein level of SLC7A11 and GPX4 was detected at 24 h after DK/212 infection. (I) A549 cells were transfected with TRIM21 Si RNA. A549 cells were treated with 2 μM ML385 at 12 h after DK/212 infection. The protein level was detected at 24 h after DK/212 infection. (J,K) A549 cells were transfected with FLAG-NRF2 plasmid. The GSH and MDA level was detected at 24 h after DK/212 infection. All data were expressed as means ± SD; the Student’s t-test was used to analyze the differences (* p < 0.05, ** p < 0.01, *** p < 0.001, ns p > 0.05.

Figure 5

TRIM21 and KEAP1 competed to bind to SQSTM1. (A) TRIM21-HA and SQSTM1-FLAG plasmids were co-transfected in 293T cells for 24 h. (B) TRIM21-HA and NRF2-FLAG plasmids were co-transfected in 293T cells for 24 h. (C) TRIM21-HA and KEAP1-FLAG plasmids were co-transfected in 293T cells for 24 h. (D) TRIM21-eGFP and SQSTM1-AsREDs plasmids were co-transfected in 293T cells for 24 h. (E) TRIM21-eGFP, SQSTM1-FLAG, and KEAP1-HA plasmids were co-transfected in 293T cells for 24 h. (F) SQSTM1-FLAG and TRIM21-HA plasmids were transfected in A549 cells. The protein level was detected at 24 h after DK/212 infection.

Figure 6

TRIM21 enhanced K63-linked polyubiquitination of SQSTM1. (A) TRIM21-eGFP, SQSTM1-FLAG, and UB-HA plasmids were co-transfected in 293T cells. (B) TRIM21-eGFP, SQSTM1-FLAG, UB (K48)-HA, and UB (K63)-HA plasmids were co-transfected in 293T cells for detection.

Figure 7

A549 cells were infected with DK/212 at an MOI of 0.1. (A) A549 cells were treated with DMSO, or Erastin treated at 12 h after DK/212 infection. (B,C) A549 cells were transfected with TRIM21 plasmid or NC plasmid. A549 cells were treated with NAC or Erastin at 12 h after DK212 infection. (* p < 0.05, ** p < 0.01, ns p > 0.05).