TRIM28 regulates SARS-CoV-2 cell entry by targeting ACE2

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019, it binds to angiotensin-converting enzyme 2 (ACE2) to enter into human cells. The expression level of ACE2 potentially determine the susceptibility and severity of COVID-19, it is thus of importance to understand the regulatory mechanism of ACE2 expression. Tripartite motif containing 28 (TRIM28) is known to be involved in multiple processes including antiviral restriction, endogenous retrovirus latency and immune response, it is recently reported to be co-expressed with SARS-CoV-2 receptor in type II pneumocytes; however, the roles of TRIM28 in ACE2 expression and SARS-CoV-2 cell entry remain unclear. This study showed that knockdown of TRIM28 induces ACE2 expression and increases pseudotyped SARS-CoV-2 cell entry of A549 cells and primary pulmonary alveolar epithelial cells (PAEpiCs). In a co-culture model of NK cells and lung epithelial cells, our results demonstrated that NK cells inhibit TRIM28 and promote ACE2 expression in lung epithelial cells, which was partially reversed by depletion of interleukin-2 and blocking of granzyme B in the co-culture medium. Furthermore, TRIM28 knockdown enhanced interferon-γ (IFN-γ)- induced ACE2 expression through a mechanism involving upregulating IFN-γ receptor 2 (IFNGR2) in both A549 and PAEpiCs. The upregulated ACE2 induced by TRIM28 knockdown and co-culture of NK cells was partially reversed by dexamethasone in A549 cells. Our study identified TRIM28 as a novel regulator of ACE2 expression and SARS-CoV-2 cell entry.

Keywords: ACE2; IFN-γ receptor; Lung epithelial cells; NK cells; SARS-CoV-2; TRIM28.

Graphical abstract

Fig. 1

TRIM28 regulates pseudotyped SARS-CoV-2 cell entry in A549 cells. (A-D) The A549 cells were transfected with siRNAs against TRIM28 (A-C) or TRIM27 (D) for 48 h. The mRNA levels of ACE2, TMPRSS2, TRIM28 and TRIM27 were determined by using qPCR and western blotting assay. Data are obtained from 3 independent experiments. **P < 0.01. (E) A549 cells were transfected with TRIM28 siRNA alone or co-transfected with ACE2 siRNA for 24 h, and then infected with LV-Spike-nCoV-Luc for another 48 h before firefly luciferase activities were measured. n = 3. **P < 0.01. (F) qPCR assay were used to confirm ACE2 knockdown efficiency. n = 3.

Fig. 2

The expression of TRIM28 and ACE2 in A549 cells co-cultured with NK-92 cells. (A-C) The NK-92 cells were placed directly on top of A549 cell layer in 12-well culture plates and co-cultured for 12 or 24 h. The mRNA expressions of TRIM28 (A) and ACE2 (B) were analyzed by qPCR method. n = 3. **P < 0.01. (C) Immunoblot analysis was performed to detect expressions of TRIM28 in A549 cells that co-cultured with NK-92 cells for 24 h. Representative blots were shown. The relative expressions of the TRIM28 proteins were quantified by Image J software. n = 3. (D, E) NK cells were cultured in medium in the presence or absence of IL-2 for 24 h, and then placed on top of A549 cell layer for another 24 h. The mRNA expressions of TRIM28 (D) and ACE2 (E) were analyzed by using qPCR method. n = 3. **P < 0.01. (F) NK-92 cell and A549 cells were co-cultured by placing the NK-92 culture inserts on top of the A549 cell layer in transwell culture plates for 24 h. qPCR assay was used to check mRNA expressions of TRIM28 and ACE2 in A549 cells. n = 3. **P < 0.01. (G) The NK-92 cells were placed directly on top of A549 cell layer and co-cultured in medium containing granzyme B inhibitor Z-AAD-CMK (100 μM) for 24 h. TRIM28 and ACE2 expressions were determined by qPCR method. n = 3. **P < 0.01. *P < 0.05.

Fig. 3

Effect of dexamethasone on TRIM28 knockdown induced ACE2 expression in A549 cells. (A-C) A549 cells were transfected with scramble or TRIM28 siRNA for 32 h and subsequently incubated with dexamethasone for another 16 h. ACE2 and TRIM28 transcripts were analyzed by using qPCR (A, B) and western blotting (C) assay. n = 3. **P < 0.01. ns. no significance. (D) Scramble and TRIM28 siRNA transfected A549 cells were treated with dexamethasone for 16 h, and then infected with LV-Spike-nCoV-Luc for another 48 h.The firefly luciferase activities were measured. n = 3. **P < 0.01. (E) The NK-92 cells were placed directly on top of A549 cell layer and co-cultured in dexamethasone containing medium for 24 h. The expression of TRIM28 was determined by qPCR method. n = 3. **P < 0.01. *P < 0.05.

Fig. 4

Effect of TRIM28 on IFN-γ induced ACE2 expression in A549 cells. (A, B) Control and TRIM28 silenced A549 cells were stimulated with IFN-γ for 16 h. qPCR assay was performed to check the expressions of ACE2 (A) and TRIM28 (B). n = 3. **P < 0.01. *P < 0.05. (C) The mRNA levels of IFNGR1 and IFNGR2 in control and TRIM28 knockdown A549 cells were examined. n = 3. **P < 0.01. (D) The protein levels of IFNGR2 in control or TRIM28 silenced cells were detected by western blot. n = 3. (E) Control or TRIM28 knockdown A549 cells were treated with IFN-γ for 15 min. Immunoblot analysis was performed to detect expressions of phosphorylated STAT1 and total STAT1. Representative blots were shown. n = 3. (F) A549 cells were transfected with scramble or IFNGR2 siRNA, and then treated with IFN-γ (20 ng/mL) for 16 h. ACE2 and IFNGR2 expressions were examined by qPCR method. n = 3. *P < 0.05.

Fig. 5

TRIM28 binds and regulates ACE2 promoter activity in A549 cells. (A) Control and TRIM28 knockdown A549 cells were transfected with the reporter plasmid of −2041 ~ +100 Luc and − 950 ~ +100 Luc. Promoter activity was measured in four independent transfection experiments. **P < 0.01. (B) ChIP assay was performed by using an anti-TRIM28 or IgG antibody. Immunoprecipitated ACE2 promoter regions were detected by qPCR analysis. n = 3. (C) The A549 cells were transfected with scrambled and TRIM28 siRNA, which is combined with ZNF90, ZNF649 or ZNF736 siRNA for 48 h. Then the mRNA expressions of ACE2 were checked by using qPCR method. n = 3. **P < 0.01. * P < 0.05. (D & E) A549 cells were transfected with either scrambled or TRIM28 siRNA alone, or co-transfected with ZNF90 siRNA for 48 h. The mRNA expressions of ACE2 (D) and ZNF90 (E) were analyzed by using qPCR method. n = 3. **P < 0.01. (F) ACE2 promoter reporter was transfected into ZNF90 knockdown A549 cells for 24 h. Promoter activity was measured in three independent transfection experiments. * P < 0.05.

Fig. 6

TRIM28 regulates ACE2 expression in human PAEpiCs. (A) ACE2 mRNA level in TRIM28 knockdown human PAEpiCs. n = 3. **P < 0.01. (B) The human PAEpiCs were transfected with either scrambled or TRIM28 siRNA alone, or co-transfected with ACE2 siRNA for 24 h followed by infection with LV-Spike-nCoV-Luc. Forty-eight hours after infection, firefly luciferase activities were measured. n = 3. **P < 0.01. (C) NK-92 cells were placed directly on top of human PAEpiC cell layer. After co-culture for 24 h, the mRNA expressions of target genes were analyzed by using qPCR method. n = 3. **P < 0.01. *P < 0.05. (D—F) Control and TRIM28 knockdown PAEpiCs were incubated with dexamethasone (1 μM) or IFN-γ (10 ng/ml) for 16 h, ACE2 (D), TRIM28 (E) and IFNGR2 (F) transcripts were analyzed by using qPCR assay. n = 3. **P < 0.01.*P < 0.05. ns. no significance.