Protein Arginylation Is Regulated during SARS-CoV-2 Infection

Abstract

Background: In 2019, the world witnessed the onset of an unprecedented pandemic. By February 2022, the infection by SARS-CoV-2 has already been responsible for the death of more than 5 million people worldwide. Recently, we and other groups discovered that SARS-CoV-2 infection induces ER stress and activation of the unfolded protein response (UPR) pathway. Degradation of misfolded/unfolded proteins is an essential element of proteostasis and occurs mainly in lysosomes or proteasomes. The N-terminal arginylation of proteins is characterized as an inducer of ubiquitination and proteasomal degradation by the N-degron pathway.

Results: The role of protein arginylation during SARS-CoV-2 infection was elucidated. Protein arginylation was studied in Vero CCL-81, macrophage-like THP1, and Calu-3 cells infected at different times. A reanalysis of in vivo and in vitro public omics data combined with immunoblotting was performed to measure levels of arginyl-tRNA-protein transferase (ATE1) and its substrates. Dysregulation of the N-degron pathway was specifically identified during coronavirus infections compared to other respiratory viruses. We demonstrated that during SARS-CoV-2 infection, there is an increase in ATE1 expression in Calu-3 and Vero CCL-81 cells. On the other hand, infected macrophages showed no enzyme regulation. ATE1 and protein arginylation was variant-dependent, as shown using P1 and P2 viral variants and HEK 293T cells transfection with the spike protein and receptor-binding domains (RBD). In addition, we report that ATE1 inhibitors, tannic acid and merbromine (MER) reduce viral load. This finding was confirmed in ATE1-silenced cells.

Conclusions: We demonstrate that ATE1 is increased during SARS-CoV-2 infection and its inhibition has potential therapeutic value.

Keywords: COVID-19; N-degron pathway; SARS-CoV-2; arginylation; viral infection.

Figure 1

N-degron pathway modulation in uninfected cell models. (A) Experimental workflow adopted to identify the modulation of protein arginylation during SARS-CoV-2 infection; (B) expression profile of enzymes participating in the N-degron pathway identified in uninfected Calu-3 (blue), Caco-2 (purple), ACE2-A549 (green), and Vero E6 (orange) cells; (C) proteins with potential to be arginylated at N-terminal (NtE, NtD, NtC, NtN, NtQ) identified and quantified in uninfected cell models; (D) expression profile of the 918 proteins with potential to be arginylated identified in uninfected Calu-3, Caco-2, ACE2-A549, and Vero E6 cells. Proteins with the potential to be arginylated were determined based on their sequences deposited in Uniprot (Homo sapiens and Chlorocebus sabaeus). The symbol “ns” indicates a non-significant statistical relationship between groups.

Figure 2

N-degron pathway modulation in infected in vitro and in vivo models. (A) Regulation of proteins involved in the N-degron pathway during SARS-Cov-2 infection. Proteins/genes were considered differentially regulated if they had a q-value < 0.05 (Benjamini–Hochberg) and were indicated by the symbol (*). The up arrows indicate proteins/genes with a higher abundance in the infected group (INF), and the down arrows indicate a lower abundance in the infected group. The color of the boxes of proteins/genes indicates the fold change (INF/CTRL) considering all the studies evaluated; the red color indicates higher abundance in the infected group and the blue color lower abundance in the INF group. The symbol (x) indicates that a protein/gene was not identified in the dataset; (B) Western blotting analysis of ATE1 protein in Calu-3 and Vero CCL-81 cells (n = 3) infected with SARS-CoV-2 (Wuhan strain) after 2 h, 6 h, 12 h, 24 h, and 48 h; (C) HEK 293T cells transfected with the SARS-CoV-2 (classical variant) spike protein; (D) modulation of ATE1 resulting from transfection in HEK 293T cells with the receptor-binding domains (RBD) of the WT (Wuhan), BETA (South Africa), P1 (Brazil), and DELTA (India) variants. Each point represents an independent experiment (n = 3). The level of significance indicates *** p < 0.001; ** p < 0.005 in relation to the control group (CTRL).

Figure 3

Multi-correlation expression analysis. (A) Proteins and genes correlated with ATE1 expression in at least three reanalyzed studies. The correlation analysis was determined by applying the Spearman test with a cut-off significance of p-value < 0.05. Only differentially regulated proteins/genes were considered for the correlation analysis; (B) gene ontology (GO) analysis of molecular functions; (C) biological processes and (D) pathways related to proteins/genes correlated with ATE1 in at least three studies; (E) correlation graph of HSPBP1 and (F) HSP90AB1 proteins indicates the positive/negative correlation with ATE1.

Figure 4

Modulation of arginylated proteins located in the endoplasmic reticulum (ER). (A) Representative Western blot images of R-BiP/BiP, R-CALR/CALR, and R-PDI/PDI proteins in Calu-3 and (B) Vero CCL-81 cells after 2 h, 6 h, 12 h, 24 h, and 48 h of infection (Wuhan strain). Each point represents an independent experiment (n = 3). The level of significance indicates: **** p < 0.0001; *** p < 0.001; ** p < 0.005; * p < 0.05 in relation to the control group (CTRL).

Figure 5

Modulation of arginylation after infection with WT (Wuhan), P1, and P2 (Brazil) variants. Calu-3 cells infected with WT, P1, and P2 variants were evaluated at 6 hpi and 48 hpi when exposed to the stress inducer thapsigargin (A) or not exposed (B). Each point represents an independent experiment (n = 3). The significance level indicates: **** p < 0.0001; *** p < 0.001; ** p < 0.005; * p < 0.05.

Figure 6

HEK 293T cells transfected with the SARS-CoV-2 (classical variant) spike protein and the receptor-binding domains (RBD). (A) Representative images of Western blot analysis of R-BiP/BiP and R-CALR/CALR proteins of cells transfected with the spike protein. (B) Representative images of Western blot analysis of R-BiP/BiP and R-CALR/CALR proteins of cells transfected with the RBD region of the wild-type (classic), Beta (South Africa), P1 (Brazil), and Delta (India) variants. Each point represents an independent experiment (n = 3). The level of significance indicates: **** p < 0.0001; *** p < 0.001; ** p < 0.005; * p < 0.05 in relation to the control group (CTRL). Each point represents an independent experiment (n = 3). ns indicates no significance.

Figure 7

ATE1 inhibition and silencing assay in Calu-3 infected cells. (A) Representative images of the Western blot analysis performed on cells exposed to the inhibitors merbromin (MER, 25 µM) and tannic acid (TA, 1 µM) for ATE1/ACTB, R-BiP/BiP, R-CALR/CALR, and R-PDI/PDI proteins; (B) PCR results for viral release after exposure to the inhibitors; (C) representative images of the Western blot analysis performed on Calu-3 cells silenced for ATE1. The proteins ATE1/ACTB, R-BiP/BiP, and R-CALR/CALR were evaluated; (D) PCR results for viral release after ATE1 silencing; (E) Western blotting of infected Calu-3 cells for INF-CTRL and INF-siATE1 groups using anti-RBD antibody. Each point represents an independent experiment (n = 3). The level of significance indicates: **** p <0.0001; *** p < 0.001; ** p < 0.005; * p < 0.05. ns indicates no significance.

Figure 8

Single-cell RNA-seq analysis of nasopharyngeal samples. (A) Single-cell RNA-seq analysis indicating cell clustering by patients and by cell type; (B) differentially regulated clusters (p-value < 0.05) between the infected (INF) and control (CTRL) groups; (C) representative images of Western blotting analysis of infected macrophages, indicating the modulation of ATE1 and arginylated and total CALR and BiP proteins. Each point represents an independent experiment (n = 3). The level of significance indicates: **** p < 0.0001; *** p < 0.001; ** p < 0.005; * p < 0.05. ns indicates no significance.