SARS-CoV-2 down-regulates ACE2 through lysosomal degradation

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes its Spike (S) glycoprotein to bind to the angiotensin-converting enzyme 2 (ACE2) receptor for cellular entry. ACE2 is a critical negative regulator of the renin-angiotensin system and plays a protective role in preventing tissue injury. Expression of ACE2 has been shown to decrease upon infection by SARS-CoV. However, whether SARS-CoV-2 down-regulates ACE2 and the underlying mechanism and biological impact of this down-regulation have not been well defined. Here we show that the SARS-CoV-2 infection down-regulates ACE2 in vivo in an animal model, and in cultured cells in vitro, by inducing clathrin- and AP2-dependent endocytosis, leading to its degradation in the lysosome. SARS-CoV-2 S-treated cells and ACE2 knockdown cells exhibit similar alterations in downstream gene expression, with a pattern indicative of activated cytokine signaling that is associated with respiratory distress and inflammatory diseases often observed in COVID-19 patients. Finally, we have identified a soluble ACE2 fragment with a stronger binding to SARS-CoV-2 S that can efficiently block ACE2 down-regulation and viral infection. Thus, our study suggests that ACE2 down-regulation represents an important mechanism underlying SARS-CoV-2-associated pathology, and blocking this process could be a promising therapeutic strategy.

FIGURE 1:

SARS-CoV-2 Spike protein down-regulates ACE2 both in vitro and in vivo. (A, B) CoV-2 S protein down-regulated ACE2 in HEK-293A^ACE2 cells. HEK-293A^ACE2 cells were treated with 25 µg of purified CoV-2 S or VSV-G for the indicated times (A) or with the indicated amount of CoV-2 S or VSV-G for 6 h (B) and subjected to Western blotting with anti-Flag. α-Tubulin was used as a loading control. (C) CoV-2 S decreased ACE2 in H1299^ACE2 and A549^ACE2 cells. Cells were treated with 25 µg of purified CoV-2 S or VSV-G for 6 h. (D) CoV-2 S decreased ACE2 in Vero and Calu-3 cells. Cells were treated with 25 µg of purified CoV-2 S or VSV-G for 6 h and subjected to Western blotting with anti-ACE2. (E) CoV-2 S did not alter ACE2 mRNA level as examined by RT-qPCR analysis in HEK-293A^ACE2 cells treated with CoV-2 S or VSV-G. Data are mean ± SEM. Each dot represents the relative mRNA expression level of ACE2 in a single sample. P values were determined using unpaired two-tailed Student’s t tests. NS, no significance. n  = 3 independent experiments. (F, G) CoV-2 S promoted ACE2 degradation. HEK-293A^ACE2 cells were treated with the indicated amount of CoV-2 S or VSV-G and 100 µg/ml cycloheximide (CHX) for 6 h (F) or with 25 µg CoV-2 S or VSV-G and 100 µg/ml CHX for the indicated time (G). ACE2 levels were measured by Western blotting with anti-Flag (top panel) and quantified in the graph shown in the bottom panel. Data are mean ± SEM. n  = 3 independent experiments. (H) CoV-2 S RBD was required for ACE2 down-regulation. HEK-293A^ACE2 cells were treated with 25 µg of CoV-2 S or CoV-2 S-∆RBD for 6 h. (I, J) Pseudovirus bearing CoV-2 S down-regulated ACE2. HEK-293A^ACE2 (I) or Calu-3 (J) cells were infected with pseudoviruses bearing CoV-2 S or VSV-G (MOI = 12) for 6 h and then subjected to Western blotting with anti-Flag (I) or anti–ACE-2 (J). (K) SARS-CoV-2 down-regulated ACE2 in cells. HEK-293A^ACE2 cells were infected with SARS-CoV-2 or γ-irradiated SARS-CoV-2 (MOI = 12) for 6 h and then subjected to Western blotting with anti-Flag. (L) SARS-CoV-2 induced lung injury as indicated by the increased inflammatory infiltration. Representative H&E stains of lung tissues harvested from hamsters infected with SARS-CoV-2 or mock virus (TCID⁵⁰ = 4 × 10³) at day 5 postinfection. Red arrows indicate the infiltrated mononuclear cells. n = 3 hamsters per group. Scale bar, 50 μm. (M) SARS-CoV-2 down-regulated ACE2 in vivo in infected lung tissues. SARS-CoV-2 or mock virus–infected hamster lung tissues were subjected to Western blotting analysis with anti-ACE2 antibody. n = 3 individual samples.

FIGURE 2:

Down-regulation of ACE2 by the S protein of different SARS-CoV-2 strains. (A, B) CoV S down-regulated ACE2 more efficiently than CoV-2 S. HEK-293A^ACE2 cells were treated with different amounts of VSV-G, CoV S, or CoV-2 S protein for 6 h (A) or with 17.5 µg of VSV-G, CoV S, or CoV-2 S protein for the indicated time (B) and then subjected to Western blotting analysis. ACE2 levels were quantified, normalized to that of α-tubulin, and are shown in the graphs. Data are mean ± SEM. n  = 3 independent experiments. (C) Strep pull-down assay was performed by incubating purified Strep-tagged CoV S or CoV-2 S with lysates of HEK-293A^ACE2 cells, and the associated ACE2 proteins were detected by Western blotting with anti-Flag. Strep-tagged S proteins were assessed by Western blotting with anti-Strep. ACE2 proteins in the cell lysates were examined by Western blotting with anti-Flag. (D) Titers of SARS-CoV-2 variants in the lungs of infected hamsters were determined on days 2 and 5 postinfection. Each hamster was infected with 4 × 10³ TCID⁵⁰ of virus. Each dot, square, or triangle represents the viral titer in each lung tissue of infected hamsters. Data are mean ± SD. n = 3, 4, or 5 hamsters per group. (E) SARS-CoV-2 variants down-regulated ACE2 in vivo in lung tissues. Lung tissues were harvested from SARS-CoV-2, SARS-CoV-2 variants, or mock virus–infected hamsters and subjected to Western blotting with anti-ACE2. n = 3 individual samples. (F, G) The S protein of CoV-2 variants down-regulated ACE2. HEK-293A^ACE2 cells were treated with various amounts of S proteins from different variants for 6 h and then subjected to Western blotting with anti-Flag. ACE2 levels were quantified and normalized to that of α-tubulin and are shown in the graph. Data are mean ± SEM. n = 3 independent experiments.

FIGURE 3:

The CoV-2 S protein down-regulates ACE2 through the lysosome. (A, B) CoV-2 S down-regulated ACE2 through the lysosome. HEK-293A^ACE2 cells were pretreated with 10 µM proteasome inhibitor MG132 or 200 nM lysosome inhibitor bafilomycin A1 (Baf A1) for 2 h, followed by 25 µg of CoV-2 S treatment for 6 h in the absence (A) or presence (B) of 100 µg/ml CHX for the indicated time. Levels of ACE2 were examined by Western blotting. (C–E) ACE2 was internalized and colocalized with lysosome markers upon CoV-2 S treatment. HEK-293A^ACE2 cells were treated with CoV-2 S for 4 h and subjected to immunostaining with anti-ACE2 (green) and anti-LAMP2 (C), anti-RCAS1 (D), or anti-AIF (E) antibodies (red). DAPI is shown in blue. Scale bar, 10 µm. (F) MDM2 was not involved in CoV-2 S-mediated ACE2 down-regulation. HEK-293A^ACE2 cells were transfected with siMDM2 or control (siCtrl) for 48 h and then treated with 25 µg of CoV-2 S protein for the indicated time. (G) HEK-293A cells stably expressing ACE2 or ACE2-K788R were treated with 25 µg of CoV-2 S protein for the indicated time, and ACE2 levels were measured by Western blotting.

FIGURE 4:

CoV-2 S induces ACE2 endocytosis. (A) ACE2 was internalized upon CoV-2 S treatment. HEK-293A cells stably expressing ACE2-GFP were treated with CoV-2 S and subjected to live-cell imaging. The durations of CoV-2 S treatment are indicated at the top. Scale bar, 10 µm. (B, C) ACE2 colocalized with early endosome markers. HEK-293A cells stably expressing ACE2-GFP were treated with CoV-2 S for the indicated time and subjected to immunostaining with anti-EEA1 (red) (B) or anti-RAB5 (red) (C) antibodies. DAPI is shown in blue. Scale bar, 10 µm. (D) ACE2 colocalized with a late endosome marker. HEK-293A cells stably expressing ACE2-GFP and mCherry-RAB7 were treated with CoV-2 S for 3 h and subjected to confocal microscopy. DAPI is shown in blue. Scale bar, 10 µm. (E) HEK-293A cells stably expressing ACE2-GFP or ACE2-Flag were treated with CoV-2 S for 3 h and subjected to immunostaining with anti-RCAS1 (red), anti-AIF (red), anti-PDI (red), or anti-ACE2 (green) antibodies. DAPI is shown in blue. Scale bar, 10 µm. (F) ACE2 colocalized with CoV-2 S. HEK-293A^ACE2 cells were treated with CoV-2 S for 3 h and subjected to immunostaining with anti-ACE2 (green) and anti-Spike (red) antibodies. DAPI is shown in blue. Scale bar, 10 µm.

FIGURE 5:

Clathrin and AP2 are required for SARS-CoV-2 S-induced ACE2 endocytosis. (A, B) Clathrin was required for ACE2 down-regulation. HEK-293A^ACE2 cells were transfected with siCAV-1, siCHC, or siCtrl for 48 h and then treated with CoV-2 S protein for 6 h. Cell lysates were subjected to Western blotting with anti-Flag, anti-CAV-1, or anti-CHC antibodies. (C) Clathrin was required for ACE2 internalization. HEK-293A^ACE2 cells were transfected with siCHC or siCtrl for 48 h, treated with CoV-2 S protein for 4 h, and then subjected to immunostaining with anti-ACE2 (green) and anti-LAMP2 (red) antibodies. DAPI is in blue. Scale bar, 10 µm. (D) μ2 was required for ACE2 down-regulation. HEK-293A^ACE2 cells were transfected with siμ2 or siCtrl for 48 h and then treated with CoV-2 S protein for 6 h. Cell lysates were subjected to Western blotting analysis with anti-Flag and anti-μ2 antibodies. (E) μ2 was required for ACE2 internalization. HEK-293A^ACE2 cells were transfected with siμ2 or siCtrl, treated with CoV-2 S protein for 4 h, and then subjected to immunostaining with anti-ACE2 (green) and anti-LAMP2 (red). DAPIis in blue. Scale bar, 10 µm. (F) ACE2 colocalized with μ2. HEK-293A cells stably expressing ACE2-GFP and μ2-mCherry were treated with CoV-2 S for 1 h and then subjected to immunostaining with anti-Spike (magenta) antibody. DAPI is shown in blue. Scale bar, 10 µm. (G) Sequence alignment of the transmembrane domain and cytoplasmic tail of ACE2 from different species. The YxxΦ motif is indicated in blue. (H) ACE2 interacted with the AP2 complex through the YASI motif. Purified Strep-tagged AP2 complex was incubated with lysates of cells stably expressing ACE2 or ACE2-AASA mutant, and the ACE2 associated with AP2 was detected by Western blotting with anti-Flag. Strep-tagged AP2 complex was assessed by Western blotting with anti-Strep, anti-mCherry, anti-HA, and anti-μ2. ACE2 proteins in the cell lysates were examined by Western blotting with anti-Flag. (I) The ACE2-AASA mutant was not down-regulated by CoV-2 S. HEK-293A cells stably expressing ACE2 or ACE2-AASA were treated with CoV-2 S for the indicated time and then subjected to Western blotting with anti-Flag. (J) The ACE2-AASA mutant was not internalized by CoV-2 S. HEK-293A cells stably expressing ACE2 or ACE2-AASA were treated with CoV-2 S for 4 h and then subjected to immunostaining with anti-ACE2 (green) and anti-LAPM2 (red) antibodies. DAPI is shown in blue. Scale bar, 10 µm.

FIGURE 6:

Effect of ACE2 down-regulation by CoV-2 S on downstream transcription responses. (A) Volcano plot showing differentially expressed genes in cells treated with CoV-2 S by RNA-seq. Each dot represents the average value of a single gene in three replicate experiments. Dashed horizontal lines mark a P value of 0.05, and dashed vertical lines indicate a Log2(fold change) of 1 and –1. Red and blue indicate up-regulated and down-regulated genes, respectively, with an absolute Log2(fold change) > 1 and a P value < 0.05. (B) DisGeNET enrichment analysis of CoV-2 S-induced up-regulated genes. Bars show the –Log10(P value). Red points show the percentage of genes enriched. (C) KEGG pathway enrichment analysis of CoV-2–induced up-regulated genes. Bars show the –Log10(P value). Red points show the percentage of genes enriched. (D) Volcano plot showing differentially expressed genes in ACE2 knockdown cells using RNA-seq. (E) DisGeNET enrichment analysis of up-regulated genes in ACE2 knockdown cells. Bars show the –Log10(P value). Red points show the percentage of genes enriched. (F) KEGG pathway enrichment analysis of up-regulated genes in ACE2 knockdown cells. Bars show the –Log10(P value). Red points show the percentage of genes enriched. (G) Venn diagram of overlapping up- or down-regulated genes identified in differential expression analysis of CoV-2 S-treated or ACE2 knockdown cells. Numbers of up-regulated or down-regulated genes in each group are indicated. (H) Heat maps of overlapping up-regulated genes in both CoV-2 S and ACE2 knockdown cells. Columns represent samples, and rows represent genes. Gene expression levels in the heat maps are z score–normalized Log2(CPM)-transformed values. CPM, count per million reads. (I) DisGeNET enrichment analysis of overlapped up-regulated genes in both CoV-2 S and ACE2 knockdown cells. Bars show the –Log10(P value). Red points show the percentage of genes enriched. (J) KEGG pathway enrichment analysis of overlapped up-regulated genes in both CoV-2 S and ACE2 knockdown cells. Bars show the –Log10(P value). Red points show the percentage of genes enriched.

FIGURE 7:

Identification of a soluble ACE2 fragment with a stronger binding to CoV-2 S. (A) The D38E mutation of ACE2 enhanced its binding to CoV-2 S. Strep-tagged CoV-2 S (CoV-2 S-Strep) was cotransfected with C9-tagged wild type (WT) or D38E mutant of ACE2 into 293T cells. Coimmunoprecipitation (co-IP) was performed with anti-Strep beads followed by Western blotting with the indicated antibodies. α-Tubulin was used as a loading control. (B) The soluble fragment of ACE2-D38E (sACE2-D38E) competed with cell surface ACE2 for CoV-2 S binding in a dose-dependent manner. CoV-2 S-Strep was purified with and immobilized on Strep beads from transfected 293T cells and incubated with lysates from HEK-293A^ACE2 cells in the absence or presence of various amounts of purified sACE2-D38E-HA (30, 60 µg). Binding of ACE2 to CoV-2 S-Strep beads was examined by Western blotting using the indicated antibodies. (C) sACE2-D38E blocked ACE2 down-regulation by CoV-2 S. HEK-293A^ACE2 cells were treated with 30 µg of purified CoV-2 S-Strep in the absence or presence of 30 or 60 µg of sACE2-D38E-HA for 6 h. ACE2-Flag levels in the whole cell lysates were analyzed by Western blotting with anti-Flag. (D) sACE2-D38E binds equally well to the S protein from WT and Alpha strains. CoV-2 S-Strep of the WT or Alpha strain was purified with and immobilized on Strep beads from transfected 293T cells and incubated with purified sACE2-D38E-HA. Western blotting was carried out using the indicated antibodies. (E) sACE2-D38E prevented the entry of pseudovirus containing CoV-2 S into cells by neutralizing virus infection. HEK-293A^ACE2 cells were infected with 1 ml of concentrated pseudovirus expressing VSV-G (left), CoV-2 S (middle), or Alpha S (right) in either the absence or presence of 60 µg of sACE2-D38E-HA or BSA for 6 h. Viral infection and entry were measured by luciferase assays (top panels). ACE2 levels were detected by Western blotting (bottom panels). Data in the graphs in E were derived from at least three independent experiments and are presented as means ± SD by Student’s t test. *P < 0.05.