The Renin-Angiotensin System Modulates SARS-CoV-2 Entry via ACE2 Receptor

Abstract

The renin-angiotensin system (RAS) plays a central role in cardiovascular regulation and has gained prominence in the pathogenesis of Coronavirus Disease 2019 (COVID-19) due to the critical function of angiotensin-converting enzyme 2 (ACE2) as the entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Angiotensin IV, but not angiotensin II, has recently been reported to enhance the binding between the viral spike protein and ACE2. To investigate the virological significance of this effect, we developed a single-round infection assay using SARS-CoV-2 viral-like particles expressing the spike protein. Our results demonstrate that while angiotensin II does not affect viral infectivity across concentrations ranging from 40 nM to 400 nM, angiotensin IV enhances viral entry at a low concentration but exhibits dose-dependent inhibition at higher concentrations. These findings highlight the unique dual role of angiotensin IV in modulating SARS-CoV-2 entry. In silico molecular docking simulations indicate that angiotensin IV was predicted to associate with the S1 domain near the receptor-binding domain in the open spike conformation. Given that reported plasma concentrations of angiotensin IV range widely from 17 pM to 81 nM, these levels may be sufficient to promote, rather than inhibit, SARS-CoV-2 infection. This study identifies a novel link between RAS-derived peptides and SARS-CoV-2 infectivity, offering new insights into COVID-19 pathophysiology and informing potential therapeutic strategies.

Keywords: angiotensin; angiotensin-converting enzyme 2; renin–angiotensin system; severe acute respiratory syndrome coronavirus 2; spike protein; viral-like particle.

Figure 1

Schematic representation of amino acid sequences and metabolic pathways of angiotensin peptides. Angiotensin I (Ang I) is converted to angiotensin II (Ang II) by angiotensin-converting enzyme (ACE). Ang II can be further metabolized by ACE2 to produce angiotensin 1–7 (Ang 1–7), or by aminopeptidase A (APA) to generate angiotensin III (Ang III). Ang III is subsequently cleaved by aminopeptidase N (APN) to produce angiotensin IV (Ang IV).

Figure 2

In silico docking models of angiotensin peptides with the SARS-CoV-2 spike protein and ACE2. (A) Predicted binding of (I) angiotensin II (green) and (II) angiotensin IV (cyan) to the SARS-CoV-2 spike receptor-binding domain (RBD, blue) in complex with ACE2 (red) based on the crystal structure (PDB: 6M0J), generated using DynamicBind. Both peptides were predicted to interact with the enzymatic groove of ACE2. Binding affinities are summarized in Table 1. (B) Simulated binding of angiotensin IV (red) to the SARS-CoV-2 spike trimer in its (I) closed (PDB: 6VXX) or (II) open (PDB: 6VYB) conformations. In these models, the RBD is shown in blue, the S1 domain in cyan, and the S2 domain in green.

Figure 3

Single-round infection assay with SARS-CoV-2 VLP in 293T-ACE2 cells. (A) Flow cytometry analysis confirming ACE2 receptor expression in HEK293T-ACE2 cells used as target cells in the infection assay. HEK293T-ACE2 and parental HEK293T cells were stained with an Alexa Fluor 647-conjugated anti-ACE2 antibody. Parental HEK293T cells showed no detectable ACE2 expression, whereas HEK293T-ACE2 cells exhibited robust surface ACE2 expression. (B) Viral infectivity was assessed in HEK293T-ACE2 cells and parental HEK293T cells using a luciferase reporter assay to confirm that SARS-CoV-2 VLP infection is ACE2-dependent. Error bars indicate standard error from six samples and statistical significance was calculated by the Wilcoxon matched-pairs signed-rank test. Each circle represents an individual trial, and the label “ns” indicates that the results of that trial was not statistically significant.

Figure 4

Effects of angiotensin II and IV on the viral entry step of SARS-CoV-2 infection. SARS2-VLPs were pre-treated with angiotensin peptides at concentrations of 40 nM, 80 nM, and 400 nM prior to infection of HEK293T-ACE2 cells. (A) Angiotensin II treatment and (B) angiotensin IV treatment were assessed for their effects on viral entry. (C) To examine the post-entry effects, HEK293T-ACE2 cells were first infected with SARS2-VLPs and incubated for 24 h. After removing residual virus by washing, cells were treated with 40 nM angiotensin IV for an additional 24 h before measuring luciferase activity. Viral entry efficiency was quantified 48 h post-infection using a luciferase reporter assay. (A) For angiotensin II, 9 independent experiments were conducted at each concentration. (B) The 40 nM angiotensin IV group included 15 independent experiments, while the 80 nM and 400 nM groups included 6 independent experiments each. (C) A total of 8 independent experiments were performed for the post-entry 40 nM angiotensin IV treatment. Statistical significance was evaluated using the Wilcoxon matched-pairs signed-rank test: 9 matched pairs for angiotensin II compared to untreated samples and 15 matched pairs for the 40 nM angiotensin IV pre-treatment group, 6 matched pairs for the 80 nM and 400 nM groups or 8 matched pairs for angiotensin IV post-treatment groups, respectively, compared to untreated samples. Error bars represent the standard error of the mean. Each circle represents an individual trial, and the label “ns” indicates that the results of that trial was not statistically significant.