Angiotensin I and II Stimulate Cell Invasion of SARS-CoV-2: Potential Mechanism via Inhibition of ACE2 Arm of RAS

Abstract

Angiotensin-converting enzyme 2 (ACE2), one of the key enzymes of the renin-angiotensin system (RAS), plays an important role in SARS-CoV-2 infection by functioning as a virus receptor. Angiotensin peptides Ang I and Ang II, the substrates of ACE2, can modulate the binding of SARS-CoV-2 Spike protein to the ACE2 receptor. In the present work, we found that co incubation of HEK-ACE2 and Vero E6 cells with the SARS-CoV-2 Spike pseudovirus (PVP) resulted in stimulation of the virus entry at low and high micromolar concentrations of Ang I and Ang II, respectively. The potency of Ang I and Ang II stimulation of virus entry corresponds to their binding affinity to ACE2 catalytic pocket with 10 times higher efficiency of Ang II. The Ang II induced mild increase of PVP infectivity at 20 microM; while at 100 microM the increase (129.74+/-3.99 %) was highly significant (p<0.001). Since the angiotensin peptides act in HEK ACE2 cells without the involvement of angiotensin type I receptors, we hypothesize that there is a steric interaction between the catalytic pocket of the ACE2 enzyme and the SARS-CoV-2 S1 binding domain. Oversaturation of the ACE2 with their angiotensin substrate might result in increased binding and entry of the SARS-CoV-2. In addition, the analysis of angiotensin peptides metabolism showed decreased ACE2 and increased ACE activity upon SARS-CoV-2 action. These effects should be taken into consideration in COVID-19 patients suffering from comorbidities such as the over-activated renin-angiotensin system as a mechanism potentially influencing the SARS-CoV-2 invasion into recipient cells.

Fig. 1

Characterization and use of HEK-ACE2 stable cell line. (a, b) Cytochemical and biochemical features of the model. The fluorescent photomicrographs of cells demonstrate the expression of ACE2 receptor on the cell surface (green: anti-ACE2 antibody, blue: nuclei stained with DAPI, scale bar: 20 μm) and Western blot analysis confirming the expression of ACE2. (c) Aa relative luminescence (RLU) recorded after incubation of HEK-ACE2 cells with PVPs for 48 h (MyBioSource) in serial dilutions (n=6). (d) The infectivity of PVPs produced in newly established cell line clones S/3 and S/7 in the cell-entry assay (n=5).

Fig. 2

The effect of Ang I and Ang II on PVP cell entry. The PVP infectivity (red line) was assessed using two cell lines (HEK-ACE2: a, b and Vero E6: c, d) at the indicated concentrations of peptides. The cell viability (blue line) was determined at the same concentrations of peptides in parallel cultures. Ang I and Ang II peptides stimulated the PVP entrance in HEK-ACE2 cells (red line, panel a, and b) at the concentration of 100 μM or higher (not shown), while the cell viability was not significantly decreased at the indicated concentrations (blue line). The increased infectivity of PVPs after the pre-treatment with Ang I (c) and Ang II (d) peptides in Vero E6 cells was observed; however, these cells showed decreased viability at the corresponding concentrations (blue line) (n=6, *** p<0.001; ** p<0.01; * p<0.05).

Fig. 3

In vitro metabolism of Ang I and Ang II in HEK-ACE2 and Vero E6 cells in absence and presence of PVP. (a) The expected products after the Ang I and Ang II cleavage by RAS enzymes. (b–e) Quantification of angiotensin peptides in cell culture (n=3, ** p<0.01; * p<0.05) as determined by liquid chromatography and mass spectrometry (Details are in Material and Methods).