Angiotensin Type 1 Receptor-Dependent Internalization of SARS-CoV-2 by Angiotensin-Converting Enzyme 2

Abstract

The current coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus, which infects the cells by interaction of its envelope S1 spike protein (S1) with ACE2 (angiotensin-converting enzyme 2).1 ACE2 is a carboxypeptidase and a negative regulator of the renin-angiotensin system, reducing the levels of Ang II (angiotensin II) and its pathological actions in cardiovascular diseases. Early on, conflicting views were expressed regarding the use of AT1R (angiotensin type 1 receptor) blockers in patients with COVID19,2 and incertitude remains regarding the role of the renin-angiotensin system in SARS-CoV-2 infection. We previously reported that cellular ACE2 activity is strictly dependent on its plasma membrane localization. The enzyme is internalized by Ang II, and this effect depends on AT1R expression. 3 In HEK293T cells, which do not endogenously express AT1R, treatment with S1 (10–300 ng/mL) failed to induce ACE2 internalization (Figure [A], top) and consequently, we detected no decrease in enzymatic activity (Figure [B], left). Upon AT1R transfection ACE2 internalization was observed following Ang II and S1 exposure (Figure [A], bottom). The effects of Ang II and S1 were not additive, similar reduction in ACE2 activity being observed after simultaneous addition of both treatments (29.6±2.5%). Like for Ang II, S1-driven internalization depended on ACE2/AT1R ratio (Figure [B]). Pretreatment with the typical AT1R blocker losartan (1 μmol/L) or the β-arrestin biased AT1R ligand TRV0274 (1 μmol/L) fully prevented the effects of Ang II or S1. These drugs have no effect on ACE2 activity in the absence of AT1R (data not shown). As observed for Ang II, S1-induced ACE2 internalization resulted in enzyme degradation (Figure [D]), an effect also prevented by losartan. Accordingly, the lysosomal inhibitor leupeptin prevented the loss of ACE2 activity (Figure [E]) and its degradation (data not shown). Hydroxychloroquine (100 μmol/L), which inhibits lysosomal fusion to endosomes and the activity of lysosomal enzymes, reversed the effects of Ang II and S1 on ACE2 activity (Figure [E]). The use of sucrose as a hyperosmolar agent prevented the loss of ACE2 activity (Figure [E]), thus confirming the importance of clathrin-coated pits in ACE2 internalization. In summary, this is the first demonstration that S1 binding to ACE2 induces enzyme internalization through clathrin-coated pits, followed by lysosomal degradation, as previously reported by our group for Ang II.3 This process requires AT1R expression and higher AT1R levels potentiate the internalization of S1, component of SARSCoV-2 envelope. One can envision that S1 stimulates the formation of AT1R/ACE2 complexes which are required for enzyme internalization, but further experiments are required to validate this hypothesis. Blockade of lysosomal function by leupeptin or hydroxychloroquine fully prevented the effects of S1 on ACE2 internalization and degradation. Importantly from a clinical point of view, our cellular experiments strongly indicate that losartan, an extensively used antihypertensive drug, blocked ACE2 internalization induced by S1. Therefore, unlike initial concerns,2 the use of AT1R blockers in COVID-19 may produce unexpected benefits. However, clinical studies are necessary to confirm these in vitro results.