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Binding to ACE2 leads to SARS-CoV-2 cell entry
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|Binding of SARS-CoV-2 to ACE2 receptor leading to acute respiratory distress associated mortality||adjacent||High||High||Young Jun Kim (send email)||Open for comment. Do not cite||Under Development|
|SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)||adjacent||Sandra Coecke (send email)||Under development: Not open for comment. Do not cite||Under Development|
|SARS-CoV-2 infection leading to hyperinflammation||adjacent||Hasmik Yepiskoposyan (send email)||Under development: Not open for comment. Do not cite|
|Binding of SARS-CoV-2 to ACE2 in enterocytes leads to intestinal barrier disruption||adjacent||High||High||Laure-Alix Clerbaux (send email)||Under development: Not open for comment. Do not cite||Under Development|
|Binding of SARS-CoV-2 to ACE2 leads to viral infection proliferation||adjacent||High||Moderate||Sally Mayasich (send email)||Under development: Not open for comment. Do not cite||Under Development|
|Binding to ACE2 leading to thrombosis and disseminated intravascular coagulation||adjacent||High||Moderate||Shihori Tanabe (send email)||Under development: Not open for comment. Do not cite||Under Development|
|Binding of SARS-CoV-2 to ACE2 leads to hyperinflammation (via cell death)||adjacent||High||High||Laure-Alix Clerbaux (send email)||Under development: Not open for comment. Do not cite|
|Homo sapiens||Homo sapiens||High||NCBI|
Life Stage Applicability
|All life stages||High|
Key Event Relationship Description
This KER deals with the evidence supporting the individual weight that the surface protein of SARS-CoV-2 spike needs to bind:ACE2, and of being cleaved in two different sites, for viral entry to occur. Viral entry is essential for initiating a cascade of events leading to COVID19.
Evidence Collection Strategy
To develop this KER, the authors have gone through the literature that came out especially since SARS-CoV-2 was detected in humans up to July 2022 to find supporting evidence linking:
1) ACE2 essentiality for viral infection
2) Mechanisms that support viral entry
2.1) involvement of host proteases
2.2) viral proteins that interact with host components to promote viral entry
Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptor (Nature 579, 270–273, 2020 ; J. Virol. 94, e00127-20; Nature 588, 327–330). The SARS-CoV-2 surface spike (S) protein mediates the binding to the receptor and requires 2 cleavage steps for viral entry to occur, as follows. The spike protein contains 1273 aminoacids divided into two subunits, S1 and S2. The subunits are cleaved by furin-like enzymes, as spike of sars-cov-2 contains an insertion 680SPRRAR↓SV687 forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. In addition, there is a second cleavage site 808PSKPSKR|SFIEDL822 just before the fusion peptide that needs to occur for viral entry. The S1 subunit contains a receptor-binding domain (RBD) encompassing the receptor-binding motif (RBM) that binds ACE2. The S2 contains a fusion peptide (FP), that penetrates into cell membranes and mediates fusion between the viral and host membranes to release viral proteins and genome. When TMPRSS2 is not available, spike it is hypothethised that the virus may use alternative proteases to get in the cells either by fusion with the plasma membrane or entry via endosomes and fusion with endocytic membranes at low pH, when proteases for priming become active, but evidence is less robust.
3) The initial delivery of SARS-CoV2 proteins and genome to the cells.
Evidence Supporting this KER
Binding of SARS-CoV-2 S protein to ACE2 receptors present in the brain (endothelial, neuronal and glial cells) :
The highest ACE2 expression level in the brain was found in the pons and medulla oblongata in the human brainstem, containing the medullary respiratory centers, and this may in part explain the susceptibility of many COVID-19 patients to severe respiratory distress (Lukiw et al., 2020). High ACE2 receptor expression was also found in the amygdala, cerebral cortex and in the regions involved in cardiovascular function and central regulation of blood pressure including the sub-fornical organ, nucleus of the tractus solitarius, paraventricular nucleus, and rostral ventrolateral medulla (Gowrisankar and Clark 2016; Xia and Lazartigues 2010). The neurons and glial cells, like astrocytes and microglia also express ACE-2, thus highlighting the vulnerability of the nervous system to SARS-CoV-2 infection. Additionally, they also express transmembrane serine protease 2 (TMPRSS2) and furin, which facilitate virus entry into the host (Jakhmola et al. 2020).
Once inside the brain, the virus can infect the neural cells, astrocytes, and microglia. These cells express ACE-2, thus initiating the viral budding cycle followed by neuronal damage and inflammation (Jakhmola et al. 2020). Specifically in the brain, ACE2 is expressed in endothelium and vascular smooth muscle cells (Hamming et al., 2004), as well as in neurons and glia (Gallagher et al., 2006; Matsushita et al., 2010; Gowrisankar and Clark, 2016; Xu et al., 2017; de Morais et al., 2018) (from Murta et al., 2020).
Astrocytes are the main source of angiotensinogen and express ATR1 and MasR; neurons express ATR1, ACE2, and MasR, and microglia respond to ATR1 activation (Shi et al., 2014; de Morais et al., 2018).
Binding of S protein to ACE2 receptors present in the intestines
Uncertainties and Inconsistencies
Known modulating factors
|Modulating Factor (MF)||MF Specification||Effect(s) on the KER||Reference(s)|
PFAS (PFOS, PFOA, PFNA, PFHxS, and GenX)
Short-term (10 days), high dose (20 mg/kg/day) exposure to PFOA leads to about 1.6 fold upregulation of the pulmonary mRNA level of Ace2 and to about 1.5 upregulation of the pulmonary mRNA level of Tmprss2 in CD1 mice. 
Long-term (12 weeks) of an environmentally relevant PFAS mixture (PFOS, PFOA, PFNA, PFHxS, and GenX; each in 2 mg/l concentration) exposure leads to downregulation of pulmonary mRNA expression of Ace2 2.5-fold in C57BL/6 J male mice. A similar decreasing trend was observed in PFAS-exposed male mice for Tmprss2. 
1. doi: 10.1016/j.toxrep.2021.11.014
2. doi: 10.1016/j.taap.2022.116284
|female sex (XX chromosomes)||
ACE2 localizes to the X sex chromosome and displays a sex-dependent expression profile with higher expression in female than in male tissues [1,2]. Estradiol inhibits TMPRSS2, needed to facilitate SARS-CoV-2 entry into the cell . Estrogen therapy has been shown to mitigate endoplasmic reticulum stress induced by SARS-CoV-2 invasion through activation of cellular unfold protein response and regulation of inositol triphosphate (IP3) and phospholipase C .
Different studies have also illustrated that estradiol increases the expression of ADAM17, leading to high-circulating soluble ACE2 potentially neutralizing SARS-CoV-2 and preventing its binding to mACE2.  Thus, Estradiol might reduce SARS-CoV-2 infectivity through modulation of cellular ACE2/TMPRSS2/ADAM17 axis expression.
1. doi: 10.1177/1933719115597760
2. doi: 10.1016/j.mce.2015.11.004
3. doi: 10.1007/s11033-021-06390-1
5. doi: 10.2217/pgs-2020-0092
Male sex (XY chromosomes)
Androgen receptors (ARs) play a key role in increasing transcription of TMPRSS2. This may explain the predominance of males to COVID-19 fatality and severity. 
6. doi: 10.1073/pnas.2021450118
|Age||Young/old people||ACE2 protein expression is increased with aging in several tissues , including lungs and particularly in patients requiring mechanical ventilation . During aging, telomere dysfunction activates a DNA damage response leading to higher ACE2 expression. Thus, telomere shortening could contribute to make elderly more susceptible to SARS-CoV-2 infection .||
1. doi: 10.1016/j.exger.2021.111507
2. doi: 10.1371/journal.pone.0247060
3. doi: 10.15252/embr.202153658
Lipids, as important structural components of cellular and sub-cellular membranes, are crucial in the infection process . Changes in intracellular cholesterol alter cell membrane composition, impacting structures such as lipid rafts, which accommodate many cell-surface receptors , including ACE2 and TMPRSS2 [3, 4].
In COVID-19. In an in vitro study, the depletion of membrane-bound cholesterol in ACE2-expressing cells led to a reduced infectivity of SARS-CoV . In vitro, higher cellular cholesterol increased uptake of SARS-CoV-2 S protein; this effect was decreased with Methyl-beta-cyclodextrin, a compound which extracts cholesterol from cell membranes . HDL scavenger receptor B type 1 (SR-B1), a receptor found in pulmonary and many other cells, could facilitate ACE2-dependent entry of SARS-CoV-2 .
1. doi: 10.1001/jama.2020.12839
2. doi: 10.3389/fcell.2020.618296
3. doi: 10.1016/j.bbrc.2008.02.023
4. doi: 10.1096/fj.202000654R
5. doi: 10.1101/2020.05.09.086249
9. doi: 10.3390/ijms21103544
In COVID-19. ACE2 is highly expressed in adipose tissue, thus excess adiposity may drive more infection (8). Obese patients have more adipose tissue and therefore more ACE2-expressing cells . SARS-CoV-2 dysregulates lipid metabolism in the host and the effect of such dysregulated lipogenesis on the regulation of ACE2, specifically in obesity . Lung epithelial cells infected with SARS-CoV-2 showed upregulation of genes associated with lipid metabolism , including the SOC3 gene. A mouse model of diet-induced obesity showed higher Ace2 expression in the lungs, which negatively correlated with the expression sterol response element binding proteins 1 and 2 (SREBP) genes. Suppression of Srebp1 showed a significant increase in Ace2 expression in the lung. Lipids, including fatty acids, could interact directly with SARS-CoV-2 influencing spike configuration and modifying the affinity for ACE2 and thus its infectivity . The dysregulated lipogenesis and the subsequently high ACE2 expression in obese patients might be one mechanism underlying the increased risk for severe complications .
Vitamin D deficiency
Vitamin D administration enhanced mRNA expression of VDR and ACE2 in a rat model of acute lung injury . In particular, vitamin D upregulates the soluble ACE2 form . Thus, low vitamin D status may impair the trapping protective mechanism of soluble ACE2 . Furthermore, vitamin D deficiency has been shown to reduce the expression of antimicrobial peptides (-defensin, cathelicidin), which act against enveloped viruses [4,5].
In COVID-19. Decreased sACE2 and cellular viral defense might be some mechanisms explaining how low vitamin D modulate SARS-CoV-2 infectibility.
1. doi: 10.1016/j.injury.2016.09.025
2. doi: 10.1152/ajplung.00071.2009
3. doi: 10.3390/ijms22105251
4. doi: 10.1007/s11154-021-09679-5
5. doi: 10.1080/14787210.2021.1941871
Gut dysbiosis (alteration of gut microbiota)
Some evidence shows that gut microbiota influences Ace2 expression in the gut. Colonic Ace2 expression decreased significantly upon microbial colonization in mice and rats [1,2]. Coprobacillus enrichment was associated with severe COVID-19 in patients  and was shown to upregulate colonic ACE2 in mice . The abundance of Bacteroides species was associated with reduced ACE2 expression in the murine gut  and negatively correlated with fecal SARS-CoV-2 load [3,5]. Thus, gut dysbiosis might lead to higher levels of ACE2 in the gut, potentially increasing the ability of SARS-CoV-2 to enter enterocytes.
1. doi: 10.1080/19490976.2021.1984105
5. doi: 10.1016/j.tifs.2020.12.009
Polymorphisms inducing amino acid residue changes of ACE2 in the binding interface would influence affinity for the viral S protein. Evidence exists that K353 and K31 in hACE2, the main hotspots that form hydrogen bonds with the main chain of N501 and Q493 in receptor-binding motif respectively, play a role in tightly binding to the S protein of SARS-CoV-2 . Around the twenty natural ACE2 variants, three alleles of 17 variants were found to affect the attachment stability . Thus, the ACE2 variants modulating the interaction between the virus and the host have been reported to be rare, consistently with the overall low appearance of ACE2 polymorphisms. In this context, it is key to approach both the ACE2 genotypes and the clinical descriptions of the phenotypes in a population-wide manner, in order to better understand how ACE2 variations are relevant in the susceptibility for SARS-CoV-2 infection . In addition, since ACE2 is X-linked, the rare variants that enhance SARS-CoV-2 binding are expected to increase susceptibility to COVID-19 in males . On the other hand, the X-chromosome inactivation of the female causes a “mosaic pattern”, which might be an advantage for females in terms of reduced viral binding . TMPRSS2 single-nucleotide polymorphisms (SNPs) were associated with a frequent “European haplotype” , which not observed in Asians, is suggested to upregulate TMPRSS2 gene expression in an androgen-specific way. Thus, there is a need for in vitro validation studies to assess the involvements of population-specific SNPs of both ACE2 and TMPRSS2 in susceptibility toward SARS-CoV-2 infection. The occurrence of a pandemic is related to the genetics of the infecting agent. In the case of SARS-CoV-2, through genomic surveillance it is possible to track the spread of SARS-CoV-2 lineages and variants, and to monitor changes to its genetic code that can influence viral entry and production. Consequently, genomic surveillance is crucial to understand how mutations occurring on SARS-CoV-2 genome influence and drive the pandemic . For example, a recent study  highlights that through genomic surveillance it is possible to trace co-infections by distinct SARS-CoV-2 genotypes, which are expected to have a different impact on factors modulating COVID-19. Genomic surveillance of SARS-CoV-2 is able to reveal tremendous genomic diversity , and coupled with language models and machine learning approaches, contributes to predicting the impact of mutations (such as those occurring in the spike protein), and thus can better address challenging aspects, like an estimation of the efficacy of therapeutic treatments .
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 doi: 10.1038/s42003-021-02030-3
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 doi: 10.3390/ijms21103474
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 doi: 10.1038/s41588-022-01033-y
 doi: 10.1038/s41598-022-13113-4
 doi: 10.3389/fgene.2022.858252
|Therapeutic intervention against COVID-19||
Casirivimab, Imdevimaband Sotrivimab
Are monoclonal antibodies designed to recognize and attach to two
different sites of the Receptor-Binding Domain (RBD) of the S protein of SARS-CoV-2,blocking the virus to enter cells [1,2,3].
2) EMA Starts Rolling Review of REGN-COV2 Antibody Combination (Casirivimab / Imdevimab). EMA 2021. Available online:
(accessed on 12 May 2022)
3) EMA Starts Rolling Review of Sotrovimab (VIR-7831) for COVID-19. EMA 2021. Available online:https://www.ema.europa.eu/en/news/ema-starts-rolling-review-sotrovimab-vir-7831-covid-19 (accessed on 12 May 2022)
Interacts directly with viral
particles and has been shown to bind to the SARS-CoV-2 S1 Spike RBD, causing significantprotein architecture alteration, impacting infectivity [1,2].
Air pollution induces Increased expression of ACE2 which may result in increased viral entry and coronavirus production.
Increased ACE2 expression has been reported in the respiratory system in response to air pollution exposure (1-4). Increased expression may affect susceptibility to SARS-CoV-2 infection. Similarly, some constituents of air pollution (PM, ozone) have been reported to increase the expression of TMPRSS2 (3, 5-6).
|Pre-existing heart failure||
ACE2 mRNA and protein levels, as well as enzymatic activity, were shown to be upregulated in explanted hearts from patients with end-stage HF, as well as in the HF rat model [1-3].
Myocytes, fibroblasts, vascular smooth muscle cells, pericytes  and endothelial cells of the coronaries  express ACE2, while myocytes in patients suffering from heart disease exhibit higher ACE2 expression .
Pericytes - the mural cells lining microvasculature, interacting with endothelial cells notably to maintain microvascular stability - exhibited the strongest ACE2 expression in HF patients , rendering these cells involved in the coronary vasculature of the myocardium, more susceptible to infection.
Furthermore, SARS-CoV-2 infects and replicates in pericytes, and a decrease in their numbers follows .
Patients with pre-existing HF showed increased ACE2 levels in myocytes and pericytes, having thereby higher risk of heart injury [7, 9].
In addition, sACE2 levels are higher in HF patients [10, 11] and sACE2 activity is increased in HF .
In contrast to a protective role of sACE2, it has been proposed that viral binding to circulating sACE2 forms SARS-CoV-2/sACE2 complexes, which might mediate infection of cells in distal tissues ; hence, pre-existing HF might disseminate SARS-CoV-2 infection.
Interestingly, the increase in sACE2 activity is associated with HF with reduced ejection fraction (HFrEF) but not with HF with preserved ejection fraction (HFpEF), suggesting (i) a rather complex role of HF in regulating ACE2-mediated infection by SARS-CoV-2  and (ii) the potential of sACE2 activity to be used as a biomarker to distinguish between the two HF types.
Lastly, it is noteworthy that Khoury et al. provided evidence in a different direction, by showing that ADAM17 and TMPRSS2  expression levels are downregulated in a HF rat model, thus potentially conferring a protective role against infection by SARS-CoV-2 in HF .
|Diet||Chemicals in foods affect ACE3 expression||
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
COVID19 References related to CNS:
de Morais SDB, et al. Integrative Physiological Aspects of Brain RAS in Hypertension. Curr Hypertens Rep. 2018 Feb 26; 20(2):10.
Gallagher PE, et al. Distinct roles for ANG II and ANG-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes. Am J Physiol Cell Physiol. 2006 Feb; 290(2):C420-6.
Gowrisankar YV, Clark MA. Angiotensin II regulation of angiotensin-converting enzymes in spontaneously hypertensive rat primary astrocyte cultures. J Neurochem. 2016 Jul; 138(1):74-85.
Hamming I et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol. 2004 Jun;203(2):631-7.
Jakhmola S, et al. SARS-CoV-2, an Underestimated Pathogen of the Nervous System. SN Compr Clin Med. 2020.
Lukiw WJ et al. SARS-CoV-2 Infectivity and Neurological Targets in the Brain. Cell Mol Neurobiol. 2020 Aug 25;1-8.
Matsushita T, et al. CSF angiotensin II and angiotensin-converting enzyme levels in anti-aquaporin-4 autoimmunity. J Neurol Sci. 2010 Aug 15; 295(1-2):41-5.
Murta et al. Severe Acute Respiratory Syndrome Coronavirus 2 Impact on the Central Nervous System: Are Astrocytes and Microglia Main Players or Merely Bystanders? ASN Neuro. 2020. PMID: 32878468
Shi A, et al. Isolation, purification and molecular mechanism of a peanut protein-derived ACE-inhibitory peptide. PLoS One. 2014; 9(10):e111188.
Xia, H. and Lazartigues, E. Angiotensin-Converting Enzyme 2: Central Regulator for Cardiovascular Function. Curr. Hypertens. 2010 Rep. 12 (3), 170– 175