Interferons and viruses induce a novel truncated ACE2 isoform and not the full-length SARS-CoV-2 receptor

Abstract

ells expressing ACE2 are potential targets of SARS-CoV-2 infection 1,2. Studies based on single-cell RNA sequencing (scRNA-seq) of lung cells have identified type II pneumocytes, ciliated cells and transient secretory cells as the main types of ACE2-expressing cell 3,4. Furthermore, ACE2 was proposed to be an ISG, on the basis of its inducible expression in cells treated with interferons (IFNs) or infected by viruses that induce IFN responses, such as influenza 4,5. These findings implied that the induction of ACE2 expression in IFN-high conditions could result in an amplified risk of SARS-CoV-2 infection 4,5. Concerns could also be raised about possible ACE2-inducing side effects of IFN-based treatments proposed for COVID-19 (refs. 6-9). ACE2 plays multiple roles in normal physiological conditions and as part of the host tissue-protective machinery in damaging conditions, including viral infections. As a terminal carboxypeptidase, ACE2 cleaves a single carboxy-terminal residue from peptide hormones such as angiotensin II and des-Arg9-bradykinin. ACE and ACE2 belong to the renin-angiotensin-aldosterone system, which regulates blood pressure and fluid-electrolyte balance; dysfunction of this system contributes to comorbidities in COVID-19 (refs. 10,11). des-Arg9-bradykinin is generated from bradykinin and belongs to the kallikrein-kinin system, which is critical in regulating vascular leakage and pulmonary edema, early signs of severe COVID-19 (refs. 12,13). High plasma angiotensin II levels were found to be responsible for coronavirus-associated acute respiratory distress syndrome (ARDS), lung damage and high mortality in mouse models 14,15 and as a predictor of lethality in avian influenza in humans 16,17. In the same conditions, ACE2, which decreases the levels of angiotensin II, was identified as a protective factor. The hijacking of the normal host tissue-protective machinery guarded by ACE2 was suggested as a mechanism through which SARS-CoV-2 could infect more cells 4,5. Thus, it is critically important to identify factors affecting ACE2 expression in normal physiological processes and during viral infections and associated pathologies, such as in COVID-19. Herein, aiming to explore the IFN-inducible expression of ACE2 and its role in SARS-CoV-2 infection, we identified a novel, truncated isoform of ACE2, which we designate as dACE2. We then showed that dACE2, but not ACE2, is induced in various human cell types by IFNs and viruses; this information is important to consider for future therapeutic strategies and understanding COVID-19 susceptibility and outcomes. Results dACE2 is a novel inducible isoform of ACE2. To address the extent to which IFNs induce the expression of ACE2 in human cells, we used our existing RNA-seq dataset (NCBI Sequence Read Archive (SRA): PRJNA512015) of a breast cancer cell line T47D infected with Sendai virus (SeV), known to be a strong inducer of IFNs and ISGs 18-20. IFNs were not expressed in T47D cells at baseline, but SeV strongly induced expression of IFNB1, a type I IFN, an...

Tissue-specific and interferon-inducible expression of nonfunctional ACE2 through endogenous retroelement co-option

“…Instead, we describe a new RNA isoform, MIRb-ACE2, that is highly responsive to IFN stimulation, but encodes a truncated and unstable protein product. In support of these findings, the new isoform is independently described in two other recent preprint reports 24,25 and matches the sequence recently deposited under GenBank accession number MT505392. We find that the MIRb-ACE2 isoform exhibits distinct patterns of expression along the aerodigestive and gastrointestinal tracts and was likely responsible for the apparent IFN inducibility of ACE2 expression reported by analysis of scRNA-seq data 17 and other similar studies 20 .…”

“…The predicted MIRb-ACE2 protein product could be detected in vitro, albeit under high levels of MIRb-ACE2 RNA expression, and it remains possible that the MIRb-ACE2 protein, or fragments thereof, are produced under certain conditions in vivo. Indeed, despite its reduced stability when compared to full-length ACE2, evidence for production of the MIRb-ACE2 protein has also been independently reported 24,25 . Nevertheless, it is worth noting that the predicted MIRb-ACE2 protein does not contain the residues required for SARS-CoV-2 spike glycoprotein binding 15 , does not bind recombinant SARS-CoV-2 S1 experimentally and is thus unlikely to contribute to viral spread.…”

“…4e). In independently reported findings 24 , endogenously produced MIRb-ACE2 protein could not be detected by western blotting using the same polyclonal anti-ACE2 serum (ab15348). However, a Myc-DDK-tagged or green fluorescent protein (GFP)-tagged MIRb-ACE2 protein product was detected following overexpression in T24 cells in the same study 24 .…”

“…In independently reported findings 24 , endogenously produced MIRb-ACE2 protein could not be detected by western blotting using the same polyclonal anti-ACE2 serum (ab15348). However, a Myc-DDK-tagged or green fluorescent protein (GFP)-tagged MIRb-ACE2 protein product was detected following overexpression in T24 cells in the same study 24 . Moreover, a separate study 25 reported detection of the putative MIRb-ACE2 protein in primary nasal epithelial cells by western blotting using the same polyclonal anti-ACE2 serum (ab15348), raising the possibility that the protein can indeed be translated.…”

Single-cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut

“…The observed upregulation of ACE2 upon infection might be tissue-specific and time-dependent. However, recently it has been proposed that ACE2 does not behave as an ISG but instead a novel form of ACE2 (dACE2) is interferon-inducible [38]. dACE2 results from transcription initiation at an internal exon leading to the production of an alternative short version.…”