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

Abstract

Angiotensin-converting enzyme 2 (ACE2) is an entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and a regulator of several physiological processes. ACE2 has recently been proposed to be interferon (IFN) inducible, suggesting that SARS-CoV-2 may exploit this phenomenon to enhance viral spread and questioning the efficacy of IFN treatment in coronavirus disease 2019. Using a recent de novo transcript assembly that captured previously unannotated transcripts, we describe a new isoform of ACE2, generated by co-option of intronic retroelements as promoter and alternative exon. The new transcript, termed MIRb-ACE2, exhibits specific expression patterns across the aerodigestive and gastrointestinal tracts and is highly responsive to IFN stimulation. In contrast, canonical ACE2 expression is unresponsive to IFN stimulation. Moreover, the MIRb-ACE2 translation product is a truncated, unstable ACE2 form, lacking domains required for SARS-CoV-2 binding and is therefore unlikely to contribute to or enhance viral infection. NATuRE GENETiCS | www.nature.com/naturegenetics Articles NATURE GENETICS which indicated peaks in the LTR16A1 retroelement and the immediately upstream MIRb retroelement in the same intronic region (Extended Data Fig. 1). FANTOM5 CAGE peak distribution over the LTR16A1 and MIRb retroelements exhibited cell-type specificity to a certain degree, with peaks residing almost exclusively in MIRb in bronchial epithelial cells but extending to LTR16A1 in HEK293 cells (Extended Data Fig. 1). Both LTR16A1 and MIRb retroelements contained multiple transcription factor binding sites, with IRF-1 and IRF-2 binding sites and TATA-box residing in MIRb (Extended Data Fig. 2). To further define the transcription start site(s), we performed 5′ rapid amplification of cDNA ends (RACE) PCR, followed by deep sequencing of the PCR products, amplified from normal human bronchial epithelial (NHBE) cells or human squamous cell carcinoma (SCC) cell lines SCC-4 and SCC-25, treated with recombinant IFN-α (Extended Data Fig. 2). Consistent with FANTOM5 CAGE data, 5′ RACE analysis showed multiple peaks in both LTR16A1 and MIRb, again with evidence of celltype specificity in their relative utilization (Extended Data Fig. 2).

Upper airway gene expression reveals suppressed immune responses to SARS-CoV-2 compared with other respiratory viruses

“…2a, 3a). However, recent reports suggest this signal is driven by an ACE2 isoform that lacks the viral binding domains and is unlikely to encode a functional receptor 19,20 .…”

Activation of Interferon-Stimulated Transcriptomes and ACE2 Isoforms in Human Airway Epithelium Is Curbed by Janus Kinase Inhibitors

“…However, the degree of induction of either form was lower than that seen for bona de ISGs. Previous studies in normal human bronchial epithelium (NHBE) did not reveal an interferon response of the native ACE2 promoter 4,5 suggesting differences between cell types or growth conditions. The mouse Ace2 gene is also induced by cytokines through a STAT5-based enhancer in the second intron 8 and a DHS site is located in the equivalent location in the human ACE2 gene in SAEC and lung tissue.…”

“…Single cell RNA sequencing (scRNA-seq) studies have revealed that ACE2 expression in type II pneumocytes is induced by interferons 3 , suggesting that the presence of an autoregulatory loop could result in increased viral infection. Recent studies 4,5 have identi ed a novel short form of ACE2, called dACE2, that originates from an intronic promoter activated by interferons, calling into question that transcription of the full length native ACE2 is under interferon control. Onabajo et al 5 used ENCODE data for chromatin modi cation marks (H3K4me3, H3K4me1 and H3K27ac) as well as DNase I hypersensitive (DHS) sites in cell lines to label putative regulatory elements at the newly identi ed exon (ex1c) located within intron 9 of the ACE2 gene.…”

Genetic Dissection of the Regulatory Mechanisms of Ace2 in the Infected Mouse Lung

“…Since our lung transcriptome quantifies steady-state expression changes of all cell types, it is conceivable that we do not detect up-regulation in a smaller population of infected cells. Besides, two recent studies ( 54 , 55 ) show alternative splicing of ACE2 in humans, up-regulated by both viruses and IFNs. Therefore, it may be important to quantify different Ace2 isoforms’ expression in infected mouse lungs by immunohistochemistry, by single-cell methods, or by in situ hybridization to resolve specificity and kinetics of responses.…”