key: cord-0899296-uftjm2pn
authors: Ng, Kevin; Attig, Jan; Bolland, William; Young, George R.; Major, Jack; Wack, Andreas; Kassiotis, George
title: Tissue-specific and interferon-inducible expression of non-functional ACE2 through endogenous retrovirus co-option
date: 2020-07-24
journal: bioRxiv
DOI: 10.1101/2020.07.24.219139
sha: 86639a1e7de7297faa0cb3629247b65a36998350
doc_id: 899296
cord_uid: uftjm2pn

Angiotensin-converting enzyme 2 (ACE2) is an entry receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), as well as a regulator of several physiological processes. ACE2 has recently been proposed to be interferon-inducible, suggesting that SARS-CoV-2 may exploit this phenomenon to enhance viral spread and questioning the efficacy of interferon treatment in Coronavirus disease 2019 (COVID-19). Using a recent de novo transcript assembly that captured previously unannotated transcripts, we describe a novel isoform of ACE2, generated by co-option of an intronic long terminal repeat (LTR) retroelement promoter. The novel transcript, termed LTR16A1-ACE2, exhibits specific expression patterns across the aerodigestive and gastrointestinal tracts and, importantly, is highly responsive to interferon stimulation. In stark contrast, expression of canonical ACE2 is completely unresponsive to interferon stimulation. Moreover, the LTR16A1-ACE2 translation product is a truncated, unstable ACE2 form, lacking domains required for SARS-CoV-2 binding and therefore unlikely to contribute to or enhance viral infection.

Interferons represent the first line of defence against viruses in humans and other jawed vertebrates (Sadler and Williams, 2008) . Recognition of viral products in an infected cell results in autocrine and paracrine signalling to induce an antiviral state characterized by expression of a module of interferon-stimulated genes (ISGs) that restrict viral replication and spread (Sadler and Williams, 2008; Stetson and Medzhitov, 2006) . Indeed, recombinant interferon is often given as first-line therapy in viral infection (Gibbert et al., 2013) and preliminary results suggest that interferon treatment may be effective against Coronavirus disease 2019 (COVID-19) (Hung et al., 2020; Wang et al., 2020) .

Interferon signalling results in rapid upregulation of several hundred ISGs, including genes that inhibit various stages of viral entry and replication as well as transcription factors that further potentiate the interferon response (Sadler and Williams, 2008; Stetson and Medzhitov, 2006) . Given that unchecked interferon signalling and inflammation can result in immunopathology, ISGs are subject to complex regulatory mechanisms (Ivashkiv and Donlin, 2014) .

At the transcriptional level, long terminal repeats (LTRs) derived from endogenous retroviruses and other LTR-retroelements serve as cis-regulatory enhancers for a number of ISGs and are required for their induction (Chuong et al., 2016) . Adding to this regulatory complexity, many LTRs are themselves promoters responsive to interferon and are upregulated following viral infection or in interferon-driven autoimmunity (Attig et al., 2017; Tokuyama et al., 2018; Young et al., 2012; Young et al., 2014) .

The co-evolution of viruses and hosts has resulted in a number of strategies by which viruses evade or subvert interferon responses (García-Sastre, 2017) . Compared with other respiratory viruses, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) elicits a weak interferon response despite strong induction of other chemokines (Blanco-Melo et al., 2020) .

Though the mechanism by which SARS-CoV-2 dampens interferon responses remains unclear, the ORF3b, ORF6, and nucleoprotein of the closely-related SARS-CoV function as interferon antagonists (Kopecky-Bromberg et al., 2007) . SARS-CoV-2 uses angiotensinconverting enzyme 2 (ACE2) as its primary receptor (Hoffmann et al., 2020; Shang et al., 2020) and recent work suggested that SARS-CoV-2 may hijack the interferon response by inducing ACE2 expression (Ziegler et al., 2020) . By integrating multiple human, macaque, and mouse single-cell RNA-sequencing (RNA-seq) datasets, Ziegler et al. identified ACE2 as a primate-specific ISG upregulated following viral infection or interferon treatment (Ziegler et al., 2020) . Use of an ISG as a viral receptor would result in a self-amplifying loop to increase local viral spread, and calls into question the efficacy and safety of recombinant interferon treatment in COVID-19 patients.

Using our recent de novo transcriptome assembly (Attig et al., 2019) , we identify a novel, truncated ACE2 transcript, termed LTR16A1-ACE2, initiated at an intronic LTR16A1 retroelement that serves as a cryptic promoter. Strikingly, we find that the truncated LTR16A1-ACE2 and not full-length ACE2 is the interferon-inducible isoform, and is strongly upregulated in viral infection and following interferon treatment. Importantly, the protein product of the LTR16A1-ACE2 transcript does not contain the amino acid residues required for SARS-CoV-2 attachment and entry and is additionally post-translationally unstable. These findings have important implications for the understanding of ACE2 expression and regulation, and thus for SARS-CoV-2 tropism and treatment.

Our recent de novo cancer transcriptome assembly (Attig et al., 2019) identified a chimeric transcript between annotated exons of ACE2 and an LTR16A1 retroelement, integrated in intron 9 of the ACE2 locus. This transcript, which we refer to here as LTR16A1-ACE2, is initiated at the LTR16A1 retroelement, which functions as a cryptic promoter and includes exons 10-19 of ACE2 ( Figure 1A ). Promoter activity of the LTR16A1 retroelement was highly supported by splice junction analysis of RNA-seq data from TCGA lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) ( Figure 1A ) and by promoter-based expression analyses of the FANTOM5 data set ( Figure S1 ).

Phylogenetic analysis of the respective LTR16A1 elements in the ACE2 loci of representative mammalian species indicated that the ancestral LTR16A1 integration predated estimated dates of mammalian radial divergence ( Figure 1B) . Indeed, comparative genomic analysis produced good alignment of the LTR16A1 integration across a variety of species, with humans, dogs, and dolphins showing above 50% sequence identity to the mammalian consensus sequence of LTR16A1 ( Figure 1B , C). Of note, the LTR16A1 integration was also present, but truncated in the mouse genome ACE2 locus ( Figure 1B , C).

The LTR16A1-ACE2 isoform is predicted to encode a truncated ACE2 product, retaining the last 449 amino acids of the canonical ACE2 protein and exonisation of the LTR16A1 element creates a novel 10 amino acid sequence (MREAGWDKGG) in the putative translation product ( Figure S2 ). Importantly, this predicted protein lacks the first 356 amino acids, including the signal peptide, substrate-binding site and domains that interact with SARS-CoV and SARS-CoV-2 spike glycoproteins ( Figure S2 ).

To assess the relative expression of ACE2 and LTR16A1-ACE2 isoforms, we quantified expression of both transcripts across tissue types in the TCGA and GTEx cohorts. Consistent with recent reports (Singh et al., 2020; Ziegler et al., 2020) , the full-length ACE2 was expressed predominantly in the healthy intestine and kidney and tumours of the same histotypes ( Figure S3 ). Expression of LTR16A1-ACE2 followed a similar overall pattern, but with notable expression also in healthy testis, likely owing to LTR element activation as part of epigenetic reprogramming during spermatogenesis.

However, despite similar histotype distribution of ACE2 and LTR16A1-ACE2 expression, the ratio of the two isoforms was characteristically different between distinct histotypes and tumour types. For example, in larger TCGA patient cohorts, LUAD samples expressed higher levels of ACE2 than of LTR16A1-ACE2 (mean ACE2/LTR16A1-ACE2 ratio = 5.63), whereas LUSC samples showed the opposite phenotype with higher expression of LTR16A1-ACE2 (mean ACE2/LTR16A1-ACE2 ratio = 0.87) (Figure 2A , B). ACE2 and LTR16A1-ACE2 expression and their ratios were not affected by patient gender, arguing against a strong effect of the X chromosomal location of ACE2 on either isoform expression (Figure 2A , B). ACE2 and LTR16A1-ACE2 exhibited characteristic expression also within tumour types with only weak correlation between the two in the same tumour type (R 2 =0.252 for LUAD; R 2 =0.337 for LUSC), suggesting partly independent regulation.

In healthy lung, expression of ACE2 and LTR16A1-ACE2 was similar to that in LUAD, with the balance slightly in favour of the full-length form (mean ACE2/LTR16A1-ACE2 ratio = 2.73) ( Figure 2C ). By contrast, healthy colon expressed considerably higher levels specifically of the full-length isoform (mean ACE2/LTR16A1-ACE2 ratio = 26.37) ( Figure 2D ). These differences in ACE2 and LTR16A1-ACE2 expression between healthy lung and colon were again independent of gender ( Figure 2C , D).

Tissue-specific patterns of ACE2 and LTR16A1-ACE2 expression suggested dependency on cell lineage or identity. Alternatively, they could reflect transient adaptations to the local microenvironment, such as oxygen or microbiota composition differences between lung and intestine, or even differences in cellular composition between the different compartments. To Together, these results uncover the transcription of a novel ACE2 isoform, initiated at an intronic LTR16A1 retroelement, in a characteristic pattern of expression, forming a gradient from the upper aerodigestive tract (highest LTR16A1-ACE2 expression) to the large intestine (highest ACE2 expression).

ACE2 has recently been described as a human interferon-stimulated gene (ISG), upregulated at the mRNA level following viral infection or interferon treatment (Ziegler et al., 2020) .

However, this conclusion was based mostly on analysis of single-cell RNA-seq data that might not have sufficient resolution to distinguish the two isoforms. Indeed, inspection of public single-cell RNA-seq data (GSE134355) (Han et al., 2020) , demonstrated the limitation of such technologies, with RNA-seq reads mapping exclusively to the shared 3' terminal exon of the ACE2 transcripts, and therefore unable to discriminate between the isoforms ( Figure S4 ).

To investigate the inducibility of the two isoforms by IFN or viral infection, we re-analysed public RNA-seq data (GSE147507) from normal human bronchial epithelial (NHBE) cells, treated with recombinant IFNβ or infected with SARS-CoV-2, Influenza A virus (IAV) or IAV lacking the viral NS1 protein (IAVΔNS1) (Blanco-Melo et al., 2020) . None of the treatments increased expression of full-length ACE2 ( Figure 4A ). In stark contrast, LTR16A1-ACE2 expression was strongly elevated by both IAVΔNS1 infection and recombinant IFNβ treatment, compared with mock treatment (p=0.0005 and p=0.0054, respectively, Student's t-test).

Similar results were also obtained with analysis of lung cancer Calu-3 cells. In the absence of stimulation, Calu-3 cells express exclusively the full-length ACE2 isoform ( Figure 4B ). SARS-CoV-2 infection did not affect levels of ACE2 expression, but noticeably induced LTR16A1-ACE2 expression ( Figure 4B ). Lastly, analysis of RNA-seq data from explanted lung tissue from a single COVID-19 patient demonstrated elevated expression of LTR16A1-ACE2, but not of ACE2, compared with healthy lung tissue ( Figure 4C ), albeit statistical comparisons were not possible in this case.

To further confirm the IFN-responsiveness exclusively of LTR16A1-ACE2 expression, we used the squamous cell carcinoma cell lines SCC-4 and SCC-25, which express both isoforms. Compared with mock treatment, addition of recombinant IFNα or IFNγ had a minimal effect on ACE2 expression in SCC-4 cells and no effect in SCC-25 cells ( Figure 4D ). This contrasted with very strong induction (~15-fold) of LTR16A1-ACE2 expression by either type of IFN in both cell lines ( Figure 4D ). Lack of ACE responsiveness to IFN stimulation was additionally confirmed at the protein level, where neither IFNα nor IFNγ affected levels of fulllength ACE2, detected by Western blotting in SCC-4 and SCC-25 cells or in A549 cells, which express neither isoform and were used as a negative control ( Figure 4E ). Of note, despite strong upregulation at the mRNA level and despite using an antibody targeting the C-terminus of ACE2 present in both protein products, we were unable to detect a truncated form that would correspond to the LTR16A1-ACE2 translation product ( Figure 4E ). To confirm the differential IFN inducibility of ACE2 and LTR16A1-ACE2 expression, we stimulated NHBE cells with IFNα, IFNβ or IFNλ, as previously described (Major et al., 2020) .

Again, treatment with none of the IFNs had any measurable effect on ACE2 expression in these primary cells ( Figure 4F ). This contrasted with robust induction of LTR16A1-ACE2 expression, particularly by IFNα ( Figure 4F ).

Collectively, these data demonstrate that type I, II and III IFNs stimulate transcription of the ACE2 isoform driven by the alternative LTR16A1, but not the canonical ACE2 promoter.

Splicing from the LTR16A1 promoter to exon 10 of ACE2 is in-frame and therefore the last 449 amino acids are shared between ACE2 protein and the putative LTR16A1-ACE2 protein.

Nevertheless, we have been unable to detect the latter in cells naturally expressing the LTR16A1-ACE2 transcript ( Figure 4E ). To explore the protein-coding potential of the LTR16A1-ACE2 transcript, we cloned the coding sequences of both isoforms into the pcDNA3.1 mammalian expression vector and transfected HEK293T cells. While ACE2transfected HEK293T cells produced readily detectable ACE2 protein, no protein of the predicted size was detectable in LTR16A1-ACE2-transfected cells ( Figure 5A ). We quantified levels of enzymatically active ACE2 and found, as expected, strong enzymatic activity in lysates from ACE2-transfected, but not LTR16A1-ACE2-transfected cells ( Figure 5B ).

To confirm the lack of protein production from LTR16A1-ACE2 transcript, we cloned the coding sequences of both isoforms into the pcDNA3.1-DYK-P2A-GFP expression vector, which adds both a FLAG tag and GFP as part of the protein product. Expression of GFP was comparable in ACE2-transfected and LTR16A1-ACE2-transfected cells, suggesting that the single RNA molecule that encodes for both the LTR16A1-ACE2 product and GFP is stable and translated ( Figure 5C ). Despite that, while full-length ACE2 was detectable across a range of plasmid concentrations, we could not detect the predicted LTR16A1-ACE2 protein product with antibodies to the FLAG tag ( Figure 5D ).

Lysine residues 625 and 702 in the full-length ACE2 protein have been described to be ubiquitinated and may contribute to its proteosomal degradation (Stukalov et al., 2020) . We generated a K625R K702R (K2R) mutant of full-length ACE2, which increased protein levels, compared to the wild-type ACE2 ( Figure 5E ). We have introduced the same mutations in the corresponding residues of the predicted LTR16A1-ACE2 protein product, K279R K356R, which were similarly accessible for ubiquitination ( Figure S5 ). However, we were unable to detect stable protein following transfection with the LTR16A1-ACE2 K2R mutant ( Figure 5E ).

Consistent with this, the addition of the proteasome inhibitor MG-132 was sufficient to increase protein levels of ACE2, but did not rescue the LTR16A1-ACE2 protein product ( Figure 5E ).

These data suggest that the latter protein is subject to post-translational degradation through a proteasome-independent mechanism and therefore unlikely to exert significant biological activity.

Regulation of ACE2 expression and function is critical both in physiology and pathology (Hamming et al., 2007) . The use of ACE2 as a primary receptor for entry by pandemic coronaviruses, SARS-CoV and SARS-CoV-2 highlighted the potential effect of changes in ACE2 expression, particularly in response to IFN, on the course or severity of COVID-19 (Ziegler et al., 2020) . Here, we showed that ACE2 transcription and protein production is not responsive to IFN. Instead, we describe a novel RNA isoform, LTR16A1-ACE2, that is highly responsive to IFN stimulation, but encodes a truncated and unstable protein product. This isoform showed 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 single-cell RNA-seq data (Ziegler et al., 2020) . We further show that transcription of this novel isoform is initiated by an intronic LTR retroelement, which functions as a cryptic, IFN-responsive promoter, adding further evidence for the widespread involvement of such retroelements in gene regulatory networks.

Indeed, endogenous retroelements comprise nearly half the human genome and can affect many host processes (Burns and Boeke, 2012; Feschotte and Gilbert, 2012; Kassiotis and Stoye, 2016) . LTR retroelements represent an abundant source of promoters, enhancers, and polyadenylation sequences that can modulate the expression and structure of neighbouring genes (Thompson et al., 2016) , as with ACE2. For instance, retroelements serve as promoters or enhancers for a number of ISGs, conferring IFN inducibility, exemplified in the case of AIM2 (Chuong et al., 2016) . Retroelements may further modify the function of ISGs and we have recently described a novel isoform of the ISG CD274 (encoding PD-L1) that produces a truncated form through retroelement exonisation (Ng et al., 2019) .

The use of the intronic LTR16A1 element as the promoter for the LTR16A1-ACE2 isoform explains its independent regulation from that of the full-length ACE2 isoform. In addition to IFN inducibility, the cryptic LTR16A1 promoter also confers tissue-specific expression, with the highest levels seen in the upper aerodigestive tract, where it can be the predominant isoform.

In contrast, the canonical ACE2 isoform far exceeds expression of the LTR16A1-ACE2 isoform in the lower gastrointestinal tract. It is theoretically possible that the balance of LTR16A1-ACE2 and full-length ACE2 isoforms plays a role in the spread of SARS-CoV-2, particularly in the upper aerodigestive tract, or that RNA or protein products of LTR16A1-ACE2 are involved in other pathological or physiological processes. However, the lack of a stable protein corresponding to the LTR16A1-ACE2 coding sequence argues that this is unlikely.

Independently of any functional significance, expression of the LTR16A1-ACE2 isoform needs to be carefully considered in studies examining ACE2 regulation at the transcriptional level (Singh et al., 2020; Ziegler et al., 2020) . The description of this novel isoform highlights the need to validate single-cell RNA-seq data with orthogonal approaches. While single-cell RNAseq initiatives are an invaluable resource and allow for rapid identification of cell types that express a gene of interest, coverage and read depth are largely insufficient to distinguish between isoforms. Technological advances to improve sequencing depth and bioinformatic tools to impute missing values are rapidly progressing; in the meantime, long-read sequencing techniques to quantify transcript isoforms and confirmation of protein expression levels can be incorporated into existing workflows.

This work established LTR16A1-ACE2 as the predominant form of ACE2 following viral infection or recombinant interferon treatment, including in the SARS-CoV-2-infected lung. The suggestion that ACE2 is an ISG raised fears that therapeutic interferon could be detrimental (Ziegler et al., 2020) ; however, we find that full-length ACE2 is not increased at the mRNA or protein level. Under none of the conditions tested, did LTR16A1-ACE2 produce any detectable protein. However, we cannot rule out the possibility that it may produce a protein product or fragments thereof under certain conditions in vivo. Nevertheless, it is worth noting that the predicted LTR16A1-ACE2 protein product does not contain the residues required for SARS-CoV-2 spike glycoprotein binding (Shang et al., 2020) and is thus unlikely to contribute to viral spread. These results reconcile the apparent discrepancy between the interferon inducibility of ACE2 with promising data showing improved outcomes in COVID-19 following interferon treatment (Hung et al., 2020; Wang et al., 2020) . 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, George Kassiotis (george.kassiotis@crick.ac.uk)

DNA constructs and other research reagents generated by the authors will be distributed upon request to other research investigators.

The published article includes all data generated or analysed during this study, and are summarized in the accompanying figures and Supplemental materials. supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), L-glutamine (2 mmol/L, Thermo Fisher Scientific), penicillin (100 U/mL, Thermo Fisher Scientific), and streptomycin (0.1 mg/mL, Thermo Fisher Scientific). NHBE cells were cultured as previously described (Major et al., 2020) 

Transcripts were previously assembled on a subset of the RNAseq data from The Cancer

Genome Atlas (TCGA) (Attig et al., 2019) . The alternative promoter within ACE2 was more highly expressed in lung squamous carcinomas than the canonical isoform, prompting us to investigate its biology. RNAseq data from TCGA, GTEx, CCLE, and other studies were aligned to the cancer-tissue transcriptome assembly using GNU parallel (Tange, 2011) and Salmon v0.12.0 (Patro et al., 2017) . Splice junctions were visualised using the Integrative Genome Viewer v2.4.19 (Thorvaldsdóttir et al., 2013) .

To identify the integration time of LTR16A1 into the ACE2 locus, we first compared the Homo sapiens LTR16A1 to the LTR16A1 consensus sequence in repBase. Based on 285 identical bases across its 396 bp length, the insertion is expected to be ~127 million years old based on the human neutral substitution rate at 2.2 × 10 -9 substitutions per site per year. To find evidence for insertion of the LTR16A1 site before the split of the major mammalian lineages, we used the UCSC liftover utility to find the ACE2 gene locus in Rhesus macaque (rheMac10 assembly), marmoset (caljac3 assembly), mouse (mm10 assembly), dog (canFam3 assembly), african elephant (loxAfr3 assembly), bottle-nose dolphin (Turtru2 assembly), cow (bosTau9 assembly), opossum (monDom5 assembly) and platypus (ornAna2). We used the MUSCLE aligner on default settings to build a global alignment of human to rhesus macaque and marmoset, and then aligned all other species to the profile, reverting the strand of the whole sequence for mouse, elephant, cow and opossum due to whole gene inversions. We then used muscle -refine on overlapping 30,000 column blocks to refine the alignment locally.

The illustration of the lineage tree including node times are taken from timetree.org.

Bulk RNA-seq data were downloaded from study GSE147507 (Blanco-Melo et al., 2020) .

Reads were adapter trimmed and filtered for minimal 35nt sequences using Trimmomatic.

Since some samples were infected with SARS-CoV-2 in vitro, we identified and removed viral reads using BowTie2 (seedlength 30nt) to align reads to the Wuhan region reference genome (MN908947). Subsequently, reads were mapped with HISAT2 (optional parameters -p 8 -q -k 5) against GRCh38 reference chromosome assembly and transcripts were quantified against our custom transcriptome assembly (Attig et al., 2019) using Salmon (v0.12.0) (Patro et al., 2017) .

For single-cell RNA-seq data analysis, we downloaded the raw paired end sequencing reads as unmapped bam files from study GSE134355 (Han et al., 2020) , which were already demultiplexed, with one individual per tissue per sample. We then used the DropSeq:picard toolbox (v2.3.0) to recapitulate processing of HCL samples as documented on 'https://github.com/ggjlab/HCL'. In summary, this includes trimming polyA ends from each primary RNA sequencing read and tagging it with the cellular and molecular adapter sequence contained in the secondary read (BASE_RANGE=1-6:22-27:43-48 and BASE_RANGE=49-54, respectively). All reads were then mapped with HISAT2 (optional parameters -p 8 -q -k 5)

against GRCh38 reference chromosome assembly. The HISAT2 index here was built with the --exon / --ss option to cover all known splice sites annotated in the GENCODE v34 basic annotation. The cellular and molecular barcode sequences were recovered using the MergeBamAlignment utility in picard.

Open reading frames encoding ACE2, LTR16A1-ACE2, and respective lysine mutants were synthesized and cloned into the pcDNA3.1-DYK-P2A-GFP mammalian expression vector.

Gene synthesis, cloning, and mutagenesis were performed by GenScript and verified by sequencing. Cells were transfected using GeneJuice (EMD Millipore) and harvested 48 hrs post-transfection for downstream assays.

For interferon stimulation experiments, 2 × 10 5 SCC-4 and SCC-25 cells were stimulated with 100 ng/mL IFN-α or IFN-γ (Abcam) or PBS for 48 hrs. For proteasome inhibition experiments, cells were cultured in 20 µM MG-132 (EMD Millipore) 24 hrs after transfection and harvested 48 hrs after transfection. NHBE cells were stimulated for 4 hrs with 1000 ng/ml IFNα, 100ng/ml IFNβ or 100ng/ml IFNλ were used in a previous study (Major et al., 2020) , and stored cDNA was analysed by RT-qPCR in this study.

Cell lysates in RIPA buffer were resuspended in SDS buffer, heat denatured at 95 o C for 10 min, run on a 4-20% gel (Biorad), transferred to a PVDF membrane (Biorad), and blocked in 5% (w/v) bovine serum albumin fraction V (Sigma-Aldrich) in TBS-T. Membranes were incubated with primary antibodies to ACE2 (1:1000; Abcam), FLAG (1:1000, clone M2; Sigma-Aldrich), HRP-conjugated secondary antibodies to rabbit and mouse (1:1000; Cell Signaling Technology), and HRP-conjugated actin (1:25000; Abcam). Blots were visualized by chemiluminescence on an Amersham Imager 600 (GE Healthcare).

Total RNA from cell lines was isolated using the QIAcube (Qiagen), and cDNA synthesis was carried out with the High Capacity Reverse Transcription Kit (Applied Biosystems) with an added RNase inhibitor (Promega). Purified cDNA was used to quantify ACE2 and LTR16A1-ACE2 using variant-specific primers.

The variant-specific forward primers were:

For both variants, the following common reverse primer was used:

Common primer | R: CCATTGTCACCTTTGTGC Interferon-inducible genes were amplified using the following primers: Values were normalised to HPRT expression using the ΔCT method.

ACE2 activity in cell lysates was measured using the SensoLyte 390 ACE2 Activity Assay (AnaSpec) according to manufacturer's instructions. Recombinant human ACE2 (Sigma-Aldrich) was used as a positive control.

For GFP detection, single-cell suspensions were run on a LSR Fortessa (BD Biosciences)

running BD FACSDiva v8.0 and analysed with FlowJo v10 (Tree Star Inc.) analysis software. Normalized data from the FANTOM Consortium and the RIKEN PMI and CLST (DGT) for transcription start sites in the proximity of the intronic LTR16A1 element in the ACE2 locus. Both the sense and antisense orientations are depicted. Data were visualized with the zenbu online viewer (https://fantom.gsc.riken.jp/zenbu) for FANTOM5 Human hg38 promoterome. Figure S2 . Protein sequence alignment of ACE2 and the predicted LTR16A1-ACE2 product.

The predicted LTR16A1-ACE2 translation product is a 459-amino acid protein lacking the indicted single peptide, domains interacting with SARS-CoV spike glycoprotein, but retaining the transmembrane domain. The novel 10-amino acid sequence created by LTR16A1 exonisation is also shown. Figure S3 . Expression of ACE2 and LTR16A1-ACE2 isoforms in cancer and healthy tissues.

Box plots of ACE2 and LTR16A1-ACE2 isoforms expression in cancer patient and healthy control samples from TCGA and GTEx. For each cancer type, 24 samples were included (a total 768 samples), whereas for respective healthy tissues a total of 813 samples were included, varying between 2 and 156 per tissue type. Figure S4 . Single-cell RNA-seq coverage of the ACE2 locus.

RNA-seq trace of two multiplexed samples from adult lung, obtained from study GSE134355. Note the lack of coverage across the entire locus with the exception of only the 3' end of the last exon, shared between the isoforms. Figure S5 . Position of the ubiquitin targets in ACE2 and LTR16A1-ACE2 protein product.

Structure of ACE2 (left) and predicted structure of the LTR16A1-ACE2 protein product (right) depicting the position of the two mutated K residues, targeted for ubiquitination.

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We are grateful for assistance from the Scientific Computing and Flow Cytometry facilities at