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...

Type-III interferons (IFNs) are important mediators of antiviral immunity. IFN-λ4 is a unique type-III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-λ4, an antiviral cytokine, are also less likely to clear HCV infection. Here, we searched for unique functional properties of IFN-λ4 that might explain its negative effect on HCV clearance. We used fresh primary human hepatocytes (PHH) treated with recombinant type-III IFNs or infected with Sendai virus (SeV) to model acute viral infection, and subsequently validated our findings in HepG2 cell line models. Endogenous IFN-λ4 protein was detectable only in SeV-infected PHH from individuals with the dG allele, where it was poorly secreted but highly functional even at concentrations below 50 pg/ml. IFN-λ4 acted faster than other type-III IFNs in inducing antiviral genes as well as negative regulators of IFN response, such as USP18 and SOCS1. Transient treatment of PHH with IFN-λ4 but not IFN-λ3 caused a strong and sustained induction of SOCS1, and refractoriness to further stimulation with IFN-λ3. Our results suggest unique functional properties of IFN-λ4 that can be important in viral clearance and other clinical conditions.

The chr12q24.13 locus encoding OAS1–OAS3 antiviral proteins has been associated with coronavirus disease 2019 (COVID-19) susceptibility. Here, we report genetic, functional and clinical insights into this locus in relation to COVID-19 severity. In our analysis of patients of European (n = 2,249) and African (n = 835) ancestries with hospitalized versus nonhospitalized COVID-19, the risk of hospitalized disease was associated with a common OAS1 haplotype, which was also associated with reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clearance in a clinical trial with pegIFN-λ1. Bioinformatic analyses and in vitro studies reveal the functional contribution of two associated OAS1 exonic variants comprising the risk haplotype. Derived human-specific alleles rs10774671-A and rs1131454-A decrease OAS1 protein abundance through allele-specific regulation of splicing and nonsense-mediated decay (NMD). We conclude that decreased OAS1 expression due to a common haplotype contributes to COVID-19 severity. Our results provide insight into molecular mechanisms through which early treatment with interferons could accelerate SARS-CoV-2 clearance and mitigate against severe COVID-19.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, primate-specific isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In vitro, dACE2, which lacks 356 N-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results reconcile current knowledge on ACE2 expression and suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, inflammatory tumor microenvironment, or viral co-infections is unlikely to affect the cellular entry of SARS-CoV-2 and promote infection.

APOBEC3A (A3A) and APOBEC3B (A3B) enzymes drive APOBEC-mediated mutagenesis. Identification of factors affecting the activity of these enzymes could help modulate mutagenesis and associated clinical outcomes. Here, we show that canonical and alternatively spliced A3A and A3B isoforms produce corresponding mutagenic and non-mutagenic enzymes. Increased expression of the mutagenic A3B isoform predicted shorter progression-free survival in bladder cancer. We demonstrate that the production of mutagenic vs. non-mutagenic A3B protein isoforms was considerably affected by inclusion/skipping of exon 5 in A3B. Furthermore, exon 5 skipping, resulting in lower levels of mutagenic A3B enzyme, could be increased in vitro. Specifically, we showed the effects of treatment with an SF3B1 inhibitor affecting spliceosome interaction with a branch point site in intron 4, or with splice-switching oligonucleotides targeting exon 5 of A3B. Our results underscore the clinical role of A3B and implicate alternative splicing of A3B as a mechanism that could be targeted to restrict APOBEC-mediated mutagenesis.

Pancreatic Cancer (PC) is a deadly disease in need of new therapeutic options. We recently developed a novel tricarbonylmethane agent (CMC2.24) as a therapeutic agent for PC, and evaluated its efficacy in preclinical models of PC. CMC2.24 inhibited the growth of various human PC cell lines in a concentration and time-dependent manner. Normal human pancreatic epithelial cells were resistant to CMC2.24, indicating selectivity. CMC2.24 reduced the growth of subcutaneous and orthotopic PC xenografts in mice by up to 65% (P < 0.02), and the growth of a human patient-derived tumor xenograft by 47.5% (P < 0.03 vs vehicle control). Mechanistically, CMC2.24 inhibited the Ras-RAF-MEK-ERK pathway. Based on Ras Pull-Down Assays, CMC2.24 inhibited Ras-GTP, the active form of Ras, in MIA PaCa-2 cells and in pancreatic acinar explants isolated from Kras mutant mice, by 90.3% and 89.1%, respectively (P < 0.01, for both). The inhibition of active Ras led to an inhibition of c-RAF, MEK, and ERK phosphorylation by 93%, 91%, and 87%, respectively (P < 0.02, for all) in PC xenografts. Furthermore, c-RAF overexpression partially rescued MIA PaCa-2 cells from the cell growth inhibition by CMC2.24. In addition, downstream of ERK, CMC2.24 inhibited STAT3 phosphorylation levels at the serine 727 residue, enhanced the levels of superoxide anion in mitochondria, and induced intrinsic apoptosis as shown by the release of cytochrome c from the mitochondria to the cytosol and the further cleavage of caspase 9 in PC cells. In conclusion, CMC2.24, a potential Ras inhibitor, is an efficacious agent for PC treatment in preclinical models, deserving further evaluation.

Genomic regions have been associated with COVID-19 susceptibility and outcomes, including the chr12q24.13 locus encoding antiviral proteins OAS1-3. Here, we report genetic, functional, and clinical insights into genetic associations within this locus. In Europeans, the risk of hospitalized vs. non-hospitalized COVID-19 was associated with a single 19Kb-haplotype comprised of 76 OAS1 variants included in a 95% credible set within a large genomic fragment introgressed from Neandertals. The risk haplotype was also associated with impaired spontaneous but not treatment-induced SARS-CoV-2 clearance in a clinical trial with pegIFN-λ1. We demonstrate that two exonic variants, rs10774671 and rs1131454, affect splicing and nonsense-mediated decay of OAS1. We suggest that genetically-regulated loss of OAS1 expression contributes to impaired spontaneous clearance of SARS-CoV-2 and elevated risk of hospitalization for COVID-19. Our results provide the rationale for further clinical studies using interferons to compensate for impaired spontaneous SARS-CoV-2 clearance, particularly in carriers of the OAS1 risk haplotypes.

IFNL3/IFNL4 polymorphisms are inversely associated with the risk of chronic hepatitis C virus (HCV) infection and cirrhosis, two major risk factors for developing hepatocellular carcinoma (HCC). To further explore these inverse associations and their molecular underpinnings, we analyzed IFNL3/IFNL4 polymorphisms represented by the IFNL4 genotype (presence of rs368234815-dG or rs12979860-T alleles) in HCV patients: 2969 from Japan and 2931 from Taiwan. IFNL4 genotype was associated with an increased risk of HCV-related HCC (OR=1.28, 95%CI=1.07-1.52, P=0.0058) in the general population of Japanese patients, but not in Taiwanese patients who achieved treatment-induced viral clearance. IFNL4 genotype was also associated with a decreased risk of cirrhosis (OR=0.66, 95%CI=0.46-0.93, P=0.018, in Taiwanese patients). We then engineered HepG2 cells to inducibly express IFN-λ4 in the presence or absence of interferon lambda receptor 1 (IFNLR1). Induction of IFN-λ4 resulted in its intracellular accumulation, mainly in lysosomes and late endosomes, and increased ER stress, leading to apoptosis and reduced proliferation. We identified the very-low-density lipoprotein receptor (VLDLR), which facilitates HCV entry into hepatocytes, as a transcript induced by IFN-λ4 but not IFN-λ3. Our results suggest that the molecular mechanisms underlying the anti-cirrhotic but pro-HCV associations observed for IFNL3/IFNL4 polymorphisms are, at least in part, contributed by intracellular accumulation of IFN-λ4 causing ER stress in hepatic cells.