key: cord-1048697-v9kveexz authors: Zhuang, Meng‐Wei; Cheng, Yun; Zhang, Jing; Jiang, Xue‐Mei; Wang, Li; Deng, Jian; Wang, Pei‐Hui title: Increasing Host Cellular Receptor—Angiotensin‐Converting Enzyme 2 (ACE2) Expression by Coronavirus may Facilitate 2019‐nCoV (or SARS‐CoV‐2) Infection date: 2020-06-04 journal: J Med Virol DOI: 10.1002/jmv.26139 sha: 3d29e8825aec5b2d18dd0e51233d872f983ce2a1 doc_id: 1048697 cord_uid: v9kveexz The ongoing outbreak of a new coronavirus (2019‐nCoV, or SARS‐CoV‐2) has caused an epidemic of the acute respiratory syndrome known as COVID‐19 in humans. SARS‐CoV‐2 rapidly spread to multiple regions of China and multiple other countries, posing a serious threat to public health. The spike (S) proteins of SARS‐CoV‐1 and SARS‐CoV‐2 may use the same host cellular receptor, angiotensin‐converting enzyme 2 (ACE2), for entering into host cells. The affinity between ACE2 and the SARS‐CoV‐2 S protein is much higher than that of ACE2 binding to the SARS‐CoV S protein, explaining why SARS‐CoV‐2 seems to be more readily transmitted from the human to human. Here, we report that ACE2 can be significantly upregulated after infection of various viruses, including SARS‐CoV‐1 and SARS‐CoV‐2, or by the stimulation with inflammatory cytokines such as interferons. We propose that SARS‐CoV‐2 may positively induce its cellular entry receptor, ACE2, to accelerate its replication and spread; high inflammatory cytokine levels increase ACE2 expression and act as high‐risk factors for developing COVID‐19, and the infection of other viruses may increase the risk of SARS‐CoV‐2 infection. Therefore, drugs targeting ACE2 may be developed for the future emerging infectious diseases caused by this cluster of coronaviruses. This article is protected by copyright. All rights reserved. during the disease. 12 Organs and tissues with high ACE2 abundance, such as the lungs, kidneys, and small intestine, are the infection targets of SARS-CoV-2. 11 These organs and tissues commonly exhibit high ACE2 expression levels, such as the kidney and small intestine. SARS-CoV-1 infects multiple cell types in several different organs; immune cells and the pulmonary epithelium are among the main sites of injury. 13 Acute kidney injury is a predictor of high mortality in SARS patients. 14 In the lungs, human ACE2 primarily occurs in type II and type I alveolar epithelial cells. ACE2 is mainly expressed in AT2 cells and is also detected in AT1 cells, airway epithelial cells, fibroblasts, endothelial cells, and macrophages. 12 Taken together, these findings reveal that the expression level of ACE2 is extremely important for the successful infection of SARS-CoV-1 and SARS-CoV-2. As the cellular receptor of SARS-CoV-1 and SARS-CoV-2, ACE2 plays an essential role during the infection of these viruses. 2, 10 The ACE2 level is the decisive factor in SARS-CoV-2 infection. 2 pcDNA6B-TBK1-FLAG, and pcDNA6B-TRIF-FLAG were constructed using standard molecular cloning methods as described previously. 15, 16 The 1582 bp promoter region of human ACE2 was cloned into pGL3-Basic (Promega, USA) to construct a luciferase reporter (pACE2-luc) using the following primers: ACE2 SARS-CoV-2 Raises ACE2 to Enhance Infection This article is protected by copyright. All rights reserved. promoter-F (5'-3'), GGGGTACCTGCGCTCAGAGAGAACGCTT; ACE2 promoter-R (5'-3'), CCCTCGAGGCCACTGTGCCCAGCCAT. Recombinant proteins of human IFN-α (P5637), human IFN-β (P5660), human IFN-γ (P5664), human TNF-α (P5318), human IL-1α (P5102), and human IL-1β (P5106) were purchase from Beyotime, China. Total RNA was isolated using TRIzol® reagent (Invitrogen, USA). VSV-GFP infection was described in our previous publications. 15, 16 Briefly, target cells were washed twice with 37°C DMEM, after which the virus, diluted in serum-free medium, was added to the cells. Virus-medium complexes were incubated with the cells in normal culture conditions. One to two hours later, virus-medium complexes were discarded and replaced with a culture medium containing 10% FBS. To determine whether the innate immune signaling pathway affects the promoter activities of ACE2, 2 × 10 5 HEK293T cells cultured in 24 well plates were transfected with the pACE2-luc luciferase reporter plasmid and the expression vector plasmids pcDNA6B-RIG-IN-FLAG, pcDNA6B-MAVS-FALG, pcDNA6B-TBK1-FLAG or pcDNA6B-TRIF-FLAG as described in our previous studies. 15, 16 The pRL-TK Renilla luciferase reporter was co-transfected to serve as an internal control. The cells were harvested and lysed at 36 hours after transfection for the assessment of luciferase activities using the Dual-Luciferase Reporter Assay System (Promega, USA). Briefly, cell lysates were loaded into a Accepted Article 96-well plate for the measurement of luciferase activity in a Centro XS 3 LB 960 microplate luminometer (BERTHOLD TECHNOLOGIES, Germany). Relative luciferase activity was calculated by normalizing firefly luciferase activity to that of Renilla luciferase. Twenty-one nasopharyngeal swab specimens were collected from fourteen COVID-19 patients who were confirmed to be SARS-CoV-2 RNA positive and SARS-CoV-2-specific IgG positive in Jinan Infectious Disease Hospital, Shandong, China as described in our previous studies. 17 Eight nasopharyngeal swab specimens were collected as controls from eight healthy individuals who were confirmed to be SARS-CoV-2 RNA negative. Total RNA was extracted from nasopharyngeal swab specimens as described in our previous studies. 17 This study was approved by the ethics commissions of Jinan Infectious Disease Hospital, Shandong, China. Three independent experiments were performed in all studies. Two-tailed unpaired Student's t-test conducted with GraphPad Prism 7.0 was used to perform statistical analysis. P < 0.05 was considered as statistically significant in all experiments. This article is protected by copyright. All rights reserved. We retrieved datasets submitted to the microarray database of NCBI and analyzed the ACE2 expression levels during virus infection. In the lungs of C57Bl/6 mice infected with 10 2 PFU SARS-CoV-1, the ACE2 mRNA level was significantly increased compared with the mock group ( Figure 1A) . 18, 19 In primary human airway epithelial cells, MERS-CoV infection increased ACE mRNA levels at 24, 36, and 48 hours post infection ( Figure 1B) . These results revealed that ACE could be upregulated by infection with SARS-CoV-1 and MERS-CoV. Next, we evaluated ACE expression after infection with other RNA viruses. Microarray data showed that the ACE2 mRNA level was strongly induced in human airway epithelial cells from 4 donors infected with rhinovirus ( Figure 2 ). 20 In wild-type H1N1 influenza-infected human bronchial epithelial cells, ACE2 expression was dramatically increased to approximately 5-fold higher than the level in the control group (Figure 3) . 21 These lines of evidence indicate that ACE2 is a virus-responsive gene and that its expression can be stimulated by various viruses. Overall, ACE2 expression can be upregulated by virus infection. This article is protected by copyright. All rights reserved. We hypothesized that ACE2 induction by viruses was caused by virus infection-induced inflammatory cytokines. After the analysis of the microarray database, we observed that ACE2 expression could be stimulated by type I IFNs including IFN-α and IFN-β, and type III IFNs, such as IFN-γ (Figure 4 We cloned the putative promoter region (from the transcription start site of human ACE2 -1281 to +300 bp) into the luciferase reporter vector pGL3-Basic to construct the pACE2-luc reporter and investigated the regulatory properties of ACE2 ( Figure 8A ). Dual-luciferase reporter assays showed that the ACE2 promoter could be strongly activated by RIG-IN, MAVS, TRIF, and TBK1 ( Figure 8B ). One of the clinical features of COVID-19 patients is the upregulation of inflammatory cytokines. Next, we treated HEK293 cells receiving pACE2-luc reporter plasmids with recombinant protein of IFN-α, IFN-β, IFN-γ, TNF-α, IL-1α, or IL-1β; twelve hours later, dual-luciferase reporter assays were SARS-CoV-2 Raises ACE2 to Enhance Infection This article is protected by copyright. All rights reserved. upregulated in SARS-CoV-2 infected individuals. To our knowledge, this is the first report that clearly showed the expression levels of ACE2 is higher in COVID-19 patients than that in healthy individuals (Figure 9 ). After this manuscript was posted at bioRxiv on February 27, 2020 ( https://www.biorxiv.org/content/10.1101/2020.02.24.963348v1), other groups independently found that ACE2 is an ISG in human airway epithelial cells and upregulated by viral infecions. 23,24 Overall, the available results indicate that ACE2 can be upregulated by infection by various viruses and stimulation by IFNs, which indicates that ACE2 is a novel ISG that can be stimulated by viruses and IFNs ( Figure 10 ). We also showed that the viral RNA mimic poly (I:C) can induce the upregulation of IFN-β and ISG56 ( Figure 5) ; importantly, as an RNA virus, SARS-CoV-2 viral RNAs are expected to be sensed by TLR3 and RIG-I/MDA-5, which will then activated the innate immune signaling cascades and induce the production of IFNs and other inflammatory cytokines, ultimately, leading to the upregulation of ACE2, which may facilitate the entry of SARS-CoV-2 into host cells ( Figure 10) . Importantly, the induction of ACE2 by inflammatory cytokines A Novel Coronavirus from Patients with Pneumonia in China A pneumonia outbreak associated with a new coronavirus of probable bat origin A new coronavirus associated with human respiratory disease in China A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster Clinical features of patients infected with 2019 novel coronavirus in Wuhan Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Nowcasting and forecasting the potential domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: a modelling study Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus The digestive system is a potential route of 2019-nCov infection: a bioinformatics analysis based on single-cell transcriptomes Single-cell Analysis of ACE2 Expression in Human Kidneys and Bladders Reveals a Potential Route of 2019-nCoV Infection SARS-CoV-2 Raises ACE2 to Enhance Infection Multiple organ infection and the pathogenesis of SARS Acute renal impairment in coronavirus-associated severe acute respiratory syndrome A novel transcript isoform of STING that sequesters cGAMP and dominantly inhibits innate nucleic acid sensing Inhibition of AIM2 inflammasome activation by a novel transcript isoform of IFI16 Long-term coexistence of SARS-CoV-2 with antibody response in COVID-19 patients Complement Activation Contributes to Severe Acute Respiratory Syndrome Coronavirus Pathogenesis. mBio Accepted Article Accepted Article