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Journal ArticleDOI

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

TL;DR: It is demonstrated that SARS-CoV-2 uses the SARS -CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming, and it is shown that the sera from convalescent SARS patients cross-neutralized Sars-2-S-driven entry.
About: This article is published in Cell.The article was published on 2020-04-16 and is currently open access. It has received 15362 citations till now. The article focuses on the topics: Proteases.
Citations
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Journal ArticleDOI
01 Apr 2020-Nature
TL;DR: Detailed virological analysis of nine cases of coronavirus disease 2019 (COVID-19) provides proof of active replication of the SARS-CoV-2 virus in tissues of the upper respiratory tract.
Abstract: Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity—but also aided in the control—of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6–8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples—in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19. Detailed virological analysis of nine cases of coronavirus disease 2019 (COVID-19) provides proof of active replication of the SARS-CoV-2 virus in tissues of the upper respiratory tract.

5,840 citations

Journal ArticleDOI
30 Mar 2020-Nature
TL;DR: High-resolution crystal structures of the receptor-binding domain of the spike protein of SARS-CoV-2 and SARS -CoV in complex with ACE2 provide insights into the binding mode of these coronaviruses and highlight essential ACE2-interacting residues.
Abstract: A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from December 2019 that quickly spread nationwide and to other countries around the world1–3. Here, to better understand the initial step of infection at an atomic level, we determined the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor4. Structural analysis identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses1–3,5. The epitopes of two SARS-CoV antibodies that target the RBD are also analysed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies. High-resolution crystal structures of the receptor-binding domain of the spike protein of SARS-CoV-2 and SARS-CoV in complex with ACE2 provide insights into the binding mode of these coronaviruses and highlight essential ACE2-interacting residues.

4,555 citations

Journal ArticleDOI
25 Aug 2020-JAMA
TL;DR: This review discusses current evidence regarding the pathophysiology, transmission, diagnosis, and management of COVID-19, the novel severe acute respiratory syndrome coronavirus 2 pandemic that has caused a worldwide sudden and substantial increase in hospitalizations for pneumonia with multiorgan disease.
Abstract: Importance The coronavirus disease 2019 (COVID-19) pandemic, due to the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a worldwide sudden and substantial increase in hospitalizations for pneumonia with multiorgan disease. This review discusses current evidence regarding the pathophysiology, transmission, diagnosis, and management of COVID-19. Observations SARS-CoV-2 is spread primarily via respiratory droplets during close face-to-face contact. Infection can be spread by asymptomatic, presymptomatic, and symptomatic carriers. The average time from exposure to symptom onset is 5 days, and 97.5% of people who develop symptoms do so within 11.5 days. The most common symptoms are fever, dry cough, and shortness of breath. Radiographic and laboratory abnormalities, such as lymphopenia and elevated lactate dehydrogenase, are common, but nonspecific. Diagnosis is made by detection of SARS-CoV-2 via reverse transcription polymerase chain reaction testing, although false-negative test results may occur in up to 20% to 67% of patients; however, this is dependent on the quality and timing of testing. Manifestations of COVID-19 include asymptomatic carriers and fulminant disease characterized by sepsis and acute respiratory failure. Approximately 5% of patients with COVID-19, and 20% of those hospitalized, experience severe symptoms necessitating intensive care. More than 75% of patients hospitalized with COVID-19 require supplemental oxygen. Treatment for individuals with COVID-19 includes best practices for supportive management of acute hypoxic respiratory failure. Emerging data indicate that dexamethasone therapy reduces 28-day mortality in patients requiring supplemental oxygen compared with usual care (21.6% vs 24.6%; age-adjusted rate ratio, 0.83 [95% CI, 0.74-0.92]) and that remdesivir improves time to recovery (hospital discharge or no supplemental oxygen requirement) from 15 to 11 days. In a randomized trial of 103 patients with COVID-19, convalescent plasma did not shorten time to recovery. Ongoing trials are testing antiviral therapies, immune modulators, and anticoagulants. The case-fatality rate for COVID-19 varies markedly by age, ranging from 0.3 deaths per 1000 cases among patients aged 5 to 17 years to 304.9 deaths per 1000 cases among patients aged 85 years or older in the US. Among patients hospitalized in the intensive care unit, the case fatality is up to 40%. At least 120 SARS-CoV-2 vaccines are under development. Until an effective vaccine is available, the primary methods to reduce spread are face masks, social distancing, and contact tracing. Monoclonal antibodies and hyperimmune globulin may provide additional preventive strategies. Conclusions and Relevance As of July 1, 2020, more than 10 million people worldwide had been infected with SARS-CoV-2. Many aspects of transmission, infection, and treatment remain unclear. Advances in prevention and effective management of COVID-19 will require basic and clinical investigation and public health and clinical interventions.

3,371 citations


Cites background from "SARS-CoV-2 Cell Entry Depends on AC..."

  • ...quartile range) interval from symptom onset to hospital admission is 7 (3-9) days....

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  • ...The mean (interquartile range) incubation period (the time from exposure to symptom onset) for COVID-19 is approximately 5 (2-7) days....

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Journal ArticleDOI
David E. Gordon, Gwendolyn M. Jang, Mehdi Bouhaddou, Jiewei Xu, Kirsten Obernier, Kris M. White1, Matthew J. O’Meara2, Veronica V. Rezelj3, Jeffrey Z. Guo, Danielle L. Swaney, Tia A. Tummino4, Ruth Hüttenhain, Robyn M. Kaake, Alicia L. Richards, Beril Tutuncuoglu, Helene Foussard, Jyoti Batra, Kelsey M. Haas, Maya Modak, Minkyu Kim, Paige Haas, Benjamin J. Polacco, Hannes Braberg, Jacqueline M. Fabius, Manon Eckhardt, Margaret Soucheray, Melanie J. Bennett, Merve Cakir, Michael McGregor, Qiongyu Li, Bjoern Meyer3, Ferdinand Roesch3, Thomas Vallet3, Alice Mac Kain3, Lisa Miorin1, Elena Moreno1, Zun Zar Chi Naing, Yuan Zhou, Shiming Peng4, Ying Shi, Ziyang Zhang, Wenqi Shen, Ilsa T Kirby, James E. Melnyk, John S. Chorba, Kevin Lou, Shizhong Dai, Inigo Barrio-Hernandez5, Danish Memon5, Claudia Hernandez-Armenta5, Jiankun Lyu4, Christopher J.P. Mathy, Tina Perica4, Kala Bharath Pilla4, Sai J. Ganesan4, Daniel J. Saltzberg4, Rakesh Ramachandran4, Xi Liu4, Sara Brin Rosenthal6, Lorenzo Calviello4, Srivats Venkataramanan4, Jose Liboy-Lugo4, Yizhu Lin4, Xi Ping Huang7, Yongfeng Liu7, Stephanie A. Wankowicz, Markus Bohn4, Maliheh Safari4, Fatima S. Ugur, Cassandra Koh3, Nastaran Sadat Savar3, Quang Dinh Tran3, Djoshkun Shengjuler3, Sabrina J. Fletcher3, Michael C. O’Neal, Yiming Cai, Jason C.J. Chang, David J. Broadhurst, Saker Klippsten, Phillip P. Sharp4, Nicole A. Wenzell4, Duygu Kuzuoğlu-Öztürk4, Hao-Yuan Wang4, Raphael Trenker4, Janet M. Young8, Devin A. Cavero4, Devin A. Cavero9, Joseph Hiatt4, Joseph Hiatt9, Theodore L. Roth, Ujjwal Rathore9, Ujjwal Rathore4, Advait Subramanian4, Julia Noack4, Mathieu Hubert3, Robert M. Stroud4, Alan D. Frankel4, Oren S. Rosenberg, Kliment A. Verba4, David A. Agard4, Melanie Ott, Michael Emerman8, Natalia Jura, Mark von Zastrow, Eric Verdin10, Eric Verdin4, Alan Ashworth4, Olivier Schwartz3, Christophe d'Enfert3, Shaeri Mukherjee4, Matthew P. Jacobson4, Harmit S. Malik8, Danica Galonić Fujimori, Trey Ideker6, Charles S. Craik, Stephen N. Floor4, James S. Fraser4, John D. Gross4, Andrej Sali, Bryan L. Roth7, Davide Ruggero, Jack Taunton4, Tanja Kortemme, Pedro Beltrao5, Marco Vignuzzi3, Adolfo García-Sastre, Kevan M. Shokat, Brian K. Shoichet4, Nevan J. Krogan 
30 Apr 2020-Nature
TL;DR: A human–SARS-CoV-2 protein interaction map highlights cellular processes that are hijacked by the virus and that can be targeted by existing drugs, including inhibitors of mRNA translation and predicted regulators of the sigma receptors.
Abstract: A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein–protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19. A human–SARS-CoV-2 protein interaction map highlights cellular processes that are hijacked by the virus and that can be targeted by existing drugs, including inhibitors of mRNA translation and predicted regulators of the sigma receptors.

3,319 citations

References
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Journal ArticleDOI
TL;DR: It is shown that TMPRSS2 might promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion.
Abstract: The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) can be proteolytically activated by cathepsins B and L upon viral uptake into target cell endosomes. In contrast, it is largely unknown whether host cell proteases located in the secretory pathway of infected cells and/or on the surface of target cells can cleave SARS S. We along with others could previously show that the type II transmembrane protease TMPRSS2 activates the influenza virus hemagglutinin and the human metapneumovirus F protein by cleavage. Here, we assessed whether SARS S is proteolytically processed by TMPRSS2. Western blot analysis revealed that SARS S was cleaved into several fragments upon coexpression of TMPRSS2 (cis-cleavage) and upon contact between SARS S-expressing cells and TMPRSS2-positive cells (trans-cleavage). cis-cleavage resulted in release of SARS S fragments into the cellular supernatant and in inhibition of antibody-mediated neutralization, most likely because SARS S fragments function as antibody decoys. trans-cleavage activated SARS S on effector cells for fusion with target cells and allowed efficient SARS S-driven viral entry into targets treated with a lysosomotropic agent or a cathepsin inhibitor. Finally, ACE2, the cellular receptor for SARS-CoV, and TMPRSS2 were found to be coexpressed by type II pneumocytes, which represent important viral target cells, suggesting that SARS S is cleaved by TMPRSS2 in the lung of SARS-CoV-infected individuals. In summary, we show that TMPRSS2 might promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion.

926 citations


"SARS-CoV-2 Cell Entry Depends on AC..." refers background or result in this paper

  • ..., 2005) and the serine protease TMPRSS2 (Glowacka et al., 2011; Matsuyama et al., 2010; Shulla et al., 2011) for S protein priming in cell lines, and inhibition of both proteases is required for robust blockade of viral entry (Kawase et al....

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  • ...In contrast, an S2 signal was largely absent in cells and particles expressing SARS-S (Figure 1B), as previously documented (Glowacka et al., 2011; Hofmann et al., 2004b)....

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  • ...272 Cell 181, 271–280, April 16, 2020 cellular serine protease TMPRSS2 for S protein priming (Glowacka et al., 2011; Matsuyama et al., 2010; Shulla et al., 2011)....

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  • ...Collectively, our present findings and previouswork highlight TMPRSS2 as a host cell factor that is critical for spreadof several clinically relevant viruses, including influenza A viruses and coronaviruses (Gierer et al., 2013; Glowacka et al., 2011; Iwata-Yoshikawa et al., 2019; Kawase et al., 2012; Matsuyama et al., 2010; Shulla et al., 2011; Zhou et al., 2015)....

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Journal ArticleDOI
TL;DR: In addition to viral spread through a respiratory route, SARS‐CoV in the intestinal tract, kidney and sweat glands may be excreted via faeces, urine and sweat, thereby leading to virus transmission.
Abstract: We previously identified the major pathological changes in the respiratory and immune systems of patients who died of severe acute respiratory syndrome (SARS) but gained little information on the organ distribution of SARS-associated coronavirus (SARS-CoV). In the present study, we used a murine monoclonal antibody specific for SARS-CoV nucleoprotein, and probes specific for a SARS-CoV RNA polymerase gene fragment, for immunohistochemistry and in situ hybridization, respectively, to detect SARS-CoV systematically in tissues from patients who died of SARS. SARS-CoV was found in lung, trachea/bronchus, stomach, small intestine, distal convoluted renal tubule, sweat gland, parathyroid, pituitary, pancreas, adrenal gland, liver and cerebrum, but was not detected in oesophagus, spleen, lymph node, bone marrow, heart, aorta, cerebellum, thyroid, testis, ovary, uterus or muscle. These results suggest that, in addition to the respiratory system, the gastrointestinal tract and other organs with detectable SARS-CoV may also be targets of SARS-CoV infection. The pathological changes in these organs may be caused directly by the cytopathic effect mediated by local replication of the SARS-CoV; or indirectly as a result of systemic responses to respiratory failure or the harmful immune response induced by viral infection. In addition to viral spread through a respiratory route, SARS-CoV in the intestinal tract, kidney and sweat glands may be excreted via faeces, urine and sweat, thereby leading to virus transmission. This study provides important information for understanding the pathogenesis of SARS-CoV infection and sheds light on possible virus transmission pathways. This data will be useful for designing new strategies for prevention and treatment of SARS.

919 citations


"SARS-CoV-2 Cell Entry Depends on AC..." refers background in this paper

  • ...However, ACE2 expression is not limited to the lung, and extrapulmonary spread of SARS-CoV in ACE2 tissues was observed (Ding et al., 2004; Gu et al., 2005; Hamming et al., 2004)....

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Journal ArticleDOI
TL;DR: Comparisons of S proteins of SARS‐CoV isolated during the 2002–2003 SARS outbreak and during the much less severe 2003–2004 outbreak, and from palm civets, provide insight into the severity of the 2002‐ 2003 SARS epidemic.
Abstract: Human angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS coronavirus (SARS-CoV) Here we identify the SARS-CoV spike (S)-protein-binding site on ACE2 We also compare S proteins of SARS-CoV isolated during the 2002-2003 SARS outbreak and during the much less severe 2003-2004 outbreak, and from palm civets, a possible source of SARS-CoV found in humans All three S proteins bound to and utilized palm-civet ACE2 efficiently, but the latter two S proteins utilized human ACE2 markedly less efficiently than did the S protein obtained during the earlier human outbreak The lower affinity of these S proteins could be complemented by altering specific residues within the S-protein-binding site of human ACE2 to those of civet ACE2, or by altering S-protein residues 479 and 487 to residues conserved during the 2002-2003 outbreak Collectively, these data describe molecular interactions important to the adaptation of SARS-CoV to human cells, and provide insight into the severity of the 2002-2003 SARS epidemic

903 citations


"SARS-CoV-2 Cell Entry Depends on AC..." refers background in this paper

  • ...The SARS-S/ACE2 interface has been elucidated at the atomic level, and the efficiency of ACE2 usage was found to be a key determinant of SARS-CoV transmissibility (Li et al., 2005a (Li et al., , 2005b ....

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Journal ArticleDOI
04 Jun 1992-Nature
TL;DR: It is reported that human aminopeptidase N, a cell-surface metalloprotease on intestinal, lung and kidney epithelial cells, is a receptor for human coronavirus strainHCV-229E, but not for HCV-OC43.
Abstract: Human coronaviruses (HCV) in two serogroups represented by HCV-229E and HCV-OC43 are an important cause of upper respiratory tract infections. Here we report that human aminopeptidase N, a cell-surface metalloprotease on intestinal, lung and kidney epithelial cells, is a receptor for human coronavirus strain HCV-229E, but not for HCV-OC43. A monoclonal antibody, RBS, blocked HCV-229E virus infection of human lung fibroblasts, immunoprecipitated aminopeptidase N and inhibited its enzymatic activity. HCV-229E-resistant murine fibroblasts became susceptible after transfection with complementary DNA encoding human aminopeptidase N. By contrast, infection of human cells with HCV-OC43 was not inhibited by antibody RBS and expression of aminopeptidase N did not enhance HCV-OC43 replication in mouse cells. A mutant aminopeptidase lacking the catalytic site of the enzyme did not bind HCV-229E or RBS and did not render murine cells susceptible to HCV-229E infection, suggesting that the virus-binding site may lie at or near the active site of the human aminopeptidase molecule.

835 citations


"SARS-CoV-2 Cell Entry Depends on AC..." refers background in this paper

  • ...Expression plasmids for vesicular stomatitis virus (VSV, serotype Indiana) glycoprotein (VSV-G), Nipah virus (NiV) fusion (F) and attachment glycoprotein (G), SARS-S (derived from the Frankfurt-1 isolate) with or without a C-terminal HA epitope tag, HCoV-229E-S, MERS-S, human and bat angiotensin converting enzyme 2 (ACE2), human aminopeptidase N (APN), human Cell 181, 271–280.e1–e5, April 16, 2020 e3 dipeptidyl-peptidase 4 (DPP4) and human TMPRSS2 have been described elsewhere (Bertram et al., 2010; Brinkmann et al., 2017; Gierer et al., 2013; Hoffmann et al., 2013; Hofmann et al., 2005; Kleine-Weber et al., 2019)....

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  • ...In agreement with these findings, directed expression of human and bat (Rhinolophus alcyone) ACE2 but not human DPP4, the entry receptor used by MERS-CoV (Raj et al., 2013), or human APN, the entry receptor used by HCoV-229E (Yeager et al., 1992), allowed SARS-2-S- and SARS-S-driven entry into otherwise non-susceptible BHK-21 cells (Figure 3A)....

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  • ..., 2013), or human APN, the entry receptor used by HCoV-229E (Yeager et al., 1992), allowed SARS-2-S- and SARS-S-driven entry into otherwise non-susceptible BHK-21 cells (Figure 3A)....

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Journal ArticleDOI
TL;DR: It is shown that SARS-CoV shares its receptor ACE2 with HCoV-NL63, which suggests that highly pathogenic variants have ample opportunity to evolve, underlining the need for vaccines against HCoVs.
Abstract: Coronavirus (CoV) infection of humans is usually not associated with severe disease. However, discovery of the severe acute respiratory syndrome (SARS) CoV revealed that highly pathogenic human CoVs (HCoVs) can evolve. The identification and characterization of new HCoVs is, therefore, an important task. Recently, a HCoV termed NL63 was discovered in patients with respiratory tract illness. Here, cell tropism and receptor usage of HCoV-NL63 were analyzed. The NL63 spike (S) protein mediated infection of different target cells compared with the closely related 229E-S protein but facilitated entry into cells known to be permissive to SARS-CoV-S-driven infection. An analysis of receptor engagement revealed that NL63-S binds angiotensin-converting enzyme (ACE) 2, the receptor for SARS-CoV, and HCoV-NL63 uses ACE2 as a receptor for infection of target cells. Potent neutralizing activity directed against NL63- but not 229E-S protein was detected in virtually all sera from patients 8 years of age or older, suggesting that HCoV-NL63 infection of humans is common and usually acquired during childhood. Here, we show that SARS-CoV shares its receptor ACE2 with HCoV-NL63. Because the two viruses differ dramatically in their ability to induce disease, analysis of HCoV-NL63 might unravel pathogenicity factors in SARS-CoV. The frequent HCoV-NL63 infection of humans suggests that highly pathogenic variants have ample opportunity to evolve, underlining the need for vaccines against HCoVs.

694 citations


"SARS-CoV-2 Cell Entry Depends on AC..." refers background in this paper

  • ...dipeptidyl-peptidase 4 (DPP4) and human TMPRSS2 have been described elsewhere (Bertram et al., 2010; Brinkmann et al., 2017; Gierer et al., 2013; Hoffmann et al., 2013; Hofmann et al., 2005; Kleine-Weber et al., 2019)....

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