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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: By sensitizing viruses to receptor-induced conformational changes, the first S cleavages expand virus tropism to cell types that are relevant to lung infection, and therefore may be significant determinants of MERS-CoV virulence.
Abstract: Middle East respiratory syndrome coronavirus (MERS-CoV) infects humans from zoonotic sources and causes severe pulmonary disease. Virions require spike (S) glycoproteins for binding to cell receptors and for catalyzing virus–cell membrane fusion. Fusion occurs only after S proteins are cleaved sequentially, first during their secretion through the exocytic organelles of virus-producing cells, and second after virus binding to target-cell receptors. To more precisely determine how sequential proteolysis contributes to CoV infection, we introduced S mutations obstructing the first cleavages. These mutations severely compromised MERS-CoV infection into human lung-derived cells, but had little effect on infection into several other cell types. These cell type-specific requirements for proteolysis correlated with S conformations during cell entry. Without the first cleavages, S proteins resisted cell receptor-induced conformational changes, which restricted the second, fusion-activating cleavages. Consistent with these findings, precleaved MERS viruses used receptor-proximal, cell-surface proteases to effect the second fusion-activating cleavages during cell entry, whereas the more rigid uncleaved MERS viruses trafficked past these cell-surface proteases and into endosomes. Uncleaved viruses were less infectious to human airway epithelial and Calu3 cell cultures because they lacked sufficient endosomal fusion-activating proteases. Thus, by sensitizing viruses to receptor-induced conformational changes, the first S cleavages expand virus tropism to cell types that are relevant to lung infection, and therefore may be significant determinants of MERS-CoV virulence.

278 citations


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

  • ...One can speculate that furin-mediated precleavage at the S1/S2 site in infected cells might promote subsequent TMPRSS2-dependent entry into target cells, as reported for MERS-CoV (Kleine-Weber et al., 2018; Park et al., 2016) ....

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Journal ArticleDOI
TL;DR: It is proposed that high-specificity, low-affinity attachment of MERS-CoV to sialoglycans during the preattachment or early attachment phase may form another determinant governing the host range and tissue tropism of this zoonotic pathogen.
Abstract: Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract both in humans and in its natural host, the dromedary camel. Virion attachment to host cells is mediated by 20-nm-long homotrimers of spike envelope protein S. The N-terminal subunit of each S protomer, called S1, folds into four distinct domains designated S1A through S1D. Binding of MERS-CoV to the cell surface entry receptor dipeptidyl peptidase 4 (DPP4) occurs via S1B. We now demonstrate that in addition to DPP4, MERS-CoV binds to sialic acid (Sia). Initially demonstrated by hemagglutination assay with human erythrocytes and intact virus, MERS-CoV Sia-binding activity was assigned to S subdomain S1A. When multivalently displayed on nanoparticles, S1 or S1A bound to human erythrocytes and to human mucin in a strictly Sia-dependent fashion. Glycan array analysis revealed a preference for α2,3-linked Sias over α2,6-linked Sias, which correlates with the differential distribution of α2,3-linked Sias and the predominant sites of MERS-CoV replication in the upper and lower respiratory tracts of camels and humans, respectively. Binding is hampered by Sia modifications such as 5-N-glycolylation and (7,)9-O-acetylation. Depletion of cell surface Sia by neuraminidase treatment inhibited MERS-CoV entry of Calu-3 human airway cells, thus providing direct evidence that virus–Sia interactions may aid in virion attachment. The combined observations lead us to propose that high-specificity, low-affinity attachment of MERS-CoV to sialoglycans during the preattachment or early attachment phase may form another determinant governing the host range and tissue tropism of this zoonotic pathogen.

266 citations


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

  • ...This could comprise binding to cellular glycans, a function ascribed to the S1 domain of certain coronaviruses (Li et al., 2017; Park et al., 2019)....

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Journal ArticleDOI
TL;DR: The results point to a central role of ACE2 in SARS-CoV infection and suggest a minor contribution of the cytoplasmic domain to receptor function, which is not important for receptor function.

257 citations


Additional excerpts

  • ..., 2013) N/A Plasmid: pCG1-hAPN (Hofmann et al., 2004a) N/A...

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Journal ArticleDOI
TL;DR: The finding that NAbs remained detectable throughout follow-up is reassuring in terms of protection provided against reinfection; however, NAb titers decreased markedly after month 16.
Abstract: In a cohort study of 56 convalescent patients with severe acute respiratory syndrome (SARS), titers of immunoglobulin G (IgG) antibodies and neutralizing antibodies (NAbs) against SARS-associated coronavirus were assessed at regular intervals (at 1, 4, 7, 10, 16, and 24 months after the onset of disease) by use of enzyme-linked immunosorbent assay and neutralization assay. IgG antibody and NAb titers were highly correlated, peaking at month 4 after the onset of disease and decreasing thereafter. IgG antibodies remained detectable in all patients until month 16, and they became undetectable in 11.8% of patients at month 24. The finding that NAbs remained detectable throughout follow-up is reassuring in terms of protection provided against reinfection; however, NAb titers decreased markedly after month 16.

256 citations


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

  • ...Convalescent SARS patients exhibit a neutralizing antibody response that can be detected even 24 months after infection (Liu et al., 2006) and that is largely directed against the S protein....

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  • ...Evidence that Antibodies Raised against SARS-CoV Will Cross-Neutralize SARS-CoV-2 Convalescent SARS patients exhibit a neutralizing antibody response directed against the viral S protein (Liu et al., 2006)....

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Journal ArticleDOI
TL;DR: The virus is endemic in camels in the Arabian Peninsula and Africa and thus poses a consistent threat of frequent reintroduction into human populations and disease prevalence will increase substantially if the virus mutates to increase human-to-human transmissibility.
Abstract: In 2012, a zoonotic coronavirus was identified as the causative agent of Middle East respiratory syndrome and was named MERS coronavirus (MERS-CoV). As of August 11, 2016, the virus has infected 1,791 patients, with a mortality rate of 35.6%. Although MERS-CoV generally causes subclinical or mild disease, infection can result in serious outcomes, including acute respiratory distress syndrome and multi-organ failure in patients with comorbidities. The virus is endemic in camels in the Arabian Peninsula and Africa and thus poses a consistent threat of frequent reintroduction into human populations. Disease prevalence will increase substantially if the virus mutates to increase human-to-human transmissibility. No therapeutics or vaccines are approved for MERS; thus, development of novel therapies is needed. Further, since many MERS cases are acquired in healthcare settings, public health measures and scrupulous attention to infection control are required to prevent additional MERS outbreaks.

236 citations


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

  • ...In contrast, the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) are transmitted from animals to humans and cause severe respiratory diseases in afflicted individuals, SARS and MERS, respectively (Fehr et al., 2017)....

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