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      Beyond Shielding: The Roles of Glycans in the SARS-CoV-2 Spike Protein

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          Abstract

          The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 28,000,000 infections and 900,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2). Similar to many other viral fusion proteins, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of the glycosylated SARS-CoV-2 S protein, both in the open and closed states, augmenting the available structural and biological data. Multiple microsecond-long, all-atom molecular dynamics simulations were used to provide an atomistic perspective on the roles of glycans and on the protein structure and dynamics. We reveal an essential structural role of N-glycans at sites N165 and N234 in modulating the conformational dynamics of the spike’s receptor binding domain (RBD), which is responsible for ACE2 recognition. This finding is corroborated by biolayer interferometry experiments, which show that deletion of these glycans through N165A and N234A mutations significantly reduces binding to ACE2 as a result of the RBD conformational shift toward the “down” state. Additionally, end-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of the SARS-CoV-2 S protein, which may be exploited in the therapeutic efforts targeting this molecular machine. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, providing a strategy to control the conformational plasticity of the RBD that could be harnessed for vaccine development.

          Abstract

          The glycan shield is a sugary barrier that helps the viral SARS-CoV-2 spikes to evade the immune system. Beyond shielding, two of the spike’s glycans are discovered to prime the virus for infection.

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          Most cited references 53

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

          Summary The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
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            A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

            Summary Background An ongoing outbreak of pneumonia associated with a novel coronavirus was reported in Wuhan city, Hubei province, China. Affected patients were geographically linked with a local wet market as a potential source. No data on person-to-person or nosocomial transmission have been published to date. Methods In this study, we report the epidemiological, clinical, laboratory, radiological, and microbiological findings of five patients in a family cluster who presented with unexplained pneumonia after returning to Shenzhen, Guangdong province, China, after a visit to Wuhan, and an additional family member who did not travel to Wuhan. Phylogenetic analysis of genetic sequences from these patients were done. Findings From Jan 10, 2020, we enrolled a family of six patients who travelled to Wuhan from Shenzhen between Dec 29, 2019 and Jan 4, 2020. Of six family members who travelled to Wuhan, five were identified as infected with the novel coronavirus. Additionally, one family member, who did not travel to Wuhan, became infected with the virus after several days of contact with four of the family members. None of the family members had contacts with Wuhan markets or animals, although two had visited a Wuhan hospital. Five family members (aged 36–66 years) presented with fever, upper or lower respiratory tract symptoms, or diarrhoea, or a combination of these 3–6 days after exposure. They presented to our hospital (The University of Hong Kong-Shenzhen Hospital, Shenzhen) 6–10 days after symptom onset. They and one asymptomatic child (aged 10 years) had radiological ground-glass lung opacities. Older patients (aged >60 years) had more systemic symptoms, extensive radiological ground-glass lung changes, lymphopenia, thrombocytopenia, and increased C-reactive protein and lactate dehydrogenase levels. The nasopharyngeal or throat swabs of these six patients were negative for known respiratory microbes by point-of-care multiplex RT-PCR, but five patients (four adults and the child) were RT-PCR positive for genes encoding the internal RNA-dependent RNA polymerase and surface Spike protein of this novel coronavirus, which were confirmed by Sanger sequencing. Phylogenetic analysis of these five patients' RT-PCR amplicons and two full genomes by next-generation sequencing showed that this is a novel coronavirus, which is closest to the bat severe acute respiatory syndrome (SARS)-related coronaviruses found in Chinese horseshoe bats. Interpretation Our findings are consistent with person-to-person transmission of this novel coronavirus in hospital and family settings, and the reports of infected travellers in other geographical regions. Funding The Shaw Foundation Hong Kong, Michael Seak-Kan Tong, Respiratory Viral Research Foundation Limited, Hui Ming, Hui Hoy and Chow Sin Lan Charity Fund Limited, Marina Man-Wai Lee, the Hong Kong Hainan Commercial Association South China Microbiology Research Fund, Sanming Project of Medicine (Shenzhen), and High Level-Hospital Program (Guangdong Health Commission).
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              Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

              Structure of the nCoV trimeric spike The World Health Organization has declared the outbreak of a novel coronavirus (2019-nCoV) to be a public health emergency of international concern. The virus binds to host cells through its trimeric spike glycoprotein, making this protein a key target for potential therapies and diagnostics. Wrapp et al. determined a 3.5-angstrom-resolution structure of the 2019-nCoV trimeric spike protein by cryo–electron microscopy. Using biophysical assays, the authors show that this protein binds at least 10 times more tightly than the corresponding spike protein of severe acute respiratory syndrome (SARS)–CoV to their common host cell receptor. They also tested three antibodies known to bind to the SARS-CoV spike protein but did not detect binding to the 2019-nCoV spike protein. These studies provide valuable information to guide the development of medical counter-measures for 2019-nCoV. Science, this issue p. 1260
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                Author and article information

                Journal
                ACS Cent Sci
                oc
                acscii
                ACS Central Science
                American Chemical Society
                2374-7943
                2374-7951
                23 September 2020
                Affiliations
                []Department of Chemistry and Biochemistry, University of California San Diego , La Jolla, California 92093, United States
                []Department of Molecular Biosciences, The University of Texas at Austin , Austin, Texas 78712, United States
                [§ ]Department of Chemistry and Hamilton Institute, Maynooth University , Dublin, Ireland
                []Biomedical Sciences Graduate Program, University of California San Diego , La Jolla, California 92093, United States
                Author notes
                Article
                10.1021/acscentsci.0c01056
                7523240
                Copyright © 2020 American Chemical Society

                This article is made available via the PMC Open Access Subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

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