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In the cell-cell fusion system, severe acute respiratory syndrome-CoV-2 S protein could effectively mediate the formation of syncytium between the effector cell and the target cell in the absence of an exogenous proteolytic enzyme, e.g., trypsin, while severe acute respiratory sy...

Inhibition of SARS-CoV-2 infection (previously 2019-nCoV) by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

biorxiv, pp.1-37, (2020)

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摘要

The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be confirmed. Therefore, we herein used a SARS-CoV-2...更多

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简介
  • In April of 2018, the World Health Organization (WHO) established a priority list of pathogens, including Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS) and Disease X, a disease with an epidemic or pandemic potential caused by an unknown pathogen1,2 (Fig.1a).

    In late December 2019, an outbreak of pneumonia with an unknown etiology in

    Wuhan, China was considered as the first Disease X following the announcement by

    WHO.
  • 2019-nCoV with 79.5% and 96% sequence identity to SARS-CoV and a bat coronavirus, SL-CoV-RaTG13, respectively 6, was renamed SARS-CoV-2 by the.
  • As of 24 February 2020, a total of 79,331 confirmed cases of COVID-19, including 2,618 deaths, were reported in China and 27 other countries serious threat to global public health and calling for the prompt development of specific anti-coronavirus therapeutics and prophylactics for treatment and prevention of COVID-19.
  • The spike (S) protein S2 subunit of SARS-CoV-2, which mediates membrane fusion, has 89.8% sequence identity and 96.9% sequence similarity angiotensin-converting enzyme 2 as the receptor to infect human cells 6
重点内容
  • In April of 2018, the World Health Organization (WHO) established a priority list of pathogens, including Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS) and Disease X, a disease with an epidemic or pandemic potential caused by an unknown pathogen1,2 (Fig.1a).

    In late December 2019, an outbreak of pneumonia with an unknown etiology in

    Wuhan, China was considered as the first Disease X following the announcement by

    World Health Organization
  • severe acute respiratory syndrome-CoV-2 infection, we found a typical syncytium phenomenon naturally formed by infected cells, which is rarely reported in severe acute respiratory syndrome-CoV infection (Fig. 1c)
  • In the cell-cell fusion system, severe acute respiratory syndrome-CoV-2 S protein could effectively mediate the formation of syncytium between the effector cell and the target cell in the absence of an exogenous proteolytic enzyme, e.g., trypsin, while severe acute respiratory syndrome-CoV S protein could not
  • Β-B coronaviruses lack the S1/S2 furin-recognition site, and their S proteins are uncleaved in the native state
  • severe acute respiratory syndrome-CoV enters into the cell mainly via the endosomal membrane fusion pathway where its S protein is cleaved by endosomal cathepsin L and activated25
  • Recently 103 severe acute respiratory syndrome-CoV-2 genomes have been identified 31, but we found that both the heptad repeat 1 and heptad repeat 2 domains among those reported genomes show promise to be developed as the first pan-CoV fusion inhibitor-based antiviral therapeutic or prophylactic for treatment or prevention of infection by the currently circulating severe acute respiratory syndrome-CoV-2 and Middle East respiratory syndrome-CoV and the future reemerging severe acute respiratory syndrome-CoV and emerging SARSr-CoVs
方法
  • Cell Lines, viruses and Peptides

    The human primary embryonic kidney cell line (293T) (CRL-3216TM), Calu-3

    (HTB-55TM), A549 (CCL-185), Vero E6 (CRL-1586TM), RD (CCL-136TM), and

    LLC-MK2 Original (CCL-7TM) cells were obtained from the American Type Culture

    Collection (ATCC).
  • Cell Lines, viruses and Peptides.
  • The human primary embryonic kidney cell line (293T) (CRL-3216TM), Calu-3.
  • Chinese Academy of Sciences (Shanghai, China), and 293T cells stably expressing human ACE2 (293T/ACE2) cells were kindly provided by Dr. Lanying Du. Lanying Du
  • All of these cell lines were maintained and grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, Carlsbad, CA, USA) containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% heat-inactivated fetal calf serum (FCS) (Gibco).
  • Wuhan Institute of Virology 6.
结果
  • The capacity of SARS-CoV-2 S protein-mediated membrane fusion

    From the GISAID Platform, the authors obtained the full-length amino-acid sequence of SARS-CoV-2 (BetaCoV 2019-2020) S protein (GenBank: QHD43416).
  • The capacity of SARS-CoV-2 S protein-mediated membrane fusion.
  • From the GISAID Platform, the authors obtained the full-length amino-acid sequence of SARS-CoV-2 (BetaCoV 2019-2020) S protein (GenBank: QHD43416).
  • Through alignment with SARS-CoV and SL-CoVs S proteins, the authors located the functional domains in SARS-CoV-2 S protein, which contains S1 subunit and S2 subunit with the cleavage site at R685/S686 15.
  • S1 subunit is located within the N-terminal 14–685 amino acids of S protein, containing.
  • S2 subunit contains fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2), transmembrane domain (TM) and cytoplasmic domain (CP) (Fig. 1b).
结论
  • At the end of 2019, the outbreak of Wuhan pneumonia with an unknown etiological agent, the first Disease X following WHO's announcement was reported to WHO.
  • . In 2018, Unlike SARS-CoV, live SARS-CoV-2-infected cells were found to form typical syncytium, suggesting that SARS-CoV-2 may mainly utilize the plasma membrane fusion pathway to enter and replicate inside host cells.
  • SARS-CoV enters into the cell mainly via the endosomal membrane fusion pathway where its S protein is cleaved by endosomal cathepsin L and activated25
表格
  • Table1: Data collection and refinement statistics
Download tables as Excel
基金
  • The recent outbreak of coronavirus disease caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health
  • Solved the X-ray crystal structure of six-helical bundle core of the HR1 and HR2 domains in SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with HR2 domain
  • Developed a pan-coronavirus fusion inhibitor, EK1, which targeted HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV
  • Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by currently circulating SARS-CoV-2 and emerging
研究对象与分析
mice: 6
For the viral control group, the same volume of 28% HBC or PBS was administered intranasally. In each group, six mice were randomly selected for euthanasia on day 5 after infection, then five mice for collecting and assessing the viral titer in mouse brain, one mouse for brain histological examination. Body weight and survival of the remaining six mice in each group were monitored for 14 days 35

mice: 6
In each group, six mice were randomly selected for euthanasia on day 5 after infection, then five mice for collecting and assessing the viral titer in mouse brain, one mouse for brain histological examination. Body weight and survival of the remaining six mice in each group were monitored for 14 days 35. Cytotoxicity of the peptides to the cells (Vero-E6, Huh-7, LLC-MK2 and RD cells) was tested by using the Cell Counting Kit-8 (CCK-8)

confirmed cases: 79331
Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by currently circulating SARS-CoV-2 and emerging SARSr-CoVs. As of 24 February 2020, a total of 79,331 confirmed cases of COVID-19, including 2,618 deaths, were reported in China and 27 other countries serious threat to global public health and thus calling for the prompt development of specific anti-coronavirus therapeutics and prophylactics for treatment and prevention of COVID-19. Coronaviruses (CoVs), the largest RNA viruses identified so far, belonging to the which mediates membrane fusion, has 89.8% sequence identity and 96.9% sequence similarity angiotensin-converting enzyme 2 (hACE2) as the receptor to infect human cells 6

mice: 5
EK1C4 harbors good therapeutic effect after a short period of HCoV-OC43 infection, possibly resulting from the establishment of HCoV-OC43 infection in mouse brain where EK1C4 cannot get through the blood brain barrier via nasal administration14. As shown in Fig. S6, high viral titer was detected in brains of all 5 mice in Pre-24 group and 4 out of 5 mice in Post-2 group, but was not detected in brain tissues of all mice in Pre-0.5, Pre-2, Pre-4, and Post-0.5 groups, while only moderate level of viral titer was detected in brain tissue in one of the 5 mice in Pre-12 group (Fig. S6 a and b). Similar to those in the viral control mice, mice in Pre-24 and Post-2 groups exhibited similar histopathological changes in brain tissues, including vacuolation, degeneration, and infiltration

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作者
Meiqin Liu
Meiqin Liu
Chao Wang
Chao Wang
Qiaoshuai Lan
Qiaoshuai Lan
Siliang Feng
Siliang Feng
Feifei Qi
Feifei Qi
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