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We evaluated entry into BHK cells and observed that transient transfection with human ACE2 rendered them susceptible to transduction with SARS-CoV-2 S-murine leukemia virus

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein.

Cell, no. 2 (2020): 281-292.e6

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摘要

The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ...更多

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简介
  • Three coronaviruses have crossed the species barrier to cause deadly pneumonia in humans since the beginning of the 21st century: severe acute respiratory syndrome coronavirus (SARS-CoV) (Drosten et al, 2003; Ksiazek et al, 2003), Middle-East respiratory syndrome coronavirus (Zaki et al, 2012) (MERS-CoV), and SARS-CoV-2 (Huang et al, 2020; Zhu et al, 2020).
  • SARS-CoV emerged in the Guangdong province of China in 2002 and spread to five continents through air travel routes, infecting 8,098 people and causing 774 deaths.
  • In 2012, MERS-CoV emerged in the Arabian Peninsula, where it remains a major public health concern, and was exported to 27 countries, infecting a total of $2,494 individuals and claiming 858 lives.
  • A previously unknown coronavirus, named SARS-CoV-2, was discovered in December 2019 in Wuhan, Hubei province of China and was sequenced and isolated by January 2020 (Zhou et al, 2020; Zhu et al, 2020).
  • On January 30, 2020, the World Health Organization declared the SARS-CoV-2 epidemic a public health emergency of international concern
重点内容
  • Three coronaviruses have crossed the species barrier to cause deadly pneumonia in humans since the beginning of the 21st century: severe acute respiratory syndrome coronavirus (SARS-CoV) (Drosten et al, 2003; Ksiazek et al, 2003), Middle-East respiratory syndrome coronavirus (Zaki et al, 2012) (MERS-CoV), and SARS-CoV-2 (Huang et al, 2020; Zhu et al, 2020)
  • We evaluated entry into BHK cells and observed that transient transfection with human ACE2 rendered them susceptible to transduction with SARS-CoV-2 S-murine leukemia virus (Figure 1B)
  • These results demonstrate human ACE2 is a functional receptor for SARS-CoV-2, in agreement with recently reported
  • We previously showed that coronavirus S glycoproteins are densely decorated by heterogeneous N-linked glycans protruding from the trimer surface (Walls et al, 2016b; Walls et al, 2019; Xiong et al, 2018)
  • SARSCoV isolates from the three phases of the 2002–2003 epidemic were more efficiently transmitted among humans and more pathogenic than the isolates associated with the 2003–2004 re-emergence that caused only a few cases, in line with their binding affinities for human ACE2 (Consortium, 2004; Kan et al, 2005; Li et al, 2005c)
  • We report here that SARS-CoV-2 uses human ACE2 as an entry receptor and recognizes it with a similar affinity to the 2002–2003 SARS-CoV isolates, which suggests it can spread efficiently in humans, in agreement with the numerous SARS-CoV-2 human-to-human transmission events reported to date
方法
  • Transient expression of SARS-CoV-2 and SARS-CoV SB The SARS-CoV SB construct was cloned from a SARS-CoV S ectodomain (Walls et al, 2019) synthesized by GeneArt (ThermoFisher Scientific) into a modified pOPING vector with an N-terminal mu-phosphatase signal peptide and a C-terminal hexa-histidine tag (G-HHHHHH).
  • The boundaries of the construct are N-terminal 328RFPN331 and C-terminal 530STNL533
  • Both constructs were produced in 500mL HEK293F cells grown in suspension using FreeStyle 293 expression medium (Life technologies) at 37C in a humidified 8% CO2 incubator rotating at 130 rpm.
结果
  • ACE2 Is an Entry Receptor for SARS-CoV-2 The SARS-CoV-2 S glycoprotein shares 76% amino acid sequence identity with the SARS-CoV S Urbani and 80% identity with bat SARSr-CoV ZXC21 S and ZC45 S glycoprotein.
  • To assess the ability of SARS-CoV-2 S to promote entry into target cells, we first compared transduction of SARS-CoV-2 S-MLV and SARS-CoV S-MLV into VeroE6 cells, that are known to express ACE2 and support SARS-CoV replication (Drosten et al, 2003; Ksiazek et al, 2003)
  • Both pseudoviruses entered cells well (Figure 1A), suggesting that SARS-CoV-2 S-MLV could use African green monkey ACE2 as entry receptor.
  • These results demonstrate hACE2 is a functional receptor for SARS-CoV-2, in agreement with recently reported
结论
  • Receptor recognition is the first step of viral infection and is a key determinant of host cell and tissue tropism.
  • Enhanced binding affinity between SARS-CoV S and hACE2 was proposed to correlate with increased virus transmissibility and disease severity in humans (Li et al, 2005c).
  • We found that the SARSCoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARSCoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry.
  • We demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARSCoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination
表格
  • Table1: Kinetic Analysis of hACE2 Binding to SARS-CoV-2 SB and SARS-CoV SB by Biolayer Interferometry
  • Table2: Conservation of N-Linked Glycosylation Sequons in SARS-CoV-2 S and SARS-CoV S
Download tables as Excel
基金
  • This study was supported by the National Institute of General Medical Sciences (R01GM120553 to D.V.), the National Institute of Allergy and Infectious Diseases (HHSN272201700059C to D.V.), a Pew Biomedical Scholars Award (D.V.), an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund (D.V.), the University of Washington Arnold and Mabel Beckman cryoEM center, the Washington Research Foundation, and the Pasteur Institute (M.A.T.)
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作者
Alexandra C Walls
Alexandra C Walls
Young-Jun Park
Young-Jun Park
Abigail Wall
Abigail Wall
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