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
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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- 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
- 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
- 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.
- 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
- 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
- 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
- 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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- Cell 180, 1–12.e1–e4, March 19, 2020 e1
- https://www.fortebio.com/products/labelfree-bli-detection/8-channel-octet-systems Part 18-5120 Lot 1910224A Cat 635683 Lot 1509573A Cat 29-0485-76 Cat 29-0915-96 Lot 10226998 Prod 01885-F Lot 020218
- Transient expression of hACE2 Expression and purification of human angiotensin-converting enzyme ectodomain (ACE2, residues 1–614) fused to the Fc region of human IgG (hFc) was performed as previously described (Walls et al., 2019) and cleavage of the Fc fragment was carried out with thrombin. Briefly, hACE2 was 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. The cultures were transfected using 293-Free transfection reagent (Millipore) with cells grown to a density of 1 million cells per mL and cultivated for 4 days. The supernatants were harvested and cells resuspended for another four days, yielding two harvests. Clarified supernatants were concentrated using Vivaflow tangential filtration cassettes (Sartorius, 10-kDa cut-off) before affinity purification using a Protein A column (GE
- e2 Cell 180, 1–12.e1–e4, March 19, 2020
- Pseudovirus production MLV-based SARS-CoV S, SARS-CoV-2 S, and SARS-CoV-2 Sfur/mut pseudotypes were prepared as previously described (Millet and Whittaker, 2016). HEK293T cells were co-transfected using Lipofectamine 2000 (Life Technologies) with an S encoding-plasmid, an MLV Gag-Pol packaging construct and the MLV transfer vector encoding a luciferase reporter, according to the manufacturer’s instructions. Cells were incubated for 5 h at 37C with transfection medium. Cells were then washed with DMEM two times and then DMEM containing 10% FBS was added for 60 h. The supernatants were then harvested and filtered through 0.45-mm membranes, concentrated with a 30kDa membrane for 10 min at 3,000 rpm and then frozen at À80C.
- Biolayer interferometry Assays were performed on an Octet Red (ForteBio) instrument at 30C with shaking at 1,000 RPM. Anti-penta His biosensors were hydrated in water for 30 min prior to a 1 min incubation in 10X kinetics buffer (undiluted). Either SARS-CoV-2 SB or SARSCoV SB were loaded at 10-20 mg/mL in 10X Kinetics Buffer for 200-900 s prior to baseline equilibration for 180 s in 10X kinetics buffer. Association of ACE2 in 10X kinetics buffer at various concentrations in a two-fold dilution series from 20nM to 2.5nM was carried out for 1,000 s prior to dissociation for 1,000 s. The data were baseline subtracted prior to fitting performed using a 1:1 binding model and the ForteBio data analysis software. Mean kon, koff values were determined with a global fit applied to all data. The experiments were done with two separate purification batches of SARS-CoV SB, SARS-CoV-2-SB, and ACE2 each in duplicate and the average is reported.
- CryoEM sample preparation and data collection 3 mL of 0.16 mg/mL SARS-CoV-2 S was loaded onto a freshly glow discharged (30 s at 20 mA) lacey carbon grid with a thin layer of evaporated continuous carbon prior to plunge freezing using a vitrobot MarkIV (ThermoFisher Scientific) using a blot force of À1 and 2.5 s blot time at 100% humidity and 25C. Data were acquired using the Leginon software (Suloway et al., 2005) to control an FEI Titan Krios transmission electron microscope operated at 300 kV and equipped with a Gatan K2 Summit direct detector and Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV. Automated data collection was carried out using Leginon at a nominal magnification of 130,000x with a pixel size of 0.525 A. The dose rate was adjusted to 8
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- Immunizations with SARS-CoV S 57BL/6J mice were obtained from the Jackson Laboratory and maintained at the Comparative Medicine Facility at the Fred Hutchinson Cancer Research Center. After collecting a pre-bleed, mice were immunized with 5mg of SARS-CoV 2P S ectodomain trimer (this construct was previously described (Walls et al., 2019)) formulated in 50ml of PBS and 50ml of Sigma Adjuvant System subcutaneously in the base of the tail at weeks 0, 4, and 12 and bleeds were collected 2 weeks after each immunization. Plasma from immunized animals was heat inactivated at 56C for 1 h and then stored at 4C until use. All experiments were conducted at the Fred Hutchinson Cancer Research Center according to approved Institutional Animal Care and Use Committee protocols.
- e4 Cell 180, 1–12.e1–e4, March 19, 2020