Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant

Abstract

Understanding the molecular mechanisms of the increased transmissibility and immune evasion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is critical to guiding current and future intervention strategies. Zhang et al . determined cryo–electron microscopy structures of the full-length spike protein trimers of the Delta, Kappa, and Gamma variants of SARS-CoV-2 and studied their function and antigenic properties. The Delta spike protein fused membranes more efficiently at low levels of the cellular receptor ACE2, and its pseudotyped viruses infected target cells substantially more rapidly than all other variants tested, possibly at least partly accounting for its heightened transmissibility. Mutations of each variant rearranged the antigenic surface of the N-terminal domain of the spike protein but only caused local changes in the receptor-binding domain, consistent with greater resistance to neutralizing antibodies. These findings elucidate the molecular events that have led these viruses to adapt in human communities and to evade host immunity. —VV

SciScore for 10.1101/2021.08.17.456689: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Western blot: Western blot was performed using an anti-SARS-COV-2 S antibody following a protocol described previously (50).	anti-SARS-COV-2 S suggested: None
Alkaline phosphatase conjugated anti-Rabbit IgG (1:5000) (Sigma-Aldrich, St. Louis, MO) was used as a secondary antibody.	anti-Rabbit IgG suggested: None
To measure binding of a full-length S protein to monoclonal antibodies, the antibody was immobilized to anti-human IgG Fc Capture (AHC) biosensor (ForteBio, Fremont, CA) following a …

SciScore for 10.1101/2021.08.17.456689: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Western blot: Western blot was performed using an anti-SARS-COV-2 S antibody following a protocol described previously (50).	anti-SARS-COV-2 S suggested: None
Alkaline phosphatase conjugated anti-Rabbit IgG (1:5000) (Sigma-Aldrich, St. Louis, MO) was used as a secondary antibody.	anti-Rabbit IgG suggested: None
To measure binding of a full-length S protein to monoclonal antibodies, the antibody was immobilized to anti-human IgG Fc Capture (AHC) biosensor (ForteBio, Fremont, CA) following a protocol recommended by the manufacturer.	anti-human IgG suggested: None
Control sensors with no S protein or antibody were also dipped in the ACE2 or S protein solutions and the running buffer as references.	ACE2 suggested: None
For ACE2615-foldon T27W staining, APC conjugated anti-HIS antibody (Miltenyi Biotec, Auburn, CA) was used as secondary antibody at 1:50 dilution.	anti-HIS suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Briefly, Expi293F cells transfected with monomeric ACE2 or dimeric ACE2 expression construct and the supernatant of the cell culture was collected.	Expi293F suggested: RRID:CVCL_D615)
Murine Leukemia Virus (MLV) particles (plasmids of the MLV components kindly provided by Dr. Gary Whittaker at Cornell University and Drs. Catherine Chen and Wei Zheng at National Center for Advancing Translational Sciences, National Institutes of Health), pseudotyped with various SARS-CoV-2 S protein constructs, were generated in HEK 293T cells, following a protocol described previously for SARS-CoV (51, 52).	HEK 293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
To prepare for infection, 7.5×103 of HEK 293 cells, stably transfected with a full-length human ACE2 expression construct, in 15 μl culture medium were plated into a 384-well white-clear plate coated with poly-D-Lysine to enhance the cell attachment.	HEK 293 suggested: None
Pseudotyped virus particles were produced in 293T/17 cells (ATCC) by co-transfection of plasmids encoding codon-optimized SARS-CoV-2 full-length S constructs, packaging plasmid pCMV DR8.2, and luciferase reporter plasmid pHR’ CMV-Luc.	293T/17 suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
The 293T cell line stably overexpressing the human ACE2 cell surface receptor protein was kindly provided by Drs.	293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
Recombinant DNA
Sentences	Resources
The S genes were fused with a C-terminal twin Strep tag (SGGGSAWSHPQFEKGGGSGGGSGGSSAWSHPQFEK) and cloned into a mammalian cell expression vector pCMV-IRES-puro (Codex BioSolutions, Inc, Gaithersburg,	pCMV-IRES-puro suggested: None
Pseudotyped virus particles were produced in 293T/17 cells (ATCC) by co-transfection of plasmids encoding codon-optimized SARS-CoV-2 full-length S constructs, packaging plasmid pCMV DR8.2, and luciferase reporter plasmid pHR’ CMV-Luc.	pCMV DR8.2, and luciferase reporter suggested: None pHR’ suggested: None
Software and Algorithms
Sentences	Resources
Expression constructs: Genes of full-length spike (S) protein from Gamma (hCoV-19/Brazil/AM-992/2020; GISAID accession ID: EPI_ISL_833172), Kappa (hCoV-19/India/MH-NEERI-NGP-40449/2021; GISAID accession ID: EPI_ISL_1547802) and Delta (hCoV-19/India/GJ-GBRC619/2021; GISAID accession ID: EPI_ISL_2020954) were synthesized by Twist Bioscience (South San Francisco, CA) or GENEWIZ (South Plainfield, NJ).	GENEWIZ suggested: (GENEWIZ, RRID:SCR_003177)
The Kd was obtained by fitting Req value and its corresponding concentration to the model: “one site-specific” using GraphPad Prism 8.0.2 according to H.J. Motulsky, Prism 5 Statistics Guide, 2007, GraphPad Software Inc.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
Automated data collection was carried out using SerialEM version 3.8.6 (53) at a nominal magnification of 105,000× and the K3 detector in counting mode (calibrated pixel size, 0.825 Å) at an exposure rate of 20.24 (for Delta), ∼20.69/20.63/27.13 (for three data sets of Gamma), or ∼21.12/20.10 (for two data sets of Kappa) electrons per pixel per second.	SerialEM suggested: (SerialEM, RRID:SCR_017293)
Image processing and 3D reconstructions: Drift correction for cryo-EM images was performed using MotionCor2 (54), and contrast transfer function (CTF) was estimated by Gctf (55) using motion-corrected sums without dose-weighting.	MotionCor2 suggested: (MotionCor2, RRID:SCR_016499)
Density maps were corrected from the modulation transfer function of the K3 detector and sharpened by applying a temperature factor that was estimated using post-processing in RELION.	RELION suggested: (RELION, RRID:SCR_016274)
Several rounds of manual building were performed in Coot (57).	Coot suggested: (Coot, RRID:SCR_014222)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

A caveat is that all our experiments were performed in vitro; additional studies with authentic viruses will be needed to confirm our findings in more clinically relevant settings. If our hypothesis is valid, what is the structural basis for the enhanced fusogenicity of the Delta S protein? All mutations but one in the Delta S are located in either the RBD or NTD. Our extensive binding studies indicate that the Delta S does not engage the receptor ACE2 more tightly than does any other variant. It is unclear what other functional roles the NTD may play in the membrane fusion process, besides protecting the nearby RBDs. If the mutations in the NTD enhance RBD exposure to potential receptors, we should have observed, in our cryo-EM study, more particles in the RBD-up conformation from the Delta data set. Thus, the structural changes in both the RBD and NTD are unlikely to explain the efficient membrane fusion by the Delta variant. The last mutation, D950N, is unique to Delta and located in HR1 of S2 near the FPPR. D950N eliminates a negative charge (three in a trimer), but we have not observed any obvious structural changes caused by this substitution in the prefusion conformation. Its location nonetheless appears to be a critical site that can influence the refolding of S2, required for membrane fusion. Although D950 is not involved in a salt bridge in the G614 trimer, it is conceivable that the local change in the electrostatic potential may destabilize the prefusion S2 in a v...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 35. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

Results from rtransparent:

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No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant

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