Trimeric receptor-binding domain of SARS-CoV-2 acts as a potent inhibitor of ACE2 receptor-mediated viral entry

Abstract

The COVID-19 pandemic has caused over four million deaths and effective methods to control CoV-2 infection, in addition to vaccines, are needed. The CoV-2 binds to the ACE2 on human cells through the receptor-binding domain (RBD) of the trimeric spike protein. Our modeling studies show that a modified trimeric RBD (tRBD) can interact with three ACE2 receptors, unlike the native spike protein, which binds to only one ACE2. We found that tRBD binds to the ACE2 with 58-fold higher affinity than monomeric RBD (mRBD) and blocks spike-dependent pseudoviral infection over 4-fold more effectively compared to the mRBD. Although mRBD failed to block CoV-2 USA-WA1/2020 infection, tRBD efficiently blocked the true virus infection in plaque assays. We show that tRBD is a potent inhibitor of CoV-2 through both competitive binding to the ACE2 and steric hindrance, and has the potential to emerge as a first-line therapeutic method to control COVID-19.

Keywords: Molecular structure; Virology.

Graphical abstract

Figure 1

Construction and expression of tRBD and mRBD in vitro (A and B) (A) Liner representation of FLAG or HIS-tagged SARS-COV-2 RBD construct sequence for the expression of tRBD (B) Expression levels of FLAG-tagged (top) or HIS-tagged (bottom) tRBD at varying timepoints after transfection of HEK293T-ACE2 cells. (C) Schematic representing protocol for tRBD and mRBD protein purification. (D) Varying concentrations of HIS-tagged commercially available recombinant SARS-COV-2 RBD protein (Lane 1–6), and conditioned media with HIS-tagged mRBD (Lane 7) or HIS-tagged tRBD (Lane 8) was separated via SDS/PAGE gel and probed with anti-HIS antibody. A linear regression curve was constructed using densitometry analysis. (E) tRBD was crosslinked with glutaraldehyde (GLA) and incubated at temperatures indicated. Crosslinked proteins were separated in SDS/PAGE gel and probed with anti-HIS antibody. (n = 2 independent samples).

Figure 2

In vitro binding ability of RBD:ACE2 (A and B) HEK293T-ACE2 cells (3 × 10⁵) or Vero E6 cells (8 × 10⁵) were plated 24 h prior in 6 and 10 cm plates, respectively. (A) HEK293T-ACE2 cells were treated with 2.5 mL of FLAG-tagged tRBD (left) or HIS-tagged tRBD (right). Vero E6 cells were treated with 5 mL of FLAG-tagged tRBD or HIS-tagged tRBD (bottom). (B) HEK293T-ACE2 cells were treated with 666.7 ng/mL of HIS-tagged mRBD (indicated as one in the figure) or tRBD (indicated as one in the figure) or 2000 ng/mL of HIS-tagged tRBD (indicated as three in the figure). Thirty minutes following incubation, cells were assessed by immunoprecipitation using (A, left) anti-FLAG or (A, right and B) NiNTA Agarose beads. (B, bottom) Densitometry analysis of three independent Western blots as in Figure 2B, top, presented as mean ± SE of mean (SEM). ∗∗p< 0.01, ns - non significant.

Figure 3

Molecular modeling and surface plasmon resonance data (A–D) Structure of (A) monomeric RBD (mRBD) and configuration of (B) trimeric RBD (tRBD). (C) Binding models of mRBD with ACE2 dimer; (D) Binding models of tRBD with ACE2. tRBD binds to two units of an ACE dimer (left) as RBDs can rotate relative to one another, or binds to three separate ACE2 dimers (right). (E) Representative binding sensorgrams for mRBD:ACE2 and tRBD:ACE2 interactions using the single cycle kinetic assay. Binding sensorgrams were generated from five independent injections of mRBD/tRBD. Black and red lines represent fitted curves generated by 1:1 binding. (F) mRBD or tRBD in running buffer (PBSP+, pH7.4 purchased from Cytiva) were flowed over three surfaces with a density of 500–1500RU biotin-ACE2 on an S series SA sensorchip in a Biacore T200 system. Mean kinetic rate constant K_a, K_d and the equilibrium dissociation constant (K_D = K_d/K_a) were measured from single-cycle kinetics and globally fitted by BiaEvaluation 3.1 software using 1:1 binding model, three sets of data were averaged and standard errors for the affinity values are shown.

Figure 4

tRBD-mediated inhibition of SARS-COV-2 pseudovirus cell entry in HEK293T-ACE2 cells (A–D) HEK293T-ACE2 cells (1.25 × 10⁴ cells/well) were treated with specified amounts of HIS-tagged mRBD or HIS-tagged tRBD. (A and B) SDS/PAGE gel was used to verify equal concentrations of mRBD or tRBD was used. (A–C) SARS-CoV-2 pseudovirus (1:0) or (D) VSVG virus (1:50) with the Luciferase-IRES-ZsGreen backbone were produced via calcium phosphate transfection and co-treated onto HEK293T-ACE2 cells. Luciferase activity was assessed and values are presented as RLU of infected cells treated with tRBD or mRBD over cells infected with pseudovirus alone. Data are representative of three independent experiments conducted in triplicates and are presented as mean ± SE of mean (SEM). Statistical significance was analyzed using two-tailed Student’s unpaired t test, ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p<0.001, ns – non significant.

Figure 5

SARS-CoV-2 Plaque reduction assay (A and B) Vero E6 cells grown in 24-well plates were incubated with undiluted (1:0) or 2-fold serially diluted mRBD (●), tRBD (▪), or media (Mk-▲) for 30 min prior to SARS-CoV-2 infection. (A) Representative images of plaque formation on Vero E6 cells in the presence of the indicated conditioned media. (B) Plaque-forming units per milliliter (PFU/mL) were measured 72 h post-infection and values of three independent experiments, each conducted in triplicate, were combined. Individual data points represent the mean titer for each experiment. Bar plots indicate the mean of the individual experiments for each condition tested ±standard errors of mean values (SEM). (C) Dose-response curve with normalized values from (B) graphed to represent the mean of three independent experiments, each value was normalized to the Mk titer of SARS-CoV-2 for the media dilution independently for each experiment performed. Error bars represent 95% confidence interval. (B and C) Significance was determined by ANOVA with Dunnett’s multiple comparison test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001, presented as mean ± SE of mean (SEM).