Biochemical Characterization of SARS-CoV-2 Spike RBD Mutations and Their Impact on ACE2 Receptor Binding

Abstract

Infection of mammalian cells by SARS-CoV-2 coronavirus requires primary interaction between the receptor binding domain (RBD) of the viral spike protein and the host cell surface receptor angiotensin-converting enzyme 2 (ACE2) glycoprotein. Several mutations in the RBD of SARS-CoV-2 spike protein have been reported for several variants and resulted in wide spread of the COVID pandemic. For instance, the double mutations L452R and E484Q present in the Indian B.1.617 variant have been suggested to cause evasion of the host immune response. The common RBD mutations N501Y and E484K were found to enhance the interaction with the ACE2 receptor. In the current study, we analyzed the biosynthesis and secretion of the RBD double mutants L452R and E484Q in comparison to the wild-type RBD and the individual mutations N501 and E484K in mammalian cells. Moreover, we evaluated the interaction of these variants with ACE2 by means of expression of the S protein and co-immunoprecipitation with ACE2. Our results revealed that the double RBD mutations L452R and E484Q resulted in a higher expression level and secretion of spike S1 protein than other mutations. In addition, an increased interaction of these mutant forms with ACE2 in Calu3 cells was observed. Altogether, our findings highlight the impact of continuous S1 mutations on the pathogenicity of SARS-CoV-2 and provide further biochemical evidence for the dominance and high transmissibility of the double Indian mutations.

Keywords: ACE2 interaction; RBD; SARS-CoV-2; double mutant; spike; transmissibility.

FIGURE 1

Schematic representation of the host ACE2 receptor and spike RBD variants analyzed in this study. (A) Three-dimensional (3D) representation of coronavirus spike protein (lower graphic) and the host ACE2 receptor (upper graphic). Spike protein forms a homotrimer with three S1-RBD heads (enclosed in dotted box). (B) Structural model of SARS-CoV-2 RBD (cyan) interacting with human ACE2 (dark green), based on PDB 6m0j (Lan et al., 2020). The area bordered by the box is magnified on the right to show the sites of mutations analyzed in the current study. (C) Linear representation of the Fc-tagged S1 cDNA plasmid used. Arrows indicate sites of mutated amino acids.

FIGURE 2

Characterization of subcellular and secretory forms of spike S1 proteins. (A) COS-1 cells were transiently transfected or non-transfected (NT) with plasmids encoding Wt-S1, dMu (L452 and E484), E484K, and N501Y S1 mutations. After 48 h of transfection, proteins were immunoprecipitated from both cell lysates, and media were then treated or not treated with Endo H. Cellular and secretory S1 proteins were immunoprecipitated and analyzed by Western blot. The upper panel shows a 130-kDa protein that shifted to a lower molecular weight following Endo H treatment. (B) Subcellular localization of S1 variants in the transiently transfected COS-1 cells. S1-Wt and mutant proteins were expressed in the COS-1 cells grown on coverslips, and their subcellular localization in the ER was investigated by immunofluorescence and visualized by confocal microscopy. The cGRP94 plasmid that encodes the ER chaperone GRP94 was used as an ER marker. Scale bars, 10 and 20 μm.

FIGURE 3

dMu, E484K, and N501Y variants of S1 are comparatively highly expressed and secreted in COS-1 cells. (A) Transfected COS-1 cells were lysed 48 h after transfection. Equal protein-containing lysates (upper left blot) and collected media (lower left blot) of each S1 mutant were pulled down using protein A-bound Sepharose (PAS) and analyzed using SDS-PAGE on 8% slab gels. To confirm these expression profiles, we carried out parallel sequential immunoprecipitation of both lysates (upper right blot) and media (lower right blot), which reflected higher intracellular and secretory protein levels in the case of S1-dMu and S1-N501Y mutants. (B) Comparison between relative expression levels of S1 variants in COS-1 cells (left bar graph) and their secretory proteins collected from the media (right bar graph) (n = 4). Data are normalized to the wild type and expressed as arbitrary units.

FIGURE 4

S1-dMu mutant interacts avidly with the ACE2 receptor in Calu3 cells. After 48 h of transfection of COS-1 cells, S1 variants were analyzed for their expression and secretion, as explained before (upper two blots). Equal amounts of COS-1 media containing secretory S1 proteins were allowed to bind to the ACE2 receptor expressed in Calu3 cells for 2 h at 4°C. Following the time specified for interaction, Calu3 cells were washed twice with PBS, pH 7.4 and lysed, as described before. Cellular lysates were analyzed for ACE2 expression where actin was used as housekeeping control protein (lowest two blots). To assess the interacting ACE2, the remaining major part of Calu3 lysate was subjected to immunoprecipitation using pAb anti-ACE2 and blotted against ACE2. Notably, the S1-dMu mutant showed stronger interaction capability with ACE2 than the other S1 proteins as revealed in the upper blot of the Calu3 cells.