Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry

Abstract

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 lifecycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2) 1-3 . The bioreporter assay is based on a Nanoluciferase complementation reporter, composed of two subunits, Large BiT and Small BiT, fused to the spike receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to RBD’s antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point towards targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.

N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

“…ACE2 binding affinity, on the other hand, was less altered in these N-glycosylation site mutants. In another recent study, RBD N-glycosylation site mutants were expressed in HEK293 cells without any obvious effects on expression levels, but decreased ACE2 binding upon removal of the N-glycans at both sites was reported (Azad et al, 2021). In agreement with the later finding, the glycan at position N343 may play an important role for the opening mechanism of the spike and stabilization of RBD in the up state that is required for ACE2 binding and cell entry (Sztain et al, 2021).…”

Synthetic Peptides That Antagonize the Angiotensin-Converting Enzyme-2 (ACE-2) Interaction with SARS-CoV-2 Receptor Binding Spike Protein

“…The various ACE2 antagonist peptides identified above were synthesized and assessed for their ability to inhibit the S-RBD interaction with ACE2 using a luciferase assay ( Figure 5 a). Taha et al 60 provides a novel bioluminescence-based sensor reporter system, the reassembly of SmBiT and LgBiT into NanoBiT when S-RBD and ACE2 interact, to probe antagonism of the protein–protein interaction. This sensitive yet robust assay, developed for the discovery of neutralizing antibodies, allowed us to rapidly test peptide-based antagonism of the SARS-CoV-2 S-RBD:ACE2 interaction.…”

“…Analysis of this data resulted in a library of six peptides that we predicted would efficiently antagonize SARS-CoV-2 S-RBD:ACE2 interaction. This library was synthesized and assayed against an in vitro bioluminescence assay 60 to determine their inhibition of the SARS-CoV-2 S-RBD:ACE2 interaction. Our data below demonstrates that all six peptides were able to strongly compete for this interaction.…”

Characterization of Critical Determinants of ACE2–SARS CoV-2 RBD Interaction

“…We also examine how specific mutations in ACE2 and RBD may alter the efficacy of drugs and neutralizing antibodies being developed for treatment and disease prevention purposes, respectively. To accomplish these aims, we use a NanoBiT SARS-CoV-2-RBD and ACE2 biosensor, previously developed by our lab, to initially characterize the RBD: ACE2 interaction (Figure 1B) [18]. The molecular basis of this technology involves the fusion of a Large Bit (LgBiT) subunit to one of the proteins of interest, and the fusion of a Small Bit subunit (SmBiT) to the second protein being investigated.…”

“…Since structural analysis has shown that certain residues in SARS-CoV-2 RBD are well conserved within SARS-CoV-1 RBD [18], we decided to also combine the various ACE2-SmBiT mutants with a LgBiT-SARS-CoV-1 RBD construct. This allowed us to also evaluate how ACE2 mutations could impact ACE2: SARS-CoV-1 RBD interaction.…”