Publication date: Jun 12, 2025

Targeted disruption of SARS-CoV entry remains a critical strategy in antiviral therapies and vaccine design. Central to this process is the viral spike (S) protein, which mediates host recognition via specific interactions with the human angiotensin-converting enzyme 2 (hACE2). Here, we expand our previous work by identifying the smallest active spike (S) protein binding motif (RBSM) and key residues of SARS-CoV (S) that specifically recognize the host receptor, human angiotensin-converting enzyme 2 (hACE2). Using the Qubevirus (Qβ) platform, we projected, analyzed and validated five essential residues (L472, N473, N479, D480, and Y491) that are critical for SARS-CoV binding and entry. Qβ phage-displayed RBSM variants disrupted hACE2 recognition and infection initiation. An engineered RBSM insert containing all five mutant residues completely abolished recognition and binding to both hACE2 and anti-RBD antibodies. Furthermore, QβRBSM1 which exhibited no cytotoxic effect on HEK293T cells, was found to reduce the infectivity of SARS-CoV pseudovirus in a competitive assay, positioning it as a blocker that inhibits the SARS-CoV entry. In addition, building upon our previous studies, we mapped three key epitopes of the S protein that recognize and bind anti-S antibodies. In this study, we determined the optimal positioning of a chimera comprising the three epitopes, fused with an LPTEG/biot-tag at the N-terminus of the Qβ-A minor coat protein for anti-S antibody titration. We determined the optimal chimera tag configuration to be epitope 3 (S) fused directly with the A at the N-terminus, followed by epitope 1 (S), epitope 2 (S), and the tag at the C-terminus. This work provides key insights into the druggability of the RBSM for developing SARS-CoV inhibitors and lays the foundation for designing a biosensor for antibody monitoring and a potential subunit vaccine.

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Original Article