Potential blocker of SARS-CoV entry and a narrow functionality of its spike protein motifs on Qubevirus platform

Targeted disruption of SARS-CoV entry remains a critical strategy in antiviral therapeutic design. Central to this process is the viral spike (S) protein, which mediates host recognition via 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_473-492) that recognize hACE2. Using the Qubevirus (Qβ) platform, we 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 exhibits no cytotoxic effect on HEK293T cells and reduces the infectivity of SARS-CoV pseudovirus in a competitive assay, as a blocker of SARS-CoV entry. In addition, building upon our previous studies, we determined the optimal positioning of a chimera comprising the three epitopes mapped, fused with an LPTEG/Biot-tag at the N-terminus of the Qβ-A₁ minor coat protein for anti-S antibody titration. We determined the optimal chimera tag configuration to be epitope 3 (S_781-800) fused directly with the A₁ at the N terminus, followed by epitope 1 (S_441-460), epitope 2 (S_601-620), 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.