Disruption of\nMolecular Interactions between the G3BP1\nStress Granule Host Protein and the Nucleocapsid (NTD-N) Protein Impedes\nSARS-CoV‑2 Virus Replication

The\nRas GTPase-activating protein SH3-domain-binding\nprotein 1\n(G3BP1) serves as a formidable barrier to viral replication by generating\nstress granules (SGs) in response to viral infections. Interestingly,\nviruses, including SARS-CoV-2, have evolved defensive mechanisms to\nhijack SG proteins like G3BP1 for the dissipation of SGs that lead\nto the evasion of the host’s immune responses. Previous research\nhas demonstrated that the interaction between the NTF2-like domain\nof G3BP1 (G3BP1<sub>NTF‑2</sub>) and the intrinsically disordered\nN-terminal domain (NTD-N<sub>1–25</sub>) of the N-protein plays\na crucial role in regulating viral replication and pathogenicity.\nInterestingly, the current study identified an additional upstream\nstretch of residues (128KDGIIWVATEG138) (N<sub>128–138</sub>) within the N-terminal domain of the N-protein (NTD-N<sub>41–174</sub>) that also forms molecular contacts with the G3BP1 protein, as revealed\nthrough <i>in silico</i> analysis, site-directed mutagenesis,\nand biochemical analysis. Remarkably, WIN-62577, and fluspirilene,\nthe small molecules targeting the conserved peptide-binding pocket\nin G3BP1<sub>NTF‑2</sub>, not only disrupted the protein–protein\ninteractions (PPIs) between NTD-N<sub>41–174</sub> and G3BP1<sub>NTF‑2</sub> but also exhibited significant antiviral efficacy\nagainst SARS-CoV-2 replication with EC<sub>50</sub> values of ∼1.8\nand ∼1.3 μM, respectively. The findings of this study,\nvalidated by biophysical thermodynamics and biochemical investigations,\nadvance the potential of developing therapeutics targeting the SG\nhost protein against SARS-CoV-2, which may also serve as a broad-spectrum\nantiviral target.