Fig. 2: 4H30 could cluster virus together to block viral entry and fusion.

a 4H30 increased virus attachment to VeroE6 cells. When cells were pretreated by 4H30 (25 μg/mL) 1 h before viral infection (1h-Pre) or when the virus was pretreated by 4H30 before viral infection (Pre-mix), the viral RNA copies in infected cell lysate were measured at 1 hpi. RNA copy (fold) was normalized to DMEM-treated virus (n = 4). b 4H30 could inhibit viral replication at 6 hpi when the virus was pretreated by 4H30 (n = 4). Viral loads in cell lysate were measured by RT-qPCR and viral RNA copy was normalized to that of 1 hpi, correspondingly. P values were generated by comparison with DMEM. c 4H30 (150, 37.5, or 0 ng) could more effectively bind to spike when compared with ACE2 (n = 8). Spike or ACE2 (100 ng) was added to coated 4H30 on the ELISA plate. Spike and ACE2 (S-ACE2, 100 ng) coated on an ELISA plate were the positive controls. *P < 0.05 when compared with ACE2. d 4H30 did not reduce ACE2 binding to spike (n = 6). Spike bound to ACE2 (150 ng) coated on ELISA plate. Ab, neutralizing antibody of the spike. **P < 0.01 when compared with PBS. e 4H30 (50 μg/mL) could significantly inhibit SARS-CoV-2 when 4H30-treated virus (10⁶ PFU/mL) was diluted by 10,000 folds for plaque reduction assay (n = 4). Cells without infection was the negative (Neg) control. *P < 0.05 when compared with PBS. f 4H30 could cross-link SARS-CoV-2 to form big viral clusters and block virus entry through the Calu-3 cell membrane. SARS-CoV-2 (green) was treated by DMEM, 4H30 or H30 before infecting cells, or cells were treated by 4H30 before viral challenge (4H30-pretreat cell). Cells without infection was the negative (Neg) control. Representative images were taken at 1 hpi in Calu-3 cells. White triangles indicated the clustered viral particles. Scale bar, 20 μm. g 4H30 could inhibit spike-ACE2-mediated cell fusion in 293T cells. 293T-ACE2 cells were cocultured with 293T with a spike (+Spike) or without spike (–Spike) under the treatment of 4H30 (40 μM), bafilomycin A1 (50 nM), or remdesivir (80 μM). Representative images were taken at 6 h post coculture. Fused cells (+Spike, > 50 μm) were larger than unfused cells (–Spike, ~10 μm), the 4H30-treated cells (+Spike+4H30) or BA1-treated cells. Scale bar, 200 μm. P values were calculated by the two-tailed Student’s t-test. Data were presented as means ± SD of indicated biological samples with more than two independent experiments.