Fig. 3: 4H30 could inhibit SARS-CoV-2 release.

a 4H30 could inhibit SARS-CoV-2 release at 10 hpi (n = 6). 4H30 was added to infected cells at 6 hpi. The supernatant viral titers were measured at 10 hpi. Viral RNA copy (%) was normalized to DMEM. Cells without infection was the negative (Neg) control. **P < 0.01 when compared with DMEM. b 4H30 inhibited SARS-CoV-2 release at 18 hpi as determined by anti-spike staining. Blue, nuclear. Red, membrane staining. Green, anti-spike staining. Scale bar, 20 μm. c Chondroitin sulfate (CS) and heparan sulfate (HS) could reduce the inhibition of 4H30 on virus release at 18 hpi (n = 6). The virus was treated with the indicated peptides or glycosaminoglycans (CS or HS) at 14 hpi and viral loads in culture supernatants were measured by RT-qPCR at 18 hpi. 4H30 + CS: 4H30 was premixed with CS. 4H30 + HS: 4H30 was premixed with HS. 4H30 + BSA: 4H30 was premixed with BSA for 30 min before adding to the virus. Viral RNA copy (%) was normalized to DMEM. **P < 0.01 and *P < 0.05 when compared with 4H30. d 4H30 could bind to CS and HS (n = 4). CS or HS (300, 75, 0 ng = BSA) was coated on ELISA plate. 4H30 binding to CS or HS was measured by anti-HBD2. 4H30 coated on an ELISA plate was used as the positive control. **P < 0.01 when compared with “0”. e Cleaving CS and HS by chondroitinase ABC (ChABC) and Heparanase (Hase) could reduce the inhibition of 4H30 on viral release (n = 4). ChABC and Hase were added to infected VeroE6 cells at 14 hpi for removing CS and HS at 37 °C for 2 h and then 4H30 was added to cells at 16 hpi and viral titers in supernatants were measured by RT-qPCR at 20 hpi. f 4H30 increasing SARS-CoV-2 attachment to A549 cells at 4 °C could be reduced by CS or HS treatment (n = 5). Virus attachment to A549 cells of SARS-CoV-2 treated by DMEM, 4H30, BSA, CS, HS, 4H30 + BSA, 4H30 + CS, and 4H30 + HS was measured by RT-qPCR. Cells without infection was the negative (Neg) control. Viral RNA copy (%) was normalized to DMEM. **P < 0.01 when compared with 4H30. g The attachment of SARS-CoV-2 to 4H30-treated A549 cells could be reduced by ChABC and Hase (n = 8). A549 cells were pretreated by buffer (Mock) or ChABC+Hase and then further treated by DMEM or 4H30. SARS-CoV-2 attachment to the treated cells was measured by RT-qPCR. Viral RNA copy (%) was normalized to DMEM. **P < 0.01 when compared with DMEM. h High concentration of CS and HS (1250 ng/mL) could reduce the antiviral activity of 4H30 in VeroE6 (n = 6). **P < 0.01 when compared with 4H30. i 4H30 could significantly inhibit viral replication in the presence of a low concentration of CS or HS (320, 80 or 0 ng/mL). Virus with the indicated treatment grew in VeroE6 cells and the supernatant virus was measured by RT-qPCR at 24 hpi (n = 6). **P < 0.01 and *P < 0.05 when compared with DMEM. 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.