Lactoferrin localizes to the cell membrane by targeting HSPGs.

<p>(<b>A</b>) LF is present on the cell surface. Oregon Green-labeled LF localization was observed under confocal microscopy. LF untreated cells were used as control. Cell membrane and nuclei were stained with DiI and Hoechst33342, respectively. Scale bar, 8 µm. (<b>B</b>) Heparin inhibits LF binding to HEK293E/ACE2-Myc cells. HEK293E/ACE2-Myc cells were incubated with 0.5 µM Oregon Green-labeled LF at 4°C for 1 h after pretreatment with heparin at the concentration of 3 µM, 10 µM or 30 µM. The MFI was measured for each group by flow cytometry as described above. Error bars represent the SD of three independent experiments. **P<0.01.</p

Lactoferrin blocks the interaction between spike protein and HEK293E/ACE2-Myc cells in an ACE2-independent fashion.

<p>(<b>A</b>) LF inhibits the binding of S1190-Fc to HEK293E/ACE2-Myc cells. Before incubation of S1190-Fc with HEK293E/ACE2-Myc cells at 4°C for 1 h, the cells were treated with LF at 37°C for 1 h at concentrations of 1 µM, 3 µM and 10 µM. Fc protein was used as a control. S1190-Fc binding to the cells was detected by flow cytometry as described in Methods. Error bars represent the SD of three independent experiments. ***P<0.001, **P<0.01 and *P<0.05. (<b>B</b>) LF does not disrupt the binding of S1190-Fc to ACE2-Myc. Error bars represent the SD of three independent experiments. ** P<0.01.</p

LF, heparin or enzymatic removal of cell surface HSPGs can prevent SARS pseudovirus entry into Vero E6 or Caco-2 cells.

<p>(<b>A</b>) Interference of the interaction between Vero E6 and SARS pseudovirus by LF, heparin and heparinase leads to reduction of viral infection. Vero E6 cells were treated by the same methods above with 10 µM LF, 10 µM heparin or 10 U of heparinase I, respectively. Then, the cells were incubated with SARS pseudovirus as described in Methods. GFP-expressing cells in the total population were analyzed by flow cytometry. The relative viral infection ratio was measured by comparing the percentage of GFP expressing cells of each group to that of the control. Error bars represent the SD of three independent experiments. *** P<0.001 and **P<0.01. (<b>B</b>) Incubation with LF or heparin, or degradation of HSPGs by heparinase inhibits SARS pseudovirus infection of Caco-2 cells. The Caco-2 cells were treated by same methods as described above. *** P<0.001, **P<0.01 and *P<0.05.</p

Incubation with heparin and degradation of HS polysaccharides on the cell surface by heparinase inhibits SARS pseudovirus infection of HEK293E/ACE2-Myc cells.

<p>(<b>A</b>) Heparin inhibits SARS pseudovirus entry into HEK293E/ACE2-Myc cells. Before incubation with SARS pseudovirus at 37°C for 4 h, HEK293E/ACE2-Myc cells were treated with heparin for 10 min at the concentration of 0.625 µM, 1.25 µM, 2.5 µM, 5 µM or 10 µM. GFP-expressing HEK293E/ACE2-Myc cells in the total population were analyzed by flow cytometry. The relative viral infection ratio was measured by comparing the percentage of GFP expressing cells of each group to that of the BSA control. Error bars represent the SD of three independent experiments. (<b>B</b>) Lysis of cell-surface HS by heparinase blocks the infection of HEK293E/ACE2-Myc cells by SARS pseudovirus. After incubation with 10 U of heparinase I or chondroitinase ABC for 1 h at 37°C, the cells were treated with SARS pseudovirus as described above. The relative viral infection ratio was calculated by the same method. *P<0.05.</p

Lactoferrin exerts its inhibitory effect on spike protein and SARS pseudovirus by binding to cell-surface HSPGs.

<p>(<b>A</b>) Heparin neutralizes the LF-mediated inhibition of S1190-Fc binding to HEK293E/ACE2-Myc cells. Before treatment with 10 µM LF for 1 h at 37°C, HEK293E/ACE2-Myc cells were incubated with heparin for 10 min at the concentration of 1 µM, 3 µM or 10 µM. S1190-Fc was added to each group to detect S1190-Fc binding to the cells as described above. Error bars represent the SD of three independent experiments. ***P<0.001, **P<0.01 and *P<0.05. (<b>B</b>) Inhibition of SARS pseudovirus infection of HEK293E/ACE2-Myc cells by LF can be partially neutralized by heparin. HEK293E/ACE2-Myc cells were treated with heparin for 10 min at the concentration of 1 µM, 3 µM or 10 µM. Then, 10 µM LF was added to each group and incubated at 37°C for 1 h. The GFP-expressing HEK293E/ACE2-Myc cells in the total population were analyzed as described above. The relative viral infection ratio was measured by comparing the percentage of GFP expressing cells of each group with that of the BSA control. Error bars represent the SD of three independent experiments. *** P<0.001, ** P<0.01 and *P<0.05.</p

Lactoferrin inhibits SARS pseudovirus infection of HEK293E/ACE2-Myc cells.

<p>(<b>A–D</b>) Fluorescence microscopy illustrates that the number of SARS pseudovirus-infected GFP-expressing HEK293E/ACE2-Myc cells decreases in the presence of LF. HEK293E/ACE2-Myc cells were treated with LF for 1 h at 37°C at the concentration of 1 µM (B), 3 µM (C) or 10 µM (D). BSA (10 µM) was used as control (A). The LF-pretreated cells were treated with SARS pseudovirus as described in Methods. (<b>E and F</b>) Western blotting reveals that LF markedly reduces GFP expression in HEK293E/ACE2-Myc cells incubated with SARS pseudovirus. Statistical analysis of the relative band density ratio of GFP to actin was performed using a <i>t</i>-test. Error bars represent the SD of three independent experiments. **P<0.01 and *P<0.05. (<b>G and H</b>) Flow cytometry demonstrates that LF is able to inhibit the infection of HEK293E/ACE2-Myc cells by SARS pseudovirus. The concentration of LF was 0.625 µM, 1.25 µM, 2.5 µM, 5 µM or 10 µM. BSA (10 µM) served as control. The percentage of GFP expressing cells in the total population was calculated by flow cytometry. The relative viral infection ratio was measured by comparing the percentage of GFP expressing cells in each group with that of the BSA control. Error bars represent the SD of three independent experiments. (<b>I</b>) No GFP expression can be detected in the cells treated with viral particles without spike protein. The percentage of GFP expressing cells in the total population was calculated by flow cytometry as described above. Error bars represent the SD of three independent experiments.</p

HSPGs are also involved in VSV-G virus cell entry.

<p>HEK293E/ACE2-Myc cells were treated with 10 µM LF, 10 µM heparin or 10 U of heparinase I, respectively, in the same way. And the same methods were used for the subsequent assay as described above. GFP-expressing cells in the total population were analyzed by flow cytometry. The relative viral infection ratio was measured by comparing the percentage of GFP expressing cells of each group to that of the control. Error bars represent the SD of three independent experiments. *** P<0.001 and **P<0.01.</p

Addition of exogenous heparin and enzymatic removal of HS chains by heparinase reduce spike protein binding to HEK293E/ACE2-Myc cells.

<p>(<b>A</b>) Heparin blocks the binding of S1190-Fc to HEK293E/ACE2-Myc cells. HEK293E/ACE2-Myc cells were incubated with heparin for 10 min at the concentration of 10 µM, 30 µM or 100 µM. S1190-Fc was added to each group and incubated at 4°C for 1 h. The MFI of each group was measured as described above. Error bars represent the SD of three independent experiments. *** P<0.001, **P<0.01 and *P<0.05. (<b>B</b>) Enzymatic degradation of cell-surface heparan sulfate (HS) chains reduces S1190-Fc binding. After treatment with 10 U of heparinase I, the cells were incubated with S1190-Fc at 4°C for 1 h. The MFI test was performed using the same method as above.</p