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In this study, Jianshe Lang et al. analyze two aspects of SARS-CoV. On the one hand, they analyze Lactoferrin (LF) as a potential SARS-CoV inhibitor. On the other hand, they introduce a novel binding mechanism, at the time, in that SARS-CoV cell binding is enhanced by Heparan Sulfate Proteoglycans (HSPG) receptor binding before functional cell entry receptors are employed (such as ACE2).
At the time, exogenous heparin and LF were already known as HSPG binders [PMID2246253] [10.1161/01.atv.14.12.2025]. This study further verified the colocalization of LF with HSPG receptors. ACE2 was considered as the primary functional entry receptor.
Mean values of triplicate samples were used. Standard deviations and p values are given.
The pseudovirus infection assays and WB assays showed a clear LF-mediated concentration-dependent inhibition on S protein-mediated viral infection ability. IC50 was ca. 0.7 µM. The mock virus control verified that the inhibition targeted the S protein. The S1190 assays showed that LF inhibited S protein-cell fusion, but the inhibition was not mediated directly by S protein-ACE2-Myc binding. It further showed that heparin neutralizes the inhibitory effect of LF. Heparin showed weaker infection inhibition despite its stronger competitive binding affinity when compared to LF. Microscopy showed the colocalization of LF and heparin on the cell membrane surface. The authors depict a figure (Fig. 3B) that supports the hypothesis that heparin competes with LF for HSPG receptor binding. Heparin sulfate degradation via Heparinase I could inhibit S1190-Fc binding and pseudovirus infection. The single dosage they used led to ca. halved infection ability. That was consistent with the curve of heparin inhibition on infection ability, indicating a lower limit can be reached in the 0.4 to 0.6 range. Chondroitin sulfate degradation did not show a significant effect. A comparison of LF, heparin, and Heparinase on SARS pseudovirus infection ability showed different results in different cell lines (i.e., Vero E6, and Caco-2). Overall, LF showed the best concentration effectivity. VSV-G infection could also be inhibited by these three agents. However, LF did show less effect than heparin and relatively less effect than heparinase when compared to the SARS pseudovirus results.
In 2011, the authors still assumed that ACE2 is the only cell surface entry receptor. There are inconsistencies in the methods and results section. It’s unclear if they used 0.43 or 0.34 µM LF for their pseudovirus assays. Not all experiments are mentioned in the methods section. Some are also introduced in the figure descriptions. The flow cytometry figure is incomplete (Fig. 1G). The confocal microscopy only verified the colocalization of LF and heparin on cell membrane surface but no ACE2-deficient cell line or pure HSPG binding assay was used as a control to prove that the interaction is purely mediated HSPGs. There is no clear explanation of how the inhibition of heparin on LF-HSPG binding was measured (Fig. 3B). Enzymatic experiments lacked different concentrations in the design. It’s unclear if a knockout effect could be reached, which seems unlikely, though, since transmembrane entry proteins aren’t blocked. Still, it would be helpful to quantify the maximum impact and the relative expression of free HSPG necessary for effective virus adsorption. They used heparin, however, to find the limits of HSPGs involvement in cell infection rate that indicate at least a two-fold virus infection due to HSPG adsorption, consistent with their enzymatic results. The finding that the three tested treatments (i.e., LF, heparin, and heparinase) showed inverse concentration correlations for SARS-CoV and VSV-G indicates that either LF or heparin could have a secondary inhibitory mechanism in either SARS-CoV or VSV-G. These comparative experiments lack different concentrations in their design.
If SARS-CoV-2 utilizes the same S protein adsorption mechanism to support cell binding, this will be important for many other studies. It seems unlikely that HSPG adsorption only supports ACE2-mediated cell entry. The authors of this study speculate that the high expression of HSPGs but low binding affinity first lets SARS-CoV anchor to HSPG and then transfer to cell entry proteins that express a higher binding affinity than HSPG, consistent with the knowledge from other viruses [10.1002/med.1026]. If SARS-CoV-2 encodes a similar “viral surfing” mechanism, this finding would also be significant for in vitro study designs since the HSPG expression of cell lines can have a substantial impact on the measurement results.
All in all, HSPG plays an essential role in the pathogenesis of SARS-CoV. Protein interaction simulation could verify if SARS-CoV-2 has a similar binding affinity to heparan sulfate for a start.
Title: Inhibition of SARS Pseudovirus Cell Entry by Lactoferrin Binding to Heparan Sulfate Proteoglycans
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Link: https://doi.org/10.1371/journal.pone.0023710
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Citation: doi:10.1371/journal.pone.0023710
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