Heparin may stop SARS-CoV-2 infecting host cells

Researchers at the University of Sheffield have developed a new assay that can be used to assess the attachment of viruses to host cells and to test potential inhibitors of viral infection.

Using the assay, the team was able to demonstrate binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein to human cells expressing angiotensin-converting enzyme 2 (ACE2).

The spike protein is the main structure that SARS-CoV-2 uses to bind to ACE2 receptors expressed on target cells, before infecting them and potentially causing coronavirus disease 2019 (COVID-19).

SARS-CoV-2 viruses binding to ACE-2 receptors on a human cell, the initial stage of COVID-19 infection. Illustration credit: Kateryna Kon / Shutterstock

The researchers also found that incubating the cells with unfractionated heparin, stopped the spike protein binding to them.

A pre-print version of the paper can be accessed in the server bioRxiv*, while the paper undergoes peer review.

Mechanism of SARS-CoV-2 infection

On binding to ACE2, the spike protein undergoes host cell proteolytic cleavage into two subunits: S1, which contains the receptor-binding domain (RBD) and S2, which enables fusion with the host cell membrane and viral entry.

“A cell-surface host serine protease, TMPRSS2 [transmembrane serine proteinase 2], is also thought to be involved in viral entry and is proposed to cleave S1 and S2, leading to activation of the fusion machinery,” write Peter Monk and colleagues.

The new assay used cells that express both ACE2 and TMPRSS2

To investigate SARS-CoV-2 binding to host cells, the team developed a new assay using the RT4 urinary bladder transitional carcinoma cell line, which expresses both ACE2 and TMPRSS2.

They found that an intact recombinant form of the viral spike protein containing both S1 and S2 (S1S2), but not the S1 domain alone, binds strongly to RT4 cells in a temperature-dependent manner.

Binding activity sharply increased at 37°C, suggesting that proteolytic cleavage was likely to be involved, says the team.

Are there any other mechanisms of viral entry?

Monk and colleagues say that most cell types only express quite low levels of ACE2, suggesting that the spike protein might also interact with other receptor sites to gain viral entry.

Certain viruses such as herpes simplex are already known to bind with host glycosaminoglycans called heparan sulfates, says the team.

In addition, a study by one group suggested that the soluble glycosaminoglycan heparin can inhibit the entry of SARS CoV-2 into “Vero” cells – a cell line derived from monkey kidney epithelia.

“These authors also showed that heparin could interact with recombinant S1 RBD and cause conformational changes, leading to the suggestion that SARS-CoV-2 might use host heparan sulfates as an additional attachment site during infection,” write the researchers.

Unfractionated heparin completely stopped the binding

Given that the new assay already seemed to mimic some features of SARS-CoV-2 infection, the researchers used it to test the effects of incubating RT4 cells with heparin at 37°C.

The team reports that unfractionated heparin (UFH) completely inhibited the binding of S1S2 to RT4 cells.

Treating the cells with two low molecular weight heparins (LMWHs) that are already in clinical use also inhibited the binding, but only partially and not as strongly.

“This suggests that heparin, particularly unfractionated forms, could be considered to reduce clinical manifestations of COVID-19 by inhibiting continuing viral infection,” write Monk and team.

Could the spike protein also bind host cell heparan sulfate?

The authors say the interaction they observed between heparin and the spike protein suggests that it might also bind to host cell heparan sulfate.

To test this hypothesis, they treated RT4 cells with a blend of heparinase I and III, enzymes that degrade heparan sulfate molecules, before testing the binding of S1S2.

The treatment did not result in any significant reduction in the binding of RT4 cells, suggesting that heparan sulfates do not play any significant role in the attachment of SARS-CoV-2 spike protein to host cells:

“Although our data supports the inhibitory activity of UFH, it does not support the conjecture that heparan sulfates are essential for viral infection,” writes the team.

What are the implications of the study?

The researchers say that LMWHs, which have already been used to treat COVID-19 patients and been shown to improve outcomes, are much smaller than UFH and have pharmacokinetics that is easier to predict.

Monk and colleagues think their work suggests that earlier use of heparin should be considered when a viral infection is still an important factor in influencing the severity of disease.

“The use of UFH rather than LMWH should also be considered, although we note that administration and the safety profile of UFH might preclude this in some cases,” they add.

Finally, the researchers say their newly developed flow cytometric assay for assessing the binding of SARS-CoV-2 spike protein to host cells lends support to a previous finding that heparin can inhibit viral attachment to monkey kidney epithelial cells.

“Our new assay could be a useful first screen for novel inhibitors of coronavirus infection,” concludes the team.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.