SARS-CoV-2 binds heparan sulfate, preventing host cell infection

The continuing global outbreak of COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has taken a tremendous toll on life and health, besides the massive national and international economic costs of lockdowns, travel restrictions, and business shutdowns. It is, therefore, urgent to find vaccines or therapeutics capable of controlling the virus. In this context, a new study published in May 2020 on the preprint server bioRxiv* shows that the SARS-CoV-2 virus can bind to a sugar molecule called heparan sulfate (HS), thereby hindering its entry into the host cell.

Illustration of SARS-CoV-2, 2019 nCoV virusImage Credit: Orpheus FX / Shutterstock
Illustration of SARS-CoV-2, 2019 nCoV virusImage Credit: Orpheus FX / Shutterstock O By

How Does SARS-Cov-2 Enter A Host Cell?

SARS-CoV-2 binds to host cells via its spike (S) protein, which attaches through its receptor-binding domain (RBD, on the S1 subunit of the protein) to a cell surface molecule on the host cell called angiotensin-converting enzyme 2 (ACE2). The virus-ACE2 receptor binding sets the stage for viral entry, along with the preactivation of furin, a proprotein convertase enzyme in the host cell that is producing the virus. Such enzymes convert precursor or inactive states of peptides and proteins to their active state.

Furin acts on a unique cleavage site between the S1 and S2 subunits of the prefusion S protein. The S1 subunit now drops off, while the S2 component undergoes a conformational change, exposing the protein sequences that promote membrane fusion to allow virus entry.

Many human coronaviruses have a co-receptor or secondary receptor such as heparan sulfate, to promote cell entry. Similarly, the earlier SARS-CoV entry into laboratory cultured cells could be markedly reduced by heparin or heparinase. These findings indicate that HS is important in the virus’s ability to infect the host cell.

The current virus also undergoes a conformational change when the RBD binds to HS. This binding is possible because of the protease-binding cleavage site at S1/S2.

What Is Heparan Sulfate?

HS is a complex carbohydrate composed of multiple units of simple sugar, with attached sulfate residues at oxygen and nitrogen sites. It is a major part of the cell membrane and the extracellular matrix in all cells above the bacterial level of organization.

The functions of HS include interacting with multiple proteins to regulate cell adhesion, the cell cycle, and inflammation. Many viruses also make use of HS as a receptor or secondary receptor.

HS is synthesized under strict control, to produce varying lengths with different patterns and numbers of attached sulfate residues. Some scientists think that this enables different cells to exhibit specific HS binding sites that attract specific HS binding proteins. These are believed to act as regulators for a host of cellular and metabolic processes.

This “HS sulfate code hypothesis” is supported by the observation that certain proteins preferentially bound specific repeating patterns of oligosaccharides. The current study seeks to identify this pattern in the S protein binding of SARS-CoV-2 to help develop drugs that can block viral binding to the host cell.

The researchers made use of the extensive library of different HS molecules with well-defined variations in chain length, backbone chain composition, and pattern of sulfate attachment. Using well over a hundred unique HS molecules containing 2-8 sugar molecules each, they studied how the virus bound to each of them.

The researchers used insect cells and laboratory culture cell lines to express the S protein and the RBD of the virus, respectively. The compounds were placed in order of increasing backbone length, and within each group, by increasing sulfate number.

What Did the Study Show About Heparan Sulfate-SARS-CoV-2 Binding?

The researchers found that the S protein and the RBD were preferentially attached to only certain HS molecules, and both showed the same pattern of preference. The preferred HS compounds had from two to four disaccharide monomers with the composition (IdoA2S-GlcNS6S), with three sulfates attached to each.

Secondly, the binding depends on the length, with the strongest binding being to the compounds with four and three monomers, respectively. The two-monomer form, and the single monomer itself, showed little affinity.

The Importance of Structure

Changing around the backbone or sulfate pattern causes a marked decline in binding, or even prevents binding altogether. The replacement of just one IdoA2S with a non-sulfated sugar reduces affinity, and further substitutions abolish it, indicating the importance of this sugar residue. The sulfate at the 2-O position is essential for binding, while the 6-O sulfate also contributes a lot.

The S protein bound with increased firmness and affinity compared to the RBD. Further studies using surface plasma resonances to confirm the location of the HS binding site led to the hypothesis that the HS binding site of the RBD allows for sequence-specific binding, while the site at the S1/S2 cleavage site increases binding affinity.

The next step was to look at how the HS with strongest binding prevented the interaction of the viral protein with heparin. The experiment showed potent competitive inhibition of viral protein (both S and RBD) binding to heparin.

The Potential for Heparin and HS in COVID-19 Prevention and Treatment

Earlier researchers have reported the highly controlled synthesis and regulatory behavior of trimeric (IdoA2S-GlcNS6S)3 in several cell processes like the activation of endothelial cells. Such activation leads to inflammation in the arterial wall and surrounding tissue.

The IdoA2S-GlcNS6S monomer pattern is present in heparin to a large extent but forms only a small component of HS. These findings are important in light of the observed connection between the inappropriate endothelial activation and the clotting abnormalities in COVID-19. This could indicate the role played by the virus-HS binding on the host cell membrane.

Heparin- or HS-based drugs could be developed to block viral binding to the cell without inducing anticoagulation. By careful selection of sites at which sulfate can be removed, it is possible to prevent the activation of anticoagulant activity while maintaining viral binding.

*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.

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