Highlights d SARS-CoV-2 spike protein interacts with heparan sulfate and ACE2 through the RBD d Heparan sulfate promotes Spike-ACE2 interaction d SARS-CoV-2 infection is co-dependent on heparan sulfate and ACE2 d Heparin and non-anticoagulant derivatives block SARS-CoV-2 binding and infection

We show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities.

Many protein ligands bind to heparan sulfate, which results in their presentation, protection, oligomerization or conformational activation. Binding depends on the pattern of sulfation and arrangement of uronic acid epimers along the chains. Sulfation at the C3 position of glucosamine is a relatively rare, yet biologically significant modification, initially described as a key determinant for binding and activation of antithrombin and later for infection by Type I Herpes Simplex virus. In mammals, a family of seven heparan sulfate 3-O-sulfotransferases installs sulfate groups at this position and constitutes the largest group of sulfotransferases involved in heparan sulfate formation. However, to date very few proteins or biological systems have been described that are influenced by 3-O-sulfation. This review describes our current understanding of the prevalence and structure of 3-O-sulfation sites, expression and substrate specificity of the 3-O-sulfotransferase family and the emerging roles of 3-O-sulfation in biology.

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The ciliopathy Joubert syndrome is marked by cerebellar vermis hypoplasia, a phenotype for which the pathogenic mechanism is unclear1–3. In order to investigate Joubert syndrome pathogenesis, we have examined mice with mutated Ahi1, the first identified Joubert syndrome gene4,5. These mice exhibit cerebellar hypoplasia with a vermis/midline fusion defect early in development. This defect is concomitant with expansion of the roof plate and is also evident in a mouse mutant for another Joubert syndrome gene, Cep2906,7. Further, fetal magnetic resonance imaging (MRI) from human subjects with Joubert syndrome reveals a similar midline cleft suggesting parallel pathogenic mechanisms. Previous evidence has suggested a role for Jouberin (Jbn), the protein encoded by Ahi1, in canonical Wnt signaling8. Consistent with this, we found decreased Wnt reporter activity at the site of hemisphere fusion in the developing cerebellum of Ahi1 mutant mice. This decrease was accompanied by reduced proliferation at the site of fusion. Finally, treatment with lithium, a Wnt pathway agonist9, partially rescued this phenotype. Our findings implicate a defect in Wnt signaling in the cerebellar midline phenotype seen in Joubert syndrome, which can be overcome with Wnt stimulation.