bioRxiv2025
Virus entry is thought to involve binding a unique receptor for cell attachment and cytosolic entry. For SARS-CoV-2 underlying the COVID-19 pandemic, angiotensin-converting enzyme 2 (ACE2) is widely considered the receptor for cell-surface attachment and cytosolic entry. Using advanced light microscopy to resolve individual virions and receptors, we found instead that heparan sulfate (HS), not ACE2, mediates SARS-CoV-2 cell-surface attachment and subsequent endocytosis. ACE2 functions only downstream of HS to enable viral genome expression. Instead of binding single HS molecules that electrostatically interact with viral surface proteins weakly, SARS-CoV-2 binds clusters of [~]6-137 HS molecules projecting 60-410 nm above the plasma membrane. These tall, HS-rich clusters, present at about one per 6 m{superscript 2}, act as docking sites for viral attachment. Blocking HS binding with the clinically used HS-binding agent pixantrone strongly inhibited an authentic pathogen, the SARS-CoV-2 Omicron JN.1 subvariant, from attaching to and infecting human airway cells. This work establishes a revised entry paradigm in which HS clusters mediate SARS-CoV-2 attachment and endocytosis, with ACE2 acting downstream, thereby identifying HS interactions as a key anti-COVID-19 strategy. This paradigm and its therapeutic implications may apply broadly beyond COVID-19 because, analogous to SARS-CoV-2, HS binds many other viruses but is only considered an attachment regulator. Statement of SignificanceViral entry, a crucial antiviral target, is typically thought to involve binding its unique receptor for the cell surface attachment and subsequent entry. We examined this concept with advanced microscopies to resolve individual receptors and SARS-CoV-2 virions responsible for the COVID-19 pandemic. We discovered two receptors for viral entry: heparan sulfate, a polysaccharide that may bind many viruses, mediates viral attachment and subsequent endocytosis, whereas angiotensin-converting enzyme 2 (ACE2), the generally assumed SARS-CoV-2 receptor, acts only downstream to facilitate viral infection. This model suggests perturbation of HS binding as a more effective anti-COVID-19 strategy than previously recognized. It may apply broadly beyond COVID-19 because, analogous to SARS-CoV-2, HS binds many other viruses but is only considered an attachment regulator.