Froese, J.; Mandalari, M.; Civera, M.; Elli, S.; Pagani, I.; Vicenzi, E.; Garcia-Monge, I.; Di Iorio, D.; Frank, S.; Bisio, A.; Lenhart, D.; Gruber, R.; Yates, E. A.; Richter, R. P.; Guerrini, M.; Wegner, S. V.; Grobe, K.

The heparan sulfate (HS)-rich extracellular matrix (ECM) serves as an initial interaction site for the homotrimeric spike (S)-protein of SARS-CoV-2 to facilitate subsequent docking to angiotensin-converting enzyme 2 (ACE2) receptors and cellular infection. Recent variants of concern (VOCs), notably Omicron, have evolved by swapping several amino acids to positively charged residues to enhance the S-protein trimer\'s interaction with the negatively charged HS polysaccharide chains in the matrix. These increased interactions, however, may reduce Omicron\'s ability to move through the HS-rich ECM to effectively find ACE2 receptors and infect cells, and raise the question of how HS-associated virus movement can be mechanistically explained. In this work, we show that Omicron S-proteins have evolved to balance HS interaction stability and dynamics, resulting in enhanced mobility on an HS-functionalized artificial matrix. Both properties are achieved by the ability of Omicrons S-proteins to cross-link at least two HS chains, providing both high avidity to retain the protein inside the HS-rich matrix, and fast dynamics, thus enabling direct S-protein switching between HS chains as a prerequisite for mobility at the cell surface. Optimized HS interactions can be targeted pharmaceutically, because an HS mimetic significantly suppressed surface binding and cellular infection specifically of the Omicron VOC. These findings suggest a robust way to interfere with SARS-CoV-2 Omicron infection and, potentially, future variants.