Among the novel mutations distinguishing SARS-CoV-2 from similar coronaviruses is a K403R substitution in the receptor-binding domain (RBD) of the viral spike (S) protein within its S1 region. This amino acid substitution occurs near the angiotensin-converting enzyme 2–binding interface and gives rise to a canonical RGD adhesion motif that is often found in native extracellular matrix proteins, including fibronectin. Here, the ability of recombinant S1-RBD to bind to cell surface integrins and trigger downstream signaling pathways was assessed and compared with RGD-containing, integrin-binding fragments of fibronectin. We determined that S1-RBD supported adhesion of fibronectin-null mouse embryonic fibroblasts as well as primary human small airway epithelial cells, while RBD-coated microparticles attached to epithelial monolayers in a cation-dependent manner. Cell adhesion to S1-RBD was RGD dependent and inhibited by blocking antibodies against α v and β 3 but not α 5 or β 1 integrins. Similarly, we observed direct binding of S1-RBD to recombinant human α v β 3 and α v β 6 integrins, but not α 5 β 1 integrins, using surface plasmon resonance. S1-RBD adhesion initiated cell spreading, focal adhesion formation, and actin stress fiber organization to a similar extent as fibronectin. Moreover, S1-RBD stimulated tyrosine phosphorylation of the adhesion mediators FAK, Src, and paxillin; triggered Akt activation; and supported cell proliferation. Thus, the RGD sequence of S1-RBD can function as an α v -selective integrin agonist. This study provides evidence that cell surface α v -containing integrins can respond functionally to spike protein and raises the possibility that S1-mediated dysregulation of extracellular matrix dynamics may contribute to the pathogenesis and/or post-acute sequelae of SARS-CoV-2 infection.
【저자키워드】 SARS-CoV-2, ACE2, angiotensin-converting enzyme 2, RBD, receptor-binding domain, Receptor binding domain, Fibronectin, Cell adhesion, SARS, Severe acute respiratory syndrome, integrin, CoV, coronavirus, SPR, surface plasmon resonance, GST, glutathione S-transferase, extracellular matrix protein, ECM, extracellular matrix, FAK, focal adhesion kinase, FN-null MEF, fibronectin-null mouse embryonic fibroblast, Focal adhesions, RGD, Arg-Gly-Asp, SAEC, small airway epithelial cell,