Integrin α₅β₁ contributes to cell fusion and inflammation mediated by SARS-CoV-2 spike via RGD-independent...

The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infects host cells by engaging its spike (S) protein with human ACE2 receptor. Recent studies suggest the involvement of integrins in SARS-CoV-2 infection through interaction with the S protein, but the underlying mechanism is not well understood. This study investigated the role of integrin α₅β₁, which recognizes the Arg-Gly-Asp (RGD) motif in its physiological ligands, in S-mediated virus entry and cell-cell fusion. Our results showed that α₅β₁ does not directly contribute to S-mediated cell entry, but it enhances S-mediated cell-cell fusion in collaboration with ACE2. This effect cannot be inhibited by the putative α₅β₁ inhibitor ATN-161 or the high-affinity RGD-mimetic inhibitor MK-0429 but requires the participation of α₅ cytoplasmic tail (CT). We detected a direct interaction between α₅β₁ and the S protein, but this interaction does not rely on the RGD-containing receptor binding domain of the S1 subunit of the S protein. Instead, it involves the S2 subunit of the S protein and α₅β₁ homo-oligomerization. Furthermore, we found that the S protein induces inflammatory responses in human endothelial cells, characterized by NF-κB activation, gasdermin D cleavage, and increased secretion of proinflammatory cytokines IL-6 and IL-1β. These effects can be attenuated by the loss of α₅ expression or inhibition of the α₅ CT binding protein phosphodiesterase-4D (PDE4D), suggesting the involvement of α₅ CT and PDE4D pathway. These findings provide molecular insights into the pathogenesis of SARS-CoV-2 mediated by a nonclassical RGD-independent ligand-binding and signaling function of integrin α₅β₁ and suggest potential targets for antiviral treatment.