Integrin activation is an essential component of SARS-CoV-2 infection.

Cellular entry of coronaviruses depends on binding of the viral spike (S) protein to a specific cellular receptor, the angiotensin-converting enzyme 2 (ACE2). Furthermore, the viral spike protein expresses an RGD motif, suggesting that cell surface integrins may be attachment co-receptors. However, using infectious SARS-CoV-2 requires a biosafety level 3 laboratory (BSL-3), which limits the techniques that can be used to study the mechanism of cell entry. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating both cell entry and productive infection. We used flow cytometry and confocal fluorescence microscopy to show that fluorescently labeled SARS-CoV-2 ^R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn ²⁺ , which activates integrins and induces integrin extension, enhances cell binding and entry of SARS-CoV-2 ^R18 in proportion to the fraction of integrins activated. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2 ^R18 with basal state integrins, but is ineffective against Mn ²⁺ -activated integrins. At the same time, RGD-integrin antagonists inhibited SARS-CoV-2 ^R18 binding regardless of integrin activity state. Integrins transmit signals bidirectionally: 'inside-out' signaling primes the ligand binding function of integrins via a talin dependent mechanism and 'outside-in' signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα ₁₃ and induces cell spreading, retraction, migration, and proliferation. Using cell-permeable peptide inhibitors of talin, and Gα ₁₃ binding to the cytoplasmic tail of an integrin's β subunit, we further demonstrate that talin-mediated signaling is essential for productive infection by SARS-CoV-2.