An RGD motif on SARS-CoV-2 Spike induces TGF-β signaling and downregulates interferon

ABSTRACT The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates canonical cell entry via ACE2 and has also been implicated as an activator of a diverse range of signaling pathways. Here, we present evidence that the RGD (Arg-Gly-Asp) motif within the receptor-binding domain (RBD) of the S1 fragment of the S protein induces TGF-β cytokine expression. RGD peptides are well characterized as ligands for a subset of integrin complexes primarily containing α5 and αV subunits. In this study, we investigate the molecular basis of TGF-β pathway activation by S protein, delivered to cells as recombinant protein, in pseudotyped virus or in virally infected cells. Activation of TGF-β signaling by the S protein requires ACE2 and leads to SMAD3-dependent expression of the pro-fibrotic marker PAI-1. Utilizing pseudotyped viruses, expression of the S protein with a mutated RGD motif abolished TGF-β signaling, as did the RGD antagonist ATN-161, implicating integrin complexes in mediating this response. We show that the S protein RGD motif suppresses IFN-β expression via TGF-β, leading to a disruption in cellular antiviral defenses, consistent with TGF-β’s role in immunosuppression. These findings further support the multifunctionality of S protein and provide mechanistic insights into its activity as a virulence factor during infection. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents an ongoing public health challenge as a cause of acute illness and post-acute sequelae of COVID-19 (PASC, or long COVID). Our study identifies the RGD integrin-binding motif in the spike (S) protein as central to the cellular response to SARS-CoV-2, leading to the expression of the pleiotropic cytokine TGF-β and disabling of antiviral immunity. This work further supports the S protein-to-integrin complex signaling axis as a potential therapeutic target. The RGD motif might also be a valid target for treating PASC given the increasing body of evidence implicating the presence of persistent S protein in the etiology of this disease.