Vascular dysregulation following SARS-CoV-2 infection involves integrin signalling


Researchers from the School of Pharmacy and Biomolecular Sciences at RCSI University of Medicine and Health Sciences-Ireland have in a new study found that integrins could be receptors for SARS-CoV-2 and that integrin signaling via a VE-Cadherin mediated pathway upon SARS-CoV-2 infection leads to vascular dysregulation.

The study findings were published on a preprint server and are currently being peer reviewed.

The interaction between the spike protein of SARS-coronavirus-2 (SARS-CoV-2) and endothelial cells has been widely demonstrated to be a critical driver in vascular dysregulation observed in COVID-19. We were the first to describe a pattern of impaired vascular functionality following SARS-CoV-2 infection, and theorized that the major endothelial adherens junction protein, VE-Cadherin, was involved1.

A plethora of data now confirms this finding, where the disruption of junction proteins leads to reduced endothelial barrier integrity and subsequent monolayer permeability, elucidating the vast cardiovascular complications and septic shock experienced in severe COVID-192. Although the canonical ACE2 receptor has been implicated in driving this reaction, another potential mechanism of action involves an integrin-mediated pathway.

These heterodimeric transmembrane proteins are key regulators of haemostasis, angiogenesis, proliferation, and inflammation. Activated through binding an RGD-containing ligand, integrins can control downstream signalling transduction cascades that tether VE-Cadherin at the cell junctions through RhoGTPase cycling3,4.

The spike protein contains an integrin-binding RGD motif that adheres to integrins αVβ3 and α5β1 on pulmonary epithelial cells and endothelial cells, where integrin antagonists Cilengitide and ATN-161 have demonstrated success in inhibiting this interaction in vitro and in vivo, thereby suggesting integrin-targeted therapeutics in COVID-191,5–8. We aimed to identify the direct pathway that associates integrins with vascular dysregulation during SARS-CoV-2 infection, and whether targeting the spike protein RGD motif is sufficient to reduce this disease phenotype.

Figure 3.
Schematic illustration depicting theorized intracellular signalling cascade following spike protein engagement with integrin.
Top panel portrays healthy endothelial cell, where VE-Cadherin and Rac1 regulate and maintain low RhoA levels through FAK and Src signalling. Rac1 and RhoA signalling is tightly controlled via integrin engagement with an extracellular ligand. Bottom panel portrays infected endothelial cell, where persistent integrin activation leads to overactive FAK and Src activity, resulting in faulty cycling between RhoA and Rac1. RhoA levels rise, leading to cadherin phosphorylation via Src and FAK. Catenins, which confine VE-Cadherin at the endothelial junctions, cannot recognize phosphorylated VE-Cadherin which results it its internalization. This causes endothelial cells to pull apart and permeability to occur, promoting vascular leakage.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel virus in the Betacoronavirus genus that causes coronavirus disease 2019 (COVID-19)1. SARS-CoV-2 was first reported in Wuhan, China, and currently persists as a global pandemic2,3. SARS-CoV-2 presents similar characteristics with the original SARS-CoV in genome structure, tissue tropism, and viral pathogenesis. However, SARS-CoV-2 is more transmissible than SARS-CoV.

Cellular entry of coronaviruses depends on binding of the viral spike (S) protein to a specific cellular receptor, the angiotensin-converting enzyme 2 (ACE2)4,5, and subsequent S protein priming by cellular protease activity such as Transmembrane Serine Protease 2 (TMPRSS2)6. Interestingly, ACE2 expression across different human tissues7 revealed low expression of ACE2 in the lungs compared to elevated expression in the kidney and heart8,9.

Nevertheless, studies have shown that type I and II interferons (IFNs) secreted during viral infection upregulate the transcription and expression of ACE210,11. Unlike its predecessor, SARS-Cov-2 expresses a novel K403R spike protein substitution encoding an Arginine-Glycine-Aspartic acid (RGD) motif12, introducing the potential for interacting with RGD-binding integrins, as likely mediators for viral cell entry and enhanced pathogenicity13.

ACE2 contains two integrin-binding domains: an RGD motif at position 204–206 and the sequence RKKKNKAR in the cytoplasmic tail at its C-terminus14. Also, ACE2 binds integrin β1 in the failing human heart14. Correlated increased expressions of β115 and ACE2 have been reported16,17. Others have shown that ACE2 interacts in cis with integrin β1 in a manner that enhances RGD-mediated cell adhesion18.

Integrins are heterodimeric transmembrane adhesion protein receptors composed of α and β subunits whose activation is tightly regulated and bidirectional19. Integrins can exist in three states characterized by their structural conformation and affinity for their ligands (Fig. 1A).

The inactive, bent-closed state (BCS) with a closed headpiece has a low affinity for extracellular matrix (ECM) ligands. The bent structure inhibits the receptors from inappropriate signaling due to random binding to extracellular matrix proteins. Integrins exhibit an extended-closed state (ECS) with a closed headpiece and higher ligand binding affinity than BCS when primed.

Active and extended-open state (EOS) presents an open headpiece and maximum affinity for ECM ligands20. Integrin function involves coordination with cytoskeletal components whose functions regulate cell adhesion and migration21,22. Changes in integrin conformation can elicit cell-signaling events that increase ligand affinity/avidity, promote cytoskeletal rearrangement, and enable virus internalization.

Ligand binding to integrins is mediated by divalent-cations bound at the Metal Ion Dependent Adhesion Site (MIDAS) domain on top of either the αI domain, in I domain-containing integrins, or the βI domain in non-αI integrins23. Physiologically, 1 mM Ca2+ and 1 mM Mg2+ in body fluid stabilize the BCS conformation. Under non-physiological conditions, 1 mM Mn2+ initiates and stabilizes ECS conformation even in the presence of Ca2+.

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Figure 1
Integrin conformational states antagonist targets and SARS-CoV-2 binding. (A) Integrin States: First, the inactive, bent-closed state (BCS), with a closed headpiece and low affinity for extracellular matrix (ECM) ligands. The bent structure inhibits the receptors from inappropriate signaling due to random binding to extracellular matrix proteins. In the BCS form, binding to large ligands is likely limited. Second, when primed, integrins exhibit an extended-closed state (ECS) with a closed headpiece and higher ligand binding affinity than BCS. Third, active and extended-open state (EOS) with an open headpiece and maximum affinity for ECM ligands. Integrin Affinity Regulation: Mn2+ binding to the MIDAS site at the αI and βI domain integrin induces integrin extension. α2β1 integrin antagonist BTT 3033 binds to the α-I domain, and stabilizes the BCS. GLP0187 blocks binding to the RGD ligand-binding domain. EOS binding to a macromolecular ligand or ECM generates a force (F) transmitted through the integrin β subunit. (B) Model of Sars-CoV-2 virion structure ( SARS-CoV-2 are spherical or ovoid particles of sizes that span the range of 60–140 nm. The SARS-CoV-2 virion consists of a lipid bilayer envelope membrane covering a large nucleoprotein (N)-encapsidated, positive-sense RNA genome. The lipid envelope is decorated with three transmembrane proteins consisting of trimeric spike proteins (S) that project above the lipid bilayer membrane and relatively small membrane (M) and envelope (E) proteins78,79. S proteins bind with high-affinity (1–50 nM)4 to the angiotensin-converting enzyme 2 (ACE2) for productive infection80. (C) Cartoon alignment of the receptor-binding domain (RBD) and RGD sequence on the trimeric spike protein, which favors engagement of activated integrin, adapted from ref.25 The illustrations were generated using Microsoft® PowerPoint Version 16.51 (21071101).

Many viruses use integrin-mediated endocytosis pathways for cell entry5,24. A recent bioinformatics-driven study predicted a model that placed integrins in a central ligating role, whereby SARS-CoV-2 could engage multiple receptors and form a multicomponent receptor complex and functional signaling platform25. Interestingly, ACE2 also has a similar MIDAS motif25. Still, it has not yet been established whether the ACE2 MIDAS domain has a potential role in creating synergy overlap between the ligand-binding profiles and regulation of ACE2 and integrins25.

Several in vitro studies have established experimental evidence in support of cognate binding interactions between SARS-CoV-2 spike proteins, integrin β126,27 and integrin β312,28. In addition, the transmembrane glycoprotein neuropilin 1 (NRP1), which is abundantly expressed in the olfactory epithelium and promotes the endocytosis of activated α5β1 integrin29–34, has been recently identified as a receptor for SARS-CoV-2 infection34,35.

In this study, we took a mechanistic approach to examine the role of integrins as effectors of SARS-CoV-2 cell entry and productive infection. First, we tested whether inducing a BCS to ECS integrin conformational change with Mn2+24,36 enhanced cell binding and entry of fluorescently tagged UV-inactivated SARS-CoV-2R18. Conversely, we used integrin extension or RGD-binding inhibitors to determine the inhibitors’ effect on cellular entry. Integrins signal bidirectionally via “inside-out” and “outside-in” signaling22,36–41.

Inside-out signaling is initiated by intracellular signaling upstream of talin, and other adaptor proteins binding to the integrin β-subunit cytoplasmic tail (β-CT), which causes integrin extension (ECS) and concomitant increases in high-affinity ligand binding21,22. Integrin engagement with macromolecular ligands stimulates the transient exchange of talin for Gα13’s occupancy of the β-CT42,43 which initiates integrin outside-in signaling.

In the context of viral infection, integrin outside-in signaling induces cell spreading, retraction, and internalization of integrin-associated ligands. We used cell-permeable inhibitors of integrin outside-in and inside-out signaling42 to test the role of canonical integrin signaling during cell entry of SARS-CoV-2R18 and infectious SARS-CoV-2. Taken together, our results demonstrate that integrins play a significant role in the infectivity of SARS-CoV-2.

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