The Intricate Role of α5β1 Integrin in SARS-CoV-2 Spike-Mediated Infection and COVID-19 Pathogenesis

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, employs angiotensin-converting enzyme 2 (ACE2) as its cell entry receptor, akin to its predecessor SARS-CoV. Nevertheless, the broader cell tropism, heightened transmission rates, and a more intricate pathogenesis of SARS-CoV-2 suggest the involvement of additional host factors and mechanisms specific to this virus (1, 2).

Dysregulated inflammatory responses, characterized by elevated levels of cytokines such as IL-6 and IL-1β, alongside vascular endothelial cell injury, are linked to the severity of COVID-19 (3, 4). While respiratory epithelial cells are the primary targets of SARS-CoV-2, dysfunction in various cell types within vascular, immune, and central nervous systems contributes to the complex pathogenesis of the disease (5–7).

Innate immune receptors, including toll-like receptors (TLRs), are implicated in COVID-19 inflammation (8). However, the specific host receptors responsible for the intricate pathogenesis of COVID-19 remain to be fully elucidated.

SARS-CoV-2 employs its spike (S) protein to bind to ACE2 for cell entry (9). Recent studies have suggested potential roles of integrins in SARS-CoV-2 infection and COVID-19 pathogenesis (10–23). Integrins, a family of cell adhesion receptors, play crucial roles in various biological processes, including immune response and blood clotting (24). Some viruses utilize integrins as receptors or coreceptors to facilitate infection (25–27).

The consideration of integrins as putative receptors for SARS-CoV-2 arose from the presence of an integrin-binding RGD (Arg-Gly-Asp) motif on the SARS-CoV-2 S (SARS2-S) protein (28, 29). Among the integrins recognizing the RGD motif, α5β1, αVβ3, and αIIbβ3 have been reported to interact with the SARS2-S protein (12, 14, 17–19). However, studies have presented inconsistencies and controversies, with some evidence obtained from cell-based assays such as cell adhesion (19, 30). The potential ligand-binding inhibitor ATN-161 for α5β1 has been utilized in SARS-CoV-2 infection and cellular function assays (10, 20, 31). Nevertheless, ATN-161 is not a direct RGD-blocking inhibitor, and its mechanism of inhibition remains undefined.

Objective

The objective of this study was to investigate the potential role of α5β1 in SARS-CoV-2 spike-mediated infection, its interaction with SARS2-S, and the resulting cellular response.

Methods

The researchers conducted a series of experiments to assess the involvement of α5β1 in virus cell entry, its interaction with SARS2-S, and the subsequent cellular response.

Results

The study findings revealed that α5β1 does not play a direct role in virus cell entry but facilitates cell–cell fusion mediated by the SARS2-S protein. A direct interaction between α5β1 and SARS2-S was detected, independent of the RGD motif. This interaction involved the non-receptor-binding S2 subunit of SARS2-S and the homo-oligomerization of α5β1.

Furthermore, the study demonstrated that SARS2-S induces proinflammatory responses in human endothelial cells, including NF-κB activation, cytokine release, and cleavage of gasdermin D (GSDMD). These processes were found to be mediated by α5β1 signaling through the PDE4D pathway.

α5β1 is widely expressed in immune cells, lung, heart, and endothelial cells, suggesting its potential role in the diverse manifestations of COVID-19. The SARS2-S-induced and α5β1-mediated cellular responses may contribute to the complex pathogenesis of COVID-19.

Discussion

The question of whether integrins serve as cell entry receptors for SARS-CoV-2 has been a contentious issue in the scientific community. Our study argues against the effective infection of cells by SARS-CoV-2 in the absence of ACE2 using integrins as receptors. Considering the widespread expression of integrins such as α5β1 and αVβ3 throughout the human body, if SARS-CoV-2 could utilize integrins to infect ACE2-null cells, it might lead to more severe disease progression. Notably, despite the similar ligand-binding properties of mouse and human integrins, mice lacking human ACE2 are not susceptible to SARS-CoV-2 infection.

Our data, based on pseudovirus and replication-competent rVSV-S, along with α5-knockout experiments, do not support the involvement of α5β1 in S-mediated cell entry. However, our findings indicate that α5β1 does contribute to S-mediated cell–cell fusion, involving the participation of α5 CT.

The rVSV-S virus has been widely accepted as a model for studying SARS2-S-mediated cell entry and inhibition, demonstrating a strong correlation with the authentic SARS-CoV-2 virus. Nonetheless, differences between VSV and SARS-CoV-2 virions, such as variations in density, distribution, stability, and potential mutations of the S proteins, may impact the cell fusion function of the S protein.

While our study partially mitigated this limitation by using the S protein expressed on the cell surface to mimic cell fusion between virus-infected cells, further comprehensive studies are essential to compare our findings with those obtained from authentic SARS-CoV-2 virus.

The RGD-based interaction between integrins and SARS2-S has been a subject of debate, with conflicting findings among different research groups. Discrepancies in studies reporting high-affinity binding between SARS2-S S1 and α5β1 or an interaction between SARS2-S RBD and αIIbβ3 highlight the complexity of this interaction.

Our data, obtained from various assays including ELISA, pull-down, competition, mutagenesis, and functional assays, do not support the RGD-dependent interaction between SARS2-S and α5β1. Instead, we propose that the interaction may depend on the S2 subunit of SARS2-S and involve α5β1 homo-oligomerization. Structural studies are warranted to gain a more comprehensive understanding of the interaction between SARS2-S and α5β1, as well as other integrins.

The lack of inhibition by ATN-161 on the interaction between α5β1 and SARS2-S, as well as on S-mediated virus infection and cell fusion, raises questions about the mechanism by which ATN-161 functions as an inhibitor. The cryo-EM structure of α5β1 in complex with Fn7-10 suggests concerns about the binding affinity of ATN-161 with α5β1. The mutation in ATN-161 may reduce its binding affinity significantly. The exact mechanism by which ATN-161 interacts with α5β1 remains unknown, and caution is advised when considering its use as a potential α5β1 antagonist.

SARS2-S has been reported to induce an inflammatory response via TLRs in macrophages, but conflicting data exist regarding which TLRs (TLR2 or TLR4) interact with SARS2-S. Integrin α5β1 has been implicated in regulating inflammatory responses, and our study using HUVEC as a model demonstrates that SARS2-S induces an inflammatory response in endothelial cells. This response is consistent with other studies and is not observed with SARS-S under our experimental conditions. The involvement of PDE4D activation in α5β1-mediated endothelial inflammatory responses aligns with previous studies, and our findings suggest a potential role for PDE4 inhibitors as a treatment for COVID-19.

In conclusion, our study provides insights into the intricate molecular interactions between SARS2-S and α5β1, shedding light on the potential role of α5β1 in SARS-CoV-2 pathogenesis. The data suggest that while α5β1 may not be a direct receptor for virus entry, it contributes to cell–cell fusion and induces proinflammatory responses in endothelial cells.

The study emphasizes the need for further investigations into the complex interplay between SARS2-S and integrins, especially α5β1, and their implications for COVID-19 pathogenesis. The identification of potential therapeutic targets, such as the S-α5β1 interaction and the downstream signaling pathways, opens avenues for the development of novel treatment strategies for COVID-19.

TABLE 1 – Integrins and their role in cell adhesion and signaling

Integrins are a family of cell surface transmembrane receptors that mediate cell-cell and cell-matrix interactions. They play crucial roles in various cellular processes, including adhesion, migration, proliferation, and differentiation. Integrins consist of two non-covalently associated subunits, α and β, each contributing to ligand-binding specificity.

Integrin-binding domains in SARS-CoV-2 viral proteins

Several studies have identified potential integrin-binding domains within the SARS-CoV-2 viral proteins. Notably, the spike protein (S protein), which mediates viral attachment and entry, possesses an RGD (Arg-Gly-Asp) motif, a recognized ligand-binding site for various integrins. Additionally, the S protein harbors other integrin-binding motifs, including the LDV (Leu-Asp-Val) and MLD (Met-Leu-Asp) sequences, which can potentially interact with specific integrins.

Evidence for integrins as alternative receptors for SARS-CoV-2

Several lines of evidence suggest integrins as potential alternative receptors for SARS-CoV-2:

• In vitro studies have demonstrated that SARS-CoV-2 can infect ACE2-null cells in the presence of specific integrin ligands, indicating the involvement of alternative receptors.
• Co-localization studies have shown that the S protein can interact with specific integrins on the cell surface, further supporting their role in viral entry.
• Neutralization assays using integrin-blocking antibodies have demonstrated reduced viral infectivity, highlighting the functional importance of integrins in viral entry.

Potential impact of integrin-mediated viral entry on disease progression

Utilizing integrins as alternative receptors might contribute to more severe disease progression for several reasons:

• Increased viral spread: Utilizing additional receptors could potentially broaden the range of susceptible cells, facilitating viral replication and dissemination throughout the body.
• Immune evasion: Integrin-mediated entry might bypass ACE2-dependent immune responses, allowing the virus to evade immune detection and clearance.
• Enhanced inflammation: Integrin signaling pathways are closely linked to inflammatory responses. Integrin-mediated viral entry might activate pro-inflammatory pathways, leading to more severe tissue damage and disease symptoms.

reference link : https://www.pnas.org/doi/10.1073/pnas.2311913120#abstract