Short linear motif candidates in the cell entry system used by SARS-CoV-2 and their potential therapeutic implications

Abstract

The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the μ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin β3 and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.

In-vivo Protection from SARS-CoV-2 infection by ATN-161 in k18-hACE2 transgenic mice

“…Post-mortem analysis of human COVID-19 patients showed immune cell accumulation in the lungs [52]. In our study, CoV-2 infection and / or pathogenesis involves these, and perhaps other integrins, that activate downstream signaling to induce lung pathology [33,61,62]. Indeed, a recent study showed that increased integrin α 5β1 and α vβ3 levels in cardiac myocytes, obtained from heart failure patients, correlates with ACE2 expression [63].…”

“…Our results suggest a critical role of integrins as an additional receptor to SARS-CoV-2 spike protein cell entry [59,60]. We observed induced expression of integrin α 5 and integrin α v in the lungs of SARS-CoV-2 infected K18-hACE2 mice whereas lung expression of hACE2 levels did not vary in SARS-CoV-2+Saline or ATN-161 treated mice, suggesting that SARS-CoV-2 infection and / or pathogenesis involves these, and perhaps other integrins, that activate downstream signaling to induce lung pathology [33,61,62]. Indeed, a recent study showed that increased integrin α 5β1 and α vβ3 levels in cardiac myocytes, obtained from heart failure patients, correlates with ACE2 expression [63].…”

“…Integrins are cell surface receptors that may bind to the SARS-CoV-2 spike protein interaction [42,43,11,33]. In our previous study, we demonstrated by ELISA that the SARS-CoV-2 spike protein and ACE2 could bind to α 5β1, and that ATN-161 could disrupt both of these interactions [26].…”

“…We further demonstrated that the small pentapeptide α 5β1 integrin inhibitor ATN-161 could inhibit this binding and also decrease SARS-CoV-2 infection and cytopathy in cultured vero-E6 cells [26]. ATN-161 has several properties that make it potentially attractive as a novel SARS-CoV-2 therapy; it is safe and welltolerated with no dose-limiting toxicity in phase I cancer clinical trials [28], has demonstrated invivo efficacy in mice against a different beta coronavirus, porcine hemagglutinating encephalomyelitis virus (PHEV) [29] as well as other disease/injury models [30,31], and can also bind to and inhibit integrin α vβ3 which is similar to α 5β1 integrin that has also been implicated in SARS-CoV-2 infection [32,33]. In the present study we examined the therapeutic potential of ATN-161 in a human ACE2 receptor-expressing K18 mouse model of SARS-CoV-2 infection.…”

Molecular relationships between SARS-CoV-2 Spike protein and LIFR, a pneumonia protective IL-6 family cytokine

“…A number of SLiMs associated with interactions involving immune signaling pathways were detected in several host proteins including STAT3, STAT5, TRAF-2 binding motifs, TRAF6 binding site for Tumor Necrosis Factor 6 and ITIM (Supplementary Table 1). The vast majority of the identified SLiMs are not directly involved in immune signaling and include the recently identified RGD motif in SARS-CoV-2 S protein that binds to host integrins 25 . Whether some of these identified motifs in the virus are functional will need experimental validation.…”