A new study by Uppsala University researchers published on the preprint server bioRxiv* in October 2020 shows possible links between the angiotensin-converting enzyme 2 (ACE2) host cell receptors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the entry of viral particles into the cell via endocytosis and autophagy. The finding could help develop newer ways to target the virus.
The virus causing the current COVID-19 pandemic binds to the ACE2 receptor to enter and infect the host cell via endocytosis triggered by this binding. Structural validation of the interaction has been provided by co-crystallization of the receptor-binding domain of the SARS-CoV-2 spike protein with the ACE2 ectodomain, as well as a cryo-EM structure with full-length ACE2.
Alternative Receptors for SARS-CoV-2
Prior research shows that ACE2 expression is low in the lungs but higher in the kidneys and heart. Thus, the extensive lung damage found in COVID-19 shows that the virus interacts with alternative receptors to enter the cell. One such potential receptor is the integrin class of molecules since the spike protein has an integrin-binding RGD motif.
Integrins and Endocytosis
Previously, it has been shown that endocytosis triggered by integrins is used by many viruses to attach to and enter cells. This has led to the structural study of the interaction between ACE2 and integrins. The pattern of integrin distribution is closely matched to the pattern of attack of the SARS-CoV-2, with integrin b1 involved in the functioning of type 2 alveolar epithelial cells.
Currently, there are two reports of patients who have multiple sclerosis and contracted COVID-19, who were given an anti-integrin a4 antibody, Natalizumab. Both showed that treatment with this antibody was correlated with mild progression of COVID-19.
Spike-Receptor Interactions and SLiMs
Other researchers have explored how the spike protein interacts with cell surface proteins, including the ACE2 receptor, to enter the host cell, using bioinformatics to understand what happens after this binding. Some have predicted the presence of several short sequences, called short linear motifs (SLiMs), in the cytoplasmic tails of ACE2 and integrins that mediate endocytosis and autophagy.
These sequences are about 3-10 residues in length and take part in binding, allowing specific proteins to interact with target proteins depending on the specific domains in each. In this case, the SLiMs on the cytosolic aspect of the host receptor may be necessary to attract other proteins triggering cellular processes linked to internalization of the receptor and the attached virus.
Some protein domains for which such SLiMs were predicted include AP2 μ2, NCK Src Homology 2 (SH2) domains, and PDZ and phosphotyrosine binding (PTB) domains, which are involved in the changes in the cell cytoskeleton, for instance, building a network of proteins, or taking part in clathrin-dependent endocytosis.
Other than these, some have predicted that both the ACE2 and the integrin b3 tail also contains an LIR motif which is regulated by phosphorylation of residues both upstream and downstream. The LIR motif links these receptors to each other and allows the virus to enter the endocytosis-lysosomal pathway.
Phosphorylation of the downstream residue Tyr785 improved binding affinity to all the peptides that were considered. Additional phosphorylation of Thr779 and Tyr785 residues in the tail SLiM sequence synergistically increased the binding of other domains, showing a phospho-dependent interaction between the ATG8 domain-containing proteins. This refers to the phosphorylation-mediated signaling that is known to be among the chief responses to SARS-CoV-2 infection.
These investigators show that Tyr781 phosphorylation in the integrin b cytoplasmic tail causes the competitive binding of the PTB domain, controlling the tail interaction with scaffolding and adaptor proteins. The phosphorylation of different residues in the SLiM of integrin b3 may allow it to act as a bimolecular switch, for instance.
Findings of Low-Affinity Binding to PDZ Domain Peptides
The current study involved the expression and purification of 18 protein domains and the affinity of binding to certain cytoplasmic regions of the receptor tails as represented by synthetic peptides. The researchers found that the C-terminal end of the ACE2 molecule binds at a low affinity to the NHERF3 PDZ1 and SHANK1 PDZ domains, which is impacted by phosphorylation.
The study also confirms phosphorylation-enhanced binding between the LIR in integrin b3 to the ATG8 domains of MAP1LC3s and GABARAPs. The SNX17 FERM domain binds to peptides containing the SLiM NPx(F/Y), again confirming the relationship between the receptor and the endosomal recycling of various proteins like integrins. Future studies will be required to elicit PTB domain specificities since there are ~60 human proteins with PTB domains.
The ACE2 tail also has a short stretch of nine residues that is predicted to have overlapping motifs for AP2 μ2, NCK SH2 and the ATG8 proteins, as well as two phosphorylation sites that act as bimolecular switches. This weak signal that promotes endocytosis in response to phosphorylation.
Thus, a group of motifs earlier predicted by bioinformatics was found to be functional and to take part in the ACE2-integrin b3 recognition that triggers viral entry into the host cell via autophagy. It shows that the ACE2 C-terminal end contains an endocytotic motif that is impaired by Tyr781 phosphorylation, suggesting that viral binding to the receptor promotes clathrin-dependent endocytosis.
Implications and Future Directions
The study shows the binding of ATG8 domains to the integrin b3 cytosolic tail following phosphorylation. This directly links the receptor to autophagy and could explain how SARS-CoV-2 could hijack this pathway for productive infection, especially since ATG8 domains mediate the recruitment of peptides containing LIR to the membranes bounding phagosomes.
The study may also explain how vesicular membranes are rearranged and integrins transported within the cell, dependent on phosphorylation of the LIR motif. The PTB domain in the Shc protein seems to be essential for anoikis, or apoptosis following detachment. This domain is bound to the integrin b3 tail, following Tyr785 phosphorylation.
Double phosphorylation of Thr779 and Tyr785 leads to the loss of such binding, even though it allows synergistic binding of MAP1LC3s and GABARAP domains that are linked to autophagy. The authors postulate, “The integrin b3 tail serves as a molecular platform signaling for either the induction of anoikis or protective autophagy.”
Further studies will be needed to confirm the biological role played by this interaction, especially because of the low affinity of this binding. However, the NHERF3 serves as a scaffold protein that regulates the surface expression of plasma membrane proteins in the apical domain of the epithelial cells, agreeing with the enrichment of ACE2 on the apex of the epithelium of the conducting airway.
Moreover, it interacts with the glutamate transporter, which carries this excitatory amino acid, and has not only the PDZ motif but an endocytic sorting signal. A comparable mechanism may mediate the surface stabilization of ACE2 via the PDZ domain and its internalization via AP2. Both NHERF3 and ACE2 are involved in similar cell pathways and may be found in the same tissues, suggesting that the observed interaction is relevant to their biological function.
The study appears to suggest specific interactions between ACE2 and PDZ, but multiple human PDZ domains possess overlapping specificities. There are still other domains with similar binding affinities that may be physiological ligands for these receptors. One instance is the SNX27 peptide that has newly been found to be essential for viral entry in the case of an engineered SARS-CoV-2 virus.
SNX27 transports molecules in a retrograde fashion from endosome to plasma membrane, helping to recycle internalized transmembrane proteins such as ACE2 back to the cell membrane and thus promotes viral entry. SNX27 also binds to the SLiM NPxY and prevents the breakdown of beta-integrins in the lysosome.
The presence of selective class I PDZ binding motif and a weak endocytic sorting signal in the cytoplasmic tail of ACE2 points to protein-protein interactions following viral attachment to the ACE2 receptor, leading to internalization. This could be true of both normal biological function and viral infection-related processes. The occurrence of a phosphorylation-dependent LIR motif in the integrin b3 tail does not prove that an autophagy LIR motif is present, but does link integrin b3 to the autophagic pathway
The other integrin b tails should also be explored for phosphorylation-modulated LIR motifs. This study thus helps to understand how the virus uses autophagosomes for propagation, subject to further biological studies.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.