Thailand Medical News Exclusive: Novel Human Host Receptors Identified For SARS-CoV-2 Cell Entry And Attachment!

SARS-CoV-2 Receptors: For a while now, the medical and research community has only focused on the ACE2 receptor being the key human host receptor for SARS-CoV-2 cell entry despite this being a fallacy. As a result, we have been inundated with a lot of studies of SARS-CoV-2 pathogenesis, medical conditions arising in the human host and even emergence of variants and their transmissibility etc all primarily based with ACE2 as the key binding receptor for SARS-CoV-2.


 
In the later part of the pandemic, studies and validation then emerged that SARS-CoV-2 could also use other human host receptors for entry or attachment including neuropilin-1 (NRP1), Heparan sulfate (HS), Basigin or EMMPRIN (CD147), Glucose regulated protein 78 (GRP78) and Dipeptidyl peptidase-4 (DPP4).
 
Unknown to many, the SARS-CoV-2 virus can actually use more than 74 identified human host receptors for viral entry and can bind to more than 1,860 other receptors and critical proteases in the human host! Thailand Medical News has retained a meticulous database on this.
 
It should be noted that as SARS-CoV-2 virus keeps on evolving and mutating at a rapid rate, it is also spawning many new sub-lineages that are getting better adapted at utilizing other human host receptors for viral entry and very little studies at present are focused on this. The research and medical communities are still stuck on the obsolete understanding that the ACE2 receptors are still the primary   SARS-CoV-2 Receptors in the virus pathogenesis!
 
Most viruses rely not only on receptors and suitable host cells but also cell surface molecules that may help viruses locate and enter host cells but do not play a decisive role in the infection process and are therefore called attachment factors or coreceptors.
 
SARS-CoV-2 infection relies on receptors and cell surface molecules for host cell entry. These receptors fall into two categories: entry receptors and attachment factors. While ACE2 is the primary entry receptor for SARS-CoV-2, its limited distribution in tissues cannot fully explain the virus's multiorgan tropism. Thus, other receptors aiding viral entry are being explored. Research indicates that the virus's interaction with host cells involves multiple transmembrane proteins, enhancing its attachment and invasion capabilities. This goes beyond a sole receptor-S protein interaction.
 
This article highlights just some novel viral receptors, their roles in SARS-CoV-2 infection, and their potential significance in treating COVID-19. Understanding these receptors' functions could shed light on the virus's pathogenesis and improve therapeutic strategies.
 
Tyrosine-Protein Kinase Receptor UFO or AXL (Entry Receptor)
The limited expression of ACE2 receptors in the lungs and trachea raises the possibility of alternative receptors facilitating respiratory cell infection.
 
One receptor that was uncovered as playing a role for SARS-CoV-2 cell entry in the respiratory cells in the lungs is the tyrosine-protein kinase receptor UFO (AXL).
 
AXL exhibited high expression in H1299 and BEAS-2B cells, enabling direct binding with the virus's S protein N-terminal domain (NTD). This interaction prompted cellular entry through reticulin-mediated endocytosis, followed by early endosomal colocalization.
 
Notably, ACE2 and AXL did not cross-inhibit each other, as ACE2 knockout had no impact on SARS-CoV-2 infection in H1299 cells, whereas AXL knockout substantially attenuated viral infection. AXL's status as a fresh SARS-CoV-2 receptor was unequivocal, enabling independent viral infection of the respiratory system without ACE2 dependency. In ACE2 and AXL double-knockout HEK293T cells, AXL overexpression markedly bolstered viral infection. Inhibition of AXL-mediated infection was achieved through soluble recombinant AXL or NTD protein, confirming ACE2-independent AXL-mediated viral entry.
 
In sum, AXL is an innovative SARS-CoV-2 entry receptor significantly enhancing viral invasion and replication, particularly relevant in respiratory infection. Additionally, AXL presents a potential target for future COVID-19 targeted therapies.
 
Asialoglycoprotein Receptor 1 or ASGR1 And Kringle Containing Transmembrane Protein 1 or KREMEN (Entry Receptor)
A genome-wide secretory omics interaction screening approach to identify receptors or ligands for target proteins under physiological conditions unveiled the interaction between asialoglycoprotein receptor 1 (ASGR1) and kringle containing transmembrane protein 1 (KREMEN1) with diverse domains of the SARS-CoV-2 S protein. ASGR1 displayed binding capabilities to RBD and NTD, whereas KREMEN1 engaged with RBD, NTD, and S2 subunits. Confirmatory in vitro infection tests demonstrated that overexpression of ASGR1 and KREMEN1 could enhance SARS-CoV-2 pseudoviral infection within ACE2 knockout cells, indicating their ACE2-independent role as entry receptors that directly facilitate SARS-CoV-2 infection.
 
Importantly, ASGR1 and KREMEN1 exhibited virus-specificity, as they had no impact on SARS-CoV and MERS-CoV infections, thus highlighting them as unique receptors for SARS-CoV-2.
 
Collectively termed ASK entry receptors, ACE2, ASGR1, and KREMEN1 collectively mediate SARS-CoV-2 entry, underpinning the virus's cell and tissue tropism.
 
Past studies demonstrated that the ASK receptors hold a stronger correlation with virus susceptibility than any single-entry receptor, both at cellular and tissue levels. SARS-CoV-2 employs distinct receptors for different cell types; ASGR1 predominates in liver cell lines, KREMEN1 in lung cell lines, and ACE2 in both lung and liver cell lines. Furthermore, the study produced blocking monoclonal antibodies against ASGR1 and KREMEN1, showcasing their potential to significantly hinder SARS-CoV-2 infection in human lung tissues.
 
Strikingly, a "cocktail antibody" targeting ASK receptors displayed superior inhibition of SARS-CoV-2 infection compared to single-target antibodies, with application in lung organ protection.
 
In summary, ASGR1 and KREMEN1 function as SARS-CoV-2 entry receptors, playing a pivotal role independently of ACE2. This study enriches the understanding of SARS-CoV-2 tropism and pathogenesis, offering novel avenues for drug development against COVID-19.
 
Transferrin Receptor or TfR (Entry Receptor)
In a significant advancement within SARS-CoV-2 research, the transferrin receptor (TfR) has emerged as another influential receptor impacting the virus's host entry. Observations in SARS-CoV-2-infected monkeys and humanized ACE2 (hACE2) mice revealed a remarkable surge in TfR expression within lung tissue and trachea. A direct interaction between TfR and SARS-CoV-2 was confirmed, notably evidenced by colocalization signals in the membrane and cytoplasm of SARS-CoV-2-infected Vero-E6 cells, denoting TfR's status as a membrane receptor.
 
Crucially, colocalization signals for TfR, ACE2, and S proteins were also detected on infected cell membranes, while solely the TfR-S protein complex was observed in the cytoplasm. Hence, TfR autonomously facilitates SARS-CoV-2 transport from the cell membrane to the cytoplasm, independent of ACE2.
 
 Correspondingly, in ACE2-knockout Vero E6 and A549 cells, TfR overexpression significantly augmented viral infection, whereas TfR knockdown hindered it. This underscores TfR's capacity to enable SARS-CoV-2 entry into host cells without ACE2 dependence.
 
Notably, introducing antibodies or polypeptides designed from TfR's amino acid sequence exhibited substantial protective effects in vitro and in vivo, effectively mitigating the lung damage inflicted by SARS-CoV-2 infection. These findings firmly establish TfR as the entry receptor for SARS-CoV-2 infection, enriching our comprehension of its infection mechanism and offering fresh leads in the pursuit of COVID-19 therapeutic targets.
 
Integrin (Entry Receptor)
A significant hallmark of severe COVID-19 cases is platelet dysfunction, encompassing thrombocytopenia, microvascular clotting, and coagulation anomalies. A comprehensive exploration aimed to decipher these phenomena, found Integrin as the primary receptor protein prevalent on platelet surfaces -distinct from ACE2 expression. Notably, Integrins α5β1 and αvβ3, identified as SARS-CoV-2's entry receptors, directly engage the RGD ligand motif within the S protein's RBD domain, circumventing reliance on ACE2 for viral invasion of platelet cells. Furthermore, S protein interaction with Integrin αvβ3 prompts cellular Actin remodeling, inducing filamentous pseudopodia and consequential platelet activation, culminating in widespread microthrombosis and adverse outcomes.
 
Likewise, the intricate interplay between SARS-CoV-2 infection, T cells, and related immune responses significantly impacts COVID-19 prognoses. Severe patients often display T-cell overactivity, apoptosis, and lymphocyte depletion. Intriguingly, T cells exhibit minimal ACE2 and established SARS-CoV-2 receptor expression, propelling research into the virus's mechanisms within these cells.
 
Elucidating this, recent studies revealed that lymphocyte Integrins α4β1, α4β7, α5β1, and αLβ2, newly unveiled as SARS-CoV-2 receptors, directly recognize three binding motifs in the S protein's RBD, facilitating viral entry into T cells. Integrin activation enhances RBD binding, significantly boosting SARS-CoV-2 infection in T cells. This interaction further triggers Src and Akt protein phosphorylation, elevating membrane CD25 activation molecule expression, and transcriptionally upregulates inflammatory factors - interleukin-2, interferon-γ, and tumor necrosis factor-α - suppressing T-cell proliferation.
 
In essence, SARS-CoV-2 exploits lymphocyte Integrin as an entry point into T cells, orchestrating T-cell immune modulation and restraint of proliferation. This process contributes to the elevated inflammatory milieu and lymphocyte reduction observed in COVID-19. Given Integrin's pivotal role as an SARS-CoV-2 entry receptor in T cells, it emerges as a promising therapeutic target for managing COVID-19-related complications.
 
Kidney Injection Molecule-1 or KIM1 (Attachment factor)
Postmortem examinations and virological investigations have revealed the presence of viral inclusion bodies and particles within the kidneys of COVID-19 patients, suggesting that SARS-CoV-2 exhibits renal tissue tropism, rendering the kidney susceptible to infection alongside the lungs. Curiously, the use of ACE2 inhibitors did not yield substantial improvements in COVID-19 patient prognosis.
 
Furthermore, ACE2 expression within the kidney markedly diminished post viral infection, implying the existence of alternative SARS-CoV-2 receptors within this organ. Given this supposition, researchers advanced the notion that kidney injury molecule-1 (KIM1) serves as an attachment factor facilitating SARS-CoV-2 invasion into the kidney. Research confirmed that during kidney injury, KIM1, markedly upregulated, could engage with the SARS-CoV-2 RBD, enhancing viral attachment to cell membranes and subsequent internalization via reticulin-mediated endocytosis. Strikingly, the viral RBD demonstrated capacity to interact with KIM1 and ACE2 via distinct binding sites, potentially indicating a synergistic role of these two receptors in mediating SARS-CoV-2 infection. Notably, based on the interaction sequence between KIM1 and SARS-CoV-2, the team devised two polypeptides as infection-blocking agents. Remarkably, one of these polypeptides, AP2, encompassing 14 amino acids, effectively curtailed SARS-CoV-2 aggregation on cell surfaces.
 
Furthermore, the researchers put forth the concept of "malignant circulation" pertaining to SARS-CoV-2's renal invasion. In the initial stages, the virus predominantly binds to ACE2 (whose physiological expression level surpasses that of KIM1), yet following virus-induced acute kidney injury, elevated KIM1 expression levels potentiate secondary viral infection. This dual action of KIM1 and ACE2 exacerbates kidney injury, perpetuating a vicious cycle by further upregulating KIM1 expression.
 
In summation, KIM1 demonstrates upregulation exclusively in response to kidney injury, underscoring its relevance and specificity to renal function. Moreover, KIM1 has emerged as a novel target for SARS-CoV-2 cell entry, offering a foundational premise for the development of therapeutic agents like polypeptides, small molecules, and antibodies directed against this target to combat COVID-19.
 
Scavenger receptor class B type I or SR-B1 (Attachment factor)
Scavenger receptor class B type I (SR-B1), a fundamental component of the lipid transport system, has established recognition in hepatocytes, ovarian cells, and testicular interstitial cells, orchestrating selective cholesterol ester absorption and contributing crucially to high-density lipoprotein (HDL) metabolism and cholesterol's "reverse transport". Collaborative research outcomes have substantiated that SARS-CoV-2's S1 subunit possesses a distinct affinity for HDL, with SR-B1 serving as HDL's cell surface receptor. This positions HDL as a "bridge" connecting the virus's S protein to the SR-B1 receptor. Consequently, within ACE2-expressing host cells, elevated HDL levels significantly bolster SARS-CoV-2's adherence and invasion onto cell surfaces.
 
Conversely, in cultured cells, HDL-facilitated SARS-CoV-2 infection can be directly hindered through in vitro treatment with a monoclonal antibody targeting the HDL binding site on the S1 subunit or an SR-B1 antagonist. Furthermore, experiments involving SR-B1 overexpression and knockdown demonstrated a proportional increase and decrease in virus RNA levels within host cells, respectively. Immunohistochemical analysis unveiled coexpression of SR-B1 and ACE2 on cell surfaces in various vulnerable tissues like lung, retina, kidney, small intestine, and colon.
 
This observation implies SR-B1's potential role as an ACE2 coreceptor, heightening SARS-CoV-2's infectivity across susceptible organs and tissues. In essence, this pioneering study unveils SR-B1 as a SARS-CoV-2 attachment factor, elucidating the interplay between the virus and lipid metabolism while enhancing our comprehension of SARS-CoV-2's pathogenesis. It furthermore lays the groundwork for potential novel antiviral drug screening and development.
 
 
Myosin Heavy Chain 9 or MYH9 (Attachment factor)
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibits diverse infectivity mechanisms, contributing to its high contagion rate. In probing how the virus specifically infiltrates human lung tissue cells, researchers identified the receptor protein myosin heavy chain 9 (MYH9) binding to the S protein. Their findings revealed MYH9's direct interaction with the N-terminal domain of the S2 and S1 subunits via its C-terminal domain (PRA). CRISPR/Cas9-mediated MYH9 gene knockout in lung cancer cell lines A549 and Calu-3 significantly impeded viral infection, while MYH9 overexpression boosted infection in A549 and H1299 cells.
 
Intriguingly, endosome and myosin inhibitors effectively thwarted SARS-CoV-2 entry, whereas TMPRSS2 and Cat B/L inhibitors proved ineffective. This indicates MYH9's role in endocytosis, bypassing the TMPRSS2 and Cat B/L pathways. Moreover, MYH9 overexpression didn't enhance SARS-CoV-2 pseudoviral infection in ACE2 knockout A549 cells, highlighting ACE2's necessity for MYH9-mediated viral entry. Thus, MYH9 functions as a coreceptor, facilitating ACE2-dependent SARS-CoV-2 endocytosis, independent of TMPRSS2 and Cat B/L pathways. MYH9's pivotal role in low ACE2-expressing cells, particularly lung tissue cells, positions it as a potential target for future clinical interventions against SARS-CoV-2 infection.
 
 
C-type lectins and TTYH2 (Attachment factor)
The exaggerated immune lung inflammation resulting from SARS-CoV-2 entry is a pivotal driver of COVID-19 severity. Thus, comprehending SARS-CoV-2's immune pathogenesis is essential for crafting effective intervention strategies. Prior research revealed viral RNA within immune cells, particularly myeloid cells, from alveolar lavage fluid of COVID-19 patients. However, given the limited expression of ACE2 receptor molecules in immune cells, alternative receptor molecules potentially mediate SARS-CoV-2's interaction with immune cells. Addressing this query, researchers presented a new insight. They identified six myeloid cell membrane proteins binding to the virus S protein, serving as attachment factors. C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) mainly adhered to the non-RBD domain (NTD or CTD) of the S protein, while tweety family member 2 (TTYH2), akin to ACE2, predominantly engaged with the RBD domain.
 
However, subsequent virus verification experiments indicated that while the interaction between SARS-CoV-2 and these surface receptors did not influence viral infection or replication, it induced robust proinflammatory cytokine production (IL1B, IL8, CXCL10, CCL2, etc.) and heightened the expression of inflammation-related genes (EGR1, THBD, C4A, and SOCS3) in myeloid cells.
 
Furthermore, RNA sequencing of single cells in alveolar lavage fluid from COVID-19 patients established a close link between these inflammatory factors, symptom severity, and the expression of surface receptors on myeloid cells. The researchers also devised a bispecific nanoantibody targeting both myeloid cell and ACE2 receptors.
 
This innovative approach effectively prevented ACE2-mediated SARS-CoV-2 infection while simultaneously curbing excessive inflammatory reactions stemming from myeloid cell receptors. In essence, this groundbreaking study unveiled how SARS-CoV-2's interaction with immune cell surface receptors triggers overactive inflammatory responses. The proposed utilization of bispecific nanoantibodies to simultaneously obstruct the viral infection pathway and mitigate immune overactivation holds potential as a treatment strategy for severe COVID-19 cases.
 
Other Co-Receptors
Beyond the aforementioned attachment factors, several coreceptors have been identified as crucial players in SARS-CoV-2 infection, each exerting distinct roles in the virus-host interaction. These coreceptors hold significance in determining cellular susceptibility to the virus and ensuring appropriate attachment and invasion.
 
For instance, T-cell immunoglobulin and mucin domain 1 (TIM-1) and TIM-4, belonging to the TIM and TAM receptor families, interact with phosphatidylserine (PS) on the virus surface, enhancing infection by facilitating ACE2-dependent endocytosis fusion.
 
A disintegrin and metalloprotease 17 (ADAM17), another pivotal coreceptor, is activated by the virus's S protein to cleave ACE2. This action prompts ACE2 detachment from the cell surface, boosting host protease activity and facilitating virus-cell fusion.
 
Similarly, the attachment factor B0AT1 (SLC6A19) strengthens ACE2 stability, forming a durable ACE2-B0AT1 complex that binds two S proteins, considerably augmenting SARS-CoV-2's recognition and host cell infection.
 
Additionally, the S protein can directly engage the pattern recognition receptor toll-like receptor 4 (TLR4), heightening viral attachment and membrane concentration. This engagement triggers downstream TLR4 signaling, upregulating inflammatory factors IL-1B and IL-6, prompting an antibacterial-like immune response.
 
Conversely, certain host cell receptors inhibit viral entry. Interferon-induced transmembrane protein 3 (IFITM3), for instance, curbs SARS-CoV-2's membrane passage by regulating host cell membrane fluidity, preventing viral envelope-cell membrane fusion.
Lymphocyte antigen-6E (LY6E), a glycosyl phosphatidylinositol-anchored receptor, impedes cell infection by disrupting S protein-mediated membrane fusion and cytoskeleton rearrangement.
 
The ezrin protein receptor, encoded by EZR gene, suppresses viral infection by reducing ACE2 and TLR expression, both pivotal to SARS-CoV-2 interaction. Notably, ezrin peptides demonstrate efficacy in inhibiting viral pneumonia, offering a key strategy in preventing and treating severe COVID-19.
 
Conclusions
These newly emerging data unveils the multifaceted interactions between SARS-CoV-2 and its host. Delving deeper into this dynamic can enhance our comprehension of host tropism and pathogenesis, presenting potential targets and theoretical frameworks for COVID-19 prevention and treatment. The spectrum of receptors and coreceptors and their dualistic roles in either promoting cell entry or affecting tropism and pathogenesis shed light on the intricate interplay that governs the virus's impact on host cells.
 
Thailand Medical News will be continuing this series of articles focusing on other SARS-CoV-2 receptors, attachment receptors and also co-receptors.
 
References:
 
SnapShot: Enveloped Virus Entry
https://pubmed.ncbi.nlm.nih.gov/32763187/
 
Multifaceted Roles of TIM-Family Proteins in Virus-Host Interactions
https://pubmed.ncbi.nlm.nih.gov/31732320/
 
ACE2: The Major Cell Entry Receptor for SARS-CoV-2
https://pubmed.ncbi.nlm.nih.gov/33170317/
 
Single cell RNA sequencing of 13 human tissues identify cell types and receptors of human coronaviruses
https://pubmed.ncbi.nlm.nih.gov/32199615/
 
AXL is a candidate receptor for SARS-CoV-2 that promotes infection of pulmonary and bronchial epithelial cells
https://pubmed.ncbi.nlm.nih.gov/33420426/
 
Receptome profiling identifies KREMEN1 and ASGR1 as alternative functional receptors of SARS-CoV-2
https://pubmed.ncbi.nlm.nih.gov/34837059/
 
Transferrin receptor is another receptor for SARS-CoV-2 entry
https://www.biorxiv.org/content/10.1101/2020.10.23.350348v1
 
Direct Cryo-ET observation of platelet deformation induced by SARS-CoV-2 spike protein
https://pubmed.ncbi.nlm.nih.gov/36739444/
 
Lymphocyte integrins mediate entry and dysregulation of T cells by SARS-CoV-2
https://pubmed.ncbi.nlm.nih.gov/36849525/
 
Kidney injury molecule-1 is a potential receptor for SARS-CoV-2
https://pubmed.ncbi.nlm.nih.gov/33493263/
 
Kidney and Lung ACE2 Expression after an ACE Inhibitor or an Ang II Receptor Blocker: Implications for COVID-19
https://pubmed.ncbi.nlm.nih.gov/32669323/
HDL-scavenger receptor B type 1 facilitates SARS-CoV-2 entry
https://pubmed.ncbi.nlm.nih.gov/33244168/
 
SR-B1's Next Top Model: Structural Perspectives on the Functions of the HDL Receptor
https://pubmed.ncbi.nlm.nih.gov/35107765/
 
Nonmuscle myosin heavy chain IIA facilitates SARS-CoV-2 infection in human pulmonary cells
https://pubmed.ncbi.nlm.nih.gov/34873039/
 
Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19
https://pubmed.ncbi.nlm.nih.gov/32398875/
 
SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2
https://pubmed.ncbi.nlm.nih.gov/34048708/
 
Phosphatidylserine receptors enhance SARS-CoV-2 infection
https://pubmed.ncbi.nlm.nih.gov/34797899/
 
ADAM10 and ADAM17 promote SARS-CoV-2 cell entry and spike protein-mediated lung cell fusion
https://pubmed.ncbi.nlm.nih.gov/35527514/
 
Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2
https://pubmed.ncbi.nlm.nih.gov/32132184/
 
Publisher Correction: SARS-CoV-2 spike protein interacts with and activates TLR4
https://pubmed.ncbi.nlm.nih.gov/33907310/
 
IFITM3 Inhibits SARS-CoV-2 Infection and Is Associated with COVID-19 Susceptibility
https://pubmed.ncbi.nlm.nih.gov/36423162/
 
The Mechanism and Consequences of SARS-CoV-2 Spike-Mediated Fusion and Syncytia Formation
https://pubmed.ncbi.nlm.nih.gov/34606831/
 
Can SARS-CoV-2 Virus Use Multiple Receptors to Enter Host Cells?
https://pubmed.ncbi.nlm.nih.gov/33498183/
 
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