Cytoplasmic domain and enzymatic activity of ACE2 are not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells.
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ABSTRACT:
SUBMITTER: Yang H
PROVIDER: S-EPMC8900885 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV)-1 and -2 initiate virus infection by binding of their spike glycoprotein with the cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and enter into the host cells mainly via the clathrin-mediated endocytosis pathway. However, the internalization process post attachment with the receptor is not clear for both SARS-CoV-1 and -2. Understanding the cellular factor/s or pathways used by these CoVs for internalization might provide insights into viral pathogenesis, transmission, and development of novel therapeutics. Here, we demonstrated that the cytoplasmic tail of ACE2 is not essential for the entry of SARS-CoV-1 and -2 by using bioinformatics, mutational, confocal imaging, and pseudotyped SARS-CoVs infection studies. ACE2 cytoplasmic domain (cytACE2) contains a conserved internalization motif and eight putative phosphorylation sites. Complete cytoplasmic domain deleted ACE2 (∆cytACE2) was properly synthesized and presented on the surface of HEK293T and BHK21 cells like wtACE2. The SARS-CoVs S1 or RBD of spike protein binds and colocalizes with the receptors followed by internalization into the host cells. Moreover, pseudotyped SARS-CoVs entered into wtACE2- and ∆cytACE2-transfected cells but not into dipeptidyl peptidase 4 (DPP4)-expressing cells. Their entry was significantly inhibited by treatment with dynasore, a dynamin inhibitor, and NH4Cl, an endosomal acidification inhibitor. Furthermore, SARS-CoV antibodies and the soluble form of ACE2-treated pseudotyped SARS-CoVs were unable to enter the wtACE2 and ∆cytACE2-expressing cells. Altogether, our data show that ACE2 cytoplasmic domain signaling is not essential for the entry of SARS-CoV-1 and -2 and that SARS-CoVs entry might be mediated via known/unknown host factor/s.
| S-EPMC8304749 | biostudies-literature
Project description:The spike protein of SARS-CoV-2 binds the angiotensin-converting enzyme 2 (ACE2) on the host cell surface and subsequently enters host cells through receptor-mediated endocytosis. Additional cell receptors may be directly or indirectly involved, including integrins. The cytoplasmic tails of ACE2 and integrins contain several predicted short linear motifs (SLiMs) that may facilitate internalization of the virus as well as its subsequent propagation through processes such as autophagy. Here, we measured the binding affinity of predicted interactions between SLiMs in the cytoplasmic tails of ACE2 and integrin ?3 with proteins that mediate endocytic trafficking and autophagy. We validated that a class I PDZ-binding motif mediated binding of ACE2 to the scaffolding proteins SNX27, NHERF3, and SHANK, and that a binding site for the clathrin adaptor AP2 ?2 in ACE2 overlaps with a phospho-dependent binding site for the SH2 domains of Src family tyrosine kinases. Furthermore, we validated that an LC3-interacting region (LIR) in integrin ?3 bound to the ATG8 domains of the autophagy receptors MAP1LC3 and GABARAP in a manner enhanced by LIR-adjacent phosphorylation. Our results provide molecular links between cell receptors and mediators of endocytosis and autophagy that may facilitate viral entry and propagation.
| S-EPMC7928716 | biostudies-literature
Project description:Cell entry of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by the viral spike (S) protein. Amino acids 319-510 on the S protein have been mapped as the receptor-binding domain (RBD), which mediates binding to the SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) on SARS-CoV susceptible cells. In this study, we expressed a fusion protein containing the human codon-optimized RBD of the SARS-CoV spike protein linked to the Fc portion of human IgG1 (named RBD-Fc) in HEK293 cells. The RBD-Fc protein was purified by affinity chromatography. The flow cytometry assay showed that the purified RBD-Fc protein could bind to ACE2. We demonstrated that the RBD spike protein alone could be internalized into SARS-CoV susceptible cells together with ACE2. We also showed that the removal of N-glycans from the RBD spike protein did not abolish this phenomenon. Our discoveries may have some implications for the development of the SARS vaccine.
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Project description:Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. Our study shed more lights into the genetic determinants of ACE2 as the functional receptor of SARS-CoV-2, which facilitates our understanding of viral entry.
| S-EPMC7990223 | biostudies-literature
Project description:BackgroundAngiotensin-converting enzyme 2 (ACE2) is known as the major viral entry site for SARS-CoV-2. However, viral tissue tropism and high rate of infectivity do not directly correspond with the level of ACE2 expression in the organs. It may suggest involvement of other receptors or accessory membrane proteins in SARSCoV-2 cell entry.Methods and resultsA systematic search was carried out in PubMed/Medline, EMBASE, and Cochrane Library for studies reporting SARS-CoV-2 cell entry. We used a group of the MeSH terms including "cell entry", "surface receptor", "ACE2", and "SARS-CoV-2". We reviewed all selected papers published in English up to end of February 2022. We found several receptors or auxiliary membrane proteins (including CD147, NRP-1, CD26, AGTR2, Band3, KREMEN1, ASGR1, ANP, TMEM30A, CLEC4G, and LDLRAD3) along with ACE2 that facilitate virus entry and transmission. Expression of Band3 protein on the surface of erythrocytes and evidence of binding with S protein of SARS-CoV-2 may explain asymptomatic hypoxemia during COVID19 infection. The variants of SARS-CoV-2 including the B.1.1.7 (Alpha), B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.617.2+ (Delta+), and B.1.1.529 (Omicron) may have different potency to bond with these receptors.ConclusionsThe high rate of infectivity of SARS-CoV-2 may be due to its ability to enter the host cell through a group of cell surface receptors. These receptors are potential targets to develop novel therapeutic agents for SARS-CoV-2.
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Project description:VE-cadherin trafficking to and from the plasma membrane has emerged as a critical mechanism for regulating cadherin surface levels and adhesion strength. In addition, proteolytic processing of cadherin extracellular and cytoplasmic domains has been reported to regulate cadherin adhesion and signaling. Here we provide evidence that VE-cadherin is cleaved by calpain upon entry into clathrin-enriched domains. This cleavage event occurs between the ?-catenin and p120-binding domains within the cadherin cytoplasmic tail. Of interest, VE-cadherin mutants that are resistant to endocytosis are similarly resistant to cleavage. Furthermore, p120-catenin overexpression blocks cadherin internalization and cleavage, coupling entry into the endocytic pathway with proteolytic processing. Of importance, the cleavage of the VE-cadherin tail alters the postendocytic trafficking itinerary of the cadherin, resulting in a higher turnover rate due to decreased recycling and increased degradation. In conclusion, this study identifies a novel proteolytic event that regulates the trafficking of VE-cadherin after endocytosis.
| S-EPMC5221631 | biostudies-literature
Project description:SARS-CoV-2, the virus responsible for COVID-19, employs two key host proteins to gain entry and replicate within cells, angiotensin-converting enzyme 2 (ACE2) and the cell surface transmembrane protease serine 2 (TMPRSS2). TMPRSS2 was first characterized as an androgen-regulated gene in the prostate. Supporting a role for sex hormones, males relative to females are disproportionately affected by COVID-19 in terms of mortality and morbidity. Several studies, including one employing a large epidemiological cohort, suggested that blocking androgen signaling is protective against COVID-19. Here, we demonstrate that androgens regulate the expression of ACE2, TMPRSS2, and androgen receptor (AR) in subsets of lung epithelial cells. AR levels are markedly elevated in males relative to females greater than 70 years of age. In males greater than 70 years old, smoking was associated with elevated levels of AR and ACE2 in lung epithelial cells. Transcriptional repression of the AR enhancesome with AR or bromodomain and extraterminal domain (BET) antagonists inhibited SARS-CoV-2 infection in vitro. Taken together, these studies support further investigation of transcriptional inhibition of critical host factors in the treatment or prevention of COVID-19. These mouse data are part of a larger investigation (data not provided here) targeting the transcriptional regulation of SARS-CoV-2 entry factors ACE2 and TMPRSS2.
2020-11-30 | GSE159576 | GEO
Project description:The emergence of SARS-CoV-2 variants is a significant concern in developing effective therapeutics and vaccines in the middle of the ongoing COVID-19 pandemic. Here, we have identified a novel small molecule that inhibited the interactions between SARS-CoV-2 spike RBDs and ACE2 by modulating ACE2 without impairing its enzymatic activity necessary for normal physiological functions. Furthermore, the identified compounds suppressed viral infection in cultured cells by inhibiting the entry of ancestral and variant SARS-CoV-2. Our study suggests that targeting ACE2 could be a novel therapeutic strategy to inhibit SARS-CoV-2 entry into host cells and prevent the development of COVID-19.
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Project description:Despite the unprecedented effort of the scientific community, the novel SARS-CoV-2 virus has infected more than 46 million people worldwide, killing over one million two hundred thousand. Understanding the mechanisms by which some individuals are more susceptible to SARS-CoV-2 infection and why a subgroup of them are prone to experience severe pneumonia, and death should lead to a better approach and more effective treatments for COVID-19. Here, we focus our attention on ACE2, a primary receptor of SARS-CoV-2. We will discuss its biology, tissue expression, and post-translational regulation that determine its potential to be employed by SARS-CoV-2 for cell entry. Particular attention will be given to how the ACE2 soluble form can have a great impact on disease progression and thus be used in a potential therapeutic strategy. Furthermore, we will discuss repercussions that SARS-CoV-2/ACE2 binding has on the renin-angiotensin system and beyond. Indeed, although mostly neglected, ACE2 can also act on [des-Arg 937]-bradykinin of the kinin-kallikrein system regulating coagulation and inflammation. Thorough comprehension of the role that ACE2 plays in different pathways will be the key to assess the impact that SARS-CoV-2/ACE2 binding has on organismal physiology and will help us to find better therapies and diagnostic tools.
| S-EPMC7653219 | biostudies-literature
Project description:Integrins have been suggested to be involved in SARS-CoV-2 infection, but the underlying mechanisms remain largely unclear. This study aimed to investigate how integrins facilitate the ACE2-mediated cellular entry of SARS-CoV-2. We first tested the susceptibility of a panel of human cell lines to SARS-CoV-2 infection using the spike protein pseudotyped virus assay and examined the expression levels of integrins in these cell lines by qPCR, western blot and flow cytometry. We found that integrin αvβ1 was highly enriched in the SARS-CoV-2 susceptible cell lines. Additional studies demonstrated that RGD (403-405)→AAA mutant was defective in binding to integrin αvβ1 compared to its wild type counterpart, and anti-αvβ1 integrin antibodies significantly inhibited the entry of SARS-CoV-2 into the cells. Further studies using mouse NIH3T3 cells expressing human ACE2, integrin αv, integrin β1, and/or integrin αvβ1 suggest that integrin αvβ1 was unable to function as an independent receptor but could significantly facilitate the cellular entry of SASR-CoV-2. Finally, we observed that the Omicron exhibited a significant increase in the ACE2-mediated viral entry. Our findings may enhance our understanding of the pathogenesis of SARS-CoV-2 infection and offer potential therapeutic target for COVID-19.
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