scholarly journals Syndecan-4 Is a Key Facilitator of the SARS-CoV-2 Delta Variant’s Superior Transmission

2022 ◽  
Vol 23 (2) ◽  
pp. 796
Author(s):  
Anett Hudák ◽  
Gábor Veres ◽  
Annamária Letoha ◽  
László Szilák ◽  
Tamás Letoha
Keyword(s):  
Heparan Sulfate ◽  
Molecular Mechanisms ◽  
Spike Protein ◽  
Cell Binding ◽  
Cellular Entry ◽  
Delta Infection ◽  
Vaccination Campaigns ◽  
Cell To Cell Adhesion ◽  
Original Virus

Emerging SARS-CoV-2 variants pose threats to vaccination campaigns against COVID-19. Being more transmissible than the original virus, the SARS-CoV-2 B.1.617 lineage, named the Delta variant, swept through the world in 2021. The mutations in the Delta’s spike protein shift the protein towards a net positive electrostatic potential. To understand the key molecular drivers of the Delta infection, we investigate the cellular uptake of the Delta spike protein and Delta spike-bearing SARS-CoV-2 pseudoviruses. Specific in vitro modification of ACE2 and syndecan expression enabled us to demonstrate that syndecan-4, the syndecan isoform abundant in the lung, enhances the transmission of the Delta variant by attaching its mutated spike glycoprotein and facilitating its cellular entry. Compared to the wild-type spike, the Delta one shows a higher affinity towards heparan sulfate proteoglycans than towards ACE2. In addition to attachment to the polyanionic heparan sulfate chains, the Delta spike’s molecular interactions with syndecan-4 also involve syndecan-4’s cell-binding domain that mediates cell-to-cell adhesion. Regardless of the complexity of these interactions, exogenously added heparin blocks Delta’s cellular entry as efficiently as syndecan-4 knockdown. Therefore, a profound understanding of the molecular mechanisms underlying Delta infections enables the development of molecularly targeted yet simple strategies to reduce the Delta variant’s spread.

scholarly journals Murine Coronavirus with an Extended Host Range Uses Heparan Sulfate as an Entry Receptor

2005 ◽  
Vol 79 (22) ◽  
pp. 14451-14456 ◽  
Author(s):  
Cornelis A. M. de Haan ◽  
Zhen Li ◽  
Eddie te Lintelo ◽  
Berend Jan Bosch ◽  
Bert Jan Haijema ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Host Range ◽  
Binding Sites ◽  
Spike Protein ◽  
Heparin Binding ◽  
Furin Cleavage ◽  
Cleavage Motif ◽  
Entry Receptor ◽  
Murine Coronavirus

ABSTRACT Only a relatively few mutations in its spike protein allow the murine coronavirus to switch from a murine-restricted tropism to an extended host range by being passaged in vitro. One such virus that we studied had acquired two putative heparan sulfate-binding sites while preserving another site in the furin-cleavage motif. The adaptation of the virus through the use of heparan sulfate as an attachment/entry receptor was demonstrated by increased heparin binding as well as by inhibition of infection through treatment of cells and the virus with heparinase and heparin, respectively.

scholarly journals In vivo importance of heparan sulfate-binding glycoproteins for murid herpesvirus-4 infection

2009 ◽  
Vol 90 (3) ◽  
pp. 602-613 ◽  
Author(s):  
Laurent Gillet ◽  
Janet S. May ◽  
Philip G. Stevenson
Keyword(s):  
Heparan Sulfate ◽  
Low Dose ◽  
Envelope Glycoproteins ◽  
High Dose ◽  
Cell Binding ◽  
Host Colonization ◽  
Infection Dose ◽  
Herpesvirus 4

Many herpesviruses bind to heparan sulfate (HS). Murid herpesvirus-4 (MuHV-4) does so via its envelope glycoproteins gp70 and gH/gL. MuHV-4 gp150 further regulates an HS-independent interaction to make that HS-dependent too. Cell binding by MuHV-4 virions is consequently strongly HS-dependent. Gp70 and gH/gL show some in vitro redundancy: an antibody-mediated blockade of HS binding by one is well tolerated, whereas a blockade of both severely impairs infection. In order to understand the importance of HS binding for MuHV-4 in vivo, we generated mutants lacking both gL and gp70. As expected, gL−gp70− MuHV-4 showed very poor cell binding. It infected mice at high dose but not at low dose, indicating defective host entry. But once entry occurred, host colonization, which for MuHV-4 is relatively independent of the infection dose, was remarkably normal. The gL−gp70− entry deficit was much greater than that of gL− or gp70− single knockouts. And gp150 disruption, which allows HS-independent cell binding, largely rescued the gL−gp70− cell binding and host entry deficits. Thus, it appeared that MuHV-4 HS binding is important in vivo, principally for efficient host entry.

scholarly journals Enoxaparin and Pentosan Polysulfate Bind to the SARS-CoV-2 Spike Protein and Human ACE2 Receptor, Inhibiting Vero Cell Infection

Biomedicines ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 49
Author(s):  
Maria Ennemoser ◽  
Julia Rieger ◽  
Eva Muttenthaler ◽  
Tanja Gerlza ◽  
Kurt Zatloukal ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Viral Transmission ◽  
Full Length ◽  
Spike Protein ◽  
Target Cells ◽  
Pentosan Polysulfate ◽  
Cell Infection ◽  
Virus Surface ◽  
Kd Values

As with many other pathogens, SARS-CoV-2 cell infection is strongly dependent on the interaction of the virus-surface Spike protein with the glycosaminoglycans of target cells. The SARS-CoV-2 Spike glycoprotein was previously shown to interact with cell-surface-exposed heparan sulfate and heparin in vitro. With the aim of using Enoxaparin as a treatment for COVID-19 patients and as prophylaxis to prevent interpersonal viral transmission, we investigated GAG binding to the Spike full-length protein, as well as to its receptor binding domain (RBD) in solution by isothermal fluorescence titration. We found that Enoxaparin bound to both protein variants with similar affinities, compared to the natural GAG ligand heparan sulfate (with Kd-values in the range of 600–680 nM). Using size-defined Enoxaparin fragments, we discovered the optimum binding for dp6 or dp8 for the full-length Spike protein, whereas the RBD did not exhibit a significant chain-length-dependent affinity for heparin oligosaccharides. The soluble ACE2 receptor was found to interact with unfractionated GAGs in the low µM Kd range, but with size-defined heparins with clearly sub-µM Kd-values. Interestingly, the structural heparin analogue, pentosan polysulfate (PPS), exhibited high binding affinities to both Spike variants as well as to the ACE2 receptor. In viral infection experiments, Enoxaparin and PPS both showed a strong inhibition of infection in a concentration range of 50–500 µg/mL. Both compounds were found to retain their inhibitory effects at 500 µg/mL in a natural biomatrix-like human sputum. Our data suggest the early topical treatment of SARS-CoV-2 infections with inhaled Enoxaparin; some clinical studies in this direction are already ongoing, and they further imply an oral or nasal prophylactic inactivation of the virus by Enoxaparin or PPS for the prevention of inter-personal viral transmission.

scholarly journals Genetic Polymorphisms and Molecular Mechanisms Mediating Oncolytic Potency of Reovirus Strains

2019 ◽  
Author(s):  
Adil Mohamed ◽  
Derek R. Clements ◽  
Prathyusha Konda ◽  
Shashi A. Gujar ◽  
Patrick W. Lee ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cell Binding ◽  
Host Cell Signaling ◽  
Mammalian Orthoreovirus ◽  
Inclusion Morphology ◽  
Viral Inclusion ◽  
Commercial Sources ◽  
Reovirus Replication

ABSTRACTThe Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapeutic agent. In this study, a comprehensive phenotypic and genetic comparison of T3D virus stocks from various laboratories and commercial sources revealed that T3D laboratory strains differ substantially in their oncolytic activitiesin vitroandin vivo. Superior replication of the most-oncolytic T3D lab strain was attributed to several mechanistic advantages: virus-cell binding, viral RNA transcriptase activity, viral inclusion morphology, and differential activation of RIG-I versus NFκB-dependent signalling pathways. Viral S4, M1 and L3 gene segments were each independently associated with a distinct mechanistic advantage. Furthermore, the specific missense polymorphisms that governed replication potency were identified, and utilized to generate a hybrid of T3D laboratory strains with further-augmented replication in tumor cells. Together, the results depict an elaborate balance between reovirus replication and host-cell signaling to achieve optimal oncolytic reovirus efficacy.

scholarly journals Contribution of Syndecans to the Cellular Entry of SARS-CoV-2

2021 ◽  
Vol 22 (10) ◽  
pp. 5336
Author(s):  
Anett Hudák ◽  
Annamária Letoha ◽  
László Szilák ◽  
Tamás Letoha
Keyword(s):  
Heparan Sulfate ◽  
Dominant Role ◽  
Scientific Evidence ◽  
In Vitro Assays ◽  
Complex Nature ◽  
Cellular Targets ◽  
Medical Need ◽  
Significant Efficacy ◽  
Cellular Entry

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel emerging pathogen causing an unprecedented pandemic in 21st century medicine. Due to the significant health and economic burden of the current SARS-CoV-2 outbreak, there is a huge unmet medical need for novel interventions effectively blocking SARS-CoV-2 infection. Unknown details of SARS-CoV-2 cellular biology hamper the development of potent and highly specific SARS-CoV-2 therapeutics. Angiotensin-converting enzyme-2 (ACE2) has been reported to be the primary receptor for SARS-CoV-2 cellular entry. However, emerging scientific evidence suggests the involvement of additional membrane proteins, such as heparan sulfate proteoglycans, in SARS-CoV-2 internalization. Here, we report that syndecans, the evolutionarily conserved family of transmembrane proteoglycans, facilitate the cellular entry of SARS-CoV-2. Among syndecans, the lung abundant syndecan-4 was the most efficient in mediating SARS-CoV-2 uptake. The S1 subunit of the SARS-CoV-2 spike protein plays a dominant role in the virus’s interactions with syndecans. Besides the polyanionic heparan sulfate chains, other parts of the syndecan ectodomain, such as the cell-binding domain, also contribute to the interaction with SARS-CoV-2. During virus internalization, syndecans colocalize with ACE2, suggesting a jointly shared internalization pathway. Both ACE2 and syndecan inhibitors exhibited significant efficacy in reducing the cellular entry of SARS-CoV-2, thus supporting the complex nature of internalization. Data obtained on syndecan specific in vitro assays present syndecans as novel cellular targets of SARS-CoV-2 and offer molecularly precise yet simple strategies to overcome the complex nature of SARS-CoV-2 infection.

scholarly journals Heparan Sulfate Proteoglycan Is an Important Attachment Factor for Cell Entry of Akabane and Schmallenberg Viruses

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Shin Murakami ◽  
Akiko Takenaka-Uema ◽  
Tomoya Kobayashi ◽  
Kentaro Kato ◽  
Masayuki Shimojima ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Molecular Mechanisms ◽  
Substantial Reduction ◽  
Heparan Sulfate Proteoglycan ◽  
Economic Losses ◽  
Sulfate Proteoglycan ◽  
Content Type ◽  
Cellular Attachment ◽  
Attachment Factor

ABSTRACT Akabane virus (AKAV) and Schmallenberg virus (SBV) are members of the genus Orthobunyavirus, which are transmitted by arthropod vectors with a broad cellular tropism in vitro as well as in vivo. Both AKAV and SBV cause arthrogryposis-hydranencephaly syndrome in ruminants. The main cellular receptor and attachment factor for entry of these orthobunyaviruses are unknown. Here, we found that AKAV and SBV infections were inhibited by the addition of heparin or enzymatic removal of cell surface heparan sulfates. To confirm this finding, we prepared heparan sulfate proteoglycan (HSPG)-knockout (KO) cells by using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system and measured the quantities of binding of these viruses to cell surfaces. We observed a substantial reduction in AKAV and SBV binding to cells, limiting the infections by these viruses. These data demonstrate that HSPGs are important cellular attachment factors for AKAV and SBV, at least in vitro, to promote virus replication in susceptible cells. IMPORTANCE AKAV and SBV are the etiological agents of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic losses in the livestock industry. Here, we identified heparan sulfate proteoglycan as a major cellular attachment factor for the entry of AKAV and SBV. Moreover, we found that heparin is a strong inhibitor of AKAV and SBV infections. Revealing the molecular mechanisms of virus-host interactions is critical in order to understand virus biology and develop novel live attenuated vaccines.

scholarly journals In vitro rosetting, cytoadherence, and microagglutination properties of Plasmodium falciparum-infected erythrocytes from Gambian and Tanzanian patients

Blood ◽  
1990 ◽  
Vol 76 (9) ◽  
pp. 1845-1852 ◽  
Author(s):  
T Hasler ◽  
SM Handunnetti ◽  
JC Aguiar ◽  
MR van Schravendijk ◽  
BM Greenwood ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Infected Cell ◽  
Blood Cells ◽  
Molecular Mechanisms ◽  
Melanoma Cells ◽  
Schizont Stage ◽  
Ring Stage ◽  
Cell Binding ◽  
Blood Samples

Abstract To understand the molecular mechanisms that lead to sequestration of red blood cells infected with mature stages of Plasmodium falciparum and to examine the relevance of earlier studies on adherence properties of laboratory-derived P falciparum parasites to the natural parasite population, we analyzed Gambian and Tanzanian isolates for in vitro cytoadherence and antibody-mediated microagglutination. Eighteen cryopreserved isolates of ring-stage parasites were cultured for 20 to 30 hours in vitro, in the patients original erythrocytes, to the trophozoite and schizont stage. All parasites were positive in the microagglutination assay with at least one of four African hyperimmune sera. In a rosetting assay, only 2 of the 18 isolates were strongly positive (35% and 41% of parasitized erythrocytes with more than two uninfected cells bound). Thirteen isolates showed either intermediate (5% to 18%) or low (less than 5%) rosetting while three isolates did not form rosettes. Infected cell-binding of the different isolates to immobilized CD36 or thrombospondin, or C32 melanoma cells correlated with the percentage of mature parasites in the blood samples (r = .932 for CD36, r = .946 for thrombospondin, and r = .881 for C32 melanoma cells). There was a high correlation between binding to CD36 and thrombospondin (r = .982). The extent of infected cell rosetting with uninfected cells in these blood samples was not correlated with these other receptor properties. We also observed coexpression of rosetting and cytoadherence receptors on the same parasitized erythrocytes.

scholarly journals SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

2020 ◽  
Author(s):  
Thomas Mandel Clausen ◽  
Daniel R. Sandoval ◽  
Charlotte B. Spliid ◽  
Jessica Pihl ◽  
Chelsea D. Painter ◽  
...  
Keyword(s):  
Protein Binding ◽  
Heparan Sulfate ◽  
Docking Studies ◽  
Spike Protein ◽  
Pseudotyped Virus ◽  
Angiotensin Converting Enzyme 2 ◽  
Viral Attachment ◽  
Vitro Binding ◽  
Domain Docking

AbstractWe show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities.

scholarly journals Pretargeted cancer radioimmunotherapy and radioimaging using antibodies and antibody fragments

2018 ◽  
Author(s):  
◽  
Manankumar Shah
Keyword(s):  
Phage Display ◽  
Therapeutic Index ◽  
Single Chain ◽  
Cell Binding ◽  
Phage Display Technology ◽  
Good Target ◽  
Display Technology ◽  
Cell To Cell Adhesion ◽  
Radiolabeled Antibodies

Over the last few decades, antibodies have become the mainstay of cancer diagnosis and therapeutics. In traditional radioimmunotherapy (RIT), tumor targeting antibodies are directly conjugated with radioisotopes and depending on the radionuclides's properties, the direct labeled antibodies can be used for diagnostic or therapeutic purposes. However, one of the major challenges of using radiolabeled antibodies for therapy is their long serum half-lives. It generally takes 5-7 days for antibodies to achieve maximum tumor binding. This slow blood clearance results in high normal tissue irradiation and a poor therapeutic index. This is exemplified by the fact that to date, only two radiolabeled antibodies have been approved by the FDA for radioimmunotherapy of cancer. … Thomsen-Friedenreich (TF) is a disaccharide (Galactose [beta]1-3 N-acetylgalactosamine) antigen, which is present on about [about]90% of carcinomas. The TF expression on the tumor cell is correlated with poor prognosis and tumor propagation. TF antigen is also involved in cell to cell adhesion and metastasis, making it a very good target for cancer imaging and therapy. Using phage display technology, TF binding scFv fragments were selected from the McCafferty antibody library. The selected scFv clones were characterized in vitro for their TF specificity and cell binding properties by ELISA and flow-cytometry assay. The selected TF specific clone (9C-scFv) was radiolabeled with [99m]Tc by directly conjugating [99m]Tc to the C-terminal 6x His-tag. The [99m]Tc-labeled 9C-scFv was injected in mice bearing MDA-MB-231 human breast cancer xenografts. The SPECT/CT images, acquired 4 hours post injection, revealed a moderate tumor uptake of radiolabeled scFvs with significant accumulation in the liver and kidneys. The phage display derived single-chain scFv fragments against the TF antigen demonstrated potential for development as an imaging agent but requires more work to achieve favorable pharmacokinetics.

scholarly journals Contribution of syndecans to the cellular entry of SARS-CoV-2

2020 ◽  
Author(s):  
Anett Hudák ◽  
László Szilák ◽  
Tamás Letoha
Keyword(s):  
Heparan Sulfate ◽  
Dominant Role ◽  
Scientific Evidence ◽  
In Vitro Assays ◽  
Complex Nature ◽  
Heparin Binding ◽  
Cellular Targets ◽  
Significant Efficacy ◽  
Cellular Entry

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel emerging pathogen causing an unprecedented pandemic in 21st century medicine. Due to the significant health and economic burden of the current SARS-CoV-2 outbreak, there is a huge unmet medical need for novel interventions effectively blocking SARS-CoV-2 infection. Unknown details of SARS-CoV-2 cellular biology significantly hamper the development of potent and highly specific SARS-CoV-2 therapeutics. Angiotensin-converting enzyme-2 (ACE2) has been reported to be the primary receptor for SARS-CoV-2 cellular entry. However, emerging scientific evidence suggests the involvement of additional membrane proteins, such as heparan sulfate proteoglycans, in SARS-CoV-2 internalization. Here we report that syndecans, the evolutionarily conserved family of transmembrane proteoglycans facilitate the cellular entry of SARS-CoV-2. Among syndecans, syndecan-4 was the most efficient in mediating SARS-CoV-2 uptake, yet overexpression of other isoforms, including the neuronal syndecan-3, also increased SARS-CoV-2 internalization. The S1 subunit of the SARS-CoV-2 spike protein plays a dominant role in the virus’s interactions with syndecans. Besides the polyanionic heparan sulfate chains - the established binding sites for several viruses - other parts of the syndecan ectodomain, such as the cell-binding domain, also contribute to the interaction with SARS-CoV-2. During virus internalization, syndecans colocalize with ACE2, suggesting a jointly shared internalization pathway. Both ACE2 and syndecan inhibitors exhibited significant efficacy in reducing cellular entry of SARS-CoV-2, thus supporting the complex nature of internalization. Among these inhibitors, a peptide compromising the spike protein’s heparin-binding PRRAR motif significantly reduced SARS-CoV-2 cellular uptake, highlighting the need to go beyond the ACE2 paradigm for developing efficient therapeutics against SARS-CoV-2. Data obtained on syndecan specific in vitro assays present syndecans as novel cellular targets of SARS-CoV-2 and offers molecularly precise, yet simple strategies in overcoming the complex nature of SARS-CoV-2 infection.

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