scholarly journals Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver TransductionIn Vivo

2015 ◽  
Vol 90 (1) ◽  
pp. 412-420 ◽  
Author(s):  
Anne K. Zaiss ◽  
Erin M. Foley ◽  
Roger Lawrence ◽  
Lina S. Schneider ◽  
Hamidreza Hoveida ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Coagulation Factor ◽  
Heparan Sulfate Proteoglycans ◽  
Factor X ◽  
Primary Hepatocytes ◽  
Adeno Associated Virus ◽  
Fold Reduction ◽  
Adenovirus 5

ABSTRACTAdeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytesin vivothrough cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lackExt1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes.Ext1HEPmutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyteExt1gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes fromExt1HEPmice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers ofExt1HEPmice. FX remained essential for Ad5 transductionin vivoinExt1HEPmice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transductionin vivo. This study reopens the question of how adenovirus enters cellsin vivo.IMPORTANCEOur understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytesin vivothrough heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis was genetically abolished, demonstrated that HSPGs are not likely to function as hepatocyte Ad5 receptorsin vivo. The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These results reopen the question of the identity of the Ad5 receptorin vivoand emphasize the necessity of demonstrating the nature of the receptor by genetic means, both for understanding Ad5 entry into cellsin vivoand for optimization of Ad5 vectors as therapeutic agents.

scholarly journals Cellular receptors for enterovirus A71

2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Kyousuke Kobayashi ◽  
Satoshi Koike
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Neurological Diseases ◽  
Enterovirus 71 ◽  
Trna Synthetase ◽  
Heparan Sulfate Proteoglycans ◽  
Health Concern ◽  
Dependent Manner ◽  
Surface Binding

AbstractEnterovirus 71 (EV-A71) is one of the major causative agents of hand, foot, and mouth disease. EV-A71 infection is sometimes associated with severe neurological diseases such as acute encephalitis, acute flaccid paralysis, and cardiopulmonary failure. Therefore, EV-A71 is a serious public health concern. Scavenger receptor class B, member 2 (SCARB2) is a type III transmembrane protein that belongs to the CD36 family and is a major receptor for EV-A71. SCARB2 supports attachment and internalization of the virus and initiates conformational changes that lead to uncoating of viral RNA in the cytoplasm. The three-dimensional structure of the virus-receptor complex was elucidated by cryo-electron microscopy. Two α-helices in the head domain of SCARB2 bind to the G-H loop of VP1 and the E-F loop of VP2 capsid proteins of EV-A71. Uncoating takes place in a SCARB2- and low pH-dependent manner. In addition to SCARB2, other molecules support cell surface binding of EV-A71. Heparan sulfate proteoglycans, P-selectin glycoprotein ligand-1, sialylated glycan, annexin II, vimentin, fibronectin, and prohibitin enhance viral infection by retaining the virus on the cell surface. These molecules are known as “attachment receptors” because they cannot initiate uncoating. In vivo, SCARB2 expression was observed in EV-A71 antigen-positive neurons and epithelial cells in the crypts of the palatine tonsils in patients that died of EV-A71 infection. Adult mice are not susceptible to infection by EV-A71, but transgenic mice that express human SCARB2 become susceptible to EV-A71 infection and develop neurological diseases similar to those observed in humans. Attachment receptors may also be involved in EV-A71 infection in vivo. Although heparan sulfate proteoglycans are expressed by many cultured cell lines and enhance infection by a subset of EV-A71 strains, they are not expressed by cells that express SCARB2 at high levels in vivo. Thus, heparan sulfate-positive cells merely adsorb the virus and do not contribute to replication or dissemination of the virus in vivo. In addition to these attachment receptors, cyclophilin A and human tryptophanyl aminoacyl-tRNA synthetase act as an uncoating regulator and an entry mediator that can confer susceptibility to non-susceptibile cells in the absence of SCARB2, respectively. The roles of attachment receptors and other molecules in EV-A71 pathogenesis remain to be elucidated.

scholarly journals Unacylated ghrelin binds heparan-sulfate proteoglycans which modulate its function

2020 ◽  
Author(s):  
Patric Jd Delhanty ◽  
Martin Huisman ◽  
Karina Prins ◽  
Jacobie Steenbergen ◽  
Rosinda Mies ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Mcf7 Cell ◽  
Biological Effects ◽  
Heparan Sulfate Proteoglycans ◽  
Surface Proteins ◽  
Cell Surface Receptor ◽  
Cell Surfaces ◽  
Growth Hormone Secretagogue

Acylated ghrelin (AG) is a gut-derived peptide with growth hormone secretagogue (GHS), orexigenic and other physiological activities mediated by GHS receptor-1a (GHSR). Ghrelin occurs in unacylated form (UAG) with activities opposing AG, although its mechanism of action is unknown. UAG does not antagonize AG at GHSR, and has biological effects on cells that lack this receptor. Because UAG binds to cells, it has been hypothesized that UAG acts via a cell-surface receptor, although this has not been confirmed. This study aimed to identify cell surface proteins to which UAG binds that could modulate or mediate its biological effects. The MCF7 cell-line was used as a model because UAG induces ERK signaling in these cells in the absence of GHSR. Using ligand-receptor capture and LC-MS/MS we identified specific heparan-sulfate proteoglycans (HSPGs) to which UAG interacts on cell surfaces. In line with this, UAG, as well as AG, bind with high affinity to heparin, and heparin and heparinase treatment suppress, whereas HSPG overexpression increases, UAG binding to MCF7 cell surfaces. Moreover, heparin suppresses the ERK response to UAG. However, conversion of the lysines in UAG to alanine, which prevent its binding to heparin and cell surface HSPGs, does not prevent its activation of ERK. Our data show that the interaction of UAG with HSPGs modulates its biological activity in cells. More broadly, the interaction of UAG and AG with HSPGs could be important for the specificity and potency of their biological action in vivo.

scholarly journals Manipulating Adenovirus Hexon Hypervariable Loops Dictates Immune Neutralisation and Coagulation Factor X-dependent Cell Interaction In Vitro and In Vivo

2015 ◽  
Vol 11 (2) ◽  
pp. e1004673 ◽  
Author(s):  
Jiangtao Ma ◽  
Margaret R. Duffy ◽  
Lin Deng ◽  
Rachel S. Dakin ◽  
Taco Uil ◽  
...  
Keyword(s):  
Coagulation Factor ◽  
Cell Interaction ◽  
Factor X ◽  
Dependent Cell

scholarly journals Vaccinia Virus Envelope H3L Protein Binds to Cell Surface Heparan Sulfate and Is Important for Intracellular Mature Virion Morphogenesis and Virus Infection In Vitro and In Vivo

2000 ◽  
Vol 74 (7) ◽  
pp. 3353-3365 ◽  
Author(s):  
Chi-Long Lin ◽  
Che-Sheng Chung ◽  
Hans G. Heine ◽  
Wen Chang
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Heparan Sulfate ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virion Morphogenesis

ABSTRACT An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L−) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L− mutant virus. IMV from the H3L− mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L− mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.

scholarly journals Distinct glycosaminoglycan chain length and sulfation patterns required for cellular uptake of Tau, Aβ, and α-Synuclein

2017 ◽  
Author(s):  
Barbara E. Stopschinski ◽  
Brandon B. Holmes ◽  
Gregory M. Miller ◽  
Jaime Vaquer-Alicea ◽  
Linda C. Hsieh-Wilson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Neurodegenerative Diseases ◽  
Heparan Sulfate ◽  
Chain Length ◽  
Cellular Uptake ◽  
Genetic Manipulation ◽  
Heparan Sulfate Proteoglycans ◽  
Cell Uptake ◽  
Major Genes

AbstractTranscellular propagation of aggregate “seeds” has been proposed to mediate progression of neurodegenerative diseases in tauopathies and α-synucleinopathies. We have previously determined that tau and α-synuclein aggregates bind heparan sulfate proteoglycans (HSPGs) on the cell surface. This mediates uptake and intracellular seeding. The specificity and mode of binding to HSPGs has been unknown. We used modified heparins to determine the size and sulfation requirements of glycosaminoglycan (GAGs) binding to aggregates in biochemical and cell uptake and seeding assays. Aggregates of tau require a precise GAG architecture with defined sulfate moieties in the N- and 6-O-positions, whereas α-synuclein and Aβ rely slightly more on overall charge on the GAGs. To determine the genetic requirements for aggregate uptake, we individually knocked out the major genes of the HSPG synthesis pathway using CRISPR/Cas9 in HEK293T cells. Knockout of EXT1, EXT2 and EXTL3, N-sulfotransferase (NDST1), and 6-O-sulfotransferase (HS6ST2) significantly reduced tau uptake. α-Synuclein was not sensitive to HS6ST2 knockout. Good correlation between pharmacologic and genetic manipulation of GAG binding by tau and α-synuclein indicates specificity that may help elucidate a path to mechanism-based inhibition of transcellular propagation of pathology.

scholarly journals Proteolytic inactivation of blood coagulation factor IX by thrombin

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1302-1308 ◽  
Author(s):  
W Kisiel ◽  
KJ Smith ◽  
BA McMullen
Keyword(s):  
Human Factor ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Light Chains ◽  
Factor X ◽  
Amino Terminal ◽  
Coagulation Factor Ix ◽  
Human Factor Ix ◽  
Coagulant Activity

Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.

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