scholarly journals A Single Mutation in the VP1 Gene of Enterovirus 71 Enhances Viral Binding to Heparan Sulfate and Impairs Viral Pathogenicity in Mice

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 883
Author(s):  
Xianliang Ke ◽  
Yuan Zhang ◽  
Yan Liu ◽  
Yuanjiu Miao ◽  
Caishang Zheng ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Enterovirus 71 ◽  
Mouse Tissue ◽  
Human Hand ◽  
Binding Property ◽  
Single Mutation ◽  
Dependent Manner ◽  
Mouse Tissues ◽  
Viral Virulence

Enterovirus 71 (EV71) is the major causative pathogen of human hand, foot, and mouth disease (hHFMD) and has evolved to use various cellular receptors for infection. However, the relationship between receptor preference and EV71 virulence has not been fully revealed. By using reverse genetics, we identified that a single E98K mutation in VP1 is responsible for rapid viral replication in vitro. The E98K mutation enhanced binding of EV71-GZCII to cells in a heparan sulfate (HS)-dependent manner, and it attenuated the virulence of EV71-GZCII in BALB/c mice, indicating that the HS-binding property is negatively associated with viral virulence. HS is widely expressed in vascular endothelial cells in different mouse tissues, and weak colocalization of HS with scavenger receptor B2 (SCARB2) was detected. The cGZCII-98K virus bound more efficiently to mouse tissue homogenates, and the cGZCII-98K virus titers in mouse tissues and blood were much lower than the cGZCII virus titers. Together, these findings suggest that the enhanced adsorption of the cGZCII-98K virus, which likely occurs through HS, is unable to support the efficient replication of EV71 in vivo. Our study confirmed the role of HS-binding sites in EV71 infection and highlighted the importance of the HS receptor in EV71 pathogenesis.

scholarly journals Synthesis and Broad Antiviral Activity of Novel 2-aryl-isoindolin-1-ones towards Diverse Enterovirus A71 Clinical Isolates

Molecules ◽  
2019 ◽  
Vol 24 (5) ◽  
pp. 985 ◽  
Author(s):  
Yixuan Wang ◽  
Huiqiang Wang ◽  
Xinbei Jiang ◽  
Zhi Jiang ◽  
Tingting Guo ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Viral Disease ◽  
Clinical Isolates ◽  
Dependent Manner ◽  
Potent Antiviral Activity ◽  
Sar Studies ◽  
Enterovirus A71

Enterovirus 71 (EV-A71) is the main causative pathogen of childhood hand, foot and mouth disease. Effective medicine is currently unavailable for the treatment of this viral disease. Using the fragment-hopping strategy, a series of 2-aryl-isoindolin-1-one compounds were designed, synthesized and investigated for their in vitro antiviral activity towards multiple EV-A71 clinical isolates (H, BrCr, Shenzhen98, Jiangsu52) in Vero cell culture in this study. The structure–activity relationship (SAR) studies identified 2-phenyl-isoindolin-1-ones as a new potent chemotype with potent antiviral activity against EV-A71. Ten out of the 24 tested compounds showed significant antiviral activity (EC50 < 10 µM) towards four EV-A71 strains. Compounds A3 and A4 exhibited broad and potent antiviral activity with the 50% effective concentration (EC50) values in the range of 1.23–1.76 μM. Moreover, the selectivity indices of A3 and A4 were significantly higher than those of the reference compound, pirodavir. The western blotting experiment indicated that the viral VP1 was significantly decreased at both the protein and RNA level in a dose-dependent manner following treatment with compound A3. Moreover, compound A3 inhibited the viral replication by acting on the virus entry stage. In summary, this study led to the discovery of 2-aryl-isoindolin-1-ones as a promising scaffold with potent anti-EV-A71 activities, which deserves further in-depth studies.

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.

In vitro evaluation of the antiviral activity of heparan sulfate mimetic compounds against Enterovirus 71

2012 ◽  
Vol 169 (1) ◽  
pp. 22-29 ◽  
Author(s):  
Hamid Reza Pourianfar ◽  
Chit Laa Poh ◽  
John Fecondo ◽  
Lara Grollo
Keyword(s):  
Antiviral Activity ◽  
Heparan Sulfate ◽  
Enterovirus 71 ◽  
In Vitro Evaluation

scholarly journals Glycocalyx components affect platelet function, whole blood coagulation, and fibrinolysis: an in vitro study suggesting a link to trauma-induced coagulopathy

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Martin W. Britten ◽  
Laura Lümers ◽  
Kenji Tominaga ◽  
Jürgen Peters ◽  
Daniel Dirkmann
Keyword(s):  
Platelet Aggregation ◽  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
Blood Coagulation ◽  
Platelet Function ◽  
Whole Blood ◽  
Dependent Manner ◽  
Trauma Induced Coagulopathy ◽  
Coagulation And Fibrinolysis

Abstract Background The mechanisms of trauma induced coagulopathy (TIC) are considered multifactorial. Amongst others, however, shedding of the endothelial glycocalyx resulting in increased concentrations of glycocalyx fragments in plasma might also play a role. Thus, we hypothesized that shedded glycocalyx components affect coagulation and may act as humoral mediators of TIC. Methods To investigate effects of heparan sulfate, chondroitin sulfate, syndecan-1, versican, and thrombomodulin we added these fragments to in vitro assays of whole blood from healthy volunteers to yield concentrations observed in trauma patients. Platelet function, whole blood coagulation, and fibrinolysis were measured by standard coagulation tests, impedance aggregometry (IA), and viscoelastic tests (VET). To assess dose-response relationships, we performed IA with increasing concentrations of versican and VET with increasing concentrations of thrombomodulin. Results Intrinsically activated clotting times (i.e., activated partial thromboplastin time and intrinsically activated VET with and without heparinase) were unaffected by any glycocalyx fragment. Thrombomodulin, however, significantly and dose-dependently diminished fibrinolysis as assessed by VET with exogenously added rt-PA, and increased rt-PA-induced lysis Indices after 30 (up to 108% of control, p <  0,0001), 45 (up to 368% of control, p <  0,0001), and 60 min (up to 950% of control, p <  0,0001) in VET. Versican impaired platelet aggregation in response to arachidonic acid (up to − 37,6%, p <  0,0001), ADP (up to − 14,5%, p <  0,0001), and collagen (up to − 31,8%, p <  0,0001) in a dose-dependent manner, but did not affect TRAP-6 induced platelet aggregation. Clotting time in extrinsically activated VET was shortened by heparan sulfate (− 7,2%, p = 0,024), chondroitin sulfate (− 11,6%, p = 0,016), versican (− 13%, p = 0,012%), and when combined (− 7,2%, p = 0,007). Conclusions Glycocalyx components exert distinct inhibitory effects on platelet function, coagulation, and fibrinolysis. These data do not support a ‘heparin-like auto-anticoagulation’ by shed glycosaminoglycans but suggest a possible role of versican in trauma-induced thrombocytopathy and of thrombomodulin in trauma-associated impairment of endogenous fibrinolysis.

scholarly journals Cloning and characterization of mouse extracellular-signal-regulated protein kinase 3 as a unique gene product of 100 kDa

2000 ◽  
Vol 346 (1) ◽  
pp. 169-175 ◽  
Author(s):  
Benjamin TURGEON ◽  
Marc K. SABA-EL-LEIL ◽  
Sylvain MELOCHE
Keyword(s):  
Protein Kinase ◽  
Gene Product ◽  
Mammalian Cells ◽  
Chromosome 9 ◽  
Mouse Tissue ◽  
Mouse Development ◽  
Extracellular Signal ◽  
Mouse Tissues

MAP (mitogen-activated protein) kinases are a family of serine/threonine kinases that have a pivotal role in signal transduction. Here we report the cloning and characterization of a mouse homologue of extracellular-signal-regulated protein kinase (ERK)3. The mouse Erk3 cDNA encodes a predicted protein of 720 residues, which displays 94% identity with human ERK3. Transcription and translation of this cDNA in vitro generates a 100 kDa protein similar to the human gene product ERK3. Immunoblot analysis with an antibody raised against a unique sequence of ERK3 also recognizes a 100 kDa protein in mouse tissues. A single transcript of Erk3 was detected in every adult mouse tissue examined, with the highest expression being found in the brain. Interestingly, expression of Erk3 mRNA is acutely regulated during mouse development, with a peak of expression observed at embryonic day 11. The mouse Erk3 gene was mapped to a single locus on central mouse chromosome 9, adjacent to the dilute mutation locus and in a region syntenic to human chromosome 15q21. Finally, we provide several lines of evidence to support the existence of a unique Erk3 gene product of 100 kDa in mammalian cells.

scholarly journals Syndecan-1 shedding facilitates the resolution of neutrophilic inflammation by removing sequestered CXC chemokines

Blood ◽  
2009 ◽  
Vol 114 (14) ◽  
pp. 3033-3043 ◽  
Author(s):  
Kazutaka Hayashida ◽  
William C. Parks ◽  
Pyong Woo Park
Keyword(s):  
Heparan Sulfate ◽  
Inflammatory Responses ◽  
Organ Damage ◽  
Dependent Manner ◽  
Wild Type ◽  
Neutrophilic Inflammation ◽  
Multiple Organs

Heparan sulfate binds to and regulates many inflammatory mediators in vitro, suggesting that it serves an important role in directing the progression and outcome of inflammatory responses in vivo. Here, we evaluated the role of syndecan-1, a major heparan sulfate proteoglycan, in modulating multiorgan host injury responses in murine endotoxemia. The extent of systemic inflammation was similar between endotoxemic syndecan-1–null and wild-type mice. However, high levels of CXC chemokines (KC and MIP-2), particularly at later times after LPS, were specifically sustained in multiple organs in syndecan-1–null mice and associated with exaggerated neutrophilic inflammation, organ damage, and lethality. Syndecan-1 shedding was activated in several organs of endotoxemic wild-type mice, and this associated closely with the removal of tissue-bound CXC chemokines and resolution of accumulated neutrophils. Moreover, administration of a shedding inhibitor exacerbated disease by impeding the removal of CXC chemokines and neutrophils, whereas administration of heparan sulfate inhibited the accumulation of CXC chemokines and neutrophils in tissues and attenuated multiorgan injury and lethality. These data show that syndecan-1 shedding is a critical endogenous mechanism that facilitates the resolution of neutrophilic inflammation by aiding the clearance of proinflammatory chemokines in a heparan sulfate–dependent manner.

scholarly journals Cell-type specific circadian bioluminescence rhythms recorded from Dbp reporter mice reveal circadian oscillator misalignment

2021 ◽  
Author(s):  
Ciearra B. Smith ◽  
Vincent van der Vinne ◽  
Eleanor McCartney ◽  
Adam C. Stowie ◽  
Tanya L. Leise ◽  
...  
Keyword(s):  
Locomotor Activity ◽  
Circadian Rhythms ◽  
Luciferase Expression ◽  
Dependent Manner ◽  
Cell Type ◽  
Reporter Mice ◽  
Mouse Tissues ◽  
Cell Type Specific

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre-recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vitro and In vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei in vitro. In vivo studies show stable Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ liver reporter mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies provide clear evidence for circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

scholarly journals SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D To Facilitate Viral Replication

2016 ◽  
Vol 90 (23) ◽  
pp. 10472-10485 ◽  
Author(s):  
Yan Liu ◽  
Zhenhua Zheng ◽  
Bo Shu ◽  
Jin Meng ◽  
Yuan Zhang ◽  
...  
Keyword(s):  
Virus Replication ◽  
Protein Localization ◽  
Enterovirus 71 ◽  
Cellular Protein ◽  
Site Directed Mutagenesis ◽  
Ev71 Infection ◽  
Host Immune System ◽  
Dependent Manner ◽  
Viral Polymerase

ABSTRACT Accumulating evidence suggests that viruses hijack cellular proteins to circumvent the host immune system. Ubiquitination and SUMOylation are extensively studied posttranslational modifications (PTMs) that play critical roles in diverse biological processes. Cross talk between ubiquitination and SUMOylation of both host and viral proteins has been reported to result in distinct functional consequences. Enterovirus 71 (EV71), an RNA virus belonging to the family Picornaviridae , is a common cause of hand, foot, and mouth disease. Little is known concerning how host PTM systems interact with enteroviruses. Here, we demonstrate that the 3D protein, an RNA-dependent RNA polymerase (RdRp) of EV71, is modified by small ubiquitin-like modifier 1 (SUMO-1) both during infection and in vitro . Residues K159 and L150/D151/L152 were responsible for 3D SUMOylation as determined by bioinformatics prediction combined with site-directed mutagenesis. Also, primer-dependent polymerase assays indicated that mutation of SUMOylation sites impaired 3D polymerase activity and virus replication. Moreover, 3D is ubiquitinated in a SUMO-dependent manner, and SUMOylation is crucial for 3D stability, which may be due to the interplay between the two PTMs. Importantly, increasing the level of SUMO-1 in EV71-infected cells augmented the SUMOylation and ubiquitination levels of 3D, leading to enhanced replication of EV71. These results together suggested that SUMO and ubiquitin cooperatively regulated EV71 infection, either by SUMO-ubiquitin hybrid chains or by ubiquitin conjugating to the exposed lysine residue through SUMOylation. Our study provides new insight into how a virus utilizes cellular pathways to facilitate its replication. IMPORTANCE Infection with enterovirus 71 (EV71) often causes neurological diseases in children, and EV71 is responsible for the majority of fatalities. Based on a better understanding of interplay between virus and host cell, antiviral drugs against enteroviruses may be developed. As a dynamic cellular process of posttranslational modification, SUMOylation regulates global cellular protein localization, interaction, stability, and enzymatic activity. However, little is known concerning how SUMOylation directly influences virus replication by targeting viral polymerase. Here, we found that EV71 polymerase 3D was SUMOylated during EV71 infection and in vitro . Moreover, the SUMOylation sites were determined, and in vitro polymerase assays indicated that mutations at SUMOylation sites could impair polymerase synthesis. Importantly, 3D is ubiquitinated in a SUMOylation-dependent manner that enhances the stability of the viral polymerase. Our findings indicate that the two modifications likely cooperatively enhance virus replication. Our study may offer a new therapeutic strategy against virus replication.

Cytotoxic activity of the maytansinoid immunoconjugate B-B4–DM1 against CD138+ multiple myeloma cells

Blood ◽  
2004 ◽  
Vol 104 (12) ◽  
pp. 3688-3696 ◽  
Author(s):  
Pierfrancesco Tassone ◽  
Victor S. Goldmacher ◽  
Paola Neri ◽  
Antonella Gozzini ◽  
Masood A. Shammas ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Antitumor Activity ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Scid Mice ◽  
Dependent Manner ◽  
In Vivo Antitumor Activity ◽  
Mouse Tissues

We tested the in vitro and in vivo antitumor activity of the maytansinoid DM1 (N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine), a potent antimicrotubule agent, covalently linked to the murine monoclonal antibody (mAb) B-B4 targeting syndecan-1 (CD138). We evaluated the in vitro activity of B-B4–DM1 against a panel of CD138+ and CD138- cell lines, as well as CD138+ patient multiple myeloma (MM) cells. Treatment with B-B4–DM1 selectively decreased growth and survival of MM cell lines, patient MM cells, and MM cells adherent to bone marrow stromal cells. We further examined the activity of B-B4–DM1 in 3 human MM models in mice: (1) severe combined immunodeficient (SCID) mice bearing subcutaneous xenografts; (2) SCID mice bearing green fluorescent protein–positive (GFP+) xenografts; and (3) SCID mice implanted with human fetal bone (SCID-hu) and subsequently injected with patient MM cells. Tumor regression and inhibition of tumor growth, improvement in overall survival, and reduction in levels of circulating human paraprotein were observed in mice treated with B-B4–DM1. Although immunohistochemical analysis demonstrates restricted CD138 expression in human tissues, the lack of B-B4 reactivity with mouse tissues precludes evaluation of its toxicity in these models. In conclusion, B-B4–DM1 is a potent anti-MM agent that kills cells in an antigen-dependent manner in vitro and mediates in vivo antitumor activity at doses that are well tolerated, providing the rationale for clinical trials of this immunoconjugate in MM.

scholarly journals A role of topoisomerase II in linking DNA replication to chromosome condensation

2003 ◽  
Vol 160 (5) ◽  
pp. 645-655 ◽  
Author(s):  
Olivier Cuvier ◽  
Tatsuya Hirano
Keyword(s):  
Dna Replication ◽  
Topoisomerase Ii ◽  
Atp Hydrolysis ◽  
Early Stage ◽  
Chromosome Condensation ◽  
Binding Property ◽  
Dependent Manner ◽  
Mitotic Chromosomes ◽  
Topo Ii

The condensin complex and topoisomerase II (topo II) have different biochemical activities in vitro, and both are required for mitotic chromosome condensation. We have used Xenopus egg extracts to investigate the functional interplay between condensin and topo II in chromosome condensation. When unreplicated chromatin is directly converted into chromosomes with single chromatids, the two proteins must function together, although they are independently targeted to chromosomes. In contrast, the requirement for topo II is temporarily separable from that of condensin when chromosome assembly is induced after DNA replication. This experimental setting allows us to find that, in the absence of condensin, topo II becomes enriched in an axial structure within uncondensed chromatin. Subsequent addition of condensin converts this structure into mitotic chromosomes in an ATP hydrolysis–dependent manner. Strikingly, preventing DNA replication by the addition of geminin or aphidicolin disturbs the formation of topo II–containing axes and alters the binding property of topo II with chromatin. Our results suggest that topo II plays an important role in an early stage of chromosome condensation, and that this function of topo II is tightly coupled with prior DNA replication.

Sign in / Sign up

Export Citation Format

Share Document