scholarly journals A Single Mutation in the E2 Glycoprotein Important for Neurovirulence Influences Binding of Sindbis Virus to Neuroblastoma Cells

2002 ◽  
Vol 76 (12) ◽  
pp. 6302-6310 ◽  
Author(s):  
Peiyu Lee ◽  
Ronald Knight ◽  
Jolanda M. Smit ◽  
Jan Wilschut ◽  
Diane E. Griffin
Keyword(s):  
Virus Strain ◽  
Sindbis Virus ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Neural Cells ◽  
Single Mutation ◽  
Virus Binding ◽  
Critical Determinant ◽  
Bhk Cells ◽  
E2 Glycoprotein

ABSTRACT The amino acid at position 55 of the E2 glycoprotein (E255) of Sindbis virus (SV) is a critical determinant of SV neurovirulence in mice. Recombinant virus strain TE (E255 = histidine) differs only at this position from virus strain 633 (E255= glutamine), yet TE is considerably more neurovirulent than 633. TE replicates better than 633 in a neuroblastoma cell line (N18), but similarly in BHK cells. Immunofluorescence staining showed that most N18 cells were infected by TE at a multiplicity of infection (MOI) of 50 to 500 and by 633 only at an MOI of 5,000, while both viruses infected essentially 100% of BHK cells at an MOI of 5. When exposed to pH 5, TE and 633 viruses fused to similar extents with liposomes derived from BHK or N18 cell lipids, but fusion with N18-derived liposomes was less extensive (15 to 20%) than fusion with BHK-derived liposomes (∼50%). Binding of TE and 633 to N18, but not BHK, cells was dependent on the medium used for virus binding. Differences between TE and 633 binding to N18 cells were evident in Dulbecco's modified Eagle medium (DMEM), but not in RPMI. In DMEM, the binding efficiency of 633 decreased significantly as the pH was raised from 6.5 to 8.0, while that of TE did not change. The same pattern was observed with RPMI when the ionic strength of RPMI was increased to that of DMEM. TE bound better to heparin-Sepharose than 633, but this difference was not pH dependent. Growth of N18 and BHK cells in sodium chlorate to eliminate all sulfation decreased virus-cell binding, suggesting the involvement of sulfated molecules on the cell surface. Taken together, the presence of glutamine at E255 impairs SV binding to neural cells under conditions characteristic of interstitial fluid. We conclude that mutation to histidine participates in or stabilizes the interaction between the virus and the surface of neural cells, contributing to greater neurovirulence.

scholarly journals Role of N-Linked Glycosylation for Sindbis Virus Infection and Replication in Vertebrate and Invertebrate Systems

2009 ◽  
Vol 83 (11) ◽  
pp. 5640-5647 ◽  
Author(s):  
Ronald L. Knight ◽  
Kimberly L. W. Schultz ◽  
Rebekah J. Kent ◽  
Meera Venkatesan ◽  
Diane E. Griffin
Keyword(s):  
Mammalian Cells ◽  
Sindbis Virus ◽  
Glycosylation Site ◽  
Surface Glycoprotein ◽  
Heparin Binding ◽  
Virus Binding ◽  
Bhk Cells ◽  
Mosquito Cells ◽  
Intrathoracic Inoculation

ABSTRACT Each Sindbis virus (SINV) surface glycoprotein has two sites for N-linked glycosylation (E1 positions 139 and 245 [E1-139 and E1-245] and E2 positions 196 and 318 [E2-196 and E2-318]). Studies of SINV strain TE12 mutants with each site eliminated identified the locations of carbohydrates by cryo-electron microscopy (S. V. Pletnev et al., Cell 105:127-136, 2001). In the current study, the effects of altered glycosylation on virion infectivity, growth in cells of vertebrates and invertebrates, heparin binding, virulence in mice, and replication in mosquitoes were assessed. Particle-to-PFU ratios for E1-139 and E2-196 mutant strains were similar to that for TE12, but this ratio for the E1-245 mutant was 100-fold lower than that for TE12. Elimination of either E2 glycosylation site increased virus binding to heparin and increased replication in BHK cells. Elimination of either E1 glycosylation site had no effect on heparin binding but resulted in an approximately 10-fold decrease in virus yield from BHK cells compared to the TE12 amount. No differences in pE2 processing were detected. E2-196 and E2-318 mutants were more virulent in mice after intracerebral inoculation, while E1-139 and E1-245 mutants were less virulent. The E1-245 mutant showed impaired replication in C7/10 mosquito cells and in Culex quinquefasciatus after intrathoracic inoculation. We conclude that the increased replication and virulence of E2-196 and E2-318 mutants are primarily due to increased efficiency of binding to heparan sulfate on mammalian cells. Lack of glycosylation at E1-139 or E1-245 impairs replication in vertebrate cells, while E1-245 also severely affects replication in invertebrate cells.

scholarly journals Amino acid changes in the Sindbis virus E2 glycoprotein that increase neurovirulence improve entry into neuroblastoma cells.

1997 ◽  
Vol 71 (8) ◽  
pp. 6106-6112 ◽  
Author(s):  
P C Tucker ◽  
S H Lee ◽  
N Bui ◽  
D Martinie ◽  
D E Griffin
Keyword(s):  
Amino Acid ◽  
Sindbis Virus ◽  
Neuroblastoma Cells ◽  
E2 Glycoprotein

scholarly journals Infection of Neonatal Mice with Sindbis Virus Results in a Systemic Inflammatory Response Syndrome

1999 ◽  
Vol 73 (12) ◽  
pp. 10387-10398 ◽  
Author(s):  
W. B. Klimstra ◽  
K. D. Ryman ◽  
K. A. Bernard ◽  
K. B. Nguyen ◽  
C. A. Biron ◽  
...  
Keyword(s):  
Cell Culture ◽  
Systemic Inflammatory Response Syndrome ◽  
Inflammatory Response ◽  
Virus Strain ◽  
Cdna Clone ◽  
Sindbis Virus ◽  
Systemic Inflammatory Response ◽  
Single Mutation ◽  
Adaptive Mutations ◽  
Nontranslated Region

ABSTRACT Laboratory strains of viruses may contain cell culture-adaptive mutations which result in significant quantitative and qualitative alterations in pathogenesis compared to natural virus isolates. This report suggests that this is the case with Sindbis virus strain AR339. A cDNA clone comprising a consensus sequence of Sindbis virus strain AR339 has been constructed (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357–7366, 1998). This clone (pTR339) regenerates a sequence predicted to be very close to that of the original AR339 isolate by eliminating several cell culture-adaptive mutations present in individual laboratory strains of the virus (K. L. McKnight et al., J. Virol. 70:1981–1989, 1996). It thus provides a unique reagent for study of the pathogenesis of Sindbis virus strain AR339 in mice. Neonatal mouse pathogenesis of virus (TR339) generated from the pTR339 clone was compared with that of virus from a cDNA clone of the cell culture-passaged laboratory AR339 strain, TRSB, and virus from a clone of a more highly cell culture-adapted strain, HRsp (Toto 50). The sequence of TRSB differs from the consensus at three coding positions, while Toto 50 differs at eight codons and one nucleotide in the 5′ nontranslated region. Both cell culture-adapted strains contain mutations associated with heparan sulfate (HS)-dependent attachment to cells (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357–7366, 1998). TR339 caused 100% mortality with an average survival time (AST) of 1.7 ± 0.25 days. While TRSB also caused 100% mortality, the AST was extended to 2.9 ± 0.52 days. The more extensively cell culture-adapted virus Toto 50 caused only 30% mortality with an AST extended to 11.0 ± 4.8 days. TRSB and TR339 induced high serum levels of alpha/beta interferon, gamma interferon, tumor necrosis factor alpha, interleukin-6, and corticosterone and induced pathology reminiscent of lipopolysaccharide-induced endotoxic shock, a type of systemic inflammatory response syndrome. However, the reduced intensity of this response in TRSB-infected mice correlated with the increased AST. Toto 50 failed to induce the shock-like cytokine cascade. In situ hybridization studies indicated that TR339 and TRSB replicated in identical tissues, but the TRSB signal was less widespread at early times postinfection. While Toto 50 also replicated in similar tissues, the extent of replication was severely restricted and mice developed lesions characteristic of encephalitis. A single mutation in TRSB at E2 position 1 (Arg) conferred HS-dependent attachment to cells and was associated with reduced cytokine induction and extended AST in vivo.

p-Trifluoroacetophenone Oxime Ester Derivatives: Synthesis, Antimicrobial and Cytotoxic Evaluation and Molecular Modeling Studies

2020 ◽  
Vol 17 (2) ◽  
pp. 169-183 ◽  
Author(s):  
İrem Bozbey ◽  
Suat Sari ◽  
Emine Şalva ◽  
Didem Kart ◽  
Arzu Karakurt
Keyword(s):  
Molecular Modeling ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Cytotoxic Agents ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma ◽  
Modeling Studies ◽  
Broth Microdilution Method ◽  
Molecular Modeling Studies

Background: Azole antifungals are among the first-line drugs clinically used for the treatment of systemic candidiasis, a deadly type of fungal infection that threatens mostly immunecompromised and hospitalized patients. Some azole derivatives were also reported to have antiproliferative effects on cancer cells. Objective: In this study, 1-(4-trifluoromethylphenyl)-2-(1H-imidazol-1-yl)ethanone (3), its oxime (4), and a series of its novel oxime ester derivatives (5a-v) were synthesized and tested for their in vitro antimicrobial activities against certain ATCC standard strains of Candida sp. fungi and bacteria. The compounds were also tested for their cytotoxic effects against mouse fibroblast and human neuroblastoma cell lines. Molecular modeling studies were performed to provide insights into their possible mechanisms for antifungal and antibacterial actions. Methods: The compounds were synthesized by the reaction of various oximes with acyl chlorides. Antimicrobial activity of the compounds was determined according to the broth microdilution method. For the determination of cytotoxic effect, we used MTS assay. Molecular docking and QM/MM studies were performed to predict the binding mechanisms of the active compounds in the catalytic site of C. albicans CYP51 (CACYP51) and S. aureus flavohemoglobin (SAFH), the latter of which was created via homology modeling. Results: 5d, 5l, and 5t showed moderate antifungal activity against C. albicans, while 3, 5c, and 5r showed significant antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa. Most of the compounds showed approximately 40-50% inhibition against the human neuroblastoma cells at 100 µM. In this line, 3 was the most potent with an IC50 value of 82.18 μM followed by 5a, 5o, and 5t. 3 and 5a were highly selective to the neuroblastoma cells. Molecular modelling results supported the hypothesis that our compounds were inhibitors of CAYP51 and SAFH. Conclusion: This study supports that oxime ester derivatives may be used for the development of new antimicrobial and cytotoxic agents.

scholarly journals Plasticity in Neuroblastoma Cell Identity Defines a Noradrenergic-to-Mesenchymal Transition (NMT)

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2904
Author(s):  
Margot Gautier ◽  
Cécile Thirant ◽  
Olivier Delattre ◽  
Isabelle Janoueix-Lerosey
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Biochemical Properties ◽  
Poor Overall Survival ◽  
Cell Identity ◽  
Mesenchymal Transition ◽  
Neuroblastoma Cell Lines

Neuroblastoma, a pediatric cancer of the peripheral sympathetic nervous system, is characterized by an important clinical heterogeneity, and high-risk tumors are associated with a poor overall survival. Neuroblastoma cells may present with diverse morphological and biochemical properties in vitro, and seminal observations suggested that interconversion between two phenotypes called N-type and S-type may occur. In 2017, two main studies provided novel insights into these subtypes through the characterization of the transcriptomic and epigenetic landscapes of a panel of neuroblastoma cell lines. In this review, we focus on the available data that define neuroblastoma cell identity and propose to use the term noradrenergic (NOR) and mesenchymal (MES) to refer to these identities. We also address the question of transdifferentiation between both states and suggest that the plasticity between the NOR identity and the MES identity defines a noradrenergic-to-mesenchymal transition, reminiscent of but different from the well-established epithelial-to-mesenchymal transition.

scholarly journals Regulation of c-myb expression in human neuroblastoma cells during retinoic acid-induced differentiation.

1988 ◽  
Vol 8 (4) ◽  
pp. 1677-1683 ◽  
Author(s):  
C J Thiele ◽  
P S Cohen ◽  
M A Israel
Keyword(s):  
Retinoic Acid ◽  
Fetal Development ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma ◽  
Hematopoietic Lineage ◽  
Specific Manner ◽  
Neuroblastoma Cell Lines

We detected expression of the c-myb proto-oncogene, which was initially thought to be expressed in a tissue-specific manner in cells of hematopoietic lineage, in human tissues of neuronal origin. Since the level of c-myb expression declined during fetal development, we studied the regulation of its expression in human neuroblastoma cell lines induced to differentiate by retinoic acid. The expression of c-myb declined during the maturation of neuroblastoma cells, and this change was mediated by a decrease in c-myb transcription.

scholarly journals Transplantation of Human Neuroblastoma Cells, Catecholaminergic and Non-Catecholaminergic: Effects on Rotational Behavoir in Parkinson's Rat Model

1992 ◽  
Vol 3 (2-3) ◽  
pp. 139-150 ◽  
Author(s):  
Jacob S. Manaster ◽  
Tony Feuerman ◽  
C. Patrick Reynolds ◽  
Charles H. Markham
Keyword(s):  
Cell Lines ◽  
Rat Model ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Neuroblastoma Cell Line ◽  
Rotational Behavior ◽  
Human Neuroblastoma ◽  
Donor Cells ◽  
Catecholamine Fluorescence ◽  
Motor Improvement

Cultured human catecholaminergic and noncatecholaminergic donor cells were used in neural transplantation experiments in a rat model of Parkinson's disease. Using two different human catecholaminergic neuroblastoma cell lines, one control non-catecholaminergic neuroblastoma cell line, and one sham control (tissue culture medium), transplants were made into the striatum using a modified Ungerstedt hemiparkinsonian rat model. Significant decreases in apomorphine-induced rotational behavior were produced by two of three catecholaminergic cell lines. Grafted cells staining positively for tyrosine hydroxylase (TH) and catecholamine fluorescence indicated viable catecholamine activity in the two cell lines which produced reductions in rotational behavior. Catecholamine fluorescence was not detected in either of the two controls. These data suggest a link between catecholamine secretion by transplanted cells and motor improvement using a rat rotational behavior model.

scholarly journals Proteomic Discovery of VEEV E2-Host Partner Interactions Identifies GRP78 Inhibitor HA15 as a Potential Therapeutic for Alphavirus Infections

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 283
Author(s):  
Michael D. Barrera ◽  
Victoria Callahan ◽  
Ivan Akhrymuk ◽  
Nishank Bhalla ◽  
Weidong Zhou ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Trafficking ◽  
Viral Protein ◽  
Sindbis Virus ◽  
Encephalitis Virus ◽  
Host Proteins ◽  
Atp Production ◽  
Equine Encephalitis Virus ◽  
E2 Glycoprotein ◽  
Late Stages

Alphaviruses are a genus of the Togaviridae family and are widely distributed across the globe. Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), cause encephalitis and neurological sequelae while chikungunya virus (CHIKV) and Sindbis virus (SINV) cause arthralgia. There are currently no approved therapeutics or vaccines available for alphaviruses. In order to identify novel therapeutics, a V5 epitope tag was inserted into the N-terminus of the VEEV E2 glycoprotein and used to identify host-viral protein interactions. Host proteins involved in protein folding, metabolism/ATP production, translation, cytoskeleton, complement, vesicle transport and ubiquitination were identified as VEEV E2 interactors. Multiple inhibitors targeting these host proteins were tested to determine their effect on VEEV replication. The compound HA15, a GRP78 inhibitor, was found to be an effective inhibitor of VEEV, EEEV, CHIKV, and SINV. VEEV E2 interaction with GRP78 was confirmed through coimmunoprecipitation and colocalization experiments. Mechanism of action studies found that HA15 does not affect viral RNA replication but instead affects late stages of the viral life cycle, which is consistent with GRP78 promoting viral assembly or viral protein trafficking.

Tissue transglutaminase and apoptosis: sense and antisense transfection studies with human neuroblastoma cells

1994 ◽  
Vol 14 (10) ◽  
pp. 6584-6596
Author(s):  
G Melino ◽  
M Annicchiarico-Petruzzelli ◽  
L Piredda ◽  
E Candi ◽  
V Gentile ◽  
...  
Keyword(s):  
Cell Death ◽  
Structural Changes ◽  
Tissue Transglutaminase ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Neuroblastoma Cell Line ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma ◽  
Transfected Cells ◽  
Protein Levels

In this report, we show that the overexpression of tissue transglutaminase (tTG) in the human neuroblastoma cell line SK-N-BE(2) renders these neural crest-derived cells highly susceptible to death by apoptosis. Cells transfected with a full-length tTG cDNA, under the control of a constitutive promoter, show a drastic reduction in proliferative capacity paralleled by a large increase in cell death rate. The dying tTG-transfected cells exhibit both cytoplasmic and nuclear changes characteristic of cells undergoing apoptosis. The tTG-transfected cells express high Bcl-2 protein levels as well as phenotypic neural cell adhesion molecule markers (NCAM and neurofilaments) of cells differentiating along the neuronal pathway. In keeping with these findings, transfection of neuroblastoma cells with an expression vector containing segments of the human tTG cDNA in antisense orientation resulted in a pronounced decrease of both spontaneous and retinoic acid (RA)-induced apoptosis. We also present evidence that (i) the apoptotic program of these neuroectodermal cells is strictly regulated by RA and (ii) cell death by apoptosis in the human neuroblastoma SK-N-BE(2) cells preferentially occurs in the substrate-adherent phenotype. For the first time, we report here a direct effect of tTG in the phenotypic maturation toward apoptosis. These results indicate that the tTG-dependent irreversible cross-linking of intracellular protein represents an important biochemical event in the induction of the structural changes featuring cells dying by apoptosis.

Sign in / Sign up

Export Citation Format

Share Document