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Article ; Online: MERS-CoV and SARS-CoV-2 membrane proteins are modified with polylactosamine chains.

Juckel, Dylan / Desmarets, Lowiese / Danneels, Adeline / Rouillé, Yves / Dubuisson, Jean / Belouzard, Sandrine

2023 Volume 104, Issue 10

Abstract: Coronaviruses are positive-stranded RNA enveloped viruses. The helical nucleocapsid is surrounded by a lipid bilayer in which are anchored three viral proteins: the spike (S), membrane (M) and envelope (E) proteins. The M protein is the major component ... ...

Abstract	Coronaviruses are positive-stranded RNA enveloped viruses. The helical nucleocapsid is surrounded by a lipid bilayer in which are anchored three viral proteins: the spike (S), membrane (M) and envelope (E) proteins. The M protein is the major component of the viral envelope and is believed to be its building block. The M protein of Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a short N-terminal domain with an N-glycosylation site. We investigated their N-glycosylation and show that polylactosamine chains are conjugated to SARS-CoV-2 and MERS-CoV M proteins in transfected and infected cells. Acidic residues present in the first transmembrane segments of the proteins are required for their glycosylation. No specific signal to specify polylactosamine conjugation could be identified and high mannose-conjugated protein was incorporated into virus-like particles.
MeSH term(s)	Humans ; Middle East Respiratory Syndrome Coronavirus/genetics ; SARS-CoV-2/metabolism ; COVID-19 ; Viral Matrix Proteins/genetics ; Membrane Proteins ; Spike Glycoprotein, Coronavirus/genetics ; Spike Glycoprotein, Coronavirus/metabolism
Chemical Substances	polylactosamine (100787-31-3) ; Viral Matrix Proteins ; Membrane Proteins ; Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2
Language	English
Publishing date	2023-10-06
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	219316-4
ISSN	1465-2099 ; 0022-1317
ISSN (online)	1465-2099
ISSN	0022-1317
DOI	10.1099/jgv.0.001900
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Article ; Online: QuantIF: An ImageJ Macro to Automatically Determine the Percentage of Infected Cells after Immunofluorescence.

Handala, Lynda / Fiore, Tony / Rouillé, Yves / Helle, Francois

Viruses

2019 Volume 11, Issue 2

Abstract: Counting labeled cells, after immunofluorescence or expression of a genetically fluorescent reporter protein, is frequently used to quantify viral infection. However, this can be very tedious without a high content screening apparatus. For this reason, ... ...

Abstract	Counting labeled cells, after immunofluorescence or expression of a genetically fluorescent reporter protein, is frequently used to quantify viral infection. However, this can be very tedious without a high content screening apparatus. For this reason, we have developed QuantIF, an ImageJ macro that automatically determines the total number of cells and the number of labeled cells from two images of the same field, using DAPI- and specific-stainings, respectively. QuantIF can automatically analyze hundreds of images, taking approximately one second for each field. It is freely available as
MeSH term(s)	Algorithms ; Cells, Cultured/virology ; Enterovirus ; Fluorescent Antibody Technique ; Hepacivirus ; Humans ; Image Processing, Computer-Assisted/methods ; Software ; Yellow fever virus
Language	English
Publishing date	2019-02-19
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v11020165
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Article: Comparative Analysis of Hepatitis C Virus NS5A Dynamics and Localization in Assembly-Deficient Mutants.

Riva, Laura / Spriet, Corentin / Barois, Nicolas / Popescu, Costin-Ioan / Dubuisson, Jean / Rouillé, Yves

Pathogens (Basel, Switzerland)

2021 Volume 10, Issue 2

Abstract: The hepatitis C virus (HCV) life cycle is a tightly regulated process, during which structural and non-structural proteins cooperate. However, the interplay between HCV proteins during genomic RNA replication and progeny virion assembly is not completely ...

Abstract	The hepatitis C virus (HCV) life cycle is a tightly regulated process, during which structural and non-structural proteins cooperate. However, the interplay between HCV proteins during genomic RNA replication and progeny virion assembly is not completely understood. Here, we studied the dynamics and intracellular localization of non-structural 5A protein (NS5A), which is a protein involved both in genome replication and encapsidation. An NS5A-eGFP (enhanced green fluorescent protein) tagged version of the strain JFH-1-derived wild-type HCV was compared to the corresponding assembly-deficient viruses Δcore, NS5A basic cluster 352-533 mutant (BCM), and serine cluster 451 + 454 + 457 mutant (SC). These analyses highlighted an increase of NS5A motility when the viral protein core was lacking. Although to a lesser extent, NS5A motility was also increased in the BCM virus, which is characterized by a lack of interaction of NS5A with the viral RNA, impairing HCV genome encapsidation. This observation suggests that the more static NS5A population is mainly involved in viral assembly rather than in RNA replication. Finally, NS4B exhibited a reduced co-localization with NS5A and lipid droplets for both Δcore and SC mutants, which is characterized by the absence of interaction of NS5A with core. This observation strongly suggests that NS5A is involved in targeting NS4B to lipid droplets (LDs). In summary, this work contributes to a better understanding of the interplay between HCV proteins during the viral life cycle.
Language	English
Publishing date	2021-02-04
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2695572-6
ISSN	2076-0817
ISSN	2076-0817
DOI	10.3390/pathogens10020172
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Article: A reporter cell line for the automated quantification of SARS-CoV-2 infection in living cells.

Desmarets, Lowiese / Callens, Nathalie / Hoffmann, Eik / Danneels, Adeline / Lavie, Muriel / Couturier, Cyril / Dubuisson, Jean / Belouzard, Sandrine / Rouillé, Yves

Frontiers in microbiology

2022 Volume 13, Page(s) 1031204

Abstract: The SARS-CoV-2 pandemic and the urgent need for massive antiviral testing highlighted the lack of a good cell-based assay that allowed for a fast, automated screening of antivirals in high-throughput content with minimal handling requirements in a BSL-3 ... ...

Abstract	The SARS-CoV-2 pandemic and the urgent need for massive antiviral testing highlighted the lack of a good cell-based assay that allowed for a fast, automated screening of antivirals in high-throughput content with minimal handling requirements in a BSL-3 environment. The present paper describes the construction of a green fluorescent substrate that, upon cleavage by the SARS-CoV-2 main protease, re-localizes from the cytoplasm in non-infected cells to the nucleus in infected cells. The construction was stably expressed, together with a red fluorescent nuclear marker, in a highly susceptible clone derived from Vero-81 cells. With this fluorescent reporter cell line, named F1G-red, SARS-CoV-2 infection can be scored automatically in living cells by comparing the patterns of green and red fluorescence signals acquired by automated confocal microscopy in a 384-well plate format. We show the F1G-red system is sensitive to several SARS-CoV-2 variants of concern and that it can be used to assess antiviral activities of compounds in dose-response experiments. This high-throughput system will provide a reliable tool for antiviral screening against SARS-CoV-2.
Language	English
Publishing date	2022-09-29
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2587354-4
ISSN	1664-302X
ISSN	1664-302X
DOI	10.3389/fmicb.2022.1031204
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Article ; Online: Sac1 phosphatidylinositol 4‐phosphate phosphatase is a novel host cell factor regulating hepatitis B virus particles assembly and release

Popescu, Mirela‐Andreea / Patriche, David / Dobrica, Mihaela‐Olivia / Pantazica, Ana‐Maria / Flintoaca (Alexandru), Petruta‐Ramona / Rouillé, Yves / Popescu, Costin‐Ioan / Branza‐Nichita, Norica

The FEBS Journal. 2022 Dec., v. 289, no. 23 p.7486-7499

2022

Abstract: The life‐cycle of the Hepatitis B Virus (HBV), an enveloped DNA virus affecting the lives of more than 296 million chronicallyinfected people, is tightly dependent on the lipid metabolism of the host cell. Fatty acids and cholesterol are among the lipid ... ...

Abstract	The life‐cycle of the Hepatitis B Virus (HBV), an enveloped DNA virus affecting the lives of more than 296 million chronicallyinfected people, is tightly dependent on the lipid metabolism of the host cell. Fatty acids and cholesterol are among the lipid factors with documented roles in regulating HBV replication and infection, respectively, but little is known about the phosphoinositide metabolism in these processes. In this study, we investigated the role of Sac1, a highly conserved phosphatidylinositol‐4‐phosphate (PI4P) phosphatase, with essential functions in phospholipid metabolism, in HBV assembly, and release. PI4P is one of the most abundant cellular phosphoinositide with complex functions at the level of the secretory pathway. Owing to the highly specific phosphatase activity toward PI4P, Sac1 controls the levels and restricts the localization of this lipid particularly at the trans‐Golgi network, where it regulates sphingolipid synthesis, proteins sorting, and vesicles budding, by recruiting specific adaptor proteins. As a complete loss of Sac1 function compromises cell viability, in this work, we first developed and characterized several HBV replication‐permissive cellular models with a moderate, transient, or stable downregulation of Sac1 expression. Our results show that Sac1 depletion in hepatic cells results in increased levels and redistribution of intracellular PI4P pools and impaired trafficking of the HBV envelope proteins to the endosomal vesicular network. Importantly, virus envelopment and release from these cells are significantly inhibited, revealing novel roles for Sac1, as a key host cell factor regulating morphogenesis of a DNA virus.
Keywords	DNA viruses ; Hepatitis B virus ; cell viability ; cholesterol ; host-cell factors ; lipid metabolism ; morphogenesis ; phospholipids ; sphingolipids ; viruses
Language	English
Dates of publication	2022-12
Size	p. 7486-7499.
Publishing place	John Wiley & Sons, Ltd
Document type	Article ; Online
Note	JOURNAL ARTICLE
ZDB-ID	2173655-8
ISSN	1742-4658 ; 1742-464X
ISSN (online)	1742-4658
ISSN	1742-464X
DOI	10.1111/febs.16575
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Lichen or Associated Micro-Organism Compounds Are Active against Human Coronaviruses.

Desmarets, Lowiese / Millot, Marion / Chollet-Krugler, Marylène / Boustie, Joël / Camuzet, Charline / François, Nathan / Rouillé, Yves / Belouzard, Sandrine / Tomasi, Sophie / Mambu, Lengo / Séron, Karin

Viruses

2023 Volume 15, Issue 9

Abstract: 1) Background: Since the emergence of SARS-CoV-2, responsible for the COVID-19 pandemic, efforts have been made to identify antiviral compounds against human coronaviruses. With the aim of increasing the diversity of molecule scaffolds, 42 natural ... ...

Abstract	(1) Background: Since the emergence of SARS-CoV-2, responsible for the COVID-19 pandemic, efforts have been made to identify antiviral compounds against human coronaviruses. With the aim of increasing the diversity of molecule scaffolds, 42 natural compounds, of which 28 were isolated from lichens and 14 from their associated microorganisms (bacteria and fungi), were screened against human coronavirus HCoV-229E. (2) Methods: Antiviral assays were performed using HCoV-229E in Huh-7 and Huh-7/TMPRSS2 cells and SARS-CoV-2 in a Vero-81-derived clone with a GFP reporter probe. (3) Results: Four lichen compounds, including chloroatranol, emodin, perlatolic acid and vulpinic acid, displayed high activities against HCoV-229E (IC
MeSH term(s)	Humans ; Lichens ; Pandemics ; COVID-19 ; SARS-CoV-2 ; Antiviral Agents/pharmacology ; Coronavirus 229E, Human
Chemical Substances	perlatolic acid ; Antiviral Agents
Language	English
Publishing date	2023-08-31
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v15091859
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Article ; Online: Secretory Vesicles Are the Principal Means of SARS-CoV-2 Egress.

Eymieux, Sébastien / Uzbekov, Rustem / Rouillé, Yves / Blanchard, Emmanuelle / Hourioux, Christophe / Dubuisson, Jean / Belouzard, Sandrine / Roingeard, Philippe

Cells

2021 Volume 10, Issue 8

Abstract: The mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress, similar to those of other coronaviruses, remain poorly understood. The virus buds in intracellular compartments and is therefore thought to be released by the ... ...

Abstract	The mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress, similar to those of other coronaviruses, remain poorly understood. The virus buds in intracellular compartments and is therefore thought to be released by the biosynthetic secretory pathway. However, several studies have recently challenged this hypothesis. It has been suggested that coronaviruses, including SARS-CoV-2, use lysosomes for egress. In addition, a focused ion-beam scanning electron microscope (FIB/SEM) study suggested the existence of exit tunnels linking cellular compartments rich in viral particles to the extracellular space resembling those observed for the human immunodeficiency (HIV) in macrophages. Here, we analysed serial sections of Vero cells infected with SARS-CoV-2 by transmission electron microscopy (TEM). We found that SARS-CoV-2 was more likely to exit the cell in small secretory vesicles. Virus trafficking within the cells involves small vesicles, with each generally containing a single virus particle. These vesicles then fuse with the plasma membrane to release the virus into the extracellular space. This work sheds new light on the late stages of the SARS-CoV-2 infectious cycle of potential value for guiding the development of new antiviral strategies.
Language	English
Publishing date	2021-08-10
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2661518-6
ISSN	2073-4409 ; 2073-4409
ISSN (online)	2073-4409
ISSN	2073-4409
DOI	10.3390/cells10082047
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Article ; Online: Ultrastructural modifications induced by SARS-CoV-2 in Vero cells: a kinetic analysis of viral factory formation, viral particle morphogenesis and virion release.

Eymieux, Sébastien / Rouillé, Yves / Terrier, Olivier / Seron, Karin / Blanchard, Emmanuelle / Rosa-Calatrava, Manuel / Dubuisson, Jean / Belouzard, Sandrine / Roingeard, Philippe

Cellular and molecular life sciences : CMLS

2021 Volume 78, Issue 7, Page(s) 3565–3576

Abstract: Many studies on SARS-CoV-2 have been performed over short-time scale, but few have focused on the ultrastructural characteristics of infected cells. We used TEM to perform kinetic analysis of the ultrastructure of SARS-CoV-2-infected cells. Early ... ...

Abstract	Many studies on SARS-CoV-2 have been performed over short-time scale, but few have focused on the ultrastructural characteristics of infected cells. We used TEM to perform kinetic analysis of the ultrastructure of SARS-CoV-2-infected cells. Early infection events were characterized by the presence of clusters of single-membrane vesicles and stacks of membrane containing nuclear pores called annulate lamellae (AL). A large network of host cell-derived organelles transformed into virus factories was subsequently observed in the cells. As previously described for other RNA viruses, these replication factories consisted of double-membrane vesicles (DMVs) located close to the nucleus. Viruses released at the cell surface by exocytosis harbored the typical crown of spike proteins, but viral particles without spikes were also observed in intracellular compartments, possibly reflecting incorrect assembly or a cell degradation process.
MeSH term(s)	Animals ; COVID-19/pathology ; Cell Line ; Chlorocebus aethiops ; Microscopy, Electron, Transmission ; SARS-CoV-2/growth & development ; Spike Glycoprotein, Coronavirus/metabolism ; Vero Cells ; Viral Replication Compartments/physiology ; Viral Replication Compartments/ultrastructure ; Virus Release/physiology ; Virus Replication/physiology
Chemical Substances	Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2
Language	English
Publishing date	2021-01-15
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	1358415-7
ISSN	1420-9071 ; 1420-682X
ISSN (online)	1420-9071
ISSN	1420-682X
DOI	10.1007/s00018-020-03745-y
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Article ; Online: Shotgun metagenomics and systemic targeted metabolomics highlight indole-3-propionic acid as a protective gut microbial metabolite against influenza infection.

Heumel, Séverine / de Rezende Rodovalho, Vinícius / Urien, Charlotte / Specque, Florian / Brito Rodrigues, Patrícia / Robil, Cyril / Delval, Lou / Sencio, Valentin / Descat, Amandine / Deruyter, Lucie / Ferreira, Stéphanie / Gomes Machado, Marina / Barthelemy, Adeline / Angulo, Fabiola Silva / Haas, Joel T / Goosens, Jean François / Wolowczuk, Isabelle / Grangette, Corinne / Rouillé, Yves /

Grimaud, Ghjuvan / Lenski, Marie / Hennart, Benjamin / Ramirez Vinolo, Marco Aurélio / Trottein, François

Gut microbes

2024 Volume 16, Issue 1, Page(s) 2325067

Abstract: The gut-to-lung axis is critical during respiratory infections, including influenza A virus (IAV) infection. In the present study, we used high-resolution shotgun metagenomics and targeted metabolomic analysis to characterize influenza-associated changes ...

Abstract	The gut-to-lung axis is critical during respiratory infections, including influenza A virus (IAV) infection. In the present study, we used high-resolution shotgun metagenomics and targeted metabolomic analysis to characterize influenza-associated changes in the composition and metabolism of the mouse gut microbiota. We observed several taxonomic-level changes on day (D)7 post-infection, including a marked reduction in the abundance of members of the
MeSH term(s)	Humans ; Animals ; Mice ; Propionates ; Influenza, Human ; Gastrointestinal Microbiome ; Tryptophan ; Actinobacteria ; Inflammation ; Polyamines
Chemical Substances	propionic acid (JHU490RVYR) ; Propionates ; Tryptophan (8DUH1N11BX) ; Polyamines
Language	English
Publishing date	2024-03-06
Publishing country	United States
Document type	Journal Article
ZDB-ID	2575755-6
ISSN	1949-0984 ; 1949-0984
ISSN (online)	1949-0984
ISSN	1949-0984
DOI	10.1080/19490976.2024.2325067
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Article: Hepatitis C virus alters the morphology and function of peroxisomes.

Martin de Fourchambault, Esther / Callens, Nathalie / Saliou, Jean-Michel / Fourcot, Marie / Delos, Oceane / Barois, Nicolas / Thorel, Quentin / Ramirez, Santseharay / Bukh, Jens / Cocquerel, Laurence / Bertrand-Michel, Justine / Marot, Guillemette / Sebti, Yasmine / Dubuisson, Jean / Rouillé, Yves

Frontiers in microbiology

2023 Volume 14, Page(s) 1254728

Abstract: Despite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver disease induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid ... ...

Abstract	Despite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver disease induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid metabolism and redox balance, but the mechanisms leading to HCV-induced pathogenesis are still poorly understood. In an APEX2-based proximity biotinylation screen, we identified ACBD5, a peroxisome membrane protein, as located in the vicinity of HCV replication complexes. Confocal microscopy confirmed the relocation of peroxisomes near HCV replication complexes and indicated that their morphology and number are altered in approximately 30% of infected Huh-7 cells. Peroxisomes are small versatile organelles involved among other functions in lipid metabolism and ROS regulation. To determine their importance in the HCV life cycle, we generated Huh-7 cells devoid of peroxisomes by inactivating the PEX5 and PEX3 genes using CRISPR/Cas9 and found that the absence of peroxisomes had no impact on replication kinetics or infectious titers of HCV strains JFH1 and DBN3a. The impact of HCV on peroxisomal functions was assessed using sub-genomic replicons. An increase of ROS was measured in peroxisomes of replicon-containing cells, correlated with a significant decrease of catalase activity with the DBN3a strain. In contrast, HCV replication had little to no impact on cytoplasmic and mitochondrial ROS, suggesting that the redox balance of peroxisomes is specifically impaired in cells replicating HCV. Our study provides evidence that peroxisome function and morphology are altered in HCV-infected cells.
Language	English
Publishing date	2023-09-21
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2587354-4
ISSN	1664-302X
ISSN	1664-302X
DOI	10.3389/fmicb.2023.1254728
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