8 results on '"Mickaël, Bouvet"'
Search Results
2. First Days in the Life of Naive Human B Lymphocytes Infected with Epstein-Barr Virus
- Author
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Dagmar Pich, Paulina Mrozek-Gorska, Mickaël Bouvet, Atsuko Sugimoto, Ezgi Akidil, Adam Grundhoff, Stephan Hamperl, Paul D. Ling, and Wolfgang Hammerschmidt
- Subjects
B lymphocytes ,human herpesviruses ,reprogramming ,transformation ,Microbiology ,QR1-502 - Abstract
ABSTRACT Epstein-Barr virus (EBV) infects and activates resting human B lymphocytes, reprograms them, induces their proliferation, and establishes a latent infection in them. In established EBV-infected cell lines, many viral latent genes are expressed. Their roles in supporting the continuous proliferation of EBV-infected B cells in vitro are known, but their functions in the early, prelatent phase of infection have not been investigated systematically. In studies during the first 8 days of infection using derivatives of EBV with mutations in single genes of EBVs, we found only Epstein-Barr nuclear antigen 2 (EBNA2) to be essential for activating naive human B lymphocytes, inducing their growth in cell volume, driving them into rapid cell divisions, and preventing cell death in a subset of infected cells. EBNA-LP, latent membrane protein 2A (LMP2A), and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding Epstein-Barr virus with small RNA (EBERs) had no discernible phenotype compared with wild-type EBV. B cells infected with a double mutant of EBNA3A and 3C had an unexpected proliferative advantage and did not regulate the DNA damage response (DDR) of the infected host cell in the prelatent phase. Even EBNA1, which has very critical long-term functions in maintaining and replicating the viral genomic DNA in established cell lines, was dispensable for the early activation of infected cells. Our findings document that the virus dose is a decisive parameter and indicate that EBNA2 governs the infected cells initially and implements a strictly controlled temporal program independent of other viral latent genes. It thus appears that EBNA2 is sufficient to control all requirements for clonal cellular expansion and to reprogram human B lymphocytes from energetically quiescent to activated cells. IMPORTANCE The preferred target of Epstein-Barr virus (EBV) is human resting B lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial increase in cell volume, followed by cellular DNA synthesis after 3 days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to 3 days later, the cells decelerate and turn into stably proliferating lymphoblast cell lines. With the aid of 16 different recombinant EBV strains, we investigated the individual contributions of EBV’s multiple latent genes during early B-cell infection and found that many do not exert a detectable phenotype or contribute little to EBV’s prelatent phase. The exception is EBNA2 that is essential in governing all aspects of B-cell reprogramming. EBV relies on EBNA2 to turn the infected B lymphocytes into proliferating lymphoblasts preparing the infected host cell for the ensuing stable, latent phase of viral infection. In the early steps of B-cell reprogramming, viral latent genes other than EBNA2 are dispensable, but some, EBNA-LP, for example, support the viral program and presumably stabilize the infected cells once viral latency is established.
- Published
- 2019
- Full Text
- View/download PDF
3. Multiple viral micrornas regulate interferon release and signaling early during infection with epstein-barr virus
- Author
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Yan Chen, Yen-Fu Adam Chen, Rebecca L. Skalsky, Mickaël Bouvet, Christine Göbel, Dagmar Pich, Takanobu Tagawa, Wolfgang Hammerschmidt, Manuel Albanese, Stefanie Voigt, and Devin N. Fachko
- Subjects
Epstein-Barr Virus Infections ,Herpesvirus 4, Human ,medicine.disease_cause ,0302 clinical medicine ,Interferon ,hemic and lymphatic diseases ,0303 health sciences ,B-Lymphocytes ,microRNA ,interferon ,B Cells ,Epstein-barr Virus ,Immune Evasion ,Interferons ,Microrna ,Plasmacytoid Dendritic Cells ,QR1-502 ,interferons ,plasmacytoid dendritic cells ,Host-Pathogen Interactions ,RNA, Viral ,Signal Transduction ,medicine.drug ,Research Article ,Gene Expression Regulation, Viral ,Biology ,Antiviral Agents ,Microbiology ,Virus ,03 medical and health sciences ,Immune system ,EBV ,Virology ,medicine ,Humans ,Epstein-Barr virus ,Secretion ,Transcription factor ,Gene ,030304 developmental biology ,miRNA ,immune evasion ,B cells ,Interferon-alpha ,pDCs ,TLR9 ,Interferon-beta ,ncRNA ,Epstein–Barr virus ,MicroRNAs ,Toll-Like Receptor 7 ,Commentary ,030215 immunology - Abstract
Acute antiviral functions of all nucleated cells rely on type I interferon (IFN-I) pathways triggered upon viral infection. Host responses encompass the sensing of incoming viruses, the activation of specific transcription factors that induce the transcription of IFN-I genes, the secretion of different IFN-I types and their recognition by the heterodimeric IFN-α/β receptor, the subsequent activation of JAK/STAT signaling pathways, and, finally, the transcription of many IFN-stimulated genes (ISGs)., Epstein-Barr virus (EBV), a human herpesvirus, encodes 44 microRNAs (miRNAs), which regulate many genes with various functions in EBV-infected cells. Multiple target genes of the EBV miRNAs have been identified, some of which play important roles in adaptive antiviral immune responses. Using EBV mutant derivatives, we identified additional roles of viral miRNAs in governing versatile type I interferon (IFN) responses upon infection of human primary mature B cells. We also found that Epstein-Barr virus-encoded small RNAs (EBERs) and LF2, viral genes with previously reported functions in inducing or regulating IFN-I pathways, had negligible or even contrary effects on secreted IFN-α in our model. Data mining and Ago PAR-CLIP experiments uncovered more than a dozen previously uncharacterized, direct cellular targets of EBV miRNA associated with type I IFN pathways. We also identified indirect targets of EBV miRNAs in B cells, such as TRL7 and TLR9, in the prelatent phase of infection. The presence of epigenetically naive, non-CpG methylated viral DNA was essential to induce IFN-α secretion during EBV infection in a TLR9-dependent manner. In a newly established fusion assay, we verified that EBV virions enter a subset of plasmacytoid dendritic cells (pDCs) and determined that these infected pDCs are the primary producers of IFN-α in EBV-infected peripheral blood mononuclear cells. Our findings document that many EBV-encoded miRNAs regulate type I IFN response in newly EBV infected primary human B cells in the prelatent phase of infection and dampen the acute release of IFN-α in pDCs upon their encounter with EBV.
- Published
- 2021
4. Correction: Reconstitution of SARS-Coronavirus mRNA Cap Methylation.
- Author
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Mickaël Bouvet, Claire Debarnot, Isabelle Imbert, Barbara Selisko, Eric J. Snijder, Bruno Canard, and Etienne Decroly
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Published
- 2010
- Full Text
- View/download PDF
5. In vitro reconstitution of SARS-coronavirus mRNA cap methylation.
- Author
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Mickaël Bouvet, Claire Debarnot, Isabelle Imbert, Barbara Selisko, Eric J Snijder, Bruno Canard, and Etienne Decroly
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
SARS-coronavirus (SARS-CoV) genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 5' end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16) constitute an unusually large replicase complex, which includes two methyltransferases putatively involved in viral mRNA cap formation. The S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) activity was recently attributed to nsp14, whereas nsp16 has been predicted to be the AdoMet-dependent (nucleoside-2'O)-methyltransferase. Here, we have reconstituted complete SARS-CoV mRNA cap methylation in vitro. We show that mRNA cap methylation requires a third viral protein, nsp10, which acts as an essential trigger to complete RNA cap-1 formation. The obligate sequence of methylation events is initiated by nsp14, which first methylates capped RNA transcripts to generate cap-0 (7Me)GpppA-RNAs. The latter are then selectively 2'O-methylated by the 2'O-MTase nsp16 in complex with its activator nsp10 to give rise to cap-1 (7Me)GpppA(2'OMe)-RNAs. Furthermore, sensitive in vitro inhibition assays of both activities show that aurintricarboxylic acid, active in SARS-CoV infected cells, targets both MTases with IC(50) values in the micromolar range, providing a validated basis for anti-coronavirus drug design.
- Published
- 2010
- Full Text
- View/download PDF
6. First Days in the Life of Naive Human B Lymphocytes Infected with Epstein-Barr Virus
- Author
-
Mickaël Bouvet, Dagmar Pich, Atsuko Sugimoto, Paul D. Ling, Paulina Mrozek-Gorska, Wolfgang Hammerschmidt, Ezgi Akidil, Stephan Hamperl, and Adam Grundhoff
- Subjects
Gene Expression Regulation, Viral ,Herpesvirus 4, Human ,viruses ,Cell ,Biology ,medicine.disease_cause ,Microbiology ,Virus ,Host-Microbe Biology ,03 medical and health sciences ,human herpesviruses ,Viral Proteins ,Antigen ,Virology ,hemic and lymphatic diseases ,medicine ,Humans ,Gene ,Cells, Cultured ,030304 developmental biology ,Cell Proliferation ,0303 health sciences ,B-Lymphocytes ,030306 microbiology ,Lymphoblast ,transformation ,reprogramming ,Cell Transformation, Viral ,Epstein–Barr virus ,QR1-502 ,3. Good health ,Virus Latency ,MicroRNAs ,medicine.anatomical_structure ,Epstein-Barr Virus Nuclear Antigens ,Cell culture ,Reprogramming ,Research Article ,B lymphocytes - Abstract
The preferred target of Epstein-Barr virus (EBV) is human resting B lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial increase in cell volume, followed by cellular DNA synthesis after 3 days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to 3 days later, the cells decelerate and turn into stably proliferating lymphoblast cell lines. With the aid of 16 different recombinant EBV strains, we investigated the individual contributions of EBV’s multiple latent genes during early B-cell infection and found that many do not exert a detectable phenotype or contribute little to EBV’s prelatent phase. The exception is EBNA2 that is essential in governing all aspects of B-cell reprogramming. EBV relies on EBNA2 to turn the infected B lymphocytes into proliferating lymphoblasts preparing the infected host cell for the ensuing stable, latent phase of viral infection. In the early steps of B-cell reprogramming, viral latent genes other than EBNA2 are dispensable, but some, EBNA-LP, for example, support the viral program and presumably stabilize the infected cells once viral latency is established., Epstein-Barr virus (EBV) infects and activates resting human B lymphocytes, reprograms them, induces their proliferation, and establishes a latent infection in them. In established EBV-infected cell lines, many viral latent genes are expressed. Their roles in supporting the continuous proliferation of EBV-infected B cells in vitro are known, but their functions in the early, prelatent phase of infection have not been investigated systematically. In studies during the first 8 days of infection using derivatives of EBV with mutations in single genes of EBVs, we found only Epstein-Barr nuclear antigen 2 (EBNA2) to be essential for activating naive human B lymphocytes, inducing their growth in cell volume, driving them into rapid cell divisions, and preventing cell death in a subset of infected cells. EBNA-LP, latent membrane protein 2A (LMP2A), and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding Epstein-Barr virus with small RNA (EBERs) had no discernible phenotype compared with wild-type EBV. B cells infected with a double mutant of EBNA3A and 3C had an unexpected proliferative advantage and did not regulate the DNA damage response (DDR) of the infected host cell in the prelatent phase. Even EBNA1, which has very critical long-term functions in maintaining and replicating the viral genomic DNA in established cell lines, was dispensable for the early activation of infected cells. Our findings document that the virus dose is a decisive parameter and indicate that EBNA2 governs the infected cells initially and implements a strictly controlled temporal program independent of other viral latent genes. It thus appears that EBNA2 is sufficient to control all requirements for clonal cellular expansion and to reprogram human B lymphocytes from energetically quiescent to activated cells.
- Published
- 2019
7. RNA 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex
- Author
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Bruno Canard, Laure Gluais, Etienne Decroly, Mickaël Bouvet, Lorenzo Subissi, and Isabelle Imbert
- Subjects
0303 health sciences ,Multidisciplinary ,Base Pair Mismatch ,030302 biochemistry & molecular biology ,RNA-dependent RNA polymerase ,RNA ,Biology ,Biological Sciences ,Viral Nonstructural Proteins ,Molecular biology ,3. Good health ,03 medical and health sciences ,Exon ,RNA silencing ,Open Reading Frames ,Severe acute respiratory syndrome-related coronavirus ,Exoribonuclease ,Transcription preinitiation complex ,Exoribonucleases ,Exoribonuclease complex ,RNA, Viral ,DNA mismatch repair ,RNA Processing, Post-Transcriptional ,030304 developmental biology - Abstract
The replication/transcription complex of severe acute respiratory syndrome coronavirus is composed of at least 16 nonstructural proteins (nsp1–16) encoded by the ORF-1a/1b. This complex includes replication enzymes commonly found in positive-strand RNA viruses, but also a set of RNA-processing activities unique to some nidoviruses. The nsp14 protein carries both exoribonuclease (ExoN) and (guanine-N7)-methyltransferase (N7-MTase) activities. The nsp14 ExoN activity ensures a yet-uncharacterized function in the virus life cycle and must be regulated to avoid nonspecific RNA degradation. In this work, we show that the association of nsp10 with nsp14 stimulates >35-fold the ExoN activity of the latter while playing no effect on N7-MTase activity. Nsp10 mutants unable to interact with nsp14 are not proficient for ExoN activation. The nsp10/nsp14 complex hydrolyzes double-stranded RNA in a 3′ to 5′ direction as well as a single mismatched nucleotide at the 3′-end mimicking an erroneous replication product. In contrast, di-, tri-, and longer unpaired ribonucleotide stretches, as well as 3′-modified RNAs, resist nsp10/nsp14-mediated excision. In addition to the activation of nsp16-mediated 2′-O-MTase activity, nsp10 also activates nsp14 in an RNA processing function potentially connected to a replicative mismatch repair mechanism.
- Published
- 2012
- Full Text
- View/download PDF
8. Crystallization and diffraction analysis of the SARS coronavirus nsp10-nsp16 complex
- Author
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Claire Debarnot, Bruno Canard, Isabelle Varlet, François Ferron, Laure Gluais, Mickaël Bouvet, Etienne Decroly, Julien Lescar, Nicolas Papageorgiou, Isabelle Imbert, Centre de recherche en neurobiologie - neurophysiologie de Marseille (CRN2M), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Division Eau et Environnement (LCPC/EAU), Laboratoire Central des Ponts et Chaussées (LCPC)-PRES Université Nantes Angers Le Mans (UNAM), and Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Methyltransferase ,viruses ,Molecular Sequence Data ,Biophysics ,MESH: SARS Virus ,RNA-dependent RNA polymerase ,Biology ,Viral Nonstructural Proteins ,Crystallography, X-Ray ,Biochemistry ,law.invention ,03 medical and health sciences ,Structural Biology ,law ,MESH: Methyltransferases ,Genetics ,Viral structural protein ,Humans ,Cap formation ,MESH: Cloning, Molecular ,Crystallization ,Cloning, Molecular ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,skin and connective tissue diseases ,Gene ,030304 developmental biology ,MESH: Crystallization ,0303 health sciences ,Messenger RNA ,MESH: Humans ,MESH: Molecular Sequence Data ,030302 biochemistry & molecular biology ,fungi ,RNA virus ,Methyltransferases ,Condensed Matter Physics ,biology.organism_classification ,RNA-Dependent RNA Polymerase ,MESH: Crystallography, X-Ray ,Virology ,3. Good health ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,body regions ,Severe acute respiratory syndrome-related coronavirus ,Crystallization Communications ,MESH: Viral Nonstructural Proteins ,MESH: RNA Replicase - Abstract
International audience; To date, the SARS coronavirus is the only known highly pathogenic human coronavirus. In 2003, it was responsible for a large outbreak associated with a 10% fatality rate. This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1-16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. The crystal structure of nsp16 is unknown. Nsp16 is an RNA-cap AdoMet-dependent (nucleoside-2'-O-)-methyltransferase that is only active in the presence of nsp10. In this paper, the expression, purification and crystallization of nsp10 in complex with nsp16 are reported. The crystals diffracted to a resolution of 1.9 Å resolution and crystal structure determination is in progress.
- Published
- 2011
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