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Accueil > Départements > Virologie > Didier PONCET : Biologie Moléculaire des Rotavirus

Les publications

2016


  • M. Gratia, P. Vende, A. Charpilienne, H. C. Baron, C. Laroche, E. Sarot, S. Pyronnet, M. Duarte, et D. Poncet, « Challenging the Roles of NSP3 and Untranslated Regions in Rotavirus mRNA Translation », PloS One, vol. 11, nᵒ 1, p. e0145998, 2016.
    Résumé : Rotavirus NSP3 is a translational surrogate of the PABP-poly(A) complex for rotavirus mRNAs. To further explore the effects of NSP3 and untranslated regions (UTRs) on rotavirus mRNAs translation, we used a quantitative in vivo assay with simultaneous cytoplasmic NSP3 expression (wild-type or deletion mutant) and electroporated rotavirus-like and standard synthetic mRNAs. This assay shows that the last four GACC nucleotides of viral mRNA are essential for efficient translation and that both the NSP3 eIF4G- and RNA-binding domains are required. We also show efficient translation of rotavirus-like mRNAs even with a 5'UTR as short as 5 nucleotides, while more than eleven nucleotides are required for the 3'UTR. Despite the weak requirement for a long 5'UTR, a good AUG environment remains a requirement for rotavirus mRNAs translation.
    Mots-clés : 3' Untranslated Regions, 5' Untranslated Regions, Animals, Base Sequence, Cell Line, Cricetinae, Mutagenesis, Site-Directed, Protein Biosynthesis, RNA, Messenger, ROTA, Sequence Homology, Nucleic Acid, Transcription, Genetic, Viral Nonstructural Proteins, VIRO.

2015


  • M. Gratia, E. Sarot, P. Vende, A. Charpilienne, C. H. Baron, M. Duarte, S. Pyronnet, et D. Poncet, « Rotavirus NSP3 Is a Translational Surrogate of the Poly(A) Binding Protein-Poly(A) Complex », Journal of Virology, vol. 89, nᵒ 17, p. 8773-8782, 2015.
    Résumé : Through its interaction with the 5' translation initiation factor eIF4G, poly(A) binding protein (PABP) facilitates the translation of 5'-capped and 3'-poly(A)-tailed mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in a 3' GACC motif that is recognized by the viral protein NSP3, which competes with PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are efficiently translated, while host poly(A)-tailed mRNA translation is, in contrast, severely impaired. To explore the roles of NSP3 in these two opposing events, the translational capabilities of three capped mRNAs, distinguished by either a GACC, a poly(A), or a non-GACC and nonpoly(A) 3' end, have been monitored after electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 (stably or transiently transfected cells). In infected cells, we found that the magnitudes of translation induction (GACC-tailed mRNA) and translation reduction [poly(A)-tailed mRNA] both depended on the rotavirus strain used but that translation reduction not genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to slightly favor the translation of both poly(A)- and nonpoly(A)-tailed mRNAs, likely by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is a translational surrogate of the PABP-poly(A) complex; therefore, it cannot by itself be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon rotavirus infection. IMPORTANCE: To control host cell physiology and to circumvent innate immunity, many viruses have evolved powerful mechanisms aimed at inhibiting host mRNA translation while stimulating translation of their own mRNAs. How rotavirus tackles this challenge is still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of cellular poly(A) mRNA translation differs with respect to rotavirus strains but is not genetically linked to NSP3. Using cells expressing rotavirus NSP3, we show that NSP3 alone not only dramatically enhances rotavirus-like mRNA translation but also enhances poly(A) mRNA translation rather than inhibiting it, likely by stabilizing the eIF4E-eIF4G complex. Thus, the inhibition of cellular polyadenylated mRNA translation during rotavirus infection cannot be attributed solely to NSP3 and is more likely the result of global competition between viral and host mRNAs for the cellular translation machinery.
    Mots-clés : Animals, Cell Line, Cricetinae, Electroporation, Eukaryotic Initiation Factor-4E, Eukaryotic Initiation Factor-4G, HeLa Cells, Humans, Macaca mulatta, Poly A, Poly(A)-Binding Proteins, Polyadenylation, Protein Binding, Protein Biosynthesis, RNA, Messenger, RNA, Viral, ROTA, Rotavirus, Rotavirus Infections, Transfection, Viral Nonstructural Proteins, VIRO.

  • F. Thiam, A. Charpilienne, D. Poncet, E. Kohli, et C. Basset, « B subunits of cholera toxin and thermolabile enterotoxin of Escherichia coli have similar adjuvant effect as whole molecules on rotavirus 2/6-VLP specific antibody responses and induce a Th17-like response after intrarectal immunization », Microbial Pathogenesis, vol. 89, p. 27-34, 2015.
    Résumé : The purpose of this study was to evaluate the adjuvant effect of the B subunits of cholera toxin (CT) and the thermolabile enterotoxin of Escherichia coli (LT) by the intrarectal route of immunization and compare them to the whole molecules CT and LT-R192G, a non toxic mutant of LT, using 2/6-VLP as an antigen, in mice. All molecules induced similar antigen specific antibody titers in serum and feces, whereas different T cell profiles were observed. CTB and LTB, conversely to CT and LT-R192G, did not induce detectable production of IL-2 by antigen specific T cells. Moreover, CTB, conversely to LT-R192G, CT and LTB, did not induce antigen specific CD4+CD25+Foxp3- and Foxp3+ T cells, thus showing different effects between the B subunits themselves. However, all molecules induced an antigen specific Th17 response. In conclusion, B subunits are potent adjuvants on B cell responses by the intrarectal route. Although their impact on T cell responses are different, all molecules induce a 2/6-VLP-specific Th17 T cell response that may play a major role in helping B cell responses and thus in adjuvanticity and protection.
    Mots-clés : Adjuvants, Immunologic, Administration, Rectal, Animals, Antibodies, Viral, Antibody Formation, B subunit, Bacterial Toxins, Cholera Toxin, Enterotoxins, Escherichia coli Proteins, Feces, Immunization, Immunologic Memory, Interleukin-2, Intrarectal, LT-R192G, Mice, ROTA, Rotavirus, Rotavirus Vaccines, Th17 Cells, Vaccines, Virus-Like Particle, VIRO.
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Publications majeures avant 2015
- Virus Res. 2013 Sep ;176(1-2):144-54
Identification of mutations in the genome of rotavirus SA11 temperature-sensitive mutants D, H, I and J by whole genome sequences analysis and assignment of tsI to gene 7 encoding NSP3. Authors : Vende P, Gratia M, Duarte MD, Charpilienne A, Saguy M, Poncet D
- Vaccine. 2013 Apr 08 ;31(15):1924-30
Different profile and distribution of antigen specific T cells induced by intranasal and intrarectal immunization with rotavirus 2/6-VLP with and without LT-R192G. Authors : Alkadah A, Thiam F, Mounier M, Charpilienne A, Poncet D, Kohli E, Basset C
- FASEB J. 2013 Mar ;27(3):1074-83
The rotavirus nonstructural protein NSP5 coordinates a [2Fe-2S] iron-sulfur cluster that modulates interaction to RNA. Authors : Martin D, Charpilienne A, Parent A, Boussac A, D’Autreaux B, Poupon J, Poncet D
- J Mol Biol. 2011 Oct 14 ;413(1):209-21
Structural organisation of the rotavirus nonstructural protein NSP5. Authors : Martin D, Ouldali M, Ménétrey J, Poncet D
- J Immunol. 2010 Nov 01 ;185(9):5377-83
Rapid generation of rotavirus-specific human monoclonal antibodies from small-intestinal mucosa. Authors : Di Niro R, Mesin L, Raki M, Zheng NY, Lund-Johansen F, Lundin KE, Charpilienne A, Poncet D, Wilson PC, Sollid LM
- Toxins (Basel). 2010 Aug ;2(8):2007-27
Unexpected modulation of recall B and T cell responses after immunization with rotavirus-like particles in the presence of LT-R192G. Authors : Thiam F, Martino CD, Bon F, Charpilienne A, Cachia C, Poncet D, Clements JD, Basset C, Kohli E
- J Virol. 2010 Jul ;84(13):6711-9
Rearranged genomic RNA segments offer a new approach to the reverse genetics of rotaviruses. Authors : Troupin C, Dehée A, Schnuriger A, Vende P, Poncet D, Garbarg-Chenon A
- J Virol. 2010 Mar ;84(5):2522-32
Sequestration of free tubulin molecules by the viral protein NSP2 induces microtubule depolymerization during rotavirus infection. Authors : Martin D, Duarte M, Lepault J, Poncet D

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