title: Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength.
abstract: Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3'end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region.
authors.affiliation: CIRI, International Center for Infectiology Research, Université de Lyon, Lyon, France.|INSERM, U1111, Lyon, France.|Ecole Normale Supérieure de Lyon, Lyon, France.|Université Claude Bernard Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France.|CNRS, UMR5308, Lyon, France.
authors.forename: Louis-Marie
authors.initials: LM
authors.lastname: Bloyet
authors.affiliation: CIRI, International Center for Infectiology Research, Université de Lyon, Lyon, France.|INSERM, U1111, Lyon, France.|Ecole Normale Supérieure de Lyon, Lyon, France.|Université Claude Bernard Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France.|CNRS, UMR5308, Lyon, France.
authors.forename: Joanna
authors.initials: J
authors.lastname: Brunel
authors.affiliation: Aix-Marseille University, Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257, Marseille, France.|CNRS, AFMB UMR 7257, Marseille, France.
authors.forename: Marion
authors.initials: M
authors.lastname: Dosnon
authors.affiliation: Aix Marseille University, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille (CRCM), Marseille, France.|CNRS, CRCM UMR 7258, Marseille, France.|INSERM, CRCM U1068, Marseille, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Hamon
authors.affiliation: Aix-Marseille University, Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257, Marseille, France.|CNRS, AFMB UMR 7257, Marseille, France.
authors.forename: Jenny
authors.initials: J
authors.lastname: Erales
authors.affiliation: Aix-Marseille University, Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257, Marseille, France.|CNRS, AFMB UMR 7257, Marseille, France.
authors.forename: Antoine
authors.identifier: http://orcid.org/0000-0001-5000-7903
authors.initials: A
authors.lastname: Gruet
authors.affiliation: CIRI, International Center for Infectiology Research, Université de Lyon, Lyon, France.|INSERM, U1111, Lyon, France.|Ecole Normale Supérieure de Lyon, Lyon, France.|Université Claude Bernard Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France.|CNRS, UMR5308, Lyon, France.
authors.forename: Carine
authors.identifier: http://orcid.org/0000-0002-7783-2820
authors.initials: C
authors.lastname: Lazert
authors.affiliation: Aix-Marseille University, Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257, Marseille, France.|CNRS, AFMB UMR 7257, Marseille, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Bignon
authors.affiliation: Aix Marseille University, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille (CRCM), Marseille, France.|CNRS, CRCM UMR 7258, Marseille, France.|INSERM, CRCM U1068, Marseille, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Roche
authors.affiliation: Aix-Marseille University, Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257, Marseille, France.|CNRS, AFMB UMR 7257, Marseille, France.
authors.forename: Sonia
authors.identifier: http://orcid.org/0000-0002-6829-6771
authors.initials: S
authors.lastname: Longhi
authors.affiliation: CIRI, International Center for Infectiology Research, Université de Lyon, Lyon, France.|INSERM, U1111, Lyon, France.|Ecole Normale Supérieure de Lyon, Lyon, France.|Université Claude Bernard Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France.|CNRS, UMR5308, Lyon, France.
authors.forename: Denis
authors.identifier: http://orcid.org/0000-0001-5539-456X
authors.initials: D
authors.lastname: Gerlier
mesh_terms: D002151:Calorimetry; D002942:Circular Dichroism; D021901:DNA, Intergenic; D006801:Humans; D013058:Mass Spectrometry; D008457:Measles / Q000378:metabolism / Q000821:virology*; D008459:Measles virus / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D021901:DNA, Intergenic; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D014764:Viral Proteins; C058314:nucleoprotein, Measles virus