title: Sequence of events in measles virus replication: role of phosphoprotein-nucleocapsid interactions.
abstract: The genome of nonsegmented negative-strand RNA viruses is tightly embedded within a nucleocapsid made of a nucleoprotein (N) homopolymer. To ensure processive RNA synthesis, the viral polymerase L in complex with its cofactor phosphoprotein (P) binds the nucleocapsid that constitutes the functional template. Measles virus P and N interact through two binding sites. While binding of the P amino terminus with the core of N (NCORE) prevents illegitimate encapsidation of cellular RNA, the interaction between their C-terminal domains, P(XD) and N(TAIL) is required for viral RNA synthesis. To investigate the binding dynamics between the two latter domains, the P(XD) F497 residue that makes multiple hydrophobic intramolecular interactions was mutated. Using a quantitative mammalian protein complementation assay and recombinant viruses, we found that an increase in P(XD)-to-N(TAIL) binding strength is associated with a slower transcript accumulation rate and that abolishing the interaction renders the polymerase nonfunctional. The use of a newly developed system allowing conditional expression of wild-type or mutated P genes, revealed that the loss of the P(XD)-N(TAIL) interaction results in reduced transcription by preformed transcriptases, suggesting reduced engagement on the genomic template. These intracellular data indicate that the viral polymerase entry into and progression along its genomic template relies on a protein-protein interaction that serves as a tightly controlled dynamic anchor.
  
  
  OBJECTIVE
  Mononegavirales have a unique machinery to replicate RNA. Processivity of their polymerase is only achieved when the genome template is entirely embedded into a helical homopolymer of nucleoproteins that constitutes the nucleocapsid. The polymerase binds to the nucleocapsid template through the phosphoprotein. How the polymerase complex enters and travels along the nucleocapsid template to ensure uninterrupted synthesis of up to ∼ 6,700-nucleotide messenger RNAs from six to ten consecutive genes is unknown. Using a quantitative protein complementation assay and a biGene-biSilencing system allowing conditional expression of two P genes copies, the role of the P-to-N interaction in polymerase function was further characterized. We report here a dynamic protein anchoring mechanism that differs from all other known polymerases that rely only onto a sustained and direct binding to their nucleic acid template.
authors.affiliation: Centre International de Recherche en Infectiologie, INSERM, U1111, CNRS, UMR5308, Université Lyon 1, ENS Lyon, CERVI, Lyon, France.
authors.forename: Joanna
authors.initials: J
authors.lastname: Brunel
authors.affiliation: Centre International de Recherche en Infectiologie, INSERM, U1111, CNRS, UMR5308, Université Lyon 1, ENS Lyon, CERVI, Lyon, France.
authors.forename: Damien
authors.initials: D
authors.lastname: Chopy
authors.affiliation: Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques UMR 7257, Marseille, France.
authors.forename: Marion
authors.initials: M
authors.lastname: Dosnon
authors.affiliation: Centre International de Recherche en Infectiologie, INSERM, U1111, CNRS, UMR5308, Université Lyon 1, ENS Lyon, CERVI, Lyon, France.
authors.forename: Louis-Marie
authors.initials: LM
authors.lastname: Bloyet
authors.affiliation: Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Patricia
authors.initials: P
authors.lastname: Devaux
authors.affiliation: Centre International de Recherche en Infectiologie, INSERM, U1111, CNRS, UMR5308, Université Lyon 1, ENS Lyon, CERVI, Lyon, France.
authors.forename: Erica
authors.initials: E
authors.lastname: Urzua
authors.affiliation: Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Roberto
authors.initials: R
authors.lastname: Cattaneo
authors.affiliation: Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques UMR 7257, Marseille, France.
authors.forename: Sonia
authors.initials: S
authors.lastname: Longhi
authors.affiliation: Centre International de Recherche en Infectiologie, INSERM, U1111, CNRS, UMR5308, Université Lyon 1, ENS Lyon, CERVI, Lyon, France denis.gerlier@inserm.fr.
authors.forename: Denis
authors.initials: D
authors.lastname: Gerlier
mesh_terms: D015967:Gene Expression Regulation, Viral; D006801:Humans; D008457:Measles / Q000821:virology*; D008459:Measles virus / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: C055859:P protein, Sendai virus; D010750:Phosphoproteins; D014764:Viral Proteins