title: Identification and characterization of the binding site of the respiratory syncytial virus phosphoprotein to RNA-free nucleoprotein.
abstract: The RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N, thus forming a helical nucleocapsid. Polymerization of N along the genomic and antigenomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy with Paramyxoviridae and Rhabdoviridae, it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N(0)-P complex). Here, we have engineered a mutant form of N that is monomeric, is unable to bind RNA, still interacts with P, and could thus mimic the N(0) monomer. We used this N mutant, designated N(mono), as a substitute for N(0) in order to characterize the P regions involved in the N(0)-P complex formation. Using a series of P fragments, we determined by glutathione S-transferase (GST) pulldown assays that the N and C termini of P are able to interact with N(mono). We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and N(mono) in vitro. Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N(0)-P interaction could constitute a potential antiviral strategy.
  
  
  OBJECTIVE
  Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or efficient antiviral treatment is available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. RSV phosphoprotein P, the main RNA polymerase cofactor, is believed to function as a chaperon protein, maintaining N as a nonassembled, RNA-free protein (N(0)) competent for RNA encapsidation. In this paper, we provide the first evidence, to our knowledge, that the N terminus of P contains a domain that binds specifically to this RNA-free form of N. We further show that overexpression of a small peptide spanning this region of P can inhibit viral RNA synthesis. These findings extend our understanding of the function of RSV RNA polymerase and point to a new target for the development of drugs against this virus.
authors.affiliation: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Galloux
authors.affiliation: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France.
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Gabiane
authors.affiliation: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France EA3647-EPIM, UFR des Sciences de la Santé Simone Veil-UVSQ, Montigny-Le-Bretonneux, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Sourimant
authors.affiliation: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France.
authors.forename: Charles-Adrien
authors.initials: CA
authors.lastname: Richard
authors.affiliation: Institut Pasteur, Plate-forme de Biophysique des Macromolécules et de leurs Interactions, Paris, France CNRS-UMR3528, Paris, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: England
authors.affiliation: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Moudjou
authors.affiliation: IBBMC, Université Paris-XI, Orsay, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Aumont-Nicaise
authors.affiliation: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France.
authors.forename: Jenna
authors.initials: J
authors.lastname: Fix
authors.affiliation: EA3647-EPIM, UFR des Sciences de la Santé Simone Veil-UVSQ, Montigny-Le-Bretonneux, France AP-HP, Laboratoire de Microbiologie, Hôpital Ambroise Paré, Boulogne-Billancourt, France.
authors.forename: Marie-Anne
authors.initials: MA
authors.lastname: Rameix-Welti
authors.affiliation: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France jean-francois.eleouet@jouy.inra.fr.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Eléouët
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002498:Centrifugation / Q000379:methods; D006224:Cricetinae; D004252:DNA Mutational Analysis; D016297:Mutagenesis, Site-Directed; D019590:Nucleocapsid Proteins / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000502:physiology*; D020349:Surface Plasmon Resonance; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D019590:Nucleocapsid Proteins; C529603:P protein, human respiratory syncytial virus; D015678:Viral Structural Proteins