title: Fine mapping and characterization of the L-polymerase-binding domain of the respiratory syncytial virus phosphoprotein.
abstract: The minimum requirement for an active RNA-dependent RNA polymerase of respiratory syncytial virus (RSV) is a complex made of two viral proteins, the polymerase large protein (L) and the phosphoprotein (P). Here we have investigated the domain on P that is responsible for this critical P-L interaction. By use of recombinant proteins and serial deletions, an L binding site was mapped in the C-terminal region of P, just upstream of the N-RNA binding site. The role of this molecular recognition element of about 30 amino acid residues in the L-P interaction and RNA polymerase activity was evaluated in cellula using an RSV minigenome system and site-directed mutagenesis. The results highlighted the critical role of hydrophobic residues located in this region.
  
  
  OBJECTIVE
  Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine and no good antivirals against RSV are available, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. Like all negative-strand RNA viruses, RSV codes for its own machinery to replicate and transcribe its genome. The core of this machinery is composed of two proteins, the phosphoprotein (P) and the large protein (L). Here, using recombinant proteins, we have mapped and characterized the P domain responsible for this L-P interaction and the formation of an active L-P complex. These findings extend our understanding of the mechanism of action of RSV RNA polymerase and allow us to define a new target for the development of drugs against RSV.
authors: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France INSERM U1173, UFR Simone Veil, Versailles-Saint-Quentin University, Saint-Quentin en Yvelines, France.
authors: Julien
authors: J
authors: Sourimant
authors: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France INSERM U1173, UFR Simone Veil, Versailles-Saint-Quentin University, Saint-Quentin en Yvelines, France AP-HP, Hôpital Ambroise Paré, Laboratoire de Microbiologie, Boulogne-Billancourt, France.
authors: Marie-Anne
authors: MA
authors: Rameix-Welti
authors: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France.
authors: Anne-Laure
authors: AL
authors: Gaillard
authors: INRA, UMR1319 Micalis, Plateforme d'Analyse Protéomique de Paris Sud-Ouest, Jouy-en-Josas, France.
authors: Didier
authors: D
authors: Chevret
authors: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France.
authors: Marie
authors: M
authors: Galloux
authors: INSERM U1173, UFR Simone Veil, Versailles-Saint-Quentin University, Saint-Quentin en Yvelines, France AP-HP, Hôpital Ambroise Paré, Laboratoire de Microbiologie, Boulogne-Billancourt, France.
authors: Elyanne
authors: E
authors: Gault
authors: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France jean-francois.eleouet@jouy.inra.fr.
authors: Jean-François
authors: JF
authors: Eléouët
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D015151:Immunoblotting; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D046912:Multiprotein Complexes; D010750:Phosphoproteins; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases
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