title: The nine C-terminal amino acids of the respiratory syncytial virus protein P are necessary and sufficient for binding to ribonucleoprotein complexes in which six ribonucleotides are contacted per N protein protomer.
abstract: The respiratory syncytial virus (RSV) phosphoprotein (P) is a major polymerase co-factor that interacts with both the large polymerase fragment (L) and the nucleoprotein (N). The N-binding domain of RSV P has been investigated by co-expression of RSV P and N proteins in Escherichia coli. Pull-down assays performed with a series of truncated forms of P fused to glutathione S-transferase (GST) revealed that the region comprising the last nine C-terminal amino acid residues of P (233-DNDLSLEDF-241) is sufficient for efficient binding to N. Site-directed mutagenesis shows that the last four residues of this peptide are crucial for binding and must be present at the end of a flexible C-terminal tail. The presence of the P oligomerization domain (residues 100-160) was an important stabilizing factor for the interaction. The tetrameric full-length P fused to GST was able to pull down both helical and ring structures, whereas a monomeric C-terminal fragment of P (residues 161-241) fused to GST pulled down exclusively RNA-N rings. Electron-microscopy analysis of the purified rings showed the presence of two types of complex: undecamers (11N) and decamers (10N). Mass-spectrometry analysis of the RNA extracted from rings after RNase A treatment showed two peaks of 22,900 and 24,820 Da, corresponding to a mean RNA length of 67 and 73 bases, respectively. These results suggest strongly that each N subunit contacts 6 nt, with an extra three or four bases further protected from nuclease digestion by the ring structure at both the 5' and 3' ends.
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
authors.forename: Thi-Lan
authors.initials: TL
authors.lastname: Tran
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Castagné
authors.affiliation: Medical Research Council Virology Unit, Church Street, Glasgow G11 5JR, UK.
authors.forename: David
authors.initials: D
authors.lastname: Bhella
authors.affiliation: Laboratoire de Virologie Moléculaire et Structurale, UMR 2472-1157 CNRS-INRA and IFR 115, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Paloma F
authors.initials: PF
authors.lastname: Varela
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Bernard
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
authors.forename: Stefan
authors.initials: S
authors.lastname: Chilmonczyk
authors.affiliation: Institute of Medical Physics and Biophysics, Westfälische Wilhelms-Universität, Münster, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Berkenkamp
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Benhamo
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
authors.forename: Katarina
authors.initials: K
authors.lastname: Grznarova
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
authors.forename: Jeanne
authors.initials: J
authors.lastname: Grosclaude
authors.affiliation: Unité de Biochimie et Structure des Protéines, INRA, 78350 Jouy-en-Josas, France.
authors.forename: Claude
authors.initials: C
authors.lastname: Nespoulos
authors.affiliation: Laboratoire de Virologie Moléculaire et Structurale, UMR 2472-1157 CNRS-INRA and IFR 115, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Felix A
authors.initials: FA
authors.lastname: Rey
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Eléouët
mesh_terms: D019943:Amino Acid Substitution; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012329:RNA, Bacterial / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012136:Respiratory Syncytial Viruses / Q000737:chemistry*; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D010750:Phosphoproteins; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins