1 Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France 2 Medical Research Council Virology Unit, Church Street, Glasgow G11 5JR, UK 3 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 4 Institute of Medical Physics and Biophysics, Westfälische Wilhelms-Universität, Münster, Germany 5 Unité de Biochimie et Structure des Protéines, INRA, 78350 Jouy-en-Josas, France Correspondence Jean-François Eléouët jean-francois.eleouet{at}jouy.inra.fr 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. These authors contributed equally to this work. Present address: Laboratoire de Biologie, Synchrotron SOLEIL, L'Orme des Merisiers, Saint Aubin – BP 48, 91192 Gif-sur-Yvette Cedex, France. Present address: Unité de Virologie Structurale, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.