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.
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.
Tran
Thi-Lan
TL
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
Castagné
Nathalie
N
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
Bhella
David
D
Medical Research Council Virology Unit, Church Street, Glasgow G11 5JR, UK.
Varela
Paloma F
PF
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.
Bernard
Julie
J
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
Chilmonczyk
Stefan
S
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
Berkenkamp
Stefan
S
Institute of Medical Physics and Biophysics, Westfälische Wilhelms-Universität, Münster, Germany.
Benhamo
Vanessa
V
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
Grznarova
Katarina
K
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
Grosclaude
Jeanne
J
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
Nespoulos
Claude
C
Unité de Biochimie et Structure des Protéines, INRA, 78350 Jouy-en-Josas, France.
Rey
Felix A
FA
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.
Eléouët
Jean-François
JF
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
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
D010446:Peptide Fragments; D010750:Phosphoproteins; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins