@article{mbs:/content/journal/jgv/10.1099/vir.0.82282-0, author = "Tran, Thi-Lan and Castagné, Nathalie and Bhella, David and Varela, Paloma F. and Bernard, Julie and Chilmonczyk, Stefan and Berkenkamp, Stefan and Benhamo, Vanessa and Grznarova, Katarina and Grosclaude, Jeanne and Nespoulos, Claude and Rey, Felix A. and Eléouët, Jean-François", 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", journal= "Journal of General Virology", year = "2007", volume = "88", number = "1", pages = "196-206", doi = "https://doi.org/10.1099/vir.0.82282-0", url = "https://www.microbiologyresearch.org/content/journal/jgv/10.1099/vir.0.82282-0", publisher = "Microbiology Society", issn = "1465-2099", type = "Journal Article", 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.", }