Quantitative investigation of the affinity of human respiratory syncytial virus phosphoprotein C-terminus binding to nucleocapsid proteinReport as inadecuate

Quantitative investigation of the affinity of human respiratory syncytial virus phosphoprotein C-terminus binding to nucleocapsid protein - Download this document for free, or read online. Document in PDF available to download.

Virology Journal

, 11:191

Negative-strand RNA viruses


BackgroundThere are no approved small molecule drug therapies for human respiratory syncytial virus hRSV, a cause of morbidity and mortality in at-risk newborns, the immunocompromised, and the elderly. We have investigated as a potential novel hRSV drug target the protein-protein interaction between the C-terminus of the viral phosphoprotein P and the viral nucleocapsid protein N, components of the ribonucleoprotein complex that contains, replicates, and transcribes the viral RNA genome. Earlier work by others established that the 9 C-terminal residues of P are necessary and sufficient for binding to N.

MethodsWe used a fluorescence anisotropy assay, surface plasmon resonance and 2-D NMR to quantify the affinities of peptides based on the C terminus of P for RNA-free, monomeric N-terminal-truncated N13-391. We calculated the contributions to the free energies of binding of P to N13-391 attributable to the C-terminal 11 residues, phosphorylation of the C-terminal 2 serine residues, the C-terminal Asp-Phe, and the phenyl ring of the C-terminal Phe.

ResultsBinding studies confirmed the crucial role of the phosphorylated C-terminal peptide DpSDNDLpSLEDF for binding of P to RNA-free, monomeric N13-391, contributing over 90% of the binding free energy at low ionic strength. The phenyl ring of the C-terminal Phe residue contributed an estimated -2.7 kcal-mole of the free energy of binding, the C-terminal Asp-Phe residues contributed -3.8 kcal-mole, the sequence DSDNDLSLE contributed -3.1 kcal-mole, and phosphorylation of the 2 Ser residues contributed -1.8 kcal-mole. Due to the high negative charge of the C-terminal peptide, the affinity of the P C-terminus for N13-391 decreased as the ionic strength increased.

ConclusionsThe results support the idea that the interaction of the C-terminal residues of P with N constitutes a protein-protein interaction hotspot that may be a suitable target for small-molecule drugs that inhibit viral genome replication and transcription.

KeywordsRespiratory syncytial virus Nucleocapsid Phosphoprotein Phosphorylation Protein-protein interaction Fluorescence anisotropy Surface plasmon resonance Nuclear magnetic resonance spectroscopy AbbreviationsPPhosphoprotein


N13-391Nucleocapsid with N-terminal truncation of 12 residues

N31-252Nucleocapsid construct consisting of residues 31-252

hRSVHuman respiratory syncytial virus


BP1BODIPY-FL labeled peptide 1 DSDNDLSLEDF

BP5BODIPY-FL labeled peptide 5 DpSDNDLpSLEDF

SPRSurface plasmon resonance

HPLCHigh-performance liquid chromatography


2-D NMRTwo-dimensional nuclear magnetic resonance spectroscopy

LC-MSLiquid chromatography-mass spectrometry

TCEPTris2-carboxyethylphosphine hydrochloride


RUResonance units

DMSODimethyl sulfoxide

TROSYTransverse relaxation optimized spectroscopy

TEVTobacco etch virus



SDS-PAGESodium dodecyl sulfate polyacrylamide gel electrophoresis

FBSFetal bovine serum

PMSFPhenylmethylsulfonyl fluoride

Electronic supplementary materialThe online version of this article doi:10.1186-s12985-014-0191-2 contains supplementary material, which is available to authorized users.

Download fulltext PDF

Author: Adam B Shapiro - Ning Gao - Nichole O’Connell - Jun Hu - Jason Thresher - Rong-Fang Gu - Ross Overman - Ian M Hardern

Source: https://link.springer.com/

Related documents