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A Druggable Pocket at the Nucleocapsid/Phosphoprotein Interaction Site of Human Respiratory Syncytial Virus.

ABSTRACT: Presently, respiratory syncytial virus (RSV), the main cause of severe respiratory infections in infants, cannot be treated efficiently with antivirals. However, its RNA-dependent polymerase complex offers potential targets for RSV-specific drugs. This includes the recognition of its template, the ribonucleoprotein complex (RNP), consisting of genomic RNA encapsidated by the RSV nucleoprotein, N. This recognition proceeds via interaction between the phosphoprotein P, which is the main polymerase cofactor, and N. The determinant role of the C terminus of P, and more particularly of the last residue, F241, in RNP binding and viral RNA synthesis has been assessed previously. Here, we provide detailed structural insight into this crucial interaction for RSV polymerase activity. We solved the crystallographic structures of complexes between the N-terminal domain of N (N-NTD) and C-terminal peptides of P and characterized binding by biophysical approaches. Our results provide a rationale for the pivotal role of F241, which inserts into a well-defined N-NTD pocket. This primary binding site is completed by transient contacts with upstream P residues outside the pocket. Based on the structural information of the N-NTD:P complex, we identified inhibitors of this interaction, selected by in silico screening of small compounds, that efficiently bind to N and compete with P in vitro. One of the compounds displayed inhibitory activity on RSV replication, thereby strengthening the relevance of N-NTD for structure-based design of RSV-specific antivirals.Respiratory syncytial virus (RSV) is a widespread pathogen that is a leading cause of acute lower respiratory infections in infants worldwide. RSV cannot be treated efficiently with antivirals, and no vaccine is presently available, with the development of pediatric vaccines being particularly challenging. Therefore, there is a need for new therapeutic strategies that specifically target RSV. The interaction between the RSV phosphoprotein P and the ribonucleoprotein complex is critical for viral replication. In this study, we identified the main structural determinants of this interaction, and we used them to screen potential inhibitors in silico. We found a family of molecules that were efficient competitors of P in vitro and showed inhibitory activity on RSV replication in cellular assays. These compounds provide a basis for a pharmacophore model that must be improved but that holds promises for the design of new RSV-specific antivirals.

SUBMITTER: Ouizougun-Oubari M

PROVIDER: S-EPMC4621127 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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A Druggable Pocket at the Nucleocapsid/Phosphoprotein Interaction Site of Human Respiratory Syncytial Virus.

Ouizougun-Oubari Mohamed M Pereira Nelson N Tarus Bogdan B Galloux Marie M Lassoued Safa S Fix Jenna J Tortorici M Alejandra MA Hoos Sylviane S Baron Bruno B England Patrick P Desmaële Didier D Couvreur Patrick P Bontems François F Rey Félix A FA Eléouët Jean-François JF Sizun Christina C Slama-Schwok Anny A Duquerroy Stéphane S

Journal of virology 20150805 21

<h4>Unlabelled</h4>Presently, respiratory syncytial virus (RSV), the main cause of severe respiratory infections in infants, cannot be treated efficiently with antivirals. However, its RNA-dependent polymerase complex offers potential targets for RSV-specific drugs. This includes the recognition of its template, the ribonucleoprotein complex (RNP), consisting of genomic RNA encapsidated by the RSV nucleoprotein, N. This recognition proceeds via interaction between the phosphoprotein P, which is ...[more]

PMID: 26246564

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Project description:BACKGROUND: There 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. METHODS: We 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 N(13-391). We calculated the contributions to the free energies of binding of P to N(13-391) 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. RESULTS: Binding studies confirmed the crucial role of the phosphorylated C-terminal peptide D(pS)DNDL(pS)LEDF for binding of P to RNA-free, monomeric N(13-391), 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 N(13-391) decreased as the ionic strength increased. CONCLUSIONS: The 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.

Project description:UnlabelledThe RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N, thus forming a helical nucleocapsid. Polymerization of N along the genomic and antigenomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy with Paramyxoviridae and Rhabdoviridae, it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N(0)-P complex). Here, we have engineered a mutant form of N that is monomeric, is unable to bind RNA, still interacts with P, and could thus mimic the N(0) monomer. We used this N mutant, designated N(mono), as a substitute for N(0) in order to characterize the P regions involved in the N(0)-P complex formation. Using a series of P fragments, we determined by glutathione S-transferase (GST) pulldown assays that the N and C termini of P are able to interact with N(mono). We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and N(mono) in vitro. Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N(0)-P interaction could constitute a potential antiviral strategy.ImportanceRespiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or efficient antiviral treatment is available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. RSV phosphoprotein P, the main RNA polymerase cofactor, is believed to function as a chaperon protein, maintaining N as a nonassembled, RNA-free protein (N(0)) competent for RNA encapsidation. In this paper, we provide the first evidence, to our knowledge, that the N terminus of P contains a domain that binds specifically to this RNA-free form of N. We further show that overexpression of a small peptide spanning this region of P can inhibit viral RNA synthesis. These findings extend our understanding of the function of RSV RNA polymerase and point to a new target for the development of drugs against this virus.

Dataset Information

A Druggable Pocket at the Nucleocapsid/Phosphoprotein Interaction Site of Human Respiratory Syncytial Virus.

Publications

A Druggable Pocket at the Nucleocapsid/Phosphoprotein Interaction Site of Human Respiratory Syncytial Virus.

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