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. IMPORTANCERespiratory 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. H uman respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory infections in infants worldwide (1).No RSV vaccine is presently available, and the development of pediatric vaccines is particularly challenging. Currently, antiviral therapy is limited to palivizumab, a humanized mouse monoclonal antibody that targets the RSV fusion protein and is licensed for prophylactic use, and ribavirin, which has been used to treat severe infections despite its toxicity, its teratogenicity, and the limited evidence...

New Insights into Structural Disorder in Human Respiratory Syncytial Virus Phosphoprotein and Implications for Binding of Protein Partners

“…4C). By fitting the CSP data with a two-site fast exchange model, a K d of 25-50 M was determined, in agreement with a K d of 30 M previously determined for the N NTD ⅐P CD complex by isothermal titration calorimetry (39).…”

“…4C). Surprisingly, no signal was recovered for the very C-terminal Phe 241 residue, which in X-ray structures appears to be the main structural determinant for NC⅐P complex formation, by tightly inserting into a hydrophobic pocket at the surface of N NTD (39). Our results thus suggest the possibility of additional binding modes corresponding to weaker interactions.…”

“…Thrombin was removed with streptavidin resin (Novagen) according to the manufacturer's instructions. Purification of N protein was carried out as described previously for N mono (24), N NTD (39), and N-RNA rings (13). His tags were not removed.…”

“…Except for Phe 241 , P NCBD is disordered in its bound form, as shown by X-ray crystal structures of N NTD complexed to C-terminal P peptides (39). Although Phe 241 is essential for NC⅐P binding, fuzzy electrostatic interactions mediated by acidic residues as well as phosphorylation of serines in P NCBD significantly contribute to the affinity of P (14,39,51). Our NMR data suggest that even Phe 241 , the linchpin for P binding to the NC, could explore different binding sites.…”

“…We therefore proceeded with the monomeric N-terminal domain of N (N NTD ), which was shown to be relevant for NC⅐P binding (14,39). By titrating N NTD into 15 N-P CD , linear chemical shift perturbations (CSPs) were observed for P NCBD (e.g.…”