Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells

Oliveira, Andressa Peres de; Simabuco, Fernando Moreira; Tamura, Rodrigo Esaki; Guerrero, Manuel Ceballos; Ribeiro, Paulo Guilherme Guimarães; Libermann, Towia A.; Zerbini, Luiz F.; Ventura, Armando Morais

doi:10.1016/j.virusres.2013.07.010

Cited by 38 publications

(44 citation statements)

References 47 publications

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“…Such a pool of monomeric N molecules could be available either for nucleocapsid assembly or membrane binding and targeting to the cell surface. Alternatively, the recent description of novel binding partners for N protein, such as the methylosome protein (WDR77) and the Heat-shock protein 70 (Hsp70) (50), raises the possibility that these proteins could contribute to preventing N protein from either being incorporated within ribonucleoprotein complexes or self-aggregating. Elucidation of the mechanism responsible for this phenomenon would require further research.…”

Section: Discussionmentioning

confidence: 99%

Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells

Céspedes

Bueno

Ramírez

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Human respiratory syncytial virus (hRSV) is the leading cause of bronchiolitis and pneumonia in young children worldwide. The recurrent hRSV outbreaks and reinfections are the cause of a significant public health burden and associate with an inefficient antiviral immunity, even after disease resolution. Although several mouse-and human cell-based studies have shown that hRSV infection prevents naïve T-cell activation by antigen-presenting cells, the mechanism underlying such inhibition remains unknown. Here, we show that the hRSV nucleoprotein (N) could be at least partially responsible for inhibiting T-cell activation during infection by this virus. Early after infection, the N protein was expressed on the surface of epithelial and dendritic cells, after interacting with trans-Golgi and lysosomal compartments. Further, experiments on supported lipid bilayers loaded with peptide-MHC (pMHC) complexes showed that surface-anchored N protein prevented immunological synapse assembly by naive CD4 + T cells and, to a lesser extent, by antigen-experienced T-cell blasts. Synapse assembly inhibition was in part due to reduced T-cell receptor (TCR) signaling and pMHC clustering at the T-cell− bilayer interface, suggesting that N protein interferes with pMHC− TCR interactions. Moreover, N protein colocalized with the TCR independently of pMHC, consistent with a possible interaction with TCR complex components. Based on these data, we conclude that hRSV N protein expression at the surface of infected cells inhibits T-cell activation. Our study defines this protein as a major virulence factor that contributes to impairing acquired immunity and enhances susceptibility to reinfection by hRSV.

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Section: Discussionmentioning

confidence: 99%

Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells

Céspedes

Bueno

Ramírez

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Efficient and specific recognition of the RNP template by the RdRp is critical for viral RNA synthesis. It is mediated by P, a multifunctional protein capable of interacting with multiple partners: L (6), N (7), and M2-1 (8), but also cellular proteins (9). P positions the RdRp complex on the RNP and is likely involved in translocation along the RNP (10).…”

mentioning

confidence: 99%

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

Ouizougun-Oubari

Pereira

Tarus

et al. 2015

J Virol

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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...

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“…Myosin 5 beta), have been shown to be essential for RSV assembly (24) with budding of released virus believed to be Vps4-independent and to require Rab11a FIP2 protein (25). However, only Importin-␤1 (5) and CRM1 (8) (see above) are known to be direct interactors of M. A proteomic screen for cellular interactors of RSV M, N, and F proteins identified only limited numbers of proteins, none of which could be validated to bind directly to M (26). Overall, the network of RSV-cell interactions is still mostly unknown, with limited targets identified.…”

mentioning

confidence: 99%

New Host Factors Important for Respiratory Syncytial Virus (RSV) Replication Revealed by a Novel Microfluidics Screen for Interactors of Matrix (M) Protein*

Kipper

Hamad

Caly

et al. 2015

Molecular & Cellular Proteomics

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Although human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and elderly worldwide, there is no licensed RSV vaccine or effective drug treatment available. The RSV Matrix protein plays key roles in virus life cycle, being found in the nucleus early in infection in a transcriptional inhibitory role, and later localizing in viral inclusion bodies before coordinating viral assembly and budding at the plasma membrane. In this study, we used a novel, high throughput microfluidics platform and custom human open reading frame library to identify novel host cell binding partners of RSV matrix. Novel interactors identified included proteins involved in host transcription regulation, the innate immunity response, cytoskeletal regulation, membrane remodeling, and cellular trafficking. A number of these interactions were confirmed by immunoprecipitation and cellular colocalization approaches. Importantly, the physiological significance of matrix interaction with the actin-binding protein cofilin 1, caveolae protein Caveolin 2, and the zinc finger protein ZNF502 was confirmed. siRNA knockdown of the host protein levels resulted in reduced RSV virus production in infected cells. These results have important implications for future antiviral strategies aimed at targets of RSV matrix in the host cell.

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Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells

Cited by 38 publications

References 47 publications

Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells

Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells

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

New Host Factors Important for Respiratory Syncytial Virus (RSV) Replication Revealed by a Novel Microfluidics Screen for Interactors of Matrix (M) Protein*

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