Biochemical characterization of the respiratory syncytial virus P–P and P–N protein complexes and localization of the P protein oligomerization domain

Castagné, Nathalie; Barbier, Alexandra; Bernard, Julie; Rézaei, Human; Huet, Jean‐Claude; Henry, Céline; Costa, Bruno Da; Éléouët, Jean-François

doi:10.1099/vir.0.79830-0

Cited by 99 publications

(120 citation statements)

References 29 publications

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“…Similarly to other paramyxoviruses (46)(47)(48), the hRSV phosphoprotein (P) could work as a chaperone to keep a pool of soluble monomeric N protein that does not bind to RNA (49). Such a pool of monomeric N molecules could be available either for nucleocapsid assembly or membrane binding and targeting to the cell surface.…”

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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“…Human RSV P (strain Long) is phosphorylated mainly at S232 (7,8), although other minor phosphorylation sites have been identified (S30, S39, S45, T46, S54, T108, S116, S117, S119, S237) (9,10). The precise role of phosphorylation for P activity remains unclear, since (i) unphosphorylated P is competent for oligomerization and binding to N-RNA (11,12) and (ii) substitution of all the phosphorylated residues has little effect on viral transcription and replication (13,14). However, P phosphorylation could play a role in NC encapsidation in viral particles (10,13).…”

mentioning

confidence: 99%

“…Although there is no sequence similarity between the P proteins of Pneumovirinae and other related families and subfamilies in the Mononegavirales order, all share similar organization, structure, and functions. RSV P forms homotetramers of elongated shape and has a modular organization with a protease-resistant central domain containing a predicted coiled coil (aa 120 to 160) involved in oligomerization, flanked by two intrinsically disordered regions (aa 1 to 120 and 161 to 241) (11,(15)(16)(17). The C-terminal region of P (aa 231 to 241) is involved in the interaction with the NC (12,18).…”

mentioning

confidence: 99%

Identification and Characterization of the Binding Site of the Respiratory Syncytial Virus Phosphoprotein to RNA-Free Nucleoprotein

Galloux

Gabiane

Sourimant

et al. 2015

J Virol

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113

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The 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. T he human respiratory syncytial virus (HRSV) is the leading cause of severe respiratory tract infections in newborn children worldwide (1). HRSV infects close to 100% of infants within the first 2 years of life and is the main cause of bronchiolitis. It is also recognized as a significant cause of severe respiratory infections in the elderly. The virus belongs to the Mononegavirales order and constitutes the prototype virus of the Pneumovirus genus of the Paramyxoviridae family. As f...

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“…Previous data characterized in vitro an oligomerization domain in P protein central region and indicated that P oligomers could also occur in HRSV infected cells (2,9,10).…”

Section: Discussionmentioning

confidence: 99%

Structural analysis of human respiratory syncytial virus P protein: identification of intrinsically disordered domains

Simabuco¹,

Asara²,

Guerrero³

et al. 2011

Braz. J. Microbiol.

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Human Respiratory Syncytial Virus P protein plus the viral RNA, N and L viral proteins, constitute the viral replication complex. In this report we describe that HRSV P protein has putative intrinsically disordered domains predicted by in silico methods. These two domains, located at the amino and caboxi terminus, were identified by mass spectrometry analysis of peptides obtained from degradation fragments observed in purified P protein expressed in bacteria. The degradation is not occurring at the central oligomerization domain, since we also demonstrate that the purified fragments are able to oligomerize, similarly to the protein expressed in cells infected by HRSV. Disordered domains can play a role in protein interaction, and the present data contribute to the comprehension of HRSV P protein interactions in the viral replication complex.

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Biochemical characterization of the respiratory syncytial virus P–P and P–N protein complexes and localization of the P protein oligomerization domain

Cited by 99 publications

References 29 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

Identification and Characterization of the Binding Site of the Respiratory Syncytial Virus Phosphoprotein to RNA-Free Nucleoprotein

Structural analysis of human respiratory syncytial virus P protein: identification of intrinsically disordered domains

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