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Le virus respiratoire syncytial (VRS) est le principal agent responsable de maladies respiratoires sévères (bronchiolites, pneumonies) chez les nouveau-nés. Il pose de sérieux problèmes également chez les personnes âgées et les immunodéprimées. En l’absence de vaccin efficace contre ce virus, le développement rationnel de traitements antiviraux constitue un enjeu de taille. Le VRS est un virus enveloppé dont le génome est constitué d’un ARN simple brin de polarité négative, codant pour 11 protéines. Le génome est transcrit et répliqué par le complexe ARN polymérase viral. Ce complexe est composé de la nucléoprotéine N, de la polymérase L, de la phosphoprotéine P et du facteur de transcription M2-1. P est une protéine tétramérique multifonctionnelle intrinsèquement désordonnée jouant un rôle central au sein du complexe polymérase et interagissant avec N (Tran et al., 2007) (Galloux et al., 2015), L (Sourimant et al., 2015) et M2-1 (Mason et al., 2003). L’état de phosphorylation de P est régulé par les phosphatases cellulaires PP1 et PP2A (Bitko et Barik, 1998 ; Asenjo et al., 2005). M2-1 est une protéine tétramérique assurant la « processivité » de la polymérase au cours de la transcription virale. Elle interagit avec P et les ARNm viraux (Tran et al., 2009 ; Blondot et al., 2012 ; Tanner 2014). Lors d’une infection, M2-1 est majoritairement sous forme déphosphorylée ; elle est phosphorylée si elle est exprimée seule en cellule et déphosphorylée si elle est co-exprimée avec P (Cuesta et al., 2000). L'élimination de la phosphorylation par mutagenèse dirigée des résidus S58 et S61 de M2-1 altère la transcription virale (Cartee et Wertz, 2001). Toutes ces données suggèrent que l’état de phosphorylation de M2-1 influe sur la transcription virale en régulant les interactions avec P et l’ARN. Cependant, le mécanisme moléculaire régulant la phosphorylation de M2-1 reste inconnu.En cartographiant le site de P interagissant avec M2-1, de façon inattendue, nous avons observé qu’une mutation du résidu F87 situé en amont du site d’interaction avec M2-1 (région 90-110), empêchait la déphosphorylation de M2-1 sans effet sur l’interaction P-M2-1, et avait un effet drastique sur l’activité ARN polymérase. Ce résidu s’inscrit dans un motif de type « RVxF », impliqué dans la capture de la phosphatase-1 (PP1), protéine de la cellule hôte, par P. Par GST-pulldown et co-immunoprécipitation, nous avons montré que P interagit directement avec PP1 via ce domaine. De plus, par immunofluorescence et microscopie, nous avons observé que P recrute PP1 dans les corps d’inclusion. Enfin nous montrons que l’emploi d’inhibiteurs spécifiques contre PP1 entraîne une accumulation de M2-1 phosphorylée et diminue l’efficacité de la réplication du VRS. Nous en déduisons ainsi que le complexe P-PP1 régule la déphosphorylation de M2-1 et la transcription du VRS. C'est la première étude montrant que la phosphoprotéine du VRS recrute une phosphatase cellulaire pour moduler l'état de phosphorylation d’un de ses partenaires viraux.

Each year, the infection of infants with RSV is the cause of a large number of hospitalizations and deathsworldwide. The current standard of care, Synagis, is used as a preventive medicine for premature children only, and there is no drug available to treat the patients who are suffering from this infection. Targeting the polymerase replication complex is a strategy that has been used successfully to develop drugs against other viruses such as HIV, HCV and HBV. In this project, we wish to inhibit the replication complex by disrupting the N0/P interaction. N0 is the nucleoprotein species that is bound as a complex to the phosphoprotein prior to nucleic acid encapsidation. Recently, the N-terminal region (1-29) of the RSV P protein has been identified as sufficient to bind to N0, and the importance of each P residues has been assessed through alanine scanning mutagenesis experiments. Additionally, NMR studies have provided evidence that P (13-25) has a propensity to fold into an alpha helix in solution. We used these data to design stabilized alpha-helical peptides, with the aim to screen for dominant negative inhibitors of the RSV N0/P interaction. The peptideswere stabilized as macrocyclic peptides using the stapled peptide technology, because this technique can improve their potency, proteolytic stability and cellular permeability. The putative P alpha-helix was plotted onto an alpha-helical wheel, and the amino-acids located on the non-interacting site of the helix were selected for modification with the non-natural amino-acids required for stapling. We will present the results of this stapled peptide scan by reporting i) the conformational analysis by circular dichroism, ii) the biological activity in a novel N0-P fluorescence polarization assay, a viral replication assay and a cytotoxicity assay and iii) the in vivo proof of concept by intranasal administration of a hit peptide, HEVS 124, to BALB/c mice inoculated with a Luc-encoding RSV.

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National audience

International audience; Nonenveloped virus must penetrate the cellular membrane to access the cytoplasm without the benefit of membrane fusion. For birnavirus, one of the peptides present in the virus capsid, pep46 for infectious bursal disease virus, is able to induce pores into membranes as an intermediate step of the birnavirus-penetration pathway. Using osmotic protection experiments, we demonstrate here that pep46 and its pore-forming N-terminal moiety (pep22) form pores of different diameters, 5-8 and 2-4 nm, respectively, showing that both pep46 moieties participate to pore formation. The solution structures of pep46, pep22, and pep24 (the pep46 C-terminal moiety) in different hydrophobic environments and micelles determined by (1)H NMR studies provide structural insights of the pep46 domain interaction. In CDCl(3)/CD(3)OH mixture and in dodecylphosphocholine micelles, the N-terminal domain of pep46 is structured in a long kinked helix, although the C terminus is structured in one or two helices depending upon the solvents used. We also show that the folding and the proline isomerization status of pep46 depend on the type of hydrophobic environment. NMR spectroscopy with labeled phospholipid micelles, differential scanning calorimetry, and plasmon waveguide resonance studies show the peptides lie parallel to the lipid-water interface, perturbing the fatty acid chain packing. All these data lead to a model in which the two domains of pep46 interact with the membrane to form pores.

International audience; The translocation of the catalytic domain through the membrane of the endosome to the cell cytoplasm is a key step of intoxication by botulinum neurotoxin (BoNT). This step is mediated by the translocation (T) domain upon endosome acidification, although the mechanism of interaction of the T domain with the membrane is still poorly understood. Using physicochemical approaches and spectroscopic methods, we studied the interaction of the BoNT/A T domain with the membrane as a function of pH. We found that the interaction with membranes does not involve major secondary or tertiary structural changes, as reported for other toxins like diphtheria toxin. The T domain becomes insoluble around its pI value and then penetrates into the membrane. At that stage, the T domain becomes able to permeabilize lipid vesicles. This occurs for pH values lower than 5.5, in agreement with the pH encountered by the toxin within endosomes. Electrostatic interactions are also important for the process. The role of the so-called belt region was investigated with four variant proteins presenting different lengths of the N-extremity of the T domain. We observed that this part of the T domain, which contains numerous negatively charged residues, limits the protein-membrane interaction. Indeed, interaction with the membrane of the protein deleted of this extremity takes place for higher pH values than for the entire T domain. Overall, the data suggest that acidification eliminates repulsive electrostatic interactions between the T domain and the membrane, allowing its penetration into the membrane without triggering detectable structural changes.

Diplôme : Dr. d'Université

National audience; Les particules des virus à ARN double brin (ARNdb) sont compétentes pour la transcription et doivent traverser une membrane cellulaire pour fonctionner dans le cytoplasme de la cellule cible. Parmi ces virus, les birnavirus sont singuliers car ils possèdent une simple capside icosaédrique de triangulation T = 13 et ne contiennent pas la capside interne caractéristique observée dans la plupart de ces virus. Nous avons récemment élucidé la structure de sous-particules virales ainsi que de particules virales complètes d’un birnavirus aviaire [1]. Nos résultats révèlent des relations structurales inattendues entre virus icosaédriques et nous permettent de proposer un lien phylogénétique entre certains virus à ARNdb et des virus à ARN de polarité positive.

Throughout the last several decades, vaccination has been key to prevent and eradicate infectious diseases. However, many pathogens (e.g., respiratory syncytial virus [RSV], influenza, dengue, and others) have resisted vaccine development efforts, largely because of the failure to induce potent antibody responses targeting conserved epitopes. Deep profiling of human B cells often reveals potent neutralizing antibodies that emerge from natural infection, but these specificities are generally subdominant (i.e., are present in low titers). A major challenge for next-generation vaccines is to overcome established immunodominance hierarchies and focus antibody responses on crucial neutralization epitopes. Here, we show that a computationally designed epitope-focused immunogen presenting a single RSV neutralization epitope elicits superior epitope-specific responses compared to the viral fusion protein. In addition, the epitope-focused immunogen efficiently boosts antibodies targeting the palivizumab epitope, resulting in enhanced neutralization. Overall, we show that epitope-focused immunogens can boost subdominant neutralizing antibody responses in vivo and reshape established antibody hierarchies.

Acute lower respiratory tract infections (ALRI) caused by respiratory syncytial virus (RSV) are associated with a severe disease burden among infants and elderly patients. Treatment options are limited. While numerous drug candidates with different viral targets are under development, the utility of RSV entry inhibitors is challenged by a low resistance barrier and by single mutations causing cross-resistance against a wide spectrum of fusion inhibitor chemotypes. We developed a cell-based screening assay for discovery of compounds inhibiting infection with primary RSV isolates. Using this system, we identified labyrinthopeptin A1 and A2 (Laby A1/A2), lantibiotics isolated from Actinomadura namibiensis, as effective RSV cell entry inhibitors with IC50s of 0.39 μM and 4.97 μM, respectively, and with favourable therapeutic index (>200 and > 20, respectively). Both molecules were active against multiple RSV strains including primary isolates and their antiviral activity against RSV was confirmed in primary human airway cells ex vivo and a murine model in vivo. Laby A1/A2 were antiviral in prophylactic and therapeutic treatment regimens and displayed synergistic activity when applied in combination with each other. Mechanistic studies showed that Laby A1/A2 exert virolytic activity likely by binding to phosphatidylethanolamine moieties within the viral membrane and by disrupting virus particle membrane integrity. Probably due to its specific mode of action, Laby A1/A2 antiviral activity was not affected by common resistance mutations to known RSV entry inhibitors. Taken together, Laby A1/A2 represent promising candidates for development as RSV inhibitors. Moreover, the cell-based screening system with primary RSV isolates described here should be useful to identify further antiviral agents.

International audience; As all the viruses belonging to the Mononegavirales order, the non-segmented negative strand RNA genome of respiratory syncytial virus (RSV) is encapsidated by the viral nucleoprotein N. N protein polymerizes along the genomic and anti-genomic RNAs during replication. This requires the maintenance of the neosynthesized N protein in a monomeric and RNA-free form by the viral phosphoprotein P that plays the role of a chaperone protein, forming a soluble N0-P complex. We have previously demonstrated that residues 1-30 of P specifically bind to N0. Here, to isolate a stable N0-P complex suitable for structural studies, we used the N-terminal peptide of P (P40) to purify truncated forms of the N protein. We show that to purify a stable N0-P-like complex, a deletion of the first 30 N-terminal residues of N (NΔ30) is required to impair N oligomerization, whereas the presence of a full-length C-arm of N is required to inhibit RNA binding. We generated structural models of the RSV N0-P with biophysical approaches, including hydrodynamic measurements and small-angle X-ray scattering (SAXS), coupled with biochemical and functional analyses of human RSV (hRSV) NΔ30 mutants. These models suggest a strong structural homology between the hRSV and the human metapneumovirus (hMPV) N0-P complexes. In both complexes, the P40-binding sites on N0 appear to be similar, and the C-arm of N provides a high flexibility and a propensity to interact with the N RNA groove. These findings reveal two potential sites to target on N0-P for the development of RSV antivirals.