Screening antivirals with a mCherry-expressing recombinant bovine respiratory syncytial virus: a proof of concept using cyclopamine

Bovine respiratory syncytial virus (BRSV) is a pathogenic pneumovirus and a major cause of acute respiratory infections in calves. Although different vaccines are available against BRSV, their efficiency remains limited, and no efficient and large-scale treatment exists. Here, we developed a new reverse genetics system for BRSV expressing the red fluorescent protein mCherry, based on a field strain isolated from a sick calf in Sweden. Although this recombinant fluorescent virus replicated slightly less efficiently compared to the wild type virus, both viruses were shown to be sensitive to the natural steroidal alkaloid cyclopamine, which was previously shown to inhibit human RSV replication. Our data thus point to the potential of this recombinant fluorescent BRSV as a powerful tool in preclinical drug discovery to enable high throughput compound screening

Longitudinal study of the immune response and memory following natural bovine respiratory syncytial virus infections in cattle of different age

Human and bovine respiratory syncytial virus (HRSV and BRSV) are closely genetically related and cause respiratory disease in their respective host. Whereas HRSV vaccines are still under development, a multitude of BRSV vaccines are used to reduce clinical signs. To enable the design of vaccination protocols to entirely stop virus circulation, we aimed to investigate the duration, character and efficacy of the immune responses induced by natural infections. The systemic humoral immunity was monitored every two months during two years in 33 dairy cattle in different age cohorts following a natural BRSV outbreak, and again in selected individuals before and after a second outbreak, four years later. Local humoral and systemic cellular responses were also monitored, although less extensively. Based on clinical observations and economic losses linked to decreased milk production, the outbreaks were classified as moderate. Following the first outbreak, most but not all animals developed neutralising antibody responses, BRSV-specific IgG1, IgG2 and HRSV F- and HRSV N-reactive responses that lasted at least two years, and in some cases at least four years. In contrast, no systemic T cell responses were detected and only weak IgA responses were detected in some animals. Seronegative sentinels remained negative, inferring that no new infections occurred between the outbreaks. During the second outbreak, reinfections with clinical signs and virus shedding occurred, but the signs were milder, and the virus shedding was significantly lower than in naïve animals. Whereas the primary infection induced similar antibody titres against the prefusion and the post fusion form of the BRSV F protein, memory responses were significantly stronger against prefusion F. In conclusion, even if natural infections induce a long-lasting immunity, it would probably be necessary to boost memory responses between outbreaks, to stop the circulation of the virus and limit the potential role of previously infected adult cattle in the chain of BRSV transmission

Etude fonctionnelle de la protéine M2-1 du virus respiratoire syncytial à l'aide d'un miniréplicon en cellules eucaryotes

Le virus respiratoire syncytial (VRS) est le principal agent responsable de maladies respiratoires graves (bronchiolites, pneumonies) chez l’Homme (nouveau-nés, personnes âgées ou immunodéprimées). En l’absence de vaccins efficaces contre ce virus, le développement rationnel de traitements antiviraux constitue un enjeu de taille. Appartenant à la famille des Paramyxoviridae, 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 répliqué et transcrit par le complexe ARN polymérase dépendante de l’ARN viral. Ce complexe est composé de la nucléoprotéine N, de la polymérase L, de la phosphoprotéine P et du facteur de transcription M2-1 qui assure la processivité de la polymérase au cours de la transcription virale. Le complexe polymérase, ne présentant pas d’équivalent dans la cellule, constitue une cible privilégiée pour le développement d’inhibiteurs. Une meilleure compréhension des mécanismes d’interaction entre les protéines du complexe polymérase est donc indispensable afin d’identifier des cibles potentielles. Le groupe de John N. Barr (Leeds University, UK) a résolu la structure cristalline tridimensionnelle de la protéine M2-1, dans sa forme tétramérique avec une résolution de 2,5Å. Nous nous sommes associés à ce laboratoire afin étudier le rôle fonctionnel de certains résidus de la protéine M2-1. Le rôle critique de certains résidus a été abordé par mutagenèse dirigée et l’utilisation d’un miniréplicon accompagné d’un contrôle d’expression par western blotting des protéines mutées. Nous avons notamment montré l’importance de deux résidus Serine phosphorylables pour l’activité de M2-1 en tant que facteur de transcription

Underlying functional bowel disorder may explain patient dissatisfaction after haemorrhoidal surgery.

International audienceAIM: The aim of this study was to assess patient dissatisfaction and functional symptoms following haemorrhoid surgery, aspects of which are seldom covered in other published series. METHOD: A self-administered questionnaire was mailed to 359 consecutive patients (prospective database; 198 men, 161 women; median follow up, 59 [1-120] months) who underwent either Milligan-Morgan haemorrhoidectomy (n=205) or stapled haemorrhoidopexy (n=154). RESULTS: The response rate was 72%; 2.4% of patients had no opinion, 13.6% were dissatisfied, 33.0% were satisfied, and 51.0% were very satisfied with the treatment. Dissatisfied patients were more likely to be women and more likely to have a long history of constipation and irritable bowel syndrome. The duration of surgery and the rates of pre- and postoperative complications did not differ between groups. Residual bleeding (49% vs 32%), prolapse (67% vs 31%) and pain (91% vs 55%) occurred more frequently in the dissatisfied group compared with the satisfied group (P<0.001). Incontinence (4 [0-16] vs 1 [0-15]; P=0.0003) and constipation (19 [1-34] vs 8 [0-31]; P<0.0001) scores were significantly higher in the dissatisfied group compared with satisfied patients. Anal pain was the predominant symptom associated with dissatisfaction in a logistic regression model. CONCLUSION: Persistent pain remains the major long-term factor associated with dissatisfaction after surgery for haemorrhoids

Interaction of the respiratory syncytial virus non-structural protein 1 with the MED25 Mediator subunit

International audienc

Fine mapping and characterization of the binding domain of the HRSV Phosphoprotein with the M2-1 protein

The RSV genome is transcribed into 10 mRNAs by the RNA-dependant RNA polymerase complex (RdRp). M2-1 protein is a transcription antiterminator which increases the processivity of the RdRp during transcription. M2-1 is recruited to RNA transcription sites by the phosphoprotein P. Since protein-protein interactions are a target for antiviral compounds, our objective is to obtain the crystallographic structure of the M2-1—P complex. The atomic structure of full-length tetrameric M2-1 is now available. However, since P is a naturally disordered protein, it is not possible to use full-length P for that purpose. The aim of this work was to finely characterise the M2-1 binding domain of P and to use this domain for co-crystallization trials. The M2-1-binding domain of P was previously mapped to residues 100-120 by internal deletions by Mason et al. By using NMR, we identified P residues ~ 90-100 as a region interacting with M2-1. Using recombinant proteins and deletions, the M2-1 binding site was finely mapped to amino acid residues 93-110. The role of amino acid residues in M2-1—P interaction was investigated by site-directed mutagenesis and pull-down assays, and the impact of these mutations on viral transcription was evaluated in cellula using an RSV minigenome. The results highlighted the critical role of some residues located in this region. The role of P oligomerization for M2-1—P interaction was also investigated

La protéine M2-1 du Virus Respiratoire Syncytial, une nouvelle cible potentiel pour des antiviraux

Structure et morphogenèse viraleStructure et morphogenèse viraleLe virus respiratoire syncytial (VRS) est le principal agent responsable de maladies respiratoires graves (bronchiolites, pneumonies) chez l’homme, principalement chez les nouveau-nés, personnes âgées ou immunodéprimées. En l’absence de vaccin efficace contre ce virus, le développement rationnel de traitements antiviraux constitue un enjeu de taille. Appartenant à la famille des Paramyxoviridae, 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 répliqué et transcrit par le complexe ARN polymérase dépendant de l’ARN viral. Ce complexe est composé de la nucléoprotéine N, de la polymérase L, de la phosphoprotéine P et du facteur de transcription M2-1 qui assure la « processivité » de la polymérase au cours de la transcription virale. Le complexe polymérase, ne présentant pas d’équivalent dans la cellule, constitue une cible privilégiée pour le développement d’inhibiteurs spécifiques. Une meilleure compréhension des mécanismes d’interaction entre les différentes protéines du complexe polymérase est donc indispensable afin d’identifier des cibles potentielles. En collaboration avec le groupe de John N. Barr (Leeds University, UK), nous avons résolu la structure cristalline tridimensionnelle de la protéine M2-1 sous forme tétramérique et avons étudié le rôle de certains résidus par mutagenèse dirigée. Nous avons notamment montré l’importance de certains résidus pour les interactions avec la phosphoprotéine et les ARN viraux.Le groupe de Ralf Altmeyer - Institut Pasteur de Shanghai, a sélectionné, après criblage, un composé inhibant la réplication du VRS in vitro. La culture du virus en présence de concentrations croissantes de ce composé, a permis de sélectionner des virus mutants résistants. Trois mutations ponctuelles ont été identifiées uniquement sur la protéine M2-1. Par génétique inverse, nous avons validé le rôle de ces résidus dans la résistance à ce composé et ainsi démontré que la protéine M2-1 est la cible de ce composé antiviral

Structure and interactions of respiratory syncytial virus non structural protein 1

International audienc

Does Intrinsic Disorder in Proteins Favor Their Interaction with Lipids?

Intrinsically disordered proteins (IDPs) are implicated in a range of human diseases, some of which are associated with the ability to bind to lipids. Although the presence of solvent-exposed hydrophobic regions in IDPs should favor their interactions with low-molecular-weight hydrophobic/amphiphilic compounds, this hypothesis has not been systematically explored as of yet. In this study, the analysis of the DisProt database with regard to the presence of lipid-binding IDPs (LBIDPs) reveals that they comprise, at least, 15% of DisProt entries. LBIDPs are classified into four groups by ligand type, functional categories, domain structure, and conformational state. 57% of LBIDPs are classified as ordered according to the CH-CDF analysis, and 70% of LBIDPs possess lengths of disordered regions below 50%. To investigate the lipid-binding properties of IDPs for which lipid binding is not reported, three proteins from different conformational groups are rationally selected. They all are shown to bind linoleic (LA) and oleic (OA) acids with capacities ranging from 9 to 34 LA/OA molecules per protein molecule. The association with LA/OA causes the formation of high-molecular-weight lipid-protein complexes. These findings suggest that lipid binding is common among IDPs, which can favor their involvement in lipid metabolism

Transcription and replication of Mononegavirales: an original molecular machine

National audienc