Biochemical characterization of the respiratory syncytial virus N0-P complex in solution

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

A persistent neutrophil-associated immune signature characterizes post-COVID-19 pulmonary sequelae

Interstitial lung disease and associated fibrosis occur in a proportion of individuals who have recovered from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection through unknown mechanisms. We studied individuals with severe coronavirus disease 2019 (COVID-19) after recovery from acute illness. Individuals with evidence of interstitial lung changes at 3 to 6 months after recovery had an up-regulated neutrophil-associated immune signature including increased chemokines, proteases, and markers of neutrophil extracellular traps that were detectable in the blood. Similar pathways were enriched in the upper airway with a concomitant increase in antiviral type I interferon signaling. Interaction analysis of the peripheral phosphoproteome identified enriched kinases critical for neutrophil inflammatory pathways. Evaluation of these individuals at 12 months after recovery indicated that a subset of the individuals had not yet achieved full normalization of radiological and functional changes. These data provide insight into mechanisms driving development of pulmonary sequelae during and after COVID-19 and provide a rational basis for development of targeted approaches to prevent long-term complications

Understanding Rhinovirus Circulation and Impact on Illness

Rhinoviruses (RVs) have been reported as one of the main viral causes for severe respiratory illnesses that may require hospitalization, competing with the burden of other respiratory viruses such as influenza and RSV in terms of severity, economic cost, and resource utilization. With three species and 169 subtypes, RV presents the greatest diversity within the Enterovirus genus, and despite the efforts of the research community to identify clinically relevant subtypes to target therapeutic strategies, the role of species and subtype in the clinical outcomes of RV infection remains unclear. This review aims to collect and organize data relevant to RV illness in order to find patterns and links with species and/or subtype, with a specific focus on species and subtype diversity in clinical studies typing of respiratory samples

Understanding Rhinovirus Circulation and Impact on Illness

Rhinoviruses (RVs) have been reported as one of the main viral causes for severe respiratory illnesses that may require hospitalization, competing with the burden of other respiratory viruses such as influenza and RSV in terms of severity, economic cost, and resource utilization. With three species and 169 subtypes, RV presents the greatest diversity within the Enterovirus genus, and despite the efforts of the research community to identify clinically relevant subtypes to target therapeutic strategies, the role of species and subtype in the clinical outcomes of RV infection remains unclear. This review aims to collect and organize data relevant to RV illness in order to find patterns and links with species and/or subtype, with a specific focus on species and subtype diversity in clinical studies typing of respiratory samples

Biochemical characterization of the respiratory syncytial virus n0-p complex in solution

As all the viruses belonging to the Mononegavirales order, the nonsegmented 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

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 phosphoprotéine P du Virus Respiratoire Syncytial recrute la protéine phosphatase-1 cellulaire afin de réguler la phosphorylation de M2-1, facteur viral de transcription, ainsi que son activité

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

Quand un virus pathogène détourne la machinerie cellulaire à son avantage

Session "Dialogues agresseurs-hôtes, adaptations réciproques"Le virus respiratoire syncytial (VRS) est le principal agent responsable de maladies respiratoires sévères (bronchiolites, pneumonies) chez l’Homme et le bovin. 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. Composé de la nucléoprotéine N, de la polymérase L, de la phosphoprotéine P et du facteur de transcription M2-1, ce complexe ne possède pas toutes les fonctions enzymatiques nécessaires à son cycle viral. Par conséquent le virus doit exploiter la cellule pour se répliquer et se répandre dans les voies respiratoires. Ce détournement des fonctions cellulaires par les virus est toujours étonnant et surprenant par son ingéniosité, mais est difficile à démasquer.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. De plus cette mutation a 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 centres réplicatifs viraux appelés « inclusion bodies » ou IBs. Enfin nous montrons que la surexpression de PP1 entraîne une accumulation de M2-1 déphosphorylée 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. En l'absence de PP1 des corps d'inclusion, M2-1 est exclue des granules associés aux IBs (IBAGs) formés par l'ARNm viral. Ces résultats suggèrent que M2-1 fonctionne non seulement en tant qu'antiterminateur de transcription, mais joue également un rôle critique post-transcriptionnel

Dephosphorylation of respiratory syncytial M2-1 protein by the cellular phosphatase PP1 is required for its mRNA binding ability

Expressing and multiplying – viral gene expressionThe M2-1 protein of respiratory syncytial virus (RSV) is essential for viral transcription. Previous reports suggested that dynamic regulation of M2-1 phosphorylation is critical for its function. M2-1 phosphorylation depends on the presence of the RSV phosphoprotein P, which is a multifunctional protein and the main cofactor of the large RNA polymerase L protein; formation of the P-M2-1 complex is required for viral transcription. However mechanisms involved in M2-1 phosphorylation and dephosphorylation in vivo have not been clarified. Using site-directed mutagenesis and NMR, we identified an RVxF-like motif located upstream of the M2-1 binding domain and involved in the capture of the host cell protein phosphatase-1 (PP1) by P and its recruitment to cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. We further show that the P-PP1 complex regulates M2-1 dephosphorylation and RSV transcription. In the absence of PP1 from inclusion bodies, M2-1 was excluded from IBs associated granules (IBAGs) formed by viral mRNA. These results suggest that M2-1 functions not only as a transcription antiterminator but plays also a critical role at late transcription steps