Latent Autoimmune Diabetes in Adults Associated with Von Recklinghausen’s Disease (Neurofibromatosis Type 1)

Introduction: Endocrine disorders during Von Recklinghausen’s Disease or neurofibromatosis type 1 (NF1) are rare and particularly observed in children. However, autoimmune diabetes mellitus (DM) remains exceptional and unusual during this phacomatosis. We report an original case of Latent Autoimmune Diabetes in Adults (LADA) associated with NF1.Case Report: A 32-year-old Tunisian male, known to have NF1 since childhood, was admitted for significant recent weight loss (10 kg in one month) with high blood glucose levels. The biological tests confirmed the diagnosis of DM with marked ketoacidosis: fast blood glucose at 16 mmol/l, postprandial glucose at 21 mmol/l, and HbA1c at 9.9%. Radiological and endoscopic investigations did not indicate pancreatic and/or duodenal tumors. Anti-GAD and anti-IA2 autoantibodies were positively confirming the diagnosis of LADA. The assessment of degenerative complications and screening for possible other autoimmune diseases were negative. The evolution was favorable under intensive insulinotherapy.Conclusion: The association of DM type 1 with NF1 remains exceptional and only four cases are found in the literature, all pediatrics. Our observation is, to our knowledge, the first reporting this association in adult (LADA with NF1)

Structure and Functional Analysis of the RNA- and Viral Phosphoprotein-Binding Domain of Respiratory Syncytial Virus M2-1 Protein

Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-158–177 core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-158–177, as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1

NMR study of the structure and interactions of proteins of human respiratory syncytial virus

Le virus respiratoire syncytial (VRS) est un membre de la famille des Paramyxoviridae, des virus simple brin d'ARN non segmentés de polarité négative. Le virus respiratoire syncytial humain (VRSh) est la principale cause des maladies respiratoires chez les enfants, et il est la priorité des cibles vaccinales. Notre objectif est d'obtenir des réponses sur la structure et la dynamique des différents composants du complexe ARN polymérase ARN dépendante du VRS (RdRp) et sur leurs interactions, en utilisant la résonance magnétique nucléaire (RMN), en espérant pouvoir proposer des molécules qui inhibent ou perturbent ces interactions afin de développer des médicaments antiviraux spécifiques. Mon travail porte sur deux protéines du complexe RdRp: la phosphoprotéine P, qui est le co-facteur principal de la polymérase et elle est nécessaire à la fois pour la transcription virale et pour la réplication, et M2-1 qui est un facteur d'antiterminaison de la transcription. Notre but est de caractériser la structure de P, par rapport à d'autres protéines P des Mononegavirales, la P du VRSh est assez courte et ne comporte pas de domaines avec structure tertiaire stable en dehors du domaine de tétramérisation central résistant à la trypsine. L'analyse de séquence de P prédit la présence d'un domaine d'oligomérisation et de grandes extensions en N-et C-terminales intrinsèquement désordonnés. Cet agencement de domaine est confirmé par RMN. En outre, nous avons utilisé l'analyse de déplacements chimiques secondaires et des mesures de relaxation nucléaire en azote 15 pour montrer que des hélices transitoires sont formées dans les extrémités N- et C-terminales de P. A l'extrémité C-terminale, des hélices presque complètement formées semblent prolonger le domaine d'oligomérisation. A l'extrémité N-terminale des hélices transitoires formées coïncident avec les sites de liaison pour la nucléoprotéine du VRS et pour la liaison au co-facteur de transcription M2-1, comme montré par des expériences d'interaction par RMN. La protéine M2-1 du VRSh est un cofacteur du complexe RdRp essentiel pour la transcription virale en augmentant la processivité de la polymérase. M2-1 est une protéine modulaire qui se lie à l'ARN et interagit également avec la phosphoprotéine virale P. Ces propriétés de liaison sont liées au domaine globulaire de M2-1. Puisque La structure du domaine globulaire de M2-1 a été déjà résolue par mon équipe par RMN, donc nous avons pu montrer le chevauchement partiel des surfaces d'interaction de l'ARN et de P, sur le fragment monomère de M2-1 (58-177) par RMN, confirmant que cette protéine se lie à la phosphoprotéine P et à l'ARN de manière compétitive. Tous les résultats de RMN sont toujours confirmés par des tests fonctionnels par nos collaborateurs à l'INRA, Jouy-en-Josas.Respiratory syncytial virus (RSV) is a member of the Paramyxoviridae family of non segmented, negative sense singlestranded RNA viruses. Human respiratory syncytial virus (hRSV) is major cause of respiratory diseases in children, and is prioritized vaccine targets. Our aim is to get answers about the structure and dynamics of different components of the RSV RNA dependent RNA polymerase complex (RdRp) and about their interactions, by using Nuclear Magnetic Resonance, as a prerequisite to rational drug design. My work focuses on two RSV proteins: the phosphoprotein, which is the main polymerase co-factor and necessary for both viral transcription and replication, and M2-1 which is the antitermination factor of transcription. Our aim is to characterize the structure of P. As compared to other P proteins of Mononegavirales, hRSV P is rather short and does not comprise domains with stable tertiary fold outside the central trypsin-resistant tetramerization domain. Sequence analysis of P predicts the presence of a helical oligomerization domain and large disordered N-and C-terminal extensions. This domain arrangement is confirmed by NMR. Moreover we used backbone chemical shift analysis and 15 N relaxation experiments to show that transient helices are formed in the N- and C-termini of P. At the C-terminus, nearly completely formed helices seem to prolong the oligomerization domain. At the N-terminus transiently formed helices coincide with the binding sites for the RSV nucleoprotein and for the transcription co-factor M2-1, as shown by NMR interaction experiments. The M2-1 protein of hRSV functions as an essential transcriptional cofactor of the viral (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P. These binding properties are related to the core region of M2-1. After solving the structure of the corresponding domain, we showed that partial overlap of the RNA and P interaction surfaces, determined by NMR on the monomeric M2-1(58-177) fragment, accounts for the previously observed competitive behavior of RNA versus P in M2-1 binding. The NMR results are always confirmed by functional tests by our collaborators at INRA, Jouy-en-Josa

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

NMR insight into transient structures and interactions within the RNA polymerase of the bronchiolitis virus.

International audienc

RSV hijacks cellular protein phosphatase 1 to regulate M2-1 phosphorylation and viral transcription

Respiratory syncytial virus (RSV) RNA synthesis occurs in cytoplasmic inclusion bodies (IBs) in which all the components of the viral RNA polymerase are concentrated. In this work, we show that RSV P protein recruits the essential RSV transcription factor M2-1 to IBs independently of the phosphorylation state of M2-1. We also show that M2-1 dephosphorylation is achieved by a complex formed between P and the cellular phosphatase PP1. We identified the PP1 binding site of P, which is an RVxF-like motif located nearby and upstream of the M2-1 binding region. NMR confirmed both P-M2-1 and P-PP1 interaction regions in P. When the P–PP1 interaction was disrupted, M2-1 remained phosphorylated and viral transcription was impaired, showing that M2-1 dephosphorylation is required, in a cyclic manner, for efficient viral transcription. IBs contain substructures called inclusion bodies associated granules (IBAGs), where M2-1 and neo-synthesized viral mRNAs concentrate. Disruption of the P–PP1 interaction was correlated with M2-1 exclusion from IBAGs, indicating that only dephosphorylated M2-1 is competent for viral mRNA binding and hence for a previously proposed post-transcriptional function

Solution structure of the core domain of RSV M2-1.

<p>(A) Cartoon representation of the NMR structure of the α-helical domain of M2-1_58–177 (model of lowest energy). The color is ramped from blue to red from the N- to the C-terminus. (B) Electrostatic surface potential of M2-1_58–177 calculated with DELPHI <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002734#ppat.1002734-Rocchia1" target="_blank">[58]</a> using PARSE parameters <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002734#ppat.1002734-Sitkoff1" target="_blank">[59]</a>. Two opposite faces of the protein are shown. The left-hand view is the same as for panel A. Colors for charges are red to blue for potential energies −6 to +6 k_BT. Basic residues belonging to the main cluster are labeled in black bold letters. Basic residues belonging to the 3 minor clusters are indicated in blue, green and purple letters. (C) Schematic representation of the tetramer of full-length M2-1.</p

Effect of mutations affecting M2-1-controlled transcription on M2-1_58–177:RNA and M2-1:P complex formation <i>in vitro</i>.

<p>(A and B) Electrophoretic mobility shift assay (EMSA) of M2-1:RNA complex formation. Eluted GST-M2-1_58–177 (WT and mutants selected using the minigenome assay, 100 µM final concentration) were incubated with yeast tRNA (∼50 µM final concentration) for 1 h at room temperature. (A) Complexes were resolved by agarose gel electrophoresis stained with ethidium bromide. (B) Proteins were revealed by amido black staining. M2-1 mutations are indicated above each lane. (C and D) GST pull-down of purified P by GST-M2-1 (WT and the same mutants as in A and B). (C) GST-M2-1 or GST were incubated alone (−) or in the presence of P (+), washed, and analyzed by SDS-PAGE. P was also run alone (lane P). (D) Coomassie blue-stained gels were scanned and M2-1:P binding was quantified using ImageJ software and corrected for nonspecific binding to GST. Errors were estimated to ±5%. (E) The surfaces formed by the 8 residues, for which mutants were analyzed, are indicated on the M2-1 structure according to their binding partner: in red (RNA binding) and blue (P binding).</p

Apparent M2-1_58–177:oligonucleotide dissociation constants determined from amide (¹H and ¹⁵N), methyl (¹H and ¹³C) and R151-H_δ chemical shift variations (at 14.1 T and 293 K).

a<p>K_d errors were estimated from standard deviations of K_d values determined for individual residues.</p>b<p>Double-stranded GE_F RNA was formed with equal amounts of the complementary GE_F-neg and GE_F-pos strands.</p

Effect of M2-1 mutations on RSV transcription and association with the nucleocapsid.

<p>(A) Analysis of RSV specific M2-1-controlled transcription with WT and M2-1 substitution mutants. BSRT7/5 cells were transfected with RSV pP, pN, pL, and pM2-1 plasmids and an RSV specific minigenome containing the firefly luciferase reporter gene, together with p-β-Gal constitutively expressing β-galactosidase. Luciferase activity, measured 24 h after transfection, was normalized by β-galactosidase activity, and the luciferase activity gained with WT M2-1 set to 100%. The mean value and confidence intervals (error bars) result from 3 separate experiments performed in duplicate. A control was run without M2-1. (B) Expression of M2-1 mutant proteins in BSRT7 cells. Cells were co-transfected with plasmids encoding M2-1 mutants and N. Cell extracts were analyzed by Western blotting with rabbit polyclonal antibodies against M2-1 and N. Expression levels of M2-1 were normalized against N expression and compared to tubulin. (C, D) Colocalization studies of M2-1 with N-P complexes. Plasmids encoding N, P, and M2-1 mutants were transfected into BSRT7/5 cells. Immunofluorescence analysis was performed on cells fixed 24 h after transfection, by using rabbit polyclonal anti-N (1∶100) or anti-P (1∶500) and Alexa Fluor 594 goat anti-rabbit (1∶1000) antibodies, and mouse monoclonal anti-M2-1 (1∶40 dilution) and Alexa Fluor 488 goat anti-mouse (1∶1000) antibodies. Horizontal bars correspond to 10 µm.</p