Jean-Christophe Le Bayon

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1 Jean-Christophe Le Bayon
Étude des interactions fonctionnelles entre les glycoprotéines d’enveloppe F et HN, impliquées dans le mécanisme d’entrée cellulaire des virus parainfluenza humains et animaux. Jean-Christophe Le Bayon VirPath CNRS FRE 3011 Dr Manuel Rosa-Calatrava Pr Bruno Lina Master 2 GBC – Juin 2009 UCBL Dear members of Jury, good afternoon, I’m Jean-Christophe Le Bayon Today I’m presenting to you the work I did the last months at the VirPath Lab, in Laennec, Lyon, supervised by Drs Rosa-Calatrava, and Terrier. This work consisted to characterized some functions of mutants of the fusion glycoprotein F of human parainfluenza virus type 2. ED 341 – E2M2

2 In a first part, I’ll present to you:
État de l’art Virus Parainfluenza de type 2 Entrée du virus Projet Caractérisation des mutations Adéquation Encadrement Projet professionnel In a first part, I’ll present to you: The Human parainfluenza virus type 2, its classification and characteristics Then I’ll focus on the first stage of virus entry into the host cell I’ll present to you the objectives of my work and the methods used to reach it As my results : -First we’ll see together the mutations we decided to perform -And the functional characterization of its Then I’ll discuss about the results we obtained and also we’ll see some future work

3 hPIV2 et NDV Famille : Paramyxoviridae Genre : Rubulavirus Avulavirus
Maladies respiratoires Pas de vaccins/antiviraux Virus enveloppé Ø 200nm ARN négatif, simple brin Terrier et al. (2008) -The Human parainfluenza virus type 2, hPIV-2, is classified in the order of the Mononegvirales. It is in the paramyxoviridae family, genus rubulavirus as mumps virus, -This Virus is responsible of respiratory infections, most of the time for the young children and also the elderly. They are no treatments available for this kind of infection !! -It is an enveloped virus, its membrane come from the cells where it replicate and bud. Behind this membrane there are the M proteins. -It has a simple stranded RNA genome, non segmented, complexed with some proteins. Altogether it forms the ribonucleoprotein. -and it can enter into the cell with its glycoproteins of fusion (F), and the GP with hemagglutnin/neuramidase fonctions (HN). (sub family paramyxovirinae, like measle (morbili), sendaï (respiro) or NewCastle Disease Virus (avula)) ((the nucleocapsid protein (N), the phosphoprotein (P) and the large RNA polymerase RNA (L) dependant) Genome 15kb environ L’entrée dans la cellule est possible grâce aux GP F et HN

4 Membrane Fusion F HN F HN Cellular membrane Takimoto et al. (2002)
Fusion peptide S HR1 F HN Extracellular domain F1 HR2 Transmembrane region Cytoplasmic tail C N F HN Cellular membrane Both are transmembrane proteins and are present at the surface of the viral envelope. The F GP is cleaved during its maturation, so a short sequence, also named fusion peptide is revealed. ->At the first step of infection, the viral particle needs the attachment of HN onto its cellular receptor in order to activate the F by a really badly known process. ->Then F anchor its fusion peptide into the target cell membrane. -> And refold itself to permit the membrane fusion. Because of these proprieties, infected cells are able to fuse together and form big multinuclear cells in vitro, also named syncytia. Takimoto et al. (2002) Viral envelope

5 Objectives Role in fusion and HN-F activation mechanisms ?
hPIV-2 variants with increased cell-cell fusion: T96A mutation (Terrier et al., 2008) PIV-5 “HN-independent” F : L22P, K132E and V290A (Ito et al., 1998, 2009) PIV5 / hPIV2 F sequences: 51% identity Functional characterization of F GP hPIV2 mutants : T96A and mutations transposed from PIV-5 Role in fusion and HN-F activation mechanisms ? In our lab, we found some hPIV2 variants which were able to form big syncytia. All these variants shared the mutation T96A present on The F2 sub-unit near a very conserved region, for all paramyxoviruses, which could be implicate in the F regulation by HN. We’ve seen that F needs HN to cause the fusion, however it exist an exception. One Japanese lab found a parainfluenza type 5, PIV-5, variant which posses several mutations which permit F to fuse with cells membrane, even HN is absent. With the L22P, K132E and V290A mutations, F may does’t need the HN activation. F is called independent. This virus is very close to hPIV2, and their respective F GP share 51% of identical aminoacids. So it was possible to transpose these mutations on F hPIV-2. We created mutants of the F GP hPIV2 which carried the mutations T96A and mutations transposed from PIV-5 in order to characterize their role in the fusion and the possible interaction on HN-F activation mechanism

6 Conclusion T96A: favorise l’activation par HN
I24P: F “indépendante” de HN K133E: interaction entre F et HN et augmentation de la fusion I294A: favorise l’activation par HN Tête Tige I24P I294A T96A K133E We determinate in this study that : Mutant T96A, found in most hPIV-2 variants, seems to be involved in a finest regulation of HN The mutation I24P seems to be involved in an increased “independence” of F. That’s the first time this results is shown on hPIV-2. Mutants K133E and, with I24P added, highlight another residue implicate in the functional interaction between F and HN and maybe highlight a functional inactivation of F by HN I294A seems to be implicate in the F activation by HN, as T96A. This implicate that different parts of the F protein are involved in the F-HN functional interaction as already described for Sendaï virus. The tridimensional structure of F was predicted by bioinformatics tools to show us the position of mutations. All are at the protein’s head surface, known to interact with HN. 2 parties distinctes de la tête de F semblent être impliquées dans l’interaction F-HN

7 Projet de thèse Expression de glycoprotéines (WB, FACS)
Interactions entre F et HN(CoIP) Comportement des mutants dans un contexte viral ou pseudo-viral (VLP and Reverse Genetics) Meilleure mesure de la fusion membranaire (RT lipid-mix assay) Caractérisation de nouveaux mutants grâce à des variants (sequencing, fusion assay) To dig these first results, we plan to : Quantify F and HN by Western-Blot to confirm our FACS results To perform a finest membrane fusion quantification by real-time lipid-mix assay To design an HN not able to promote the F GP to have a better mock and characterize, on HN, the domains of interaction with F. Co-IP could be performed. We plan to see the behavior of a F and HN mutant on a virus, by reverse genetics, or a Virus-Like Particle, to have a better model that the cell-cell fusion. And to evaluate new clinical variants to find next interesting mutations -> All these investigations could permit us to better understand the entry mechanism into the cell of the virus and may allow designing new drugs molecules in order to fight the parainfluenza infection. WB : séparation sur gel d’acrylamide des prot selon leur masse, et immuno-marquage par Ac FACS : cytometrie, permet de déterminer des caractéristiques basiques des cellules (mortes –vivantes) et reconnaitre un marquage immunofluorescent Fusion lipidique en temps réel : enveloppes lipidiques des particules marquées (quenchée) et on mesure la disparition du signal lors de fusion entre l’enveloppe de la particule et la membrane cellulaire (dequenching). CoIP : Immunoprécipitation d’une protéine, F par exemple, on vérifie parmi les molécules précipitées la présence d’un partenaire comme HN. / Méthode difficile à mettre en œuvre car c’est des protéines membranaires. / Besoin d’un « linker », petite molécule qui permet de stabiliser les liaisons inter-moléculaire (cross-linking). VLP : virus-like-particle, particules pseudos-virales, ce sont des particules virales ne contenant pas de génome et donc incapable de se répliquer. Elles permettent de constituer un modèle d’infection. / Production en transfectant des cellules par F et HN, et aussi M qui permet l’auto-assemblage de la particule virale et son bourgeonnement. / On peut aussi améliorer la production grâce à des protéines rétro-virales Gag et Env. Génétique inverse : production de virus réplicatif grâce à des seuls plasmides. / Dans le cas des paramyxovirus. 4 plasmides : 1 portant l’ensemble du génome viral afin de permettre la réplication en ARN négatif. Et des plasmides permettant l’expression des ribonucléoprotéines : la protéine se liant à l’ARN viral N, la phosphoprotéine P et l’ARN polymérase ARN dépendante L.

8 -> We first quantify the level of cell-to-cell fusion permit by F WT alone, and we arbitrarily set its value to 100%. As a control we determinate the level of expression of F at the cell-surface, the value for F WT alone was set to 100%. The mock (t minus) are cells tranfected with empty plasmid. We also transfected cells with the HN GP added, in light gray. For the WT F with HN, its level of fusion is better and also its expression. -> With the mutation T96A, we observed an increased ability to fuse in the presence of HN, despite same levels of expression at the cell surface. -> The T96A cause a better fusogenic propriety of F, but only in presence of HN. T96A seems to be implicate into the HN promotion of fusion mechanism.

9 -> We first quantify the level of cell-to-cell fusion permit by F WT alone, and we arbitrarily set its value to 100%. As a control we determinate the level of expression of F at the cell-surface, the value for F WT alone was set to 100%. The mock (t minus) are cells tranfected with empty plasmid. We also transfected cells with the HN GP added, in light gray. For the WT F with HN, its level of fusion is better and also its expression. -> With the mutation T96A, we observed an increased ability to fuse in the presence of HN, despite same levels of expression at the cell surface. -> The T96A cause a better fusogenic propriety of F, but only in presence of HN. T96A seems to be implicate into the HN promotion of fusion mechanism.

10 Contribution scientifique du projet
Comprendre le mécanisme d’entrée du virus dans la cellule → Design de nouvelles molécules anti-virales -> We first quantify the level of cell-to-cell fusion permit by F WT alone, and we arbitrarily set its value to 100%. As a control we determinate the level of expression of F at the cell-surface, the value for F WT alone was set to 100%. The mock (t minus) are cells tranfected with empty plasmid. We also transfected cells with the HN GP added, in light gray. For the WT F with HN, its level of fusion is better and also its expression. -> With the mutation T96A, we observed an increased ability to fuse in the presence of HN, despite same levels of expression at the cell surface. -> The T96A cause a better fusogenic propriety of F, but only in presence of HN. T96A seems to be implicate into the HN promotion of fusion mechanism.

11 We determinate in this study that :
Mutant T96A, found in most hPIV-2 variants, seems to be involved in a finest regulation of HN The mutation I24P seems to be involved in an increased “independence” of F. That’s the first time this results is shown on hPIV-2. Mutants K133E and, with I24P added, highlight another residue implicate in the functional interaction between F and HN and maybe highlight a functional inactivation of F by HN I294A seems to be implicate in the F activation by HN, as T96A. This implicate that different parts of the F protein are involved in the F-HN functional interaction as already described for Sendaï virus. The tridimensional structure of F was predicted by bioinformatics tools to show us the position of mutations. All are at the protein’s head surface, known to interact with HN.

12 We determinate in this study that :
Mutant T96A, found in most hPIV-2 variants, seems to be involved in a finest regulation of HN The mutation I24P seems to be involved in an increased “independence” of F. That’s the first time this results is shown on hPIV-2. Mutants K133E and, with I24P added, highlight another residue implicate in the functional interaction between F and HN and maybe highlight a functional inactivation of F by HN I294A seems to be implicate in the F activation by HN, as T96A. This implicate that different parts of the F protein are involved in the F-HN functional interaction as already described for Sendaï virus. The tridimensional structure of F was predicted by bioinformatics tools to show us the position of mutations. All are at the protein’s head surface, known to interact with HN.

13 We determinate in this study that :
Mutant T96A, found in most hPIV-2 variants, seems to be involved in a finest regulation of HN The mutation I24P seems to be involved in an increased “independence” of F. That’s the first time this results is shown on hPIV-2. Mutants K133E and, with I24P added, highlight another residue implicate in the functional interaction between F and HN and maybe highlight a functional inactivation of F by HN I294A seems to be implicate in the F activation by HN, as T96A. This implicate that different parts of the F protein are involved in the F-HN functional interaction as already described for Sendaï virus. The tridimensional structure of F was predicted by bioinformatics tools to show us the position of mutations. All are at the protein’s head surface, known to interact with HN.

14 Acknowledgments All VirPath team for their support Dr Olivier Terrier
Dr Manuel Rosa-Calatrava Pr Bruno Lina Christelle Godard and Gaëlle Cartet for their technical support Thanks for your attention, questions ? All VirPath team for their support