Functional characterization of human Parainfluenza type 2 (hPIV-2) fusion glycoprotein mutants

1. Functional characterization of human Parainfluenza type 2 (hPIV-2) fusion glycoprotein mutants Dr Manuel Rosa-Calatrava Dr Olivier Terrier Pr Bruno Lina Jean-Christophe Le Bayon Master 2 GBC – 18 Juin 2009 UCBL • VirPath • CNRSFRE 3011

3. The hPIV2 • Mononegavirales • Paramyxoviridae • Rubulavirus • Virus responsible of Respiratory diseases • Enveloped virus Ø 200nm • Simple stranded RNA / negative polarity Terrier et al. (2008) • Entry into the cell possible with glycoproteinsF and HN

4. Membrane Fusion Cellular membrane N C F2 S Fusion peptide S HR1 HN F Extracellular domain F1 HR2 Transmembrane region Cytoplasmic tail N C F HN Takimotoet al. (2002) Viral envelope

5. Objectives • Terrier et al. (2008): hPIV-2 variants capable of an increased cell-cell fusion which carried the T96Amutation • Ito et al. (1998, 2009): when presenting L22P, K132Eand V290A PIV5 F becomes independent from HN • PIV5 / hPIV2 are very closed viruses We created mutants of the F GP hPIV2 which carried the mutations T96A and mutations transposed from PIV-5 in order to characterize their possible interaction on HN-F activation mechanism

6. Methods • Bioinformatics and annotation of F hPIV2 • Cloning F and HN genes and directed mutagenesis • Flow cytometryand cell-cell luciferasefusion assay HuH7-TAT cell P TAT + substrate (luciferine) TAT Luc TAT Luc Fusion activate Luc Luc HIV-1 LTR Luc Luc P : constitutive promoter TAT : transcriptional activator pLUC: plasmid which carry : - LTR : TAT-dependant promoter - Luc: Firefllyluciferase gene Luc pLUCtransfected A549 cell

7. Choice of mutations S-S F2 F1 PF TM C N HR1 HR2 I24P T96A K133E I294A SC 16 24 34 TGIVGSDAIAGDQLLNVGV TGIVGSDAIAGDQLLNIGV LAGAGSLDLAALMQIGVIP 14 22 32 86 96 106 PLIENLSKISAVTDTKPRRER PLIENLSKISTVTDTKTRQER F hPIV-2 variant F hPIV-2 Greer F PIV-5 VR-288 124 133 142 TITAAVAIVKANANAAAIN QITAAVAIVKANANAAAIN QVTAAVALVKANKNAAAIL 120 129 132 138 284 294 305MQPGAKVIDLIAISANHKLQEV MQPGAKVIDLIAISANHKLQEVVQPATQIIDLVTISAFINNQEV 280 290 301 F hPIV-2 variant F hPIV-2 Greer F PIV-5 VR-288

8. T96A Mutation A B T96A is implicate into the HN promotion of fusion mechanism

9. PIV-5 transposed mutations • Increased potential of fusion for I24P with or without HN A • K133E is very well expressed especially without HN • I294A permit an increased fusion only with HN as T96A B

10. I24P based Mutations • I24P combination bring a better fusion effect also without HN A • With K133E big fusogenic characteristic, independent of HN B

11. Conclusion • Mutant T96A is involved in a finest up-regulation of F by HN • The mutation I24P is involved in an increased “independence” of F • Mutants K133E and I24P-K133E may highlight another functional interaction between F and HN (down regulation) • I294A seems to have the same effect as T96A • Several parts of F seems to be involved in the F-HN interaction T96A K133E Head I24P I294A Neck

12. Further investigations • Quantification of GP by Western-Blot • Finest membrane fusion quantification • Design an HN not able to promote the F GP • Behavior of a F mutant on a virus or a VLP (and mutant HN) • Evaluation of new clinical variants • Techniques: • Western-Blot/CoIP • Real-time lipid-mix assay • Virus-like particles with F and HN/Reverse genetics Design new moleculesagainst the hPIV-2 entry into the cells

13. Acknowledgments Dr Olivier Terrier Dr Manuel Rosa-Calatrava Chrystelle for the “last-minute” cells All VirPath team for their support

14. The Membrane Fusion Hickey et al. 2009

15. CLUSTERING F F + HN