Functional characterization of human parainfluenza type 2 hpiv 2 fusion glycoprotein mutants
Download
1 / 15

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


  • 64 Views
  • Uploaded on

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 CNRS FRE 3011. The h PIV2. Mononegavirales Paramyxoviridae

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' Functional characterization of human Parainfluenza type 2 (hPIV-2) fusion glycoprotein mutants' - haru


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
Functional characterization of human parainfluenza type 2 hpiv 2 fusion glycoprotein mutants

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


The h (hPIV-2) fusion glycoprotein mutantsPIV2

  • 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


Membrane Fusion (hPIV-2) fusion glycoprotein mutants

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


Objectives (hPIV-2) fusion glycoprotein mutants

  • 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


Methods (hPIV-2) fusion glycoprotein mutants

  • 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


Choice of mutations (hPIV-2) fusion glycoprotein mutants

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


T96A Mutation (hPIV-2) fusion glycoprotein mutants

A

B

T96A is implicate into the HN promotion of fusion mechanism


PIV-5 (hPIV-2) fusion glycoprotein mutants 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


I24P based Mutations (hPIV-2) fusion glycoprotein mutants

  • I24P combination bring a better fusion effect also without HN

A

  • With K133E big fusogenic characteristic, independent of HN

B


Conclusion (hPIV-2) fusion glycoprotein mutants

  • 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


Further investigations (hPIV-2) fusion glycoprotein mutants

  • 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


Acknowledgments (hPIV-2) fusion glycoprotein mutants

Dr Olivier Terrier

Dr Manuel Rosa-Calatrava

Chrystelle for the “last-minute” cells

All VirPath team for their support


The Membrane Fusion (hPIV-2) fusion glycoprotein mutants

Hickey et al. 2009


CLUSTERING (hPIV-2) fusion glycoprotein mutants

F

F + HN


ad