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

mutants

JEAN-CHRISTOPHE LE BAYONMASTER 2 GBC – 18 JUIN 2009UCBL

DR MANUEL ROSA-CALATRAVADR OLIVIER TERRIER

PR BRUNO LINA

VIRPATHCNRS FRE 3011

2

•Parainfluenza virus type 2•The outer membrane fusion•Objectives and methods

Introduction

•Choice of mutations•Functional characterization

Results

•Discussion and conclusion•Further investigations

Conclusion

3

The hPIV2Mononegavirales Paramyxoviridae Rubulavirus

Terr

ier e

t al.

(200

8)

•Virus responsible of Respiratory diseases•Enveloped virus Ø 200nm •Simple stranded RNA / negative polarity

•Entry into the cell possible with glycoproteins F and HN

MEMBRANE FUSION

F HN

Viral envelope

Cellular membrane

F1

HN

S

S

F2

C N

NC

Fusion peptide

Cytoplasmic tail

Extracellular domain

Transmembrane region

HR1

HR2

F

Taki

mot

o et

al.

(200

2)

5

OBJECTIVES

•Terrier et al. (2008): hPIV-2 variants capable of an increased cell-cell fusion which carried the T96A mutation•Ito et al. (1998, 2009): when presenting L22P, K132E and 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

•Bioinformatics and annotation of F hPIV2•Cloning F and HN genes and directed mutagenesis•Flow cytometry and cell-cell luciferase fusion assay

METHODS

PTAT

HIV-1LTR

Luc

TAT

Luc

+ substrate (luciferine)

LucLuc

LucLuc

activate

Luc

TATFusion

P : constitutive promoterTAT : transcriptional activatorpLUC : plasmid which carry :- LTR : TAT-dependant promoter- Luc : Fireflly luciferase gene

HuH7-TAT cell

pLUC transfected A549 cell

CHOICE OF MUTATIONS

7

HR2

N

F1F2

HR1

TM

S-SPF

C

SCI24P T96A I294AK133E

F hPIV-2 variantF hPIV-2 GreerF PIV-5 VR-288

16 24 34 TGIVGSDAIAGDQLLNVGV TGIVGSDAIAGDQLLNIGV LAGAGSLDLAALMQIGVIP14 22 32

86 96 106 PLIENLSKISAVTDTKPRRER PLIENLSKISTVTDTKTRQER

124 133 142 TITAAVAIVKANANAAAIN QITAAVAIVKANANAAAIN QVTAAVALVKANKNAAAIL 120 129 132 138

284 294 305 MQPGAKVIDLIAISANHKLQEV MQPGAKVIDLIAISANHKLQEV VQPATQIIDLVTISAFINNQEV 280 290 301

F hPIV-2 variantF hPIV-2 GreerF PIV-5 VR-288

T96A MUTATION

WT 96 T-0%

50%

100%

150%

200%

250%

300%

350%

100%

29%

8%

146%

330%

8%

FF+HN

Rela

tive

Luci

fera

se A

ctivi

ty

WT 96 T-0%

50%

100%

150%

200%

250%

300%

100%

246%

0%

143%

231%

0%

Rela

tive

F ex

pres

sion

A

B

T96A is implicate into the HN promotion of fusion mechanism

9

PIV-5 TRANSPOSED MUTATIONS

WT 24 133 294 T-0%

200%

400%

600%

800%

1000%

1200%

1400%

100%

83%

1153

%

384%

0%

143%

430%

626%

100%

0%

Rela

tive

F ex

pres

sion

A

B

WT 24 133 294 T-0%

200%

400%

600%

800%

1000%

1200%

1400%

1600%

100%

743%

561%

30%

8%

146%

1467

%

237%

1187

%

8%

FF+HN

Rela

tive

Luci

fera

se A

ctivi

ty

•Increased potential of fusion for I24P with or without HN

•I294A permit an increased fusion only with HN as T96A

•K133E is very well expressed especially without HN

I24P BASED MUTATIONSA

B

•I24P combination bring a better fusion effect also without HN•With K133E big fusogenic characteristic, independent of HN

WT 24 24-96

24-133

24-294

T-0%

1000%

2000%

3000%

4000%

5000%

6000%

7000%

8000%

9000%

10000%

100%

743%

513%

7941

%

476%

8%146%

1467

%

1465

%

1454

%

167%

8%

FF+HN

Reati

ve L

ucife

rase

Acti

vity

WT 24 24-96 24-133 24-294 T-0%

50%

100%

150%

200%

250%

300%

350%

400%

450%

500%

100%

83%

418%

39%

160%

0%

143%

430%

430%

55%

109%

0%

Rela

tive

F ex

pres

sion

11

Mutant T96A is involved in a finest up-regulation of F by HNThe mutation I24P is involved in an increased “independence” of FMutants K133E and I24P-K133E may highlight another functional interaction between F and HN (down regulation)I294A seems to have the same effect as T96ASeveral parts of F seems to be involved in the F-HN interaction

CONCLUSIONHead

Neck

I24PI294A

T96A

K133E

12

FURTHER INVESTIGATIONSQuantification of GP by Western-BlotFinest membrane fusion quantificationDesign an HN not able to promote the F GPBehavior of a F mutant on a virus or a VLP (and mutant HN)Evaluation of new clinical variants

Techniques:Western-Blot/CoIPReal-time lipid-mix assayVirus-like particles with F and HN/Reverse genetics

Design new molecules against the hPIV-2 entry into the cells

ACKNOWLEDGMENTS

Dr Olivier TerrierDr Manuel Rosa-CalatravaChrystelle for the “last-minute” cells

All VirPath team for their support

THE MEMBRANE FUSION

Hick

ey e

t al.

2009

15

CLUSTERINGF

F + HN