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