Characterization of mutants of the human Parainfluenza type 2 (hPIV-2) F glycoprotein
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Characterization of mutants of the human Parainfluenza type
2 (hPIV-2) F glycoprotein
JEAN-CHRISTOPHE LE BAYONMASTER 2 GBC – 18 JUIN 2009UCBL
DR MANUEL ROSA-CALATRAVA
DR OLIVIER TERRIERPR BRUNO LINA
VIRPATHCNRS FRE 3011
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Mononegavirales Paramyxoviridae Rubulavirus
Terr
ier e
t al.
(200
8)
•Virus responsible of Respiratory Respiratory diseasesdiseases•Enveloped virus Enveloped virus Ø 200nm •Simple stranded RNA / negative polarity
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•Entry into the cell possible with glycoproteins F and HNF and HN
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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)
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•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 / hPIV2PIV5 / hPIV2 are very closed viruses
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We created mutants of the F GP hPIV2 which carried the We created mutants of the F GP hPIV2 which carried the mutations T96A and mutations transposed from PIV-5 in mutations T96A and mutations transposed from PIV-5 in order to characterize their possible interaction on HN-F order to characterize their possible interaction on HN-F
activation mechanismactivation mechanism
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•Bioinformatics Bioinformatics and annotation of F hPIV2•Cloning Cloning F and HN genes and directed mutagenesismutagenesis•Flow cytometry Flow cytometry and cell-cell luciferase fusion assayfusion assay
PTAT
HIV-1LTR
Luc
TAT
Luc
+ substrate (luciferine)Luc
Luc
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
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HR2
N
F1F2
HR1
TM
S-S
PF
C
SCI24P T96A I294AK133E
F hPIV-2 variantF hPIV-2 variantF hPIV-2 GreerF hPIV-2 GreerF PIV-5 VR-288F PIV-5 VR-288
16 16 2424 34 34 TGIVTGIVGSGSDADAIIAAGDQLGDQLLLNVGVNVGV TGIVTGIVGSGSDADAIIAAGDQLGDQLLLNIGVNIGV LAGALAGAGSGSLDLDLLAAALMQALMQIIGVIPGVIP14 14 2222 32 32
86 86 9696 106 106 PLIENLSKIS PLIENLSKISAAVTDTKVTDTKPPRRRRER ER PLIENLSKIS PLIENLSKISTTVTDTKVTDTKTTRRQQERER
124 124 133 133 142142 TITITAAVAIVTAAVAIVKKANANAANAAAINAAAINN QQIITAAVAIVTAAVAIVKKANANAANAAAINAAAINN QQVVTAAVALVTAAVALVKKANANKKNAAAINAAAILL 120 120 129 129 132132 138 138
284 284 294294 305 305 MMQPQPGAKVGAKVIDLIDLIIAAISAISANHKLNHKLQEV QEV MMQPQPGAKVGAKVIDLIDLIIAAISAISANHKLNHKLQEV QEV VVQPQPATQIATQIIDLIDLVVTTISAISAFINNFINNQEV QEV 280 280 290290 301 301
F hPIV-2 variantF hPIV-2 variantF hPIV-2 GreerF hPIV-2 GreerF PIV-5 VR-288F PIV-5 VR-288
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A
B
T96A is implicate into the HN promotion of fusion mechanism
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A
B
•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
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A
B
•I24P combination bring a better fusion effect also without HN•With K133E big fusogenic characteristic, independent of HN
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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 are involved in the F-HN interaction
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Head
Neck
I24PI294A
T96A
K133E
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Quantification 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
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Dr Olivier TerrierDr Manuel Rosa-CalatravaChrystelle for the “last-minute” cells
All VirPath team for their support
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Hic
key
et a
l. 20
09
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F
F + HN