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

2

Mononegavirales Paramyxoviridae Rubulavirus

Terr

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(200

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•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

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

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o et

al.

(200

2)

•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

•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-SPF

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

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•K133E is very well expressed especially without HN•I294A permit an increased fusion only with HN as T96A

A

B

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

Mutant T96A is involved in a finest regulation of HNThe mutation I24P is involved in an increased “independence” of FMutants K133E and I24P-K133E highlight another residue implicate in the functional interaction between F and HN

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Head

Neck

I24PI294A

T96A

K133E

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 (Boris for the coke, Linda the candy distributor, Matthieu my coffee partner…)

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F

F + HN