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Materials and Methods
Cell culture
Human pulmonary carcinoma A549 cells (1) and monkey kidney LLC-MK2 (2) cells grew in (1) DMEM
medium supplemented by L-Glutamine, penicillin and spectomycin with 10% of fetal bovine serum or
(2) EMEM medium supplemented by L-Glutamine, penicillin and spectomycin with 5% of fetal bovine
serum.
Virus culture
Virus were
Cell transfection
A549 and LLC-MK2 cells were transfected using the Exgen 500 transfection reagent (Euromedex©).
At least 4µg of plasmid, into a final volume of 100µL NaCl 150mM, were mixed with 100µL of Exgen
mix (10µL Exgen 500 + 90µL NaCL 150mM). The cells were incubated with this solution and 500µL of
their optimum media without SVF.
Structure prediction
The tridimensional model of the GP F hPIV-2 was predicted with Geno3D© (http://geno3Dpbil.ibcp.fr) from the available structure of F PIV5 (RCSB accession number: 2B9B). The theoretical HR
domains were found with the LearnCoil-VMF software (http://groups.csail.mit.edu/cb/learncoilvmf/cgi-bin/vmf.cgi). Structural comparison and the location of mutations on the structure were
made with PyMol. Proteins alignments were done with the ClustalW2 software.
RNA Extraction
Total RNA was extracted from 100µL of hPIV-2 (Greer) infected cells suspension with the Stratagene’s
“Absolutely RNA®” kit, according to the kit instructions. RNA extracts were kept at -80°C before their
use in RT-PCR.
F and HN cDNA synthesis and amplification
The reverse transcription stage of F and HN (from genomic RNA to cDNA) was made using 5µL of
total RNA extract with AMV-RT buffer (Promega®), 1mM of each dNTPs (Eurogentec®), 1µg of
random hexamers (Amersham biosciences®), 10wu of AMV-RT enzyme (Promega®), 20 units of
ribonuclease inhibitor (Recombinant RNAsin®, Promega®) in a final volume of 20µL. The amplification
stage was performed by a PCR. We added 100ng of cDNA to the followed mix: PfuUltra II™ buffer
(Stratagene®), dNTPs 0.4mM each, 100nM of F or HN forward and reverse primers (synthesized by
Eurogentec®), 14wu of PfuUltra II™ enzyme (Stratagene®) in a final volume of 100µL. The reaction
was initialized 2’ at 94°C, followed by 39 cycles of 3 steps 1) denaturation step: 30”, 94°C 2) annealing
step: 1’, 58°C 3) extension step: 2’30”, 72°C. Then a final elongation (5’, 72°C) was performed, and
the solution was hold at 10°C. The primers used had the particularity to add a NotI restriction site at
the 5’ extremity, and a XhoI restriction site at the 3’ extremity (annex).
F and HN cloning
The F cDNA was cloned into the pCDNA3.1(+) (Invitrogen©) plasmid with a blunt-ends strategy (NotIXhoI cohesive-ends strategy failed). The plasmid was linearized by EcoRV (5wu for 500ng of DNA, 1H
37°C – all enzymes are Fermentas© or Euromedex©) and unphosphorylated (with CIAP – 20’ 37°C,
inactivation 10’ 65°C), the insert F, product of amplification, was phosphorylated (with T4-PNK – 20’
37°C, inactivation 10’ 65°C). The insert was ligated to the plasmid (with 16fmol of plasmid ends and
90fmol of insert, PEG6000 and 5wu of T4 DNA ligase – RT° during 2H then 4°C O/N).
The HN cDNA was also cloned into the pCDNA3.1(+) plasmid but with a directional NotI-XhoI
cohesive-ends strategy. The plasmid was linearized with NotI and XhoI (protocol Fermentas ©
DoubleDigest™, 1H30 37°C). The cDNA was first sub-cloned into a pBlueScript KS+ plasmid
(Stratagen©) with a blunt-ends strategy, using the protocol described before for F cDNA (with
52fmol of plasmid ends and 271fmol of insert), then was double-digested as the plasmid
pCDNA3.1(+). The recombinant plasmid was construct by the ligation of 88fmol of HN cDNA ends for
28fmol of pCDNA3.1(+) ends (ratio ~3:1) (with 5wu of T4 DNA ligase – RT° during 2H).
F mutagenesis
See annex for primers and amplification conditions.
The mutagenesis and amplification stage was made by a PCR. We added 30ng of template plasmid to
the followed mix: PfuUltra II™ buffer (Stratagene®), dNTPs 0.4mM each, 125ng of F or HN forward
and reverse primers (synthesized by Eurogentec®), 14wu of PfuUltra II™ enzyme (Stratagene®) in a
final volume of 50µL. The reaction was initialized 5’ at 94°C, followed by 19 cycles of 3 steps 1)
denaturation step: 1’, 94°C 2) annealing step: 1’ (see annex for annealing temperature) 3) extension
step: 8’, 68°C. Then a final elongation (16’, 68°C) was performed, and the solution was hold at 10°C.
Cell-cell fusion assay
The cell-cell fusion was quantified by a luciferase assay, which permit to calculate the luciferase
enzyme production by cells. One population of cell (target) carry the plasmid pLuc (HIV-1 long
terminal repeat (LTR)-luciferase reporter plasmid, gene under the TAT promoter), the other one is
HuH7-Tat cell (bait) which express the transcription factor Tat. When both kinds of cells fusionate,
the TAT transcription factor permit the expression of luciferase.
Target cells (2.5 x 105 cells/well seeded in six-well tissue culture dishes 24 h prior to transfection)
were transfected by at least 2µg of pcDNA3.1 plasmid, which carried mutated or WT version of F
hPIV2 or HN hPIV2 gene/cDNA, and 50ng of pLuc. Twelve hours post-transfection, target cells were
detached with PBS-EDTA 0,25mM, centrifugated (10’, 2500rpm), counted, and reseeded at the same
concentration (105 cells/well) in six-well plates. Bait HuH7-Tat cells (4 x 105 cells per well), detached in
PBS with 1/6 trypsine and washed, were then added to each well. The luciferase activity was
measured 48 and 72 hours later using a luciferase assay kit according to the manufacturer's
instructions (Promega).