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Minimal viral partners and M1 basic residues essential for influenza A(H1N1)pdm09 M1 membrane localization.

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posted on 2016-11-04, 17:33 authored by Adeline Kerviel, Shantoshini Dash, Olivier Moncorgé, Baptiste Panthu, Jan Prchal, Didier Décimo, Théophile Ohlmann, Bruno Lina, Cyril Favard, Etienne Decroly, Michèle Ottmann, Philippe Roingeard, Delphine Muriaux

HEK 293T cells were transfected with empty vector (mock) or with the pcDNA-M1 (M1 WT or mutant), pcDNA-M2 (M2), pHW2000-NS (NS1/NEP) or pHW2000-M (M) plasmids, as indicated. Cell fractionation experiments were performed 48h post-transfection. (A) Percentage of M1 detected in the nuclear, cell membrane or cytosolic fraction in each condition. M2-mut2 was used as control for low M1 membrane binding. The histograms show the result of at least three independent experiments (mean± standard deviation represented in the error bars). Differences between conditions were assessed using the Student’s t-test. (B) Cell fractionation controls. Fractions of cells co-expressing M1+M2+NS1/NEP were immunoblotted with antibodies against a membrane marker (LAMP2) and a cytosolic marker (the ribosomal S6 protein). Tubulin was used as loading control. PNS, Post-Nuclear Supernatant. (C) Expression of the indicated influenza A(H1N1)pdm09 viral proteins was checked after transfection in HEK 293T cells of the relevant plasmids by western blotting with anti-M1 (H1N1), anti-NEP and anti-NS1 antibodies. HA was detected with a serum obtained using an influenza A(H1N1)pdm09 strain isolated from a vaccinated patient.