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. 2015 Mar 27;11(3):e1004750. doi: 10.1371/journal.ppat.1004750

Fig 6. NS5 with mutation on residue 19 (M19A) showed reduced binding ability with MTP.

Fig 6

(A) Western blot analysis of indicated proteins in HEK293T cells transfected with the plasmids expressing wild type (WT)-, mutated-NS5-Flag or vector control for 24 h after immunoprecipitation with anti-Flag affinity gel. Band densities were quantified by use of MetaMorph (Molecular Devices). (B and F) HEK293T cells transfected with NS5-Flag or NS5-M19A-Flag (B) or infected with wild-type JEV (JEV-WT) or JEV-NS5-M19A (MOI = 5) (F) for 24 h underwent Qproteome Mitochondria Isolation. Western blot analysis of indicated proteins in mitochondrial and cytosolic fractions. C, cytosolic fraction; H, heavy membrane fraction/crude mitochondrial fraction. (C and D) JEV-NS5-M19A mutant virus was generated by using a JEV infectious clone. (C) Plaque morphology of wild type JEV (JEV-WT) and JEV-NS5-M19A mutant in BHK-21 cells. (D) A549 cells were infected with JEV-WT or JEV-NS5-M19A (MOI = 0.1) for the indicated times. Western blot analysis of protein levels of NS3 and actin. Plaque-forming assay of virus titration in culture supernatants (n = 3). Data are mean±SD. (E) IP analysis with V5 or HA affinity gel and Western blot analysis with the indicated antibodies in HEK293T cells adsorbed with JEV for 1 h, then transfected with HADHα-V5-His or HADHβ-HA for 24 h.