Fig 2. CVB3 VP1 may directly interact with MAT1.

(A) Plasmids pGBKT7-VP1 and pGADT7-MAT1 were co-transformed with yeast strain AH109, and the positive result selected on high-stringency medium (SD/-Ade/-His/-Leu/-Trp/X-α-Gal). Interaction is reflected by blue color, while white colonies suggest no interaction. (B) the VP1-MAT1 interaction identified by transcription-translation system in vitro. Plasmids HA-MAT1 and c-Myc-VP1 were labeled with ³⁵S-Methionine, and immunoprecipitated with antibody against c-Myc; the binding proteins with ³⁵S-Methionine were analyzed by 10% SDS-PAGE and autoradiography. (C) Co-immunoprecipitation detected the interaction of VP1 and MAT1 in CVB3 infected cells. At 3 hours post infection, the cells lysates of mock and CVB3 infected cells were incubated with or without monoclonal antibody against MAT1 or irrelevant IgG affinity gel, and analyzed by western blotting with the specific antibodies after immunoprecipitation. Original blots are shown in S16 Fig for statistical analysis. (D) MAT1 and VP1 intracellular localization in CVB3 infected HPAC cells. Representative confocal immunofluorescence microscopic images of MAT1 and VP1 stained with rabbit anti-MAT1 (green) and mouse anti-VP1 antibodies (red), respectively, the MAT1 and VP1 images were also merged; the nuclei are labeled with DAPI. Scale bar = 10 μm. Values are shown as the mean ± SEM of three independent experiments. (*P<0.05, **P<0.01).