FIG 1.

Interaction of proteins with tagged protein 3A from EV-A71-infected cells. (A) Genome organization of EV-A71 and 3A protein. Arrows indicate sites of insertion. Amino acids that encode the FLAG tag are underlined. (B) Cytopathic effect of EV-A71-3A4-FLAG recombinant virus at an MOI of 20 at 12 h postinfection (magnification, 200x). (C) Effects of EV-A71-3A4-FLAG recombinant virus on viral RNA replication and viral protein synthesis. RD cells were infected with EV-A71-3A4-FLAG virus at an MOI of 20 or were mock infected. (i) Cells were collected to detect positive-strand viral RNA or negative-strand viral RNA by slot blot at different times following viral infection. (ii and iii) Cells were collected to detect viral protein levels using Western blotting at various times points after infection. (D) Experimental strategy for the immunoaffinity assay. RD cells were infected with EV-A71-3A4-FLAG recombinant virus at an MOI of 20. Infected-cell lysates were immunoprecipitated with control IgG or anti-FLAG antibodies, washed, and eluted with sample buffer. The sample that contained eluted proteins was then boiled, subjected to 8 to 16% SDS-PAGE, and visualized by silver staining. Protein bands were excised and identified by in-gel trypsin digestion and analyzed by LC-MS/MS. (E) RD cells were infected with EV-A71-3A4-FLAG recombinant virus at an MOI of 20 for 12 h. Immunoaffinity purification via using IgG or anti-FLAG antibody with infected-cell lysates was performed. Protein eluates from the affinity beads were resolved by 8 to 16% SDS-PAGE, and the gel was silver stained. *, light chain; **, heavy chain. (F) From LC-MS/MS data, 1,576 proteins were identified. Following analysis using Proteome Discoverer Daemon 1.4 software, 984 proteins were selected and separated into two groups. The candidate proteins that were associated only with the FLAG group were selected for DAVID analysis, yielding 172 candidates, including GBF1 and ACBD3, which have been reported to be proteins that interact with 3A.