Fig. 6
Sam68 interacts with FMDV 3Cpro and 3Dpol. a Co-immunoprecipitation of FMDV 3Dpol and Sam68 during FMDV infection. BHK-21 cells either mock-infected or infected with FMDV at a MOI of 10 were lysed and the lysates were immunoprecipitated using either anti-FMDV 3Dpol or anti-Sam68 and the eluates examined by Western blot. One lane in each panel (as indicated below) was not subject to IP as a control. Equal amount of isotype control antibody served as an IP control. The eluates from the anti-3Dpol IP reaction were probed with anti-Sam68 (left panel). Conversely, the Sam68 IP eluates were probed with an anti-3Dpol (right panel). In the left panel, lane 1 corresponds to the isotype IP control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP and lane 4 is the anti-3Dpol IP eluate from FMDV-infected cell lysates. Similarly, in the right panel, lane 1 corresponds to the isotype control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP, and lane 4 is the anti-Sam68 IP eluate from FMDV-infected cell lysates. b The fragments (frag) listed in the table correspond to the amino acid (aa) sequence of FMDV 3Dpol, starting from the N-terminus: frag #1 aa 1–48, frag #2 aa 49–108, frag #3 aa 109–157, frag #4 aa 158–217, frag #5 aa 218–268, frag #6 aa 269–331, frag #7 aa 332–404, and frag #8 aa 405–470. A scrambled peptide was used as a negative control. c Computational prediction of the interaction between FMDV 3Dpol and Sam68. (i) Electrostatic surface representation of FMDV 3Dpol in the docking pose (PDB: 1U09); red color depicts the negatively charged surface, white shows the neutral surface, and blue color shows the positively charged surface. Color intensity is proportional to the surface charge. Areas under dashed lines indicate Sam68 binding interface of FMDV 3Dpol. (ii) Electrostatic surface representation of Sam68 in the docking pose. Surface charge and color annotation are same as section (i). Surface marked with dashed lines indicates FMDV 3Dpol binding interface of Sam68. (iii) Electrostatic representation of Sam68 docked to FMDV 3Dpol. [98] FMDV 3Dpol green docked on Sam68 blue in cartoon representation. The 3Dpol frag-4 residues 193–217 (orange), frag-5 residues 221, 222, 225, 226 (magenta) and frag-8 residues 453–470 (red) form the Sam68 binding interface of 3Dpol (d) LFBK cells were uninfected or infected with FMDV at a MOI of 10, and cells were harvested at 1, 3, and 5 hpi by treatment with versine. Left panel: cell lysates were IP with mouse monoclonal anti-FMDV 3Cpro, and examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Right panel: collected cells were lysed and separated into nuclear and cytoplasmic fractions, and the cytoplasmic fractions were examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Loading control is indicated confirming equivalent loading per lane