FIG 8.

C-terminal part of GBF1 can compensate for the defects in 3A-GBF1 interaction. (A) The C-terminal part of GBF1 facilitates its recruitment by 3A. Top, scheme of the full-length and 1060 truncated GBF1 constructs and amino acid sequence of 3As from the wt poliovirus and 3A-2 mutant. HeLa cells were cotransfected with plasmids expressing wt 3A or 3A-2 mutant proteins together with plasmids expressing either full-length or 1060 truncated GBF1 constructs. The next day, the cells were fixed and stained for 3A upon mild permeabilization, and the distribution of GBF1 signal in 3A-expressing cells was evaluated. Graph shows quantification of three independent experiments, with at least 200 cells counted for each sample. Statistical significance is indicated. (B) The C-terminal part of GBF1 can functionally compensate for the defect of GBF1-3A interaction in poliovirus replication. Top, scheme of a full-length GBF1 construct with substitution of amino acids 32 to 46 for those derived from a corresponding segment of BIG2; 1060 truncated GBF1 construct with wt N-terminal sequence; and 1060 truncated GBF1 construct with BIG2-derived substitution of amino acids 32 to 46. HeLa cells were transfected with the plasmids expressing indicated GBF1 constructs. The next day, the cells were transfected with a wt poliovirus replicon RNA coding for the Renilla luciferase gene, and incubated in the presence or absence of 1 μg/ml of BFA. Expression of the GBF1 constructs was verified by Western blot.