FIG 3.

The TM domain (amino acids 13 to 34) is critical for the ASFV E66L inhibition of Rluc gene expression. (A) Based on the genomic structure of E66L, we constructed a series of truncated plasmids. The recombinant plasmids were as follows: E66L(1–12), E66L(13–34), E66L(35–50), E66L(1–34), and E66L(13–50). (B) HEK-293 T and IPI-2I cells were cotransfected with pRL-SV40 (0.1 μg) and one of the above plasmids (0.5 μg). After 24 h, the cell lysates were prepared and subjected to luciferase assays. Data are presented as the mean ± SD of the results from three independent experiments; ns, not significant; ***, P < 0.001. (C) The above-described plasmids were transfected into HEK-293T and IPI-2I cells. At 24 h, the cells were fixed and IF was performed with a monoclonal antibody against the HA protein. Original magnification 200× (scale bars 100 μm). (D) EGFP was linked to the HA tag and then E66L(1–12) or E66L(35–50), and the resulting construct was named EGFP-E66L(1–12) or EGFP-E66L(35–50). (E) HEK-293 T cells were cotransfected with pRL-SV40 (0.1 μg) and one of the above plasmids (0.5 μg). After 24 h, the cell lysates were prepared and subjected to luciferase assays. Data are presented as the mean ± SD of the results from three independent experiments; ns, not significant.