FIG. 3.

MxA(L612K) inhibits reporter gene expression in a THOV minireplicon system. COS-1 cells were transfected with T7 promoter constructs coding for the components of a THOV minireplicon system as previously described (29). In this system, synthesis of CAT protein reflects the activity of the reconstituted vRNPs (30). Wild-type or mutant MxA was expressed under the control of the T7 promoter using increasing amounts of expression plasmid pBS-T7/MxA, pBS-T7/MxA(L612K), or pBS-T7/MxA(T103A). (A) CAT protein concentration as determined by a colorimetric immunoassay (Boehringer Mannheim). The amounts of CAT and luciferase activity were determined in the cell lysates. Luciferase activity was used to normalize CAT expression, and the ratio of CAT protein concentration to luciferase activity (CAT/Luc ratio) was calculated as described previously (29). The CAT/luciferase ratio of experiments without MxA were set at 1. (B) Expression of MxA proteins. Aliquots of the cell lysates (15 μg of protein per lane) were analyzed by Western blotting using a polyclonal antiserum directed against MxA.