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. 2009 Nov 4;84(2):1169–1175. doi: 10.1128/JVI.01372-09

FIG. 1.

FIG. 1.

VP24 mutants with impaired inhibition of IFN-β-induced gene expression. At 24 h posttransfection, 293T cells were mock treated or treated with 1,000 U/ml IFN-β for 16 h. Cells were then lysed, and lysates were assessed for firefly luciferase activity using a dual luciferase assay (Promega). All reporter gene assays were performed in triplicate (A, B, and D). (A) Inhibition of an ISG54 promoter-driven firefly luciferase gene by Z-VP24 and VP24 truncation mutants. 293T cells were transfected with plasmids pRLTK and pISG54FFluc plus either pCAGGS-empty, -HAVP24, -HAVP24(26-251), -HAVP24(51-251), -HAVP24(61-251), or -HAVP24(71-251). (B) Inhibition of an ISG54 promoter-driven firefly luciferase gene by Z-VP24 and VP24 single alanine mutants. 293T cells were transfected with plasmids pRLTK and pISG54FFluc plus either pCAGGS-empty; Z-VP24; or VP24-Q36A, -G47A, -W38A, -K39A, -V40A, -Y41A, -W42A, -G44A, or -I45A. (C) Schematic representation and nomenclature of VP24 mutants. Amino acid 42 and amino acids 142 to 146 of Z-VP24 were replaced with alanine residues to create VP24 mutants mut1 and mut2, respectively. Mutant VP24 proteins containing both mut1 and mut2 substitutions were designated mut3. (D) Inhibition of an ISG54 promoter-driven firefly luciferase gene by Z-VP24 and VP24 mutants. 293T cells were transfected with plasmids pRLTK and pISG54FFluc plus either pCAGGS-empty, -HA-Z-VP24, -HAmut1, -HAmut2, or -HAmut3. Whole-cell extracts were analyzed by Western blotting using antitubulin and anti-hemagglutinin (anti-HA) antibodies (right panel).