FIG. 6.

EMSA results confirmed the AP-1 site as the major KSHV-responsive cis-element in the MMP-1 promoter. (A) KSHV infection induced specific binding of the AP-1 complex to the MMP-1 promoter. Nuclear extracts from either mock-infected HUVEC (lane 2) or HUVEC infected with KSHV for 6 h (lanes 3 to 6) were subjected to EMSA using a ³²P-labeled oligonucleotide probe designed from the region mapped to be responsive to KSHV infection (−64 to −97). The labeled probe alone is shown in lane 1. In the competition assay, a 100× excess amount of unlabeled probe was added to the reaction mixture (lane 4). Addition of 1 μg of antibody to c-Fos (lane 5) but not Ets1 (lane 6) supershifted the AP-1-DNA complex band. (B to D) Mutagenesis analysis of the MMP-1 promoter to identify the major AP-1-DNA complex that was responsive to KSHV infection. Nuclear extracts from either mock-infected HUVEC (lane 2) or HUVEC infected with KSHV for 6 h (lanes 3 and 4) were subjected to EMSA using ³²P-labeled mutant probes with the putative AP-1 site (B), Ets1 site (C), or both AP-1 and Ets1 sites (D) mutated. The labeled probes alone are shown in lane 1. In the competition assay, a 100× excess amount of unlabeled probe was added to the reaction mixtures (lanes 4).