Analysis of MAVS-TRAF3 interactions and regulation by HCV NS3/4A in HEK293 cells.

<p>(<b>A</b>) HAVI-Fluc-MAVS was determined to be associated with Rluc-TRAF3 using the DLR-PD assay. HEK293 cells were co-transfected with the indicated vectors. The percentage of Rluc on the beads compared with the cell lysate was calculated by dividing the Rluc activity of Rluc-TRAF3 measured on the beads by the Rluc activity in the same amount of lysate used in the pull-down assay. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (<b>B</b>) Using the DLR-PD assay, the HAVI-Fluc-MAVS association with Rluc-TRAF3 was determined to be inhibited by HCV NS3/4A in a dose-dependent manner in HEK293 cells. Cells were co-transfected with HAVI-Fluc-MAVS (100 ng), Rluc-TRAF3 (100 ng) and DNA (100 ng) containing the NS3/4A vector (0, 10, 50 or 100 ng) and control plasmid pCI neo. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads. (<b>C</b>) Biotinylated protein pull-down assay to determine the MAVS-TRAF3 interactions and regulation by HCV NS3/4A in HEK293 cells. (<b>D</b>) Overexpression of HCV NS3/4A inhibited the MAVS-induced activation of the IFNβ-Luc promoter after normalization with RL-TK in HEK293 cells. The Fluc activity from different cell lysates was divided by that of cell lysates from mock transfected cells without MAVS activation.