Figure 7

DQBS inhibits Nef-mediated downregulation of MHC-I by preventing assembly of the SFK-ZAP-70-PI3K complex. A) MHC-I downregulation. H9 cells were infected with a recombinant vaccinia virus carrying Nef-Flag or wild-type vaccinia as a control. Cells were then treated with DQBS at concentrations of 1 μM or 10 μM for 4 h. The cells were then fixed and processed for flow cytometry using the anti-MHC-I antibody, W6/32. B) Nef-SFK-PI3K co-precipitation assay. H9 cells were infected with an Hck vaccinia virus either alone or together with the Nef-Flag virus, followed by treatment with 10 μM DQBS for 4 h prior to harvest. Immunoprecipitates were prepared with the M2 anti-Flag antibody, and associated Hck and p85 were detected by immunoblotting. Control blots with the cell lysates for p85, Hck and Nef are shown on the right. C) Zap-70 kinase activation assay. H9 cells were infected with a Zap-70 vaccinia virus either alone or together with the Nef-Flag virus, followed by treatment with 10 μM DQBS for 4 h prior to harvest. Levels of activated Zap-70 were analyzed by immunoblotting with a phosphospecific antibody for the activation loop phosphotyrosine residue (pZap-70). Control blots for Zap-70 levels, Nef and actin are also shown. D) DQBS does not directly inhibit Hck or Zap-70 kinase activity in vitro. Kinase assays were performed with recombinant purified Hck-YEEI and Zap-70 in the absence or presence of the DQBS concentrations indicated using the Z’Lyte method as described elsewhere [40, 45]. As inhibitor controls in the kinase assay, we observed potent inhibition of Hck by the pan-SFK/Abl inhibitor dasatinib [46] and of Zap-70 by the Syk/Zap-70 inhibitor, BAY 61–3606 [47] (data not shown).