Figure 5

Contribution of oligomerization to the binding of MxB to the HIV-1 core. (A) MxB oligomerization assay. Human 293T cells were cotransfected with plasmids expressing wild type MxB-HA and MxB-FLAG proteins. Cells were lysed 24 hours post-transfection and analyzed by Western blotting using anti-HA and anti-FLAG antibodies (Input). Subsequently, lysates were immunoprecipitated by using anti-FLAG agarose beads, as described in Methods. Anti-FLAG agarose beads were eluted using the 3X FLAG peptide. Elutions were analyzed by Western blotting using anti-HA and anti-FLAG antibodies (Immunoprecipitation). WB, Western blot; IP, Immunoprecipitation; WT, wild type. (B) Oligomerization of MxB variants. Human 293T cells were cotransfected with a plasmid expressing wild type MxB-HA and a plasmid either expressing wild type or the indicated mutant MxB-FLAG protein. Similarly, FLAG-tagged proteins were immunoprecipitated, and elutions were analyzed by Western blotting using anti-HA and anti-FLAG antibodies. (C) Oligomerization of the MxB’s leucine zipper domain mutant L661K. Human 293T cells were cotransfected with a plasmid expressing wild type MxB-HA and a plasmid either expressing wild type or the mutant MxB-L661K-FLAG protein. Similarly, FLAG-tagged proteins were immunoprecipitated, and elutions were analyzed by Western blotting using anti-HA and anti-FLAG antibodies. (D) The ability of MxB-L661K-FLAG to bind in vitro assembled HIV-1 CA-NC complexes was measured, as described Methods. (E) Cf2Th cells stably expressing wild type or the indicated mutant MxB protein (upper panel) were challenged with increasing amounts of HIV-1-GFP. Forty-eight hours post-infection the percentage of GFP-positive cells was measured by flow cytometry (lower panel). As a control, Cf2Th cells stably transduced with the empty vector LPCX were challenged with HIV-1. Similar results were obtained in three independent experiments and a representative experiment is shown.