FIG. 6.
Binding of TRIM5αrh B-box 2 mutants to assembled HIV-1 capsids. (A) 293T cells were transfected with plasmids expressing the indicated wild-type and mutant TRIM5αrh proteins tagged with HA epitopes. At 36 h after transfection, the cells were lysed. Serial dilutions of the cleared lysates were made by mixing with cleared lysates from untransfected 293T cells. The lysates were incubated at room temperature for 1 h with HIV-1 CA-NC complexes that had been assembled in vitro. The mixtures were applied to a 70% sucrose cushion and centrifuged. Input represents the mixtures analyzed by Western blotting before being applied to the 70% cushion. The input mixtures were Western blotted for the HA tag. The pellet from the 70% cushion (pellet) was analyzed by Western blotting with antibodies against the HA tag and HIV-1 CA-NC protein. (B) The Western blots shown in panel A were quantitated as described in Materials and Methods. The amounts of TRIM5αrh protein in the input and pellet (bound) fractions are shown in arbitrary units.