Figure 7. Rescue of HIV-1 release correlates with chain type specificity.

(A) Schematic illustration of chimeric ubiquitin ligase constructs. The C lobe of HUWE1 is indicated in yellow. (B to D) Replacement of the C lobe of the ITCH HECT domain by that of HUWE1 greatly impairs the rescue of HIV-1_ΔPTAPP by a NEDD4-2s/ITCH chimera (panel B), but not the overall ubiquitination of VLP-associated Z_WT Gag (panel C) or of virus particle-associated authentic Gag expressed by an HIV-1 provirus lacking protease (panel D). Expression vectors for HECT domain constructs were transfected in varying amounts as indicated to achieve comparable expression levels. (E) The parental N_1-245/ITCH construct, in contrast to the N_1-245/ITCH-HUWE1 HECT domain chimera, induces the attachment of K63-linked chains to Z_WT Gag. 293T cells were transfected with the Z_WT Gag construct and empty vector, or vectors expressing FLAG-tagged versions of the indicated HECT domain constructs. VLP samples were analyzed by Western blotting with anti-CA to detect unmodified and ubiquitinated versions of Z_WT Gag (left panel), and in parallel with Apu3 to detect K63-linked ubiquitin chains conjugated to Z_WT Gag (right panel). The cell lysates were also examined with anti-FLAG.