Figure 2.

Induction of Gag ubiquitination by functional L domains. 293T (A) or HeLa (B) cells were transfected with proviral constructs expressing variants of the L domain-independent Z_WT Gag molecule that have either WT p2b^gag, 2× p2b^gag, or Y/G p2b^gag (A) or p6^gag (B) attached to the C terminus. As controls, a proviral construct unable to express Gag (A) and the unmodified Z_WT construct (B) were used. To compare the levels of VLP formation, particulate material released into the medium during metabolic labeling with [³⁵S]methionine was pelleted through sucrose and analyzed directly by SDS/PAGE (Left panels). To detect ubiquitin conjugates, each Gag construct was cotransfected with an expression vector for HA-tagged ubiquitin (HA-Ub), or with the empty vector, and sucrose-purified VLPs were analyzed by immunoblotting with a HA-specific monoclonal antibody (Right panels).