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. 2005 Jan 3;168(1):89–101. doi: 10.1083/jcb.200408155

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Copyright © 2005, The Rockefeller University Press

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Figure 6. — HECT ubiquitin ligases mediate PPXY-dependent viral budding. (A) Restoration of infectious MLV release from 293T cells transfected with MLV proviral DNA and an inhibitory YFP-WWP1_WW expression plasmids. “Rescuing” plasmids encoded YFP-WWP1_WW-HECT or YFP-WWP1_{WW-HECT(C890S)} as indicated. Infectious virus release, measured using HeLa P4R5 indicator cells, is plotted as a proportion of that obtained in the absence of inhibitory YFP-WWP1_WW protein expression. Error bars indicate the SD from the mean of triplicate measurements. (B) Western blot analysis using αGFP antibodies showing expression of the inhibitory and “rescuing” YFP-fusion proteins in cell lysates. (C) Western blot analysis of cell and virion lysates using αMLV Gag antibodies upon coexpression of MLV with the inhibitory YFP-WWP1_WW and “rescuing” YFP-WWP1_WW-HECT fusion proteins. In this experiment, both the wild-type (left) and the chimeric MLV/ENaC (right) proviral plasmids were used. (D) Restoration of Gag processing and virion release by a budding-attenuated MLV mutant (DAPPYR) upon overexpression of the indicated YFP-fusion proteins (left). Wild-type MLV was used as a control (right). Cell and virion lysates were analyzed by Western blotting with αMLV Gag antibodies. The bottom left panel shows Western blot analysis of YFP-fusion protein expression in cell lysates. (E) Rescue of infectious DAPPYR mutant MLV release by WWP1 overexpression. 293T cells were transfected as in D and infectious virus release was measured using HeLa P4R5 indicator cells as in Fig. 1. Error bars indicate the SD from the mean of triplicate measurements.