FIG. 4.

Effects of WWP1 mutants C890S and C2WW on MA ubiquitination and VLP release. 293T cells were transfected with 100 ng of pCMVHT-1ΔX and the indicated amounts of pUCHR empty vector (−), pUCHRWWP1 (WT), pUCHRWWP1-C890S, or pUCHRWWP1-C2WW. Cells and cell culture supernatants (concentrated by ultracentrifugation through a 20% glycerol cushion) were lysed in radioimmunoprecipitation assay buffer, and equal amounts of extracts were size fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblots were probed with monoclonal anti-HTLV-1 p19(MA) (upper panel) and rabbit anti-Nedd4 antibodies (lower panel), followed by IRDye680-labeled antimouse and IRDye 800-labeled antirabbit antibodies. Molecular size standards (kDa) are shown on the left-hand side. The numbers under the supernatant part of the anti-MA blot indicate the fraction of the total MA (cell extract plus supernatant) released into the supernatant, derived by quantitative analysis of immunoblots.