Figure 6

Interaction of chimeras between HIV-1 and SIVcpz/gor Vpus with ß-TrCP and CD4. (A) Interaction of Vpu with ß-TrCP. 293T cells were transfected with equal amounts of plasmids expressing ß-TrCP N-terminally fused to the C-terminal fragment of click beetle green (CBG) and Vpu C-terminally fused to the N-terminal fragment of CBG. ß-catenin served as positive control. After 48 h, click beetle luciferase activity was determined in living cells by addition of D-luciferin and quantification of bioluminescence. Values represent means (±SEM) derived from five independent experiments. (B) Interaction of Vpu with CD4. 293T cells were transfected with plasmids expressing CD4 C-terminally fused to the C-terminal fragment of CBG and Vpu C-terminally fused to the N-terminal fragment of CBG and interaction efficiencies were determined as described in the methods section. (C) To exclude a bias caused by Vpu-mediated degradation of CD4, a dominant negative mutant of β-TrCP1 isoform 2 lacking the F-box (amino acids 141–193) was co-transfected in a control experiment. This mutant still binds Vpu but lacks the Fbox domain and thus fails to recruit the E3 ubiquitin ligase complex. Values represent means (±SEM) derived from three to four independent experiments.