107 CHARACTERIZATION OF THE MECHANISM UNDERLYING Vpu-MEDIATED CD4-DEGRADATION J Binette, ÉA Cohen Objectives: HIV-1 induces the down-regulation of CD4 to produce fully infectious virions. Vpu contributes to this process by inducing CD4 degradation in the ER by a mechanism that is reminiscent of ER-associated protein degradation (ERAD). Vpu connects CD4 to the ubiquitin-proteasome system via an interaction with beta-TrCP, the specificity component of an ubiquitin ligase complex (SCFbeta-TrCP). Our objective is to analyse in detail the mechanism underlying Vpu-mediated CD4 degradation.
Laboratory of Human Retrovirology, IRCM, Montréal, Québec
Methods: CD4 ubiquitination was assessed by expressing Vpu, CD4 and myc-tagged ubiquitin in 293T cells. CD4-ubiquitin conjugates were revealed by western-blotting using anti-myc antibodies. CD4 dislocation was done by cell fractionation. CD4 stability was measured by pulse-chase labelling and immunoprecipitation.
Results: We showed directly that CD4 is poly-ubiquitinated and undergoes dislocation from the ER membrane to the cytoplasm to be degraded. Mutations of lysine residues in the cytosolic tail of CD4 did not prevent CD4 degradation, suggesting that this process might involve dislocation of lysine-containing lumenal parts of CD4 across the ER membrane. A Vpu mutant (S52,56/D) unable to interact with beta-TrCP and to induce CD4 degradation increased the half-life of wt CD4 (12 hrs vs 7 hrs without Vpu), while it seemed to not stabilize CD4 mutants unable to interact with Vpu, suggesting that CD4-Vpu interaction might protect CD4 from basal degradation.
Conclusions: Our data provide evidence supporting a model whereby Vpu-mediated CD4 degradation involves dislocation of CD4 and poly-ubiquitination by SCFbeta-TrCP. The recruitment by Vpu of an ubiquitin ligase distinct from those used in ERAD combined with the stabilization of CD4 by Vpu S52,56/D suggests that Vpu might target CD4 to a dislocation complex where ERAD-specific ubiquitination components are not available. The outcome of these studies will not only shed light on processes involved in HIV-1 Vpu-mediated CD4 degradation but will also enhance our understanding of the ERAD pathway.