CD4 GLYCOPROTEIN DEGRADATION INDUCED BY HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 VPU PROTEIN REQUIRES THE FUNCTION OF PROTEASOMES AND THE UBIQUITIN-CONJUGATING PATHWAY

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a typ e I anchored integral membrane phosphoprotein with two independent fun ctions, First, it regulates virus release from a post-endoplasmic reti culum (ER) compartment by an ion channel activity mediated by its tran smembrane anchor, Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasm ic tail, In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked br peptide aldehydes that ac t as competitive inhibitors of proteasome function and also by lactacy stin, which blocks proteasome activity by covalently binding to the ca talytic beta subunits of proteasomes. The sensitivity of Vpu-induced C D4 degradation to proteasome inhibitors paralleled the inhibition of p roteasome degradation of a model ubiquitinated substrate, Characteriza tion of CD4-associated oligosaccharides indicated that CD-l rescued fr om Vpu-induced degradation by proteasome inhibitors is exported from t he ER to the Golgi complex, This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal d egradation, CD4 degradation mediated by Vpu does not require the ER ch aperone calnexin and is dependent on an intact ubiquitin-conjugating s ystem, This was demonstrated by inhibition of CD I degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activat ing enzyme E-1 or (ii) following expression of a mutant form of ubiqui tin (Lys(48) mutated to Arg(48)) known to compromise ubiquitin targeti ng by interfering with the formation of polyubiquitin complexes, CD4 d egradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation, The results c learly demonstrate a role for the cytosolic ubiquitin-proteasome pathw ay in the process of Vpu-induced CD4 degradation, In contrast to other viral proteins (human cytomegalovirus US2 and US11), hoc-ever, whose translocation of host ER molecules into the cytosol occurs in the pres ence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.