This project is aimed at elucidating the mechanisms by which the Nef and Vpu proteins of human immunodeficiency virus 1 (HIV-1) downregulate CD4 in the viral host cells, T-lymphocytes and macrophages. HIV-1 Nef is a 27-kDa myristoylated accessory protein that is produced at high levels early during infection. Nef is an important determinant of pathogenicity, as demonstrated by the finding that some long-term non-progressors (i.e., untreated infected persons who do not develop symptoms of AIDS for 10 years or longer) carry an HIV-1 strain with inactivating mutations of the Nef gene. An understanding of Nef function could thus provide new avenues for therapeutic intervention. Nef has many effects in the infected cells, the best characterized of which is the downregulation of CD4. Together with chemokine receptors, CD4 serves as a co-receptor for HIV-1 entry into cells. The downregulation of CD4 by Nef is thought to prevent superinfection and increase the release of infectious particles, thus explaining the higher virulence of Nef-carrying HIV-1 strains. In previous work, we demonstrated that the downregulation of CD4 by HIV-1 Nef requires clathrin and the heterotetrameric, clathrin-associated adaptor protein-2 (AP-2) complex in the host cells. In addition, we found that this requirement stemmed from a direct interaction of Nef with AP-2 involving a dileucine and a diacidic motif in Nef and the alpha and sigma2 subunits of AP-2. Through mutational analyses, we mapped the binding site for both Nef motifs on alpha and sigma2. Since clathrin and AP-2 mediate endocytosis from the plasma membrane, these findings indicated that Nef downregulates CD4 by accelerating its internalization form the surface of host cells. In addition, we demonstrated that Nef has a second function: targeting internalized CD4 to the multivesicular body (MVB) pathway for eventual degradation in lysosomes. We showed that this targeting depends on ESCRT complexes but, surprisingly, not on ubiquitination of lysine residues in either CD4 or Nef. At later stages of infection, the HIV-1 genome directs expression of a second protein, Vpu, which downregulates CD4 from the endoplasmic reticulum (ER). Vpu is a type I integral membrane protein that inserts into the membrane of the endoplasmic reticulum (ER) and targets newly-synthesized CD4 for degradation by cytosolic proteasomes. This targeting was thought to be distinct from the canonical ER-associated degradation (ERAD) pathway. However, in collaboration with Klaus Strebel (NIAID) and Yihong Ye (NIDDK), we recently demonstrated that CD4 downregulation by Vpu involves at least part of the ERAD machinery. In particular, we found that the p97-Npl4-Ufd1 ERAD dislocase is required for Vpu-induced CD4 targeting for proteasomal degradation. We also found that targeting involves CD4 ubiquitination dependent on not only lysine but also serine and threonine residues. Finally, we demonstrated that Vpu also induces CD4 retention in the ER largely by virtue of transmembrane domain interactions. The multiple levels at which Vpu engages these cellular quality control mechanisms underscore the importance of ensuring profound suppression of CD4 to the life cycle of HIV-1. Finally, we explored how common non-lysine-dependent ubiquitination is as a signal for ERAD. To this end, we examined the degradation of a prototypical ERAD substrate, the alpha subunit of the T-cell antigen receptor complex (TCR-alpha). TCR-alpha is a type I integral membrane protein that becomes ubiquitinated and targeted to ERAD when it fails to assemble into the TCR complex. Remarkably, TCR-alpha has a cytosolic tail of only five amino acid residues (i.e., RLWSS), none of which is the canonical ubiquitin-acceptor lysine. We found that substitution of two conserved serine residues in the cytosolic tail of TCR-alpha to alanine decreased ubiquitination, whereas placement of additional serine residues enhanced it. Moreover, replacement of the cytosolic serine residues by other ubiquitinatable residues (i.e., cysteine, threonine, or lysine) allowed ubiquitination to take place. Serine-dependent ubiquitination perfectly correlated with targeting of TCR-alpha for ERAD. In collaboration with Allan Weissman (NCI), we found that this ubiquitination was mediated by the ER-localized ubiquitin ligase, HRD1. These findings indicated that serine-dependent, HRD1-mediated ubiquitination targets TCR-alpha to the ERAD pathway. Therefore, Vpu does not induce a viral-specific modification but exploits an endogenous machinery for serine-dependent ubiquitination in order to downregulate CD4.