Primate immunodeficiency viruses target helper T-cells and macrophages/monocytes through binding of the viral envelope glycoprotein to a combination of CD4 and a chemokine receptor (CCR4 or CXCR5) on the surface of the host cells. Strikingly, infection results in rapid and sustained downregulation of CD4 and, to a lesser extent, the chemokine receptors. Downregulation of these viral co-receptors prevents superinfection, promotes virion release and interferes with the immune response, leading to the establishment of a robust infection. CD4 downregulation is so important to the life cycle of human immunodeficiency virus-1 (HIV-1) that two accessory proteins, Nef and Vpu, encoded in the viral genome are devoted to this task. Indeed, Nef and Vpu are critical for the progression from infection to AIDS, a fact that is best illustrated by the existence of long-term non-progressors that are infected with HIV-1 strains bearing inactivating mutations in the genes encoding these proteins. Therefore, pharmacologic or biologic perturbation of Nef and/or Vpu has the potential to prevent the pathogenic effects of HIV-1. To date, however, this potential has not been realized mainly because Nef and Vpu have no enzymatic activity and their mechanisms of action are insufficiently understood. In previous work, we made substantial progress towards elucidating the mechanism of CD4 downregulation by Nef. We found that Nef connects surface CD4 to both the endocytic and lysosomal targeting machineries, leading to efficient and sustained removal of CD4 from the host cells early during infection. The current project focuses on the mechanisms by which Vpu downregulates CD4 at later stages of infection. Vpu is a small transmembrane protein comprising a short luminal domain, a single transmembrane domain (TMD) and a cytosolic domain. The Vpu cytosolic domain simultaneously binds to the CD4 cytosolic tail and the SCF-beta-TrCP E3 ubiquitin ligase complex in the endoplasmic reticulum (ER), causing CD4 ubiquitination on lysine and serine/threonine residues followed by interaction with the VCP-UFD1L-NPL4 dislocase complex and subsequent targeting for degradation by the proteasome. Recognition of CD4 for Vpu-induced degradation involves interactions at the level of both the cytosolic and transmembrane domains of CD4 and Vpu. Transmembrane domain interactions promote targeting for degradation as well as retention of CD4 in the ER. Interference with transmembrane domain interactions reduce CD4 polyubiquitination as well as dislocation from the ER membrane, thus abrogating CD4 degradation in vivo. The multiple levels at which Vpu engages the cellular quality control mechanisms underscore the importance of ensuring profound suppression of CD4 to the life cycle of HIV-1. This past year we succeeded in reconstituting Vpu-induced CD4 ubiquitination in an in vitro system. Also in this system, CD4 and Vpu engaged in transmembrane domain interactions. Disruption of these interactions decreased the extent of CD4 polyubiquitination. Unexpectedly, differential processivity alone could not explain the differential fates of degraded and nondegraded CD4 variants. Instead, continuous deubiquitination was identified as a prerequisite for maximal substrate discrimination. This explains how small differences in substrate-ligase interaction can be amplified into larger differences in polyubiquitination and net degradation. In addition to shedding light on the mechanisms of Vpu-induced CD4 degradation, these results provide a conceptual framework for the general mechanisms of substrate discrimination during membrane protein quality control.

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Ren, Xuefeng; Park, Sang Yoon; Bonifacino, Juan S et al. (2014) How HIV-1 Nef hijacks the AP-2 clathrin adaptor to downregulate CD4. Elife 3:e01754
Amorim, Nathaly A; da Silva, Eulália M L; de Castro, Rodrigo O et al. (2014) Interaction of HIV-1 Nef protein with the host protein Alix promotes lysosomal targeting of CD4 receptor. J Biol Chem 289:27744-56
Zhang, Zai-Rong; Bonifacino, Juan S; Hegde, Ramanujan S (2013) Deubiquitinases Sharpen Substrate Discrimination during Membrane Protein Degradation from the ER. Cell 154:609-22
Cosson, Pierre; Perrin, Jackie; Bonifacino, Juan S (2013) Anchors aweigh: protein localization and transport mediated by transmembrane domains. Trends Cell Biol 23:511-7