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* Department of Medicine and
Cancer Institute, New York University School of Medicine, New York, NY 10016; and
Department of Pharmaceutics, Rutgers University, School of Pharmacy, Piscataway, NJ 08854
Nef is a HIV-1 accessory protein critical for the replication of the virus and the development of AIDS. The major pathological activity of Nef is the down-regulation of CD4, the primary receptor of HIV-1 infection. The mechanism underlying Nef-mediated CD4 endocytosis and degradation remains incompletely understood. Since protein ubiquitination is the predominant sorting signal in receptor endocytosis, we investigated whether Nef is ubiquitinated. The in vivo ubiquitination assay showed that both HIV-1 and SIV Nef proteins expressed in Jurkat T cells and 293T cells were multiple ubiquitinated by ubiquitin-His. The lysine-free HIV-1 Nef mutant (
10K) generated by replacing all 10 lysines with arginines was not ubiquitinated and the major ubiquitin-His attachment sites in HIV-1 Nef were determined to be lysine 144 (di-ubiquitinated) and lysine 204 (mono-ubiquitinated). Lysine-free HIV-1 Nef was completely inactive in Nef-mediated CD4 down-regulation, so was the Nef mutant with a single arginine substitution at K144 but not at K204. A mutant HIV-1 provirion NL4–3 with a single arginine substitution in Nef at K144 was also inactive in Nef-mediated CD4 down-regulation. Lysine-free Nef mutant reintroduced with lysine 144 (K10 + K144) was shown active in CD4 down-regulation. These data suggest that ubiquitination of Nef, particularly diubiquitination of the lysine 144, is necessary for Nef-mediated CD4 down-regulation.
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1 This work was supported by New York University Cancer Institute and partially supported by grants from the Association of International Cancer Research (02-265; to Y.J.J.) and from a National Institutes of Health Center for AIDS Research pilot grant to New York University and also partially supported by National Institutes of Health Grant AI 51214 (to X.Z.).
2 Address correspondence and reprint requests to Dr. Yong-Jiu Jin at the current address: Department of Oncological Sciences, Mt. Sinai School of Medicine, 1425 Madison Avenue, Room 1576, New York, NY 10029. E-mail address: Yong-Jiu.Jin{at}mssm.edu
3 Abbreviations used in this paper: Ub, ubiquitin; wt, wild type.
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