Nef is a myristoylated protein of 27 to 35 kDa that is conserved in primate lentiviruses. In vivo, Nef is required for high viral load and full pathological effects. In vitro, Nef has at least four activities: induction of CD4 and major histocompatibility complex (MHC) class I downregulation, enhancement of viral infectivity, and alteration of T-cell activation pathways. We previously reported that the Nef protein from human immunodeficiency virus type 1 interacts with a novel human thioesterase (hTE). In the present study, by mutational analysis, we identified a region of the Nef core, extending from the residues D108 to W124, that is involved both in Nef-hTE interaction and in Nef-induced CD4 downregulation. This region of Nef is located on the oligomer interface and is in close proximity to the putative CD4 binding site. One of the mutants carrying a mutation in this region, targeted to the conserved residue D123, was also found to be defective in two other functions of Nef, MHC class I downmodulation and enhancement of viral infectivity. Furthermore, mutation of this residue affected the ability of Nef to form dimers, suggesting that the oligomerization of Nef may be critical for its multiple functions.
The peroxisomal membrane is equipped with four ATP-binding cassette (ABC) 1 transporters, which include the adrenoleukodystrophy protein (ALDP) (1), the adrenoleukodystrophyrelated protein (ALDRP) (2), the 70-kDa peroxisomal membrane protein (PMP70) (3), and the PMP70-related protein (4, 5). Typical mammalian ABC transporters, like the multidrug-resistant P-glycoprotein, are single functional proteins with two related halves comprised of one hydrophobic transmembrane domain and one hydrophilic nucleotide-binding fold (NBF) (6). In contrast, the peroxisomal ABC transporters, as well as TAP1/TAP2 (7) and ABC7 (8), which are respectively located within the endoplasmic reticulum or mitochondria, are half-transporters with only one hydrophobic domain and one NBF.X-linked adrenoleukodystrophy (X-ALD) is the only genetic disease known to result from a peroxisomal ABC transporter gene defect. This neurodegenerative disorder is characterized by progressive demyelination within the central nervous system, adrenal insufficiency, and accumulation of very long-chain fatty acids because of an impaired peroxisomal -oxidation (9 -12). Although it is firmly established that the loss of ALDP function is responsible for the abnormality in VLCFA metabolism, its precise role is unknown. Similarly, no precise function has been assigned to ALDRP, PMP70, or PMP70-related proteins. ALDRP, PMP70, and PMP70-related protein present 66, 38, and 27% amino acid identity with ALDP, respectively, suggesting functional similarity of these four transporters. These proteins display specific but sometimes overlapping patterns of expression in different cell types (13-15). Because it is likely that half-transporters need to dimerize to exert their function (6), this raises the possibility that different types of peroxisomal ABC dimers could allow the import of distinct substrates. Few ABC transporters are known to dimerize. Genetic evidence suggests that the bacterial hemolysin transporter B ABC half-transporter forms homodimers (16), whereas the Drosophila white, brown, and scarlet gene products form heterodimers (17). Heterodimerization of ABC transporters has also been reported for the transporters of antigenic peptides, TAP1 and TAP2 (18), and the two yeast peroxisomal ABC proteins, Pxa1 and Pxa2 (19,20).This study used the yeast two-hybrid assay to show that the carboxyl-terminal half of ALDP, ALDRP, and PMP70 can engage in homo-and heterodimerization. These two processes were confirmed by co-immunoprecipitation methods. We examined the effect of four different ALD patient mutations upon these interactions and attempted to map the carboxyl-terminal subdomains of ALDP allowing dimerization of the protein. EXPERIMENTAL PROCEDURESAntibodies, Yeast Strains, and Cell Lines-Monoclonal antibody 1D6 against human ALDP (hALDP) and polyclonal antibodies 1664 and 7373 raised against mouse ALDP (mALDP) and mouse ALDRP (mALDRP), respectively, have been described previously (13,15,21).
Nef is a 27-kDa myristoylated protein conserved in primate lentiviruses. In vivo, simian immunodeficiency virus Nef is required in macaques to produce a high viral load and full pathological effects. Nef has at least three major effects in vitro, induction of CD4 downregulation, alteration of T cell activation pathways, and enhancement of viral infectivity. We have used the yeast two-hybrid system to identify cellular proteins that interact with HIV-1Lai Nef and could mediate Nef function. A human cDNA was isolated that encodes a new type of thioesterase, an enzyme that cleaves thioester bonds. This novel thioesterase is unlike the animal types I and II thioesterases previously cloned but is homologous to the Escherichia coli thioesterase II. Nef and this thioesterase interact in vitro and are co-immunoprecipitated by anti-Nef antibodies in CEM cells expressing Nef. Nef alleles from human immunodeficiency virus-1 (HIV-1) isolates unable to down-regulate CD4 do not react or react poorly with thioesterase. An HIV-1 NefLai mutant selected for its lack of interaction with thioesterase was also unable to down-regulate CD4 cell-surface expression. These observations suggest that this human thioesterase is a cellular mediator of Nef-induced CD4 down-regulation.
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