title: The ABCA1 domain responsible for interaction with HIV-1 Nef is conformational and not linear.
abstract: HIV-1 Nef is an accessory protein responsible for inactivation of a number of host cell proteins essential for anti-viral immune responses. In most cases, Nef binds to the target protein and directs it to a degradation pathway. Our previous studies demonstrated that Nef impairs activity of the cellular cholesterol transporter, ABCA1, and that Nef interacts with ABCA1. Mutation of the (2226)DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 disrupted interaction with Nef. Here, we tested Nef interaction with the ABCA1 C-terminal cytoplasmic fragment using yeast 2-hybrid system assay and co-immunoprecipitation analysis in human cells. Surprisingly, analysis in a yeast 2-hybrid system did not reveal any interaction between Nef and the C-terminal cytoplasmic fragment of ABCA1. Using co-immunoprecipitation from HEK 293T cells expressing these polypeptides, only a very weak interaction could be detected. The (2226)DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 found previously to be essential for interaction between ABCA1 and Nef is insufficient to bestow strong binding to Nef. Molecular modeling suggested that interaction with Nef may be mediated by a conformational epitope composed of the sequences within the cytoplasmic loop of ABCA1 and the C-terminal cytoplasmic domain. Studies are now underway to characterize this epitope.
authors.affiliation: Department of Molecular Biology, Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119899, Russia.
authors.forename: Daria
authors.initials: D
authors.lastname: Jacob
authors.affiliation: Department of Microbiology, Immunology and Tropical Medicine, GWU School of Medicine and Health Sciences, Washington, DC 20037, USA.
authors.forename: Ruth
authors.initials: R
authors.lastname: Hunegnaw
authors.affiliation: Department of Molecular Biology, Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119899, Russia.
authors.forename: Tatyana A
authors.initials: TA
authors.lastname: Sabyrzyanova
authors.affiliation: Department of Microbiology, Immunology and Tropical Medicine, GWU School of Medicine and Health Sciences, Washington, DC 20037, USA.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Pushkarsky
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov St. 32, 119991 Moscow, Russia.
authors.forename: Vladimir O
authors.initials: VO
authors.lastname: Chekhov
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov St. 32, 119991 Moscow, Russia.
authors.forename: Alexei A
authors.initials: AA
authors.lastname: Adzhubei
authors.affiliation: Department of Molecular Biology, Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119899, Russia.
authors.forename: Tatyana S
authors.initials: TS
authors.lastname: Kalebina
authors.affiliation: Department of Microbiology, Immunology and Tropical Medicine, GWU School of Medicine and Health Sciences, Washington, DC 20037, USA. Electronic address: mbukrins@gwu.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Bukrinsky
mesh_terms: D064286:ATP Binding Cassette Transporter 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D057809:HEK293 Cells; D015497:HIV-1 / Q000378:metabolism* / Q000472:pathogenicity; D054884:Host-Pathogen Interactions; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D020798:Two-Hybrid System Techniques; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D000939:Epitopes; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1
keywords: ABCA1; Co-immunoprecipitation; HIV-1; Interaction; Molecular modeling; Nef; Yeast 2-hybrid