title: HIV-1 protein Nef inhibits activity of ATP-binding cassette transporter A1 by targeting endoplasmic reticulum chaperone calnexin.
abstract: HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism.
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, D. C. 20037.
authors.forename: Lucas
authors.initials: L
authors.lastname: Jennelle
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, D. C. 20037.
authors.forename: Ruth
authors.initials: R
authors.lastname: Hunegnaw
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, D. C. 20037.
authors.forename: Larisa
authors.initials: L
authors.lastname: Dubrovsky
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, D. C. 20037.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Pushkarsky
authors.affiliation: the Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Fitzgerald
authors.affiliation: the Baker IDI Heart and Diabetes Institute, Melbourne, Victoria 3004, Australia, and.
authors.forename: Dmitri
authors.initials: D
authors.lastname: Sviridov
authors.affiliation: the George Washington Center for Microscopy and Image Analysis, Office of VP for Research, Washington, D. C. 20037.
authors.forename: Anastas
authors.initials: A
authors.lastname: Popratiloff
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, D. C. 20037.
authors.forename: Beda
authors.initials: B
authors.lastname: Brichacek
authors.affiliation: From the George Washington University School of Medicine and Health Sciences, Washington, D. C. 20037, mbukrins@gwu.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Bukrinsky
mesh_terms: D064286:ATP Binding Cassette Transporter 1 / Q000378:metabolism*; D050197:Atherosclerosis / Q000378:metabolism; D037281:Calnexin / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D057809:HEK293 Cells; D019302:HIV Envelope Protein gp160 / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D019302:HIV Envelope Protein gp160; C514797:gp160 protein, Human immunodeficiency virus 1; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D037281:Calnexin; D002784:Cholesterol
keywords: Atherosclerosis; Cholesterol Regulation; Confocal Microscopy; Endoplasmic Reticulum (ER); Human Immunodeficiency Virus (HIV)