HIV-1 Protein Nef Inhibits Activity of ATP-binding Cassette Transporter A1 by Targeting Endoplasmic Reticulum Chaperone Calnexin

Lucas Jennelle,Ruth Hunegnaw,Larisa Dubrovsky,Tatiana Pushkarsky,Michael L Fitzgerald,Dmitri Sviridov,Anastas Popratiloff,Beda Brichacek,Michael Bukrinsky

doi:10.1074/jbc.m114.583591

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.

Highlights

HIV-1 Nef inhibits activity of ATP-binding cassette transporter A1 (ABCA1) and suppresses cholesterol efflux
HIV-1 infection is associated with a high risk of developing atherosclerosis [1], and studies in animal models and in vitro demonstrated that Nef-mediated impairment of ATP-binding cassette transporter A1 (ABCA1)3 function may contribute to this side effect of HIV infection [2,3,4]
We describe a novel mechanism by which Nef interferes with activity of a host cell protein ABCA1, which is essential for the first step of reverse cholesterol transport, cholesterol efflux

Summary

Background

HIV-1 Nef inhibits activity of ABCA1 and suppresses cholesterol efflux. Results: Nef binds to calnexin and disrupts its interaction with ABCA1 but enhances calnexin interaction with viral gp160. HIV-1 infection is associated with a high risk of developing atherosclerosis [1], and studies in animal models and in vitro demonstrated that Nef-mediated impairment of ATP-binding cassette transporter A1 (ABCA1) function may contribute to this side effect of HIV infection [2,3,4]. Nef-mediated inactivation of ABCA1 causes accumulation of cholesterol in macrophages, increases the abundance of lipid rafts (sites of HIV assembly), and elevates cholesterol content of viral membranes, increasing HIV production and infectivity (2, 6 – 8) Another consequence of this activity is impairment of HDL maturation [3], which may be responsible for low HDL levels, an established risk factor for atherosclerosis in HIV-infected patients [9]. This novel mechanism of action contributes to the effect of Nef on cellular cholesterol efflux

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION