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

Jennelle, Lucas T.; Hunegnaw, Ruth; Dubrovsky, Larisa; Pushkarsky, Tatiana; Fitzgerald, Michael L.; Sviridov, Dmitri; Popratiloff, Anastas; Břicháček, Beda; Bukrinsky, Michael

doi:10.1074/jbc.m114.583591

Cited by 31 publications

(42 citation statements)

References 47 publications

(40 reference statements)

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“…Importantly, ABCC7 and ABCB1 mutants that escape CNX binding do not achieve mature glycosylation and these mutations result in reduced transporter function 8, 9 . Our recently published study demonstrated that ABCA1 interacts with CNX, and reduction of CNX expression by RNAi resulted in a significant decrease in functional activity of ABCA1, evidenced by reduced cholesterol efflux to ABCA1-specific acceptor apoA-I 5 . We also showed that Nef impairs interaction between ABCA1 and CNX, and this effect of Nef is essential for inactivation and downregulation of ABCA1 5 .…”

Section: Introductionmentioning

confidence: 93%

“…Our recently published study demonstrated that ABCA1 interacts with CNX, and reduction of CNX expression by RNAi resulted in a significant decrease in functional activity of ABCA1, evidenced by reduced cholesterol efflux to ABCA1-specific acceptor apoA-I 5 . We also showed that Nef impairs interaction between ABCA1 and CNX, and this effect of Nef is essential for inactivation and downregulation of ABCA1 5 . Importantly, inhibition of ABCA1-CNX interaction by Nef is specific, as interaction between ABCA1 and two other proteins, dystrophin and serine palmitoyltransferase, shown previously to bind ABCA1 10 , was not affected.…”

Section: Introductionmentioning

confidence: 93%

“…Studies in animal models demonstrated that this activity of Nef may be responsible for hypoalphalipoproteinemia and high risk of atherosclerosis observed in HIV-infected subjects 2–4 . Our recent study identified calnexin, an integral endoplasmic reticulum (ER) membrane lectin-like chaperone, as a key player in the mechanism of Nef-mediated inhibition of ABCA1 and cholesterol efflux 5 . Calnexin (CNX) and its homologue calreticulin (CRT) regulate folding and maturation of newly synthesized glycoproteins by engaging them in a CNX/CRT cycle 6 .…”

Section: Introductionmentioning

confidence: 99%

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Interaction Between HIV-1 Nef and Calnexin

Hunegnaw

Vassylyeva

Dubrovsky

et al. 2016

ATVB

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Objective HIV-infected patients are at an increased risk of developing atherosclerosis, in part due to downmodulation and functional impairment of ATP-Binding Cassette A1 (ABCA1) cholesterol transporter by the HIV-1 protein Nef. The mechanism of this effect involves Nef interacting with an endoplasmic reticulum (ER) chaperone calnexin and disrupting calnexin binding to ABCA1, leading to ABCA1 retention in ER, its degradation and resulting suppression of cholesterol efflux. However, molecular details of Nef-calnexin interaction remained unknown, limiting translational impact of this finding. Approach and results Here, we used molecular modeling and mutagenesis to characterize Nef-calnexin interaction and to identify small molecule compounds that could block it. We demonstrated that interaction between Nef and calnexin is direct and can be reconstituted using recombinant proteins in vitro with a binding affinity of 89.1 nM measured by surface plasmon resonance. The cytoplasmic tail of calnexin is essential and sufficient for interaction with Nef, and binds Nef with affinity of 9.4 nM. Replacing lysine residues in positions 4 and 7 of Nef with alanines abrogates Nef-calnexin interaction, prevents ABCA1 downregulation by Nef, and preserves cholesterol efflux from HIV-infected cells. Through virtual screening of the NCI library of compounds, we identified a compound, 1[(7-Oxo-7H-benz[de]anthracene-3-yl)amino]anthraquinone, which blocked Nef-calnexin interaction, partially restored ABCA1 activity in HIV-infected cells, and reduced foam cell formation in a culture of HIV-infected macrophages. Conclusion This study identifies potential targets that can be exploited to block the pathogenic effect of HIV infection on cholesterol metabolism and prevent atherosclerosis in HIV-infected subjects.

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Section: Introductionmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

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Interaction Between HIV-1 Nef and Calnexin

Hunegnaw

Vassylyeva

Dubrovsky

et al. 2016

ATVB

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“…6,7 However, the only in vivo evidence for the effects of Nef on cholesterol metabolism are studies where recombinant Nef was injected into mice. 8 These studies demonstrated an enhanced development of atherosclerosis in a mouse model of this disease (apoe -/-mice on high fat diet), and in wild-type C57BL/6 mice fed high-fat/high-cholesterol diet, Nef induced accumulation of lipid-laden macrophages in adventitia of aorta.…”

mentioning

confidence: 99%

“…Future studies would also be required to determine the chain of molecular events behind the Nef-induced accumulation of lipid droplets. Nef is known to impair the function of the ABCA1 cholesterol transporter, 6 but may also, through its association with calnexin, 7 affect other proteins involved in lipid synthesis or transport. Nef interaction with Acyl-CoA thioesterase 8 (ACOT8) 15,16 may also influence fatty acid metabolism.…”

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confidence: 99%

Short Communication: Accumulation of Neutral Lipids in Liver and Aorta of Nef-Transgenic Mice

Pushkarsky

Shilov

Kruglova

et al. 2017

AIDS Research and Human Retroviruses

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HIV-infected individuals are at high risk of developing atherosclerosis and cardiovascular disease, in part, due to HIV-induced impairment of cholesterol metabolism. In vitro studies demonstrated that HIV-1 protein Nef inhibits activity of ABCA1, the main cellular cholesterol transporter, leading to cholesterol accumulation in macrophages and conversion of these cells into foam cells, characteristic for atherosclerosis. However, the mechanisms of Nef-mediated effects on cholesterol metabolism in vivo are not well characterized. In this study, we generated Nef-transgenic mice and evaluated the accumulation of neutral lipids in liver and aorta of these animals. Nef expression was low in all transgenic mice, with some mice carrying the Nef transgene, but not expressing the Nef RNA. Using Oil Red O staining, we demonstrated increased levels of neutral lipids in liver and aorta of mice expressing Nef relative to transgenic animals, with no detectable Nef expression or control wild-type mice. These results provide direct evidence that Nef promotes cholesterol deposition in tissues.

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Analysis of ABCA1 and Cholesterol Efflux in HIV-Infected Cells

Mukhamedova

Břicháček

Darwish

et al. 2016

Methods in Molecular Biology

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Summary Cholesterol is an essential component of the cellular membranes and, by extension, of the HIV envelope membrane, which is derived from the host cell plasma membrane. Depletion of the cellular cholesterol has a inhibitory effect on HIV assembly, reduces infectivity of the produced virions, and makes the cell less susceptible to HIV infection. It is not surprising that the virus has evolved to gain access to cellular proteins regulating cholesterol metabolism. One of the key mechanisms used by HIV to maintain high levels of cholesterol in infected cells is Nef-mediated inhibition of cholesterol efflux and the cholesterol transporter responsible for this process, ABCA1. In this article, we describe methods to investigate these effects of HIV-1 infection.

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

Cited by 31 publications

References 47 publications

Interaction Between HIV-1 Nef and Calnexin

Interaction Between HIV-1 Nef and Calnexin

Short Communication: Accumulation of Neutral Lipids in Liver and Aorta of Nef-Transgenic Mice

Analysis of ABCA1 and Cholesterol Efflux in HIV-Infected Cells

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