Electronic Thesis/Dissertation
Mechanism of HIV-1 Nef-mediated Impairment of ABCA1 Open Access
HIV-infected patients are at increased risk of developing atherosclerosis, in part due to downmodulation and impairment of activity of the ATP-Binding Cassette A1 (ABCA1) cholesterol transporter by the HIV-1 protein Nef. Targeting of cholesterol to lipid rafts, regions where HIV assembles and buds, has been shown to increase in the presence of Nef. The mechanism of this effect of Nef remained unknown for several years. Initial work demonstrated that inhibition of ABCA1 activity in HIV-infected cells involves the interaction of Nef with an endoplasmic reticulum (ER) chaperone calnexin and disruption of calnexin binding to ABCA1, leading to ABCA1 retention in ER, degradation, and eventual suppression of cholesterol efflux. However, the mechanics of this Nef effect remained unclear, in particular because the substrate-binding domain of calnexin is located within the ER lumen, whereas Nef has not been documented to enter the lumen of ER. Therefore, the Nef-calnexin interaction needed to be characterized and well understood. Experiments using deletion mutants of calnexin lacking cytoplasmic or luminal domains demonstrated that Nef interacts with the cytoplasmic tail of calnexin. Computerized structural modeling and docking analyses predicted that lysine residues in positions 4 and 7 of Nef are critical for this interaction. Next, surface plasmon resonance was used to demonstrate that the interaction between Nef and calnexin is direct with a binding affinity of 0.67 µM in vitro. Using computerized docking analysis, a virtual screen for small molecules that could block this interaction was performed. Biochemical tests using compounds identified by the screen led to a compound 1[(7-Oxo-7H-benz[de]anthracene-3-yl) amino] anthraquinone, which inhibited Nef-calnexin interaction and restored ABCA1 function in HIV-1 infected macrophages. The compound also prevented accumulation of lipid droplets in infected macrophages when compared to untreated infected cells. In conclusion, effects of HIV-1 Nef on cholesterol transport can be counteracted by inhibiting a critical interaction of the viral protein with calnexin. As cardiovascular diseases like atherosclerosis have emerged as an important cause of morbidity and mortality in HIV-infected individuals, there is a great need for targeted therapeutic strategies. This study characterizes the role of HIV in altering cholesterol transport and identifies an important compound that can be developed as a drug to inhibit the pathogenic effect of HIV on cholesterol metabolism.
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Hunegnaw_gwu_0075A_13317.pdf | 2018-01-16 | Open Access |
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