Interaction Between HIV-1 Nef and Calnexin

Abstract

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.

Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells

“…Expression of Abca1 was increased after a 24 h exposure to exNef, but there was no statistical difference in Abca1 expression after 48 h of exposure between exGFP and exNef (Fig 3B). The discrepancy between ABCA1 abundance and gene expression indicates that regulation of ABCA1 abundance by exNef mainly occurs at a post-transcriptional level, as we suggested for endogenously expressed Nef [20]. On average, cholesterol efflux to apoA-I was reduced by 40±3% (mean ± SEM, p<0.05; n = 6) after 48 h exposure to exNef (Fig 3C).…”

Tetrapeptide Ac-HAEE-NH2 Protects α4β2 nAChR from Inhibition by Aβ

“…The structure of α4β2 nAChR neuronal acetylcholine receptor was modelled using as a template PDB:5KXI structure [ 75 ] solved by X-ray crystallography with a resolution of 3.941 Å. Fragments 1–24 and 365–585 of the α4 subunit, and 1–25 and 356–445 of the β2 subunit are absent in this structure. The missing fragments were modeled by the SwissModel, RaptorX, and iTasser servers, in accordance with our previously developed approach [ 76 ], which involves the construction of models by several independent servers with subsequent analysis of the quality of structures and identification of a representative model. Using this model, expert modeling of the final structure and energy minimization in the Amber12 force field was performed.…”

Analysis of HIV-1 (Human immunodeficiency virus-1, Lentivirus, Orthoretrovirinae, Retroviridae) Nef protein polymorphism of variants circulating in the former USSR countries

“…Так, на основе молекулярного моделирования разработан новый класс соединений DPPD (diphenylpyrazolodiazene), способных непосредственно связывать Nef и ингибировать репликацию ВИЧ в культуре клеток [21]. Совместно с российскими специалистами разрабатывается другой подход к ингибированию функции Nef, направленный на ограничение патогенного влияния этого белка ВИЧ на метаболизм холестерина и предотвращение атеросклероза [22]. Очевидно, что работы такого рода и в будущем будут проводиться в растущих масштабах.…”

“…Причиной нарушения функциональных свойств Nef могли быть как более или менее обширные делеции гена nef, так и единичные замены в его составе, среди которых отмечались мутации A84D, Y135F и G140R [16]. Кроме того, функционально дефектный ген мог содержать замены в составе мотивов 56 CAWLEAQ 61 и 22 Rxx 24 , а также полиморфизмы R25, RD35/36, T80, GL96/97, D108, D111, DW123/124, RY134/135, C142, EE154/155, LL164/165, DD174/175, RRE179, RF184/185 [2].…”