Abstract 151: Lysine Residues at Positions 4 and 7 on Nef are Critical for Interaction with Calnexin and Drive Inhibition of Cholesterol Transporter: ATP-Binding Cassette A1
HIV patients are at a greater risk of developing atherosclerosis than non-infected individuals, partly due to the impairment of the ATP-Binding Cassette A1 (ABCA1) cholesterol transporter by the HIV-1 viral protein Nef leading to accumulation of cholesterol inside the cell. While studying the possible mechanism of Nef-mediated disruption of cholesterol efflux, we found that ABCA1 interacts with Nef, but a direct interaction with Nef is dispensable for the inactivation of ABCA1. Using mass spectroscopy we identified calnexin as a protein that associates with both ABCA1 and Nef and provided evidence to show that in the presence of Nef, ABCA1-calnexin interaction is disrupted leading to ABCA1 retention in the ER, subsequent degradation and impairment of cholesterol efflux. However, the molecular interactions taking place remained unknown as Nef is not known to enter the ER lumen and the domain of calnexin involved in binding to substrate proteins is located within the ER lumen. We hypothesized that Nef interacts with the C-terminal cytoplasmic domain of calnexin and that inhibiting this interaction would rescue ABCA1 function and expression. Using calnexin mutants lacking a luminal or cytoplasmic domain, we identified that the C-terminal cytoplasmic domain is responsible for Nef interaction. Using structural models of Nef and calnexin, possible Nef-calnexin interaction models were built using docking servers. Interacting residues in Nef were identified by calculating intermolecular contacts in the resulting complexes. Identified residues were mutated to confirm loss of interaction and this loss of interaction was found to associate with rescue of ABCA1 expression and restoration of cholesterol efflux. In conclusion, lysine residues at positions 4 and 7 on Nef were found to be indispensable for interacting with calnexin and inactivation of ABCA1. 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 identifies important targets that can be manipulated to inhibit the pathogenic effect of HIV on cholesterol metabolism.
Author Disclosures: R. Hunegnaw: None. M. Bukrinsky: None. A. Adzhubei: None.
- © 2015 by American Heart Association, Inc.