HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered HDL profile exacerbated by downmodulation and impairment of ATP-Binding Cassette Transporter A1 (ABCA1) activity by the HIV-1 protein Nef. Nef has been shown to increase delivery of cholesterol to lipid rafts, sites of viral assembly and egress, by inhibition of ABCA1 cholesterol efflux functionality and reduction of ABCA1 protein levels through lysosomal degradation. Important mechanistic details of ABCA1 inactivation and degradation by Nef, and whether these two processes are intimately linked or separable are still to be defined. The studies presented here were designed to identify cellular co-factors for ABCA1-mediated cholesterol efflux that may be targeted by Nef to achieve ABCA1 inactivation. In these studies, a novel cellular factor, the ER-resident lectin chaperone calnexin, was shown to be involved in a physical interaction with ABCA1 that is disrupted by Nef. Nef was found to bind and redistribute calnexin and reduce binding and co-localization of ABCA1 with calnexin. In vitro knockdown of calnexin via RNAi reproduced several previously described biochemical effects of Nef, including redistribution of ABCA1, increased ABCA1 membrane localization, and reduced ABCA1 recycling. Importantly, knockdown of calnexin also resulted in reduced ABCA1-mediated cholesterol efflux, but without the Nef-mediated reduction in ABCA1 protein levels, suggesting that Nef utilizes a bipartite mechanism to inactivate and degrade ABCA1 and that these functions may be separable. Despite the lack of effect of calnexin knockdown on ABCA1 protein levels, interference with the ABCA1-calnexin interaction was critical for Nef-mediated functional impairment of ABCA1. This was shown with a Nef mutant defective in interaction with calnexin which was incapable of preventing ABCA1-calnexin interaction and was also defective in impairing ABCA1-mediated cholesterol efflux activity. Thus, these studies identified a novel mechanism by which HIV-1 Nef impairs functional activity of cholesterol transporter ABCA1 by blocking its interaction with calnexin. Calnexin acts as an ABCA1 functional chaperone, limiting total and cell surface ABCA1 expression while increasing ABCA1-mediated cholesterol efflux. Combined with the demonstration that Nef increases delivery of ABCA1 to lysosomes, these results suggest the Nef-mediated impairment of ABCA1 function involves reduced interaction with calnexin followed by delivery of ABCA1 to lysosomes for degradation.
|Advisor:||Bukrinsky, Michael I.|
|Commitee:||Kumar, Ajit, Leitenberg, David, McCaffrey, Timothy, Zeichner, Steven|
|School:||The George Washington University|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 74/08(E), Dissertation Abstracts International|
|Subjects:||Microbiology, Biochemistry, Virology|
|Keywords:||ABCA1, Calnexin, Cardiovascular disease, HIV-1 Nef, High density lipoprotein|
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