Experimental model of HeLa ABCA1-GFP was developed by Neufeld et al.1 to study intracellular localization and trafficking of ABCA1, the ATP-binding cassette protein that is involved in apoA-I-mediated cholesterol efflux, and formation of high-density lipoprotein. Our laboratory adopted this model for investigation of the role that HIV-1 plays in the pathogenesis of atherosclerosis in HIV-infected subjects. Our previous study points to HIV-1 Nef as the major factor responsible for the disruption of cholesterol efflux from infected cells by impairing the interaction between the cholesterol transporter ABCA1 and calnexin.2 As a consequence, ABCA1 is retained in the endoplasmic reticulum and eventually undergoes degradation. The continuous overexpression of HIV-1 Nef can lead to almost undetectable levels of ABCA1. To observe the dynamics of Nef-induced changes in ABCA1 abundance and localization, we transfected HeLa ABCA1-GFP cells with plasmid expressing HIV-1SF2 Nef-mRFP (kind gift of Dr. Fackler) and imaged live cells over a period of 48 h. A snapshot captured at 26 h post-transfection is presented in Figure 1. The presented image was captured with an Axio Cell Observer Spinning Disk fluorescent microscope (Carl Zeiss) equipped with Yokogawa CSU X1 spinning disk and Evolve Delta EM CCD cameras (512 × 512, Photometrics), using a Plan Apochromat 100×/1.46 oil immersion lens. The image was processed using Volocity software (PerkinElmer Life Sciences). Cells with no detectable expression of HIV-1SF2 Nef-mRFP (Nef) (marked A and B in Fig. 1) have a prominent presence of ABCA1-GFP throughout the cell and on the plasma membrane, whereas the cell exhibiting high expression of Nef (cell marked D) has only barely detectable ABCA1. Interestingly, in the cell with low Nef expression (cell marked C), ABCA1 seems to be localized primarily intracellularly, with very low presence on the plasma membrane.
FIG. 1.
Effect of Nef expression on ABCA1 localization. HeLa ABCA1-GFP cells were transfected with a vector expressing HIV-1SF2 Nef-RFP and live cells were observed by fluorescent microscopy. The upper panel shows a composite image of two channels representing ABCA1-GFP (green) and HIV-1SF2 Nef-mRFP (red), whereas panels in the bottom represent single channels. Letters and arrows in the bottom panels identify cells not expressing Nef (A and B), cell with low expression of Nef (C), and cell with high Nef expression (D). Bar size is 20 μm. Color images available online at www.liebertpub.com/aid
Acknowledgments
This study was supported by NIH Grants R01HL101274, R21AI108533, P30AI087714, S10OD010710, and S10RR025565.
Author Disclosure Statement
No competing financial interests exist.
References
- 1.Neufeld EB, Remaley AT, Demosky SJ, et al. : Cellular localization and trafficking of the human ABCA1 transporter. J Biol Chem 2001;276:27584–27590 [DOI] [PubMed] [Google Scholar]
- 2.Jennelle L, Hunegnaw R, Dubrovsky L, et al. : HIV-1 protein Nef inhibits activity of ATP-binding cassette transporter A1 by targeting endoplasmic reticulum chaperone calnexin. J Biol Chem 2014;289:28870–28884 [DOI] [PMC free article] [PubMed] [Google Scholar]