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HIV Causes Heart Disease
  Does HIV Cause Cardiovascular Disease?- (12/11/06)
Commentary by Andrew Carr on this research study.
....The data support the shift away from a paradigm of delaying or stopping ART to reduce the risk of cardiovascular disease and raise the possibility that antiretroviral drugs without direct metabolic effects may actually reduce cardiovascular risk....
HIV Causes Heart Disease
PLoS Medicine Nov 2006
Human Immunodeficiency Virus Impairs Reverse Cholesterol Transport from Macrophages
Zahedi Mujawar1, Honor Rose2, Matthew P. Morrow1, Tatiana Pushkarsky1, Larisa Dubrovsky1, Nigora Mukhamedova2, Ying Fu2, Anthony Dart2, Jan M. Orenstein1, Yuri V. Bobryshev3, Michael Bukrinsky1*, Dmitri Sviridov2
1 The George Washington University, Washington, District of Columbia, United States of America, 2 Baker Heart Research Institute, Melbourne, Victoria, Australia, 3 University of New South Wales, Sydney, New South Wales, Australia
"......Results presented in this report demonstrate that HIV-1, via the accessory protein Nef, impairs cholesterol efflux from macrophages....Other viruses (such as Herpes virus or CMV) were found in atherosclerotic plaques and were epidemiologically associated with elevated risk of development of atherosclerosis [64-66]..... ..... the effect on reverse cholesterol transport may be a common feature of viral and bacterial infection of macrophages, although mechanisms involved are likely unique for each infection..."
Several steps of HIV-1 replication critically depend on cholesterol. HIV infection is associated with profound changes in lipid and lipoprotein metabolism and an increased risk of coronary artery disease. Whereas numerous studies have investigated the role of anti-HIV drugs in lipodystrophy and dyslipidemia, the effects of HIV infection on cellular cholesterol metabolism remain uncharacterized. Here, we demonstrate that HIV-1 impairs ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux from human macrophages, a condition previously shown to be highly atherogenic. In HIV-1-infected cells, this effect was mediated by Nef. Transfection of murine macrophages with Nef impaired cholesterol efflux from these cells. At least two mechanisms were found to be responsible for this phenomenon: first, HIV infection and transfection with Nef induced post-transcriptional down-regulation of ABCA1; and second, Nef caused redistribution of ABCA1 to the plasma membrane and inhibited internalization of apolipoprotein A-I. Binding of Nef to ABCA1 was required for down-regulation and redistribution of ABCA1. HIV-infected and Nef-transfected macrophages accumulated substantial amounts of lipids, thus resembling foam cells. The contribution of HIV-infected macrophages to the pathogenesis of atherosclerosis was supported by the presence of HIV-positive foam cells in atherosclerotic plaques of HIV-infected patients. Stimulation of cholesterol efflux from macrophages significantly reduced infectivity of the virions produced by these cells, and this effect correlated with a decreased amount of virion-associated cholesterol, suggesting that impairment of cholesterol efflux is essential to ensure proper cholesterol content in nascent HIV particles. These results reveal a previously unrecognized dysregulation of intracellular lipid metabolism in HIV-infected macrophages and identify Nef and ABCA1 as the key players responsible for this effect. Our findings have implications for pathogenesis of both HIV disease and atherosclerosis, because they reveal the role of cholesterol efflux impairment in HIV infectivity and suggest a possible mechanism by which HIV infection of macrophages may contribute to increased risk of atherosclerosis in HIV-infected patients.
Funding. This work was supported in part by the National Institutes of Health (NIH) grants R03 TW006238 and R21 DK072926 (MB and DS), a grant from the Campbell Foundation (MB), grants from the American Heart Association (MB and ZM), and grants #317810 and #317811 from the National Health and Medical Research Council of Australia (DS) and Australian Postgraduate Award #1549 (HR).
Results presented in this report demonstrate that HIV-1, via the accessory protein Nef, impairs cholesterol efflux from macrophages. This finding can be interpreted as a virus-mediated switch of cholesterol trafficking from physiological efflux to virus-controlled transport, thus reducing the ability of a cell to export excessive cholesterol. Given that availability of cholesterol is critical for HIV assembly and infectivity [48], it is physiologically sensible for the virus to take over control of intracellular cholesterol metabolism.
A previous report demonstrated that Nef binds cholesterol and may deliver it to nascent virions [5]. Our study suggests that Nef-mediated impairment of cholesterol efflux is another mechanism ensuring efficient delivery of cholesterol to HIV. Importantly, this mechanism may be a necessary component of the above-mentioned Nef-mediated transport of cholesterol to virions. Indeed, prevention of cholesterol efflux impairment by LXR agonist reduces virion-associated cholesterol without interfering with Nef incorporation into the virions (unpublished data). Reduction of virion-associated cholesterol correlates with lower virion infectivity (Figure 9B and 9C).
Our results demonstrate that Nef specifically targets ABCA1. Indeed, Nef did not suppress cholesterol efflux in cells lacking ABCA1 (HeLa cells or non-activated RAW 264.7 cells), but did so in ABCA1-expressing cells, such as RAW 264.7 cells stimulated with an LXR agonist, HeLa cells transfected with ABCA1, and differentiated human macrophages. Furthermore, Nef did not suppress cholesterol efflux from ABCG1-transfected HeLa cells (Figure 2E). These findings and the fact that Nef can interact with ABCA1 (Figure 5A) suggest that there is an interplay between Nef and ABCA1 in an HIV-infected cell. The end result of this interplay would depend on relative levels of expression of Nef and ABCA1. Consistent with this suggestion, overexpression of Nef from the cytomegalovirus (CMV) promoter inhibits ABCA1-mediated cholesterol efflux stimulated with the LXR agonist (Figure 1C), whereas levels of Nef expressed from the HIV LTR are insufficient to suppress LXR agonist-stimulated cholesterol efflux in HIV-infected macrophages (Figure 9A). As a result, HIV infectivity is reduced in LXR agonist-stimulated cells (Figure 9B). Therefore, drugs stimulating cholesterol efflux may provide a dual benefit to HIV-infected patients by limiting HIV replication and reducing the risk of atherosclerosis.
Our studies show that cholesterol efflux impairment is a conserved feature of HIV-1 Nef. Indeed, we show this phenomenon using three R5 (ADA, Yu-2, and 92US660) and two X4 (SF2 and LAI) HIV-1 isolates. We demonstrate that HIV-1 Nef impairs cholesterol efflux by at least two mechanisms: it reduces ABCA1 abundance, and it causes intracellular re-distribution of ABCA1. These two mechanisms may be related, as they both depend on interaction between Nef and ABCA1 (Figures 3-5). For example, a block to intracellular trafficking of ABCA1 may re-target this protein to a degradation pathway. Alternatively, ABCA1 re-distribution and down-regulation may be two independent effects of Nef, both contributing to impairment of cholesterol efflux. Indeed, several recent reports demonstrated a role of ABCA1 trafficking between late endosomes and the cell surface in cholesterol efflux from endosomal compartment [36,38,39,49]. The effects of Nef on ABCA1 distribution and apoA-I binding and internalization are similar to the effects of cyclosporin A [43] or deletion of the PEST sequence in ABCA1 [39], both of which inhibit efflux of cholesterol from late endosomes. Therefore, both down-regulation of ABCA1 and its intracellular re-distribution can independently contribute to cholesterol efflux impairment observed in HIV-infected cells. Interestingly, Nef alone had less effect on ABCA1 abundance than HIV-1 infection (compare Figure 3A and 3B), however, it had a more profound effect on the sequestration of ABCA1 at the plasma membrane (compare Figure 4D and 4F). Although this result may be due to differences between the cell types in which these analyses were performed (primary human macrophages for HIV infection and murine macrophage cell line RAW 264.7 for Nef transfection), it is also possible that the primary effect of Nef is to sequester ABCA1 at the plasma membrane, and some other HIV protein may cooperate with Nef to stimulate down-regulation of sequestered ABCA1.
The exact molecular mechanisms responsible for the effect of Nef on intracellular trafficking and abundance of ABCA1 are yet to be fully investigated. Nef is known to regulate expression of several transmembrane proteins, including CD4 [45], MHC I [50], MHC II [50], CD28 [51], and DC-SIGN [52]. In most cases, Nef mediates internalization and degradation of the receptor [53], but in some cases (e.g., with DC-SIGN or invariant chain of MHC II), it up-regulates the cell surface expression of the protein. These effects are consistent with our findings showing ABCA1 re-localization and down-regulation, which may involve mechanisms similar to those described for Nef interactions with other proteins. It is worth noting that few of the above-mentioned studies showed co-immunoprecipitation of Nef with a target protein from HIV-infected cells, consistent with our inability to pull down Nef and ABCA1 from HIV-infected macrophages. This may be due to a transitory nature of Nef-ABCA1 interaction and low-level expression of these proteins. Analysis of this interaction in cells overexpressing both proteins demonstrated a critical role of Nef myristoylation (Figure 5A). This fatty acid may either be directly involved in binding of Nef to ABCA1, similar to the role that farnesylation of the yeast pheromone a-factor plays in its interaction with the yeast ABC transporter Ste6 [54], or it may regulate Nef-ABCA1 interaction indirectly by targeting Nef to the plasma membrane. Further analysis of the mechanisms by which Nef affects ABCA1 function would require understanding of the molecular events that regulate intracellular trafficking and degradation of ABCA1 in uninfected cells, which is incompletely characterized and is a subject of the ongoing studies.
The results of this study have potential implications for understanding pathogenesis of CAD in HIV-infected patients. These patients have a mildly elevated risk of CAD [55], which is sharply raised by treatment with certain PIs [7,9,14,19]. Increased risk of CAD after treatment with PIs led to the assumption that PIs and/or dyslipidemia are the primary source of development of atherosclerosis in HIV patients. Results presented in this report suggest that HIV-induced impairment of cholesterol efflux from macrophages may be another important contributor to the pathogenesis of CAD. Indeed, inactivation of ABCA1 in macrophages of hyperlipidemic mice significantly increased development of atherosclerosis [27], and genetic mutation inactivating ABCA1 in humans leads to Tangier disease, one of the characteristic features of which is an increased risk of CAD [28]. Impairment of reverse cholesterol transport mediated by down-regulation of ABCA1 has been described for bacterial infections and has been linked to pathogenesis of atherosclerosis (reviewed in [56]). In the case of HIV infection, this mechanism would have only a mild atherogenic effect or not at all on the background of hypocholesterolemia characteristic for untreated HIV-1 infection [55,57]. Treatment of HIV-infected patients with HAART causes a sharp rise of triglyceride-rich VLDL, resulting in enhanced lipid uptake and foam cell formation [58], and small dense LDL [59,60], which is particularly susceptible to oxidation [61], is more able to infiltrate the subendothelial space, and is a risk factor for CAD [62]. A combination of these effects of HAART and impairment of cholesterol efflux by HIV (which prevents compensatory removal of excessive cholesterol) would result in a greatly enhanced accumulation of cholesterol in HIV-infected macrophages and would potentially further increase the risk of development of atherosclerosis. It should be noted that HIV-infected macrophages, unlike T cells, survive for extended periods of time and are considered long-term reservoirs of HIV-1 [63]. As a result, infected macrophages persist, at least for some time, in HAART-treated patients, when conditions favor development of atherosclerotic plaques. We can speculate that these macrophages may contribute to initiation of atherosclerotic plaque formation, which then proceeds even in the absence of newly infected cells. This mechanism is consistent with the presence of HIV-infected macrophages in atherosclerotic plaques of HAART-treated patients observed in our study (Figure 8). However, further in vivo and clinical studies are required to evaluate the contribution of the impairment of reverse cholesterol transport to the risk of atherosclerosis in HIV patients.
Findings presented in this report provide an example of how viruses may interfere with cellular cholesterol metabolism and may potentially affect the risk of atherosclerosis. This example may be not unique to HIV. Other viruses (such as Herpes virus or CMV) were found in atherosclerotic plaques and were epidemiologically associated with elevated risk of development of atherosclerosis [64-66]. Future studies will determine whether these viruses cause disturbances in cholesterol metabolism similar to those found in this report. In support of this possibility, several reports demonstrated that bacterial and viral pathogens may modulate macrophage cholesterol efflux by down-regulating ABC transporters via LXR-dependent [24] and LXR-independent [25] pathways. The first pathway is engaged after activation of Toll-like receptors by invading viruses or bacteria. The second pathway involves the negative effect of bacterial endotoxin on ABCA1 mRNA levels in macrophages [25]. Both pathways promote conversion of macrophages into foam cells, which may acquire resistance to pathogen, but retain their atherogenic properties [12]. Therefore, the effect on reverse cholesterol transport may be a common feature of viral and bacterial infection of macrophages, although mechanisms involved are likely unique for each infection.
Macrophage cells and cholesterol play a central role in the pathogenesis of two diseases, AIDS and atherosclerosis. Macrophages are among the main targets of HIV in the body, and HIV assembly and budding, as well as infection of new target cells, all depend on plasma membrane cholesterol. Depletion of cellular cholesterol markedly and specifically reduces HIV-1 particle production [1,2], and cholesterol-sequestering drugs, such as ƒĀ-cyclodextrin, render the virus incompetent for cell entry [3,4]. Therefore, regulating cholesterol delivery to nascent virions would be highly beneficial for the virus. Some clues to potential mechanisms that may be employed by HIV to achieve this goal have been provided recently when it was demonstrated that HIV-1 accessory protein Nef binds cholesterol and may deliver it to the site of virion assembly at the plasma membrane [5]. However, little is known about the relation of this mechanism to major cellular cholesterol trafficking pathways and about the effect of HIV infection on lipid metabolism in the host cells.
Increased risk of atherosclerosis and coronary artery disease (CAD) is a recognized clinical problem in HIV-infected patients [6-10]. A key element of atherosclerotic plaque is formation of foam cells [11], the majority of which are macrophages overloaded with cholesterol. Foam cells undergo apoptosis, necrosis, and calcification, and cholesterol released from foam cells forms the lipid-rich core of the plaque. Interestingly, foam cells are less conducive than macrophages to growth of certain pathogens, such as Chlamydia pneumoniae [12], suggesting that they may contribute to antibacterial response of the organism. One of the main causes of foam cell formation and accumulation of cholesterol in the vessel wall may be dyslipidemia, especially high levels of very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), as well as low levels of high-density lipoprotein (HDL) [13], and/or impairment of intracellular cholesterol metabolism. Dyslipidemia caused by antiretroviral drugs, in particular protease inhibitors (PIs) [14], is a known risk factor in pathogenesis of CAD in HIV-infected patients. One mechanism of PI treatment-dependent dyslipidemia is inhibition of degradation of proteins involved in metabolism of cholesterol and triglycerides, such as apolipoprotein B [15] or the adipocyte determination- and differentiation-dependent factor 1/sterol regulatory element binding protein 1 [16]. In addition to causing dyslipidemia, PIs may contribute to initiation of atherosclerosis by affecting intracellular metabolism of cholesterol via up-regulation of the scavenger receptor CD36 and the subsequent accumulation of sterol in macrophages [17]. Therefore, PIs affect both extra- and intracellular pathways of cholesterol metabolism and are likely an important factor in pathogenesis of atherosclerosis in HIV-infected patients. However, a number of clinical reports found a correlation between heart disease and HIV viral load, and detected an increased risk of CAD in patients not treated with PIs or even in drug-naive HIV-infected individuals [18,19]. Recent results support the role of HIV infection as an independent risk factor of CAD [7,9,10,20-22], but the mechanisms of this atherogenic effect of HIV remain unknown.
In this report, we identify one such mechanism. We show that HIV, via its accessory protein Nef, affects normal function of ATP-binding cassette transporter A1 (ABCA1) and consequently impairs cholesterol efflux from infected macrophages. Previous studies demonstrated the key role of ABCA1 in reverse cholesterol transport and found that functional mutations in ABCA1 cause Tangier disease, which is characterized by severe HDL deficiency, accumulation of sterols in tissue macrophages, and accelerated atherosclerosis [23]. The effect of HIV-1 on ABCA1 is consistent with ABCA1 inhibition described for viral [24] and bacterial [25] infections; however, the mechanism of impairment is unique for HIV. As a result of ABCA1 inhibition, HIV-infected macrophages accumulate lipids and transform into foam cells, a step that may contribute to the increased risk of atherosclerotic plaque formation. Our results also suggest that cholesterol efflux impairment may be a mechanism that ensures access of nascent virions to intracellular cholesterol, which is critical for HIV infectivity. Therefore, pharmacological stimulation of cholesterol efflux may be considered as a novel anti-HIV therapeutic strategy.
Impairment of Cholesterol Efflux in HIV-Infected Macrophages

Cholesterol efflux is a pathway for removing excessive cholesterol from cells to extracellular acceptors. It is the first step of reverse cholesterol transport, and it plays a key role in maintaining cell cholesterol homeostasis. Impairment of cholesterol efflux leads to accumulation of intracellular cholesterol [26] and development of atherosclerosis in animal models [27] and in humans [28,29]. Analysis of cholesterol efflux from monocyte-derived macrophages infected in vitro with HIV-1 ADA [30] demonstrated a substantial inhibition of apolipoprotein A-I (apoA-I)-specific efflux (Figure 1A). A similar effect was observed in macrophages infected with two primary macrophage-tropic HIV-1 strains, Yu-2 and 92US660, indicating that impairment of cholesterol efflux is a general feature of HIV-1 replication in macrophages (Figure 1A). The level of cholesterol efflux inhibition correlated with the level of virus replication (Figure 1A). Importantly, at the time of analysis (21 d after infection), 80%-90% of the cells infected with ADA and Yu-2 viruses were p24+ (only 20% of the cells infected with 92US660 strain were p24+ at that time), indicating that reverse transcription (RT) values in these infections reflected the amount of virus produced per cell and that cholesterol efflux impairment depended on the level of virus protein expression.
Nef Is Critical for Cholesterol Efflux Impairment
Previous studies demonstrated that the HIV-1 protein Nef can directly bind cholesterol and suggested that in CD4+ T cells, Nef may be involved in transporting cholesterol to the sites of HIV assembly at the plasma membrane [5]. To test the role of Nef in the observed impairment of cholesterol efflux from macrophages, we infected macrophages with HIV-1 SF2 constructs carrying a mutated or a functional Nef gene. To ensure similar levels of infection, the constructs were pseudotyped by the glycoprotein of vesicular stomatitis virus (VSV-G), which targets HIV-1 entry to an endocytic pathway, thus eliminating the requirement for Nef in the early steps of infection [31]. Under these one-cycle replication conditions, both viruses infected about 40% of cells and produced similar levels of p24 (Figure 1B). Cholesterol efflux to apoA-I was substantially reduced in the culture infected with the Nef-positive virus (wild type [WT]), whereas in the culture infected with Nef-deficient virus (ƒĒNef), it was similar to the level observed in uninfected cells (Figure 1B). This result indicates that Nef is necessary for HIV-mediated impairment of cholesterol efflux.
To determine whether Nef is sufficient for the observed effect, we transiently transfected murine macrophages RAW 264.7 with constructs expressing SF2-derived Nef and stimulated ABCA1 expression in these cells by liver X receptor (LXR) agonist TO-901317. Cholesterol efflux to apoA-I from Nef-transfected RAW 264.7 macrophages was significantly reduced (by more than 50%) compared to cells transfected with an empty vector (mock transfection) (Figure 1C). This result indicates that expression of Nef is sufficient to impair cholesterol efflux from macrophages. Interestingly, Nef mutant Nef.G2A, defective in myristoylation and membrane localization [5], was not effective in impairing cholesterol efflux (Figure 1C) despite levels of expression being similar to that of WT Nef (Figure 1D). Cholesterol efflux impairment in Nef-transfected RAW cells was less than in HIV-infected macrophages, likely due to differences between these cell types.
Nef Specifically Targets ABCA1-Dependent Cholesterol Efflux
Specific efflux of cholesterol from cells is mediated by the members of the family of ATP-binding cassette (ABC) transporters. The ABCA1 transporter is responsible for lipidation of lipid-poor apoA-I with cellular lipids [32], whereas ABCG1 controls efflux to mature HDL [33]. Our finding that Nef inhibits cholesterol efflux to apoA-I (Figure 1) suggests that ABCA1 may be the specific target of Nef. Consistent with this notion, cholesterol efflux to HDL (controlled by ABCG1) from HIV-infected macrophages was not significantly impaired (Figure 2A). Furthermore, phospholipid efflux, which is dependent on ABCA1 [34], from Nef-transfected RAW 264.7 macrophages was reduced (Figure 2B), similar to the effect of Nef on cholesterol efflux (Figure 1C). In addition, Nef did not affect cholesterol efflux from RAW 264.7 cells in which ABCA1 expression was not stimulated (Figure 2C) and consequently was very low.
The notion that ABCA1-mediated efflux is the target of Nef was further supported by experiments in HeLa cells, which do not express ABC transporters and have very low background cholesterol efflux to apoA-I [35]. Consistent with a previous report [36], transfection of HeLa cells with ABCA1 significantly enhanced cholesterol efflux to apoA-I, whereas co-transfection with Nef derived from SF2 or LAI strains of HIV-1 brought the efflux back to the level observed in mock-transfected cells (Figure 2D). Importantly, NefLAI, which was expressed to higher levels than NefSF2 (Figure 2D), was more effective also in cholesterol efflux impairment. A similar experiment testing the effect of Nef on ABCG1-directed cholesterol efflux showed stimulation of cholesterol efflux to HDL after transfection of HeLa cells with ABCG1, but did not reveal significant inhibition by Nef (Figure 2E). Therefore, we conclude that Nef specifically targets ABCA1-dependent cholesterol efflux.
Nef Down-Regulates ABCA1
Since ABCA1 appears to be the target of Nef, we tested ABCA1 abundance (by Western blotting) and transcription (by real-time RT-PCR) in HIV-infected human macrophages and Nef-transfected RAW 264.7 cells. Analysis of ABCA1 in macrophages at the peak of HIV-1 ADA replication showed a substantial decrease of ABCA1 abundance (Figure 3A). Importantly, abundance of two other proteins involved in cholesterol efflux to HDL, ABCG1 and scavenger receptor B1 (SR-B1), was not affected by HIV infection (Figure 3A), consistent with specific targeting of the ABCA1-dependent pathway by the virus. A similar phenomenon was observed in Nef-transfected RAW 264.7 macrophages, although the effect was less pronounced (approximately 50% reduction in ABCA1 abundance when assessed by densitometry of the Western blot) (Figure 3B). The Nef.G2A mutant, which was inactive in cholesterol efflux impairment (Figure 1C), was also inactive in depleting ABCA1. RT-PCR analysis revealed a significant increase of ABCA1 mRNA in HIV-infected (Figure 3C) or Nef-transfected (Figure 3D) cells, which likely reflects a compensatory response for the loss of ABCA1 [37]. This observation rules out a suppressive effect of Nef on ABCA1 transcription and suggests a post-transcriptional down-regulation of ABCA1 by Nef. Therefore, down-regulation of ABCA1 is one of the mechanisms responsible for impairment of cholesterol efflux by HIV-1.
Nef Alters Intracellular Distribution of ABCA1
Although down-regulation of ABCA1 alone would account for a substantial part of the inhibition of cholesterol efflux, we further found that intracellular distribution of ABCA1 was also affected by HIV infection. Recent reports established that ABCA1 resides both on the plasma membrane and in endocytic vesicles [36], and demonstrated the role of endosomal ABCA1 and trafficking of ABCA1 between endosomes and plasma membrane in the apoA-I-mediated efflux of cellular lipids from the endosomal compartment [38,39]. Figure 4A and 4B show p24 staining, and Figure 4C and 4D show ABCA1 distribution in human macrophages infected with ƒĒNef and Nef-expressing HIV-1, respectively. Consistent with the findings of Neufeld and colleagues [36], ABCA1 was distributed evenly between the cytoplasm and the plasma membrane in human macrophages either uninfected (unpublished data) or infected with ƒĒNef HIV-1 (Figure 4C), as well as in mock-transfected murine RAW 264.7 cells (Figure 4E). It appears that in macrophages infected with Nef-expressing HIV-1, ABCA1 was re-localized to the cell periphery (p24+ cells in Figure 4D). This re-localization of ABCA1 to the plasma membrane was even more pronounced in Nef-transfected murine macrophages RAW 264.7 (compare Figure 4E and 4F). No re-localization of ABCA1 was observed in macrophages transfected with Nef.G2A (Figure 4G). Therefore, Nef expression induces re-localization of ABCA1, which requires myristoylation of Nef.
Previous studies demonstrated that apoA-I specifically binds to ABCA1 at the cell surface [40-42]. It was also suggested that trafficking of apoA-I to intracellular cholesterol pools correlates with trafficking of ABCA1 [39,43]. Consistent with re-localization of ABCA1 to the plasma membrane, the specific binding of [125I]apoA-I to Nef-transfected RAW 264.7 macrophages was increased (Figure 4H, left panel). However, internalization of [125I]apoA-I was almost completely blocked, supporting the model whereby Nef impairs intracellular trafficking of ABCA1 (Figure 4H, right panel). Degradation of [125I]apoA-I was negligible and was not affected by Nef (unpublished data).
Therefore, Nef-dependent changes in intracellular distribution of ABCA1 may be another mechanism responsible for impairment of cholesterol efflux.
Nef Interacts with ABCA1
Nef has been shown to modulate expression of several trans-membrane proteins. In some cases (e.g., with CD4 or major histocompatibility complex [MHC] I) Nef down-regulates the protein, and in some (e.g., with invariant chain of MHC class II or with dendritic cell-specific ICAM-3-grabbing nonintegrin [DC-SIGN]), it up-regulates the protein's surface expression ([44] and references therein). Some of these effects, including down-regulation of CD4 [45] and MHC I [46], have been shown to depend on an interaction between Nef and the target protein. We therefore tested whether Nef interacts with ABCA1. HeLa cells were co-transfected with Nef or Nef.G2A and FLAG-tagged ABCA1, ABCA1 was immunoprecipitated using anti-FLAG antibody, and immunoprecipitates were analyzed for co-precipitation of Nef. This analysis revealed that Nef co-precipitated with ABCA1, whereas Nef.G2A did not (Figure 5A, upper panel) despite equally high expression of the two forms of Nef (Figure 5A lower panel). We conclude that Nef can interact with ABCA1, and this interaction requires myristoylation of Nef and correlates with the ability of Nef to impair cholesterol efflux. The Nef-specific signal observed in this experiment required high-level expression of participating proteins, likely due to the transitory nature of Nef interaction with ABCA1.
Interaction between ABCA1 and Nef at the plasma membrane was supported by confocal microscopy, which demonstrated co-localization of Nef and ABCA1 in RAW 264.7 cells transfected with Nef.wt-expressing vector (Figure 5B). No co-localization was observed between ABCA1 and Nef.G2A (Figure 5B). This visual analysis was reinforced by an analytical quantification presented in Figure 5C. Indeed, both ABCA1 and the WT Nef proteins are found at the cell periphery, and their co-localization is indicated by overlapping green and blue peaks at either end of the graph. Moreover, both colors peak and valley in tandem, suggesting a correlation in subcellular localization of ABCA1 and WT Nef. No such correlation is observed in ABCA1 and Nef.G2A distribution. Taken together, these results suggest that interaction between ABCA1 and Nef occurs at the cell plasma membrane.
Therefore, both re-localization and down-modulation of ABCA1 depend on its interaction with Nef, which in turn requires myristoylation and membrane localization of Nef.
HIV-Infected Macrophages Transform into Foam Cells
To determine whether impairment of cholesterol efflux by HIV-1 infection leads to cholesterol accumulation and foam cell formation, we loaded macrophages (uninfected or infected with Nef-expressing or ƒĒNef HIV-1) with lipids by incubating with acetylated LDL (AcLDL) in the presence of apoA-I and stained cellular lipids with Oil Red O (Figure 6). This experiment revealed formation of typical lipid-rich cells in cultures infected with Nef-expressing HIV-1, whereas uninfected cells or macrophages infected with ƒĒNef virus accumulated substantially less cholesterol (compare Figure 6A, 6B, and 6C). Analysis by transmission electron microscopy revealed more lipid vacuoles (arrows in Figure 6E) in macrophages infected with Nef-expressing HIV-1 than in uninfected cells or cells infected with ƒĒNef HIV-1 (compare Figure 6D, 6E, and 6F). Cholesterol loading of RAW 264.7 macrophages transfected with Nef also led to accumulation of significantly larger amounts of lipids when compared to cells transfected with an empty vector (compare Figure 6H and 6I). In addition, Nef-transfected RAW 264.7 macrophages demonstrated accelerated cholesteryl ester synthesis, especially when cells were loaded with AcLDL (Figure 6G). Enhanced synthesis of cholesteryl esters is a sensitive indicator of accumulation of cholesterol inside the cells and a key element of foam cell formation.
Measurements of cholesterol mass confirmed substantially higher cholesteryl ester content in Nef-transfected RAW 264.7 macrophages compared to mock-transfected cells (Figure 7A); there was also more free cholesterol in the transfected macrophages (Figure 7B). Synthesis of triglycerides was not affected (Figure 7C), indicating that increased cholesteryl ester synthesis and content is a consequence of increased concentration of cholesterol rather than of fatty acids. The increased cholesterol content in Nef-transfected cells was not caused by differences in AcLDL uptake, as the latter was similar between Nef-transfected and mock-transfected cells (Figure 7D). To accommodate the increasing amounts of cholesteryl esters, cells would require an additional amount of phospholipids, and, indeed, the efflux of phospholipids was inhibited (Figure 2A), whereas phospholipid synthesis was accelerated in Nef-transfected cells (Figure 7E). Taken together, these results indicate that HIV-1 infection, via Nef expression, impairs reverse cholesterol transport in macrophages and leads to accumulation of lipids and formation of foam cells.
HIV-Positive Foam Cells in Atherosclerotic Plaques of HIV-Infected Patients
Our finding that HIV-1 infection of macrophages impairs cholesterol efflux from these cells suggests that HIV-infected macrophages may potentially contribute to the development of atherosclerotic plaques, especially when combined with dyslipidemia found in PI-treated patients. Immunostaining of sections of atherosclerotic plaques obtained from highly active antiretroviral therapy (HAART)-treated HIV-infected patients demonstrated the presence of p24+ macrophages (Figure 8A, 8B, 8E, and 8F). In areas surrounding lipid cores, some p24+ cells displayed a typical foam cell appearance (Figure 8B). Analysis of parallel consecutive sections stained with anti-CD68 showed that these p24+ cells were located in areas composed of CD68+ cells (Figure 8C), indicating the macrophage nature of p24+ foam cells. Double immunostaining confirmed this notion by demonstrating the association of p24 staining with CD68+ macrophages and macrophage foam cells (Figure 8E and 8F). These findings indicate that HIV-infected, cholesterol-loaded macrophages are present in the atherosclerotic plaque and therefore may potentially be involved in pathophysiological events leading to the development of atherosclerosis.
Active Cholesterol Efflux Reduces Infectivity of HIV Virions
To determine whether impairment of cholesterol efflux has a role in HIV biology, we compared infectivity of HIV virions produced from monocyte-derived macrophages stimulated or not with an LXR agonist, TO-901317. We hypothesized that if impairment of cholesterol efflux is a specific mechanism to increase HIV replication, then agents counteracting this effect should have anti-HIV activity. LXR agonists up-regulate expression of ABCA1 at a transcriptional level and stimulate cholesterol efflux from various cell types, including human monocyte-derived macrophages ([47] and Figure 9A). When added to HIV-infected macrophages at day 7 after infection and kept with cells for another 7 d (to allow ABCA1 to accumulate and overcome Nef-mediated inhibition), LXR agonist prevented the impairment of cholesterol efflux by HIV-1 infection (Figure 9A); in fact, cholesterol efflux from TO-901317-treated HIV-infected macrophages was similar to efflux from uninfected cells stimulated with the LXR agonist. The lack of HIV-specific reduction of cholesterol efflux is likely due to overproduction of ABCA1, which exceeds production of Nef. Virions were collected from TO-901317-treated and untreated cells, adjusted according to p24 content, and analyzed for infectivity using indicator P4-CCR5 cells. This analysis revealed a substantial reduction (by about 80%) of infectivity of virions produced from macrophages treated with LXR agonist (Figure 9B). Interestingly, protein composition of the virions produced from LXR agonist-treated and untreated cells was very similar (unpublished data), whereas virion-associated cholesterol was significantly diminished in virions produced from TO-901317-treated macrophages (Figure 9C). These results suggest that stimulation of cellular cholesterol efflux may be an effective approach to suppressing HIV replication.
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