HIV-1 Nef Impairs Cholesterol Efflux from Macrophages

<div><p>(A) Monocyte-derived macrophages were inoculated with indicated HIV-1 strains equalized according to RT activity and cultivated for 21 d (RT activity in the culture supernatants on day 21 is shown beneath the bars). Mock-infected cells were incubated with virus-free medium. Specific cholesterol efflux to apoA-I (30 μg/ml) was performed for 12 h and analyzed as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040365#s4" target="_blank">Materials and Methods</a>. Results are presented as percentage of efflux from mock-infected cells (taken as 100%) and are mean ± standard deviation (SD) of triplicate determinations. An asterisk (*) indicates <i>p</i> < 0.001</p> <p>(B) Monocyte-derived macrophages were inoculated with VSV-G-pseudotyped HIV-1 SF2 either deficient in Nef (SF2.ΔNef) or carrying WT Nef (SF2.wt). Specific cholesterol efflux to apoA-I (30 μg/ml) was analyzed on day 6 after inoculation using the same procedure as described in (A). p24 concentration in the culture medium is shown beneath the bars. Results are presented as percentage of efflux from mock-infected cells (taken as 100%) and are mean ± SD of triplicate determinations. An asterisk (*) indicates <i>p</i> < 0.001.</p> <p>(C) RAW 264.7 cells were transfected with plasmids expressing indicated Nef variants or an empty vector (mock-transfection). Twenty-four hours after transfection, LXR agonist, TO-901317 (1 μmol/L), was added. Cholesterol efflux to apoA-I (30 μg/ml) was performed for 3 h with cells 48 h after transfection as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040365#s4" target="_blank">Materials and Methods</a>. Means ± SD of quadruplicate determinations are shown. An asterisk (*) indicates <i>p</i> < 0.001.</p> <p>(D) Immunoblotting of RAW 264.7 cells transfected with empty vector (mock), WT Nef derived from HIV-1 strain SF2 (Nef.wt), or Nef.G2A mutant, and stained with anti-Nef antibodies.</p></div

The Effect of Nef on ABCA1 Localization

<div><p>(A–D) On day 5 after infection with VSV-G-pseudotyped Nef-expressing (B and D) or ΔNef (A and C) HIV-1 SF2, cells were co-stained with anti-p24 mouse monoclonal and anti-ABCA1 rabbit polyclonal antibodies, followed by FITC-conjugated anti-mouse (A and B) and Cy5-conjugated anti-rabbit IgG (C and D). Arrows point to cells with re-localized ABCA1. The scale bars represent 20 μm.</p> <p>(E–G) Distribution of ABCA1 revealed by staining with monoclonal anti-ABCA1 antibody and FITC-conjugated anti-mouse IgG in RAW 264.7 cells transfected with empty vector (E), WT Nef derived from SF2 HIV-1 (<i>Nef.wt,</i> panel [F]), or SF2 Nef carrying a G2A mutation (<i>Nef.G2A</i>, [G]). Insets in (E and F) show cross-section of the image reconstituted from serial sectioning. Scale bars represent 20 μm.</p> <p>(H) [¹²⁵-I]apoA-I binding (left panel) and internalization (right panel) in RAW 264.7 macrophages transfected with HIV-1 SF2-derived Nef. An asterisk (*) indicates <i>p</i> < 0.01.</p></div

Cholesterol Efflux and Infectivity of HIV Virions

<div><p>Human monocyte-derived macrophages were infected with HIV-1 ADA or mock-infected, and 7 d after infection were treated or not treated with LXR agonist, TO-901317 (500 nM), for seven more days.</p> <p>(A) Cholesterol efflux to apoA-I was measured on day 21 after infection. An asterisk (*) indicates <i>p</i> < 0.01 (versus uninfected cells not treated with TO-901317); a number sign (#) indicates <i>p</i> < 0.01 (versus HIV-infected cells not treated with TO-901317).</p> <p>(B) Virions were collected from culture supernatants of LXR agonist-treated and untreated (control) cells on day 10 and day 14 (pooled together), adjusted according to p24 content, and analyzed for infectivity on indicator P4-CCR5 cells. Experiment was performed in triplicate, and results (mean ± SD) are presented as percent infectivity of virions produced by control cells; an asterisk (*) indicates <i>p</i> < 0.001.</p> <p>(C) Incorporation of [³H]cholesterol into virions produced by LXR agonist-treated and untreated (control) cells was measured in triplicate, and results (mean ± SD) are presented relative to cholesterol in the virions produced by control cells; an asterisk (*) indicates <i>p</i> < 0.001.</p></div

Nef Targets ABCA1-Dependent Cholesterol Efflux

<div><p>(A) Cholesterol efflux to HDL (30 μg/ml) was measured from HIV-1 ADA-infected and mock-infected macrophages used also to measure efflux to apoA-I in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040365#pbio-0040365-g001" target="_blank">Figure 1</a>A.</p> <p>(B) Impairment of phospholipid efflux in Nef-transfected RAW 264.7 cells. RAW 264.7 cells were transfected with plasmid expressing HIV-1 SF2-derived Nef or empty vector (mock-transfection). Phospholipid efflux to apoA-I (30 μg/ml) was measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040365#s4" target="_blank">Methods</a>. Means ± SD of quadruplicate determinations are shown. An asterisk (*) indicates <i>p</i> < 0.001.</p> <p>(C) Nef does not decrease cholesterol efflux in RAW 264.7 cells not treated with LXR agonist. Experiment was performed using HIV-1 SF2-derived Nef as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040365#pbio-0040365-g001" target="_blank">Figure 1</a>C, except that LXR agonist was not added.</p> <p>(D) Cholesterol efflux to apoA-I from HeLa cells. HeLa cells were either mock-transfected (mock) or co-transfected with ABCA1 and empty vector (ABCA1), or vector expressing Nef derived from HIV-1 SF2 (ABCA1 + NefSF2) or LAI strains (ABCA1 + NefLAI); cholesterol efflux to apoA-I was analyzed. An asterisk (*) indicates <i>p</i> < 0.001 (versus cells without ABCA1); a number sign (#) indicates <i>p</i> < 0.001 (versus cells without Nef). Expression of Nef determined by Western blot is shown beneath the bars.</p> <p>(E) Cholesterol efflux to HDL from HeLa cells. Experiment was performed as in (D), except that ABCG1 was used instead of ABCA1, and HDL (30 μg/ml) instead of apoA-I was used as cholesterol acceptor. An asterisk (*) indicates <i>p</i> < 0.01 (versus cells without ABCG1).</p></div

Nef Induces Down-Modulation of ABCA1

<div><p>(A) Human monocyte-derived macrophages were infected with HIV-1 ADA or mock-infected and cultured for 14 d (RT in culture supernatant was 4,000 cpm/μl). ABCA1, ABCG1, SR-B1, and β-actin (loading control) were analyzed by Western blotting.</p> <p>(B) RAW 264.7 cells were transfected with vector expressing HIV-1 SF2-derived Nef (either WT or carrying a G2A mutation) or empty vector (mock). Twenty-four hours after transfection, cells were stimulated with TO-901317 (1 μM) and 24 h later, were analyzed by Western blotting for ABCA1 and β-actin (loading control).</p> <p>(C) ABCA1 RNA from HIV-infected macrophages used for Western blotting in (A) was analyzed by real-time RT-PCR. Results were adjusted according to β-actin signal and are presented in arbitrary units; an asterisk (*) indicates <i>p</i> < 0.01 (versus mock).</p> <p>(D) RNA was extracted from non-activated RAW 264.7 cells (control), mock-transfected RAW cells activated with LXR agonist TO-901317 (LXR), or cells transfected with SF2-derived Nef and activated with TO-901317 (LXR+NefSF2), and analyzed by real-time RT-PCR. Results were adjusted according to 28S RNA signal and are presented in arbitrary units; an asterisk (*) indicates <i>p</i> < 0.01 (versus LXR agonist-treated, mock-transfected cells).</p></div

Identification of HIV-1–Positive Macrophages in Atherosclerotic Plaques of HIV-Infected Subjects.

<div><p>Single (A–D) and double (E and F) immunostaining of aortic wall segments.</p> <p>(A) p24 staining. A low-magnification image showing the presence of p24⁺ cells in an area adjacent to the plaque lipid core. The scale bar represents 100 μm.</p> <p>(B) Detail of (A). p24⁺ cells show a characteristic morphology of foam cells. The scale bar represents 10 μm.</p> <p>(C) CD68 staining. CD68⁺ cells were identified in a parallel consecutive section to that shown in (A). The scale bar represents 100 μm.</p> <p>(D) Negative control (staining with an irrelevant primary antibody). The scale bar represents 100 μm.</p> <p>(E) Double immunostaining showing the co-localization of p24 (brown) with CD68 (rose). Immunostaining included a combination of a rabbit polyclonal anti-p24 antibody in the peroxidase–anti-peroxidase system with DAB chromogen yielding a brown reaction product, and a mouse monoclonal antibody to CD68 in the alkaline phosphatase–anti-alkaline phosphatase system with Fast Red chromogen, resulting in a rose precipitate. Counterstaining was with Mayer's hematoxylin. The scale bar represents 50 μm.</p> <p>(F) A detail of (E). The scale bar represents 15 μm.</p></div

Accumulation of Lipids in Cells Infected with HIV-1 or Transfected with Nef

<div><p>(A–C) Oil Red O staining of HIV-infected macrophages. Uninfected (A) macrophages or cells infected with VSV-G–pseudotyped Nef-positive (B) or ΔNef (C) HIV-1 SF2 variants were loaded with cholesterol on day 3 after infection by incubating with AcLDL in the presence of apoA-I, and lipids were stained with Oil Red O 24 h later. p24 concentration in the culture supernatant on day 3 after infection was 4.7 ng/ml for cells inoculated with Nef-positive virus and 9.8 ng/ml for the culture inoculated with ΔNef HIV-1.</p> <p>(D–F) Electron microscopy of cholesterol-loaded uninfected macrophages (D) and cells infected with Nef-positive (E) and ΔNef (F) HIV-1 AD8 performed 14 d after infection. Uninfected cells have small numbers of electron-lucent lipid vacuoles (arrows). The cytoplasm of cells infected with Nef-positive virus is filled with electron-dense lipid vacuoles (arrows). Cells infected with ΔNef virus have small numbers of electron-lucent lipid vacuoles (arrows), similar in number to those in uninfected cells. The scale bars represent 5 μm.</p> <p>(G) The effect of Nef on cholesteryl ester synthesis. The rate of cholesteryl ester synthesis in RAW 264.7 cells transfected with an empty vector (mock-transfected) or Nef-expressing construct and incubated with or without AcLDL in the presence of apoA-I or 5% human plasma is presented as mean ± SD of quadruplicate determinations. An asterisk (*) indicates <i>p</i> < 0.02.</p> <p>(H) and (I) RAW 264.7 cells were transfected with empty vector (H) or Nef-expressing construct (I), stimulated with LXR agonist, incubated with AcLDL and lipid-free apoA-I, fixed with formaldehyde, and stained with Oil Red O.</p></div

Analysis of Lipids in RAW 264.7 Macrophages Transfected with Nef

<div><p>(A) Cholesteryl ester content after 24 h incubation with AcLDL (50 μg/ml) determined by enzymatic assay; an asterisk (*) indicates <i>p</i> < 0.01.</p> <p>(B) Free cholesterol content after 24 h incubation with AcLDL (50 μg/ml) determined by enzymatic assay; an asterisk (*) indicates <i>p</i> < 0.05.</p> <p>(C) Triglyceride biosynthesis after 24 h incubation with AcLDL (50 μg/ml) measured as incorporation of [¹⁴C]oleic acid into triglycerides as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040365#s4" target="_blank">Materials and Methods</a>.</p> <p>(D) Uptake of AcLDL was calculated as a sum of ¹²⁵I-AcLDL specifically taken up and degraded by cells.</p> <p>(E) Phospholipid biosynthesis measured as incorporation of [¹⁴C]choline into phospholipid fraction as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040365#s4" target="_blank">Materials and Methods</a>; an asterisk (*) indicates <i>p</i> < 0.01.</p></div