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

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

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

Ultrastructural defects in <i>Abca12^el12/el12</i> mice.

<p>Thin sections of <i>Abca12^el12/el12</i> epidermis illustrated hyperkeratosis and expansion of the stratum granulosum (A). Nile red staining shows reduced intercellular lamellae lipids at E18.5 (B). In wild type epidermis intercellular lipid lamellae (white arrow and inset) were noted as well as LBs fusing with the surface of granular cells (red arrow) (C). Lamellar bodies in wild type and heterozygous embryonic epidermis were normally loaded with lipid (D). In mutant skin, LBs lacked lamellar cargo (E, F arrowheads) but fused with the granular cell membrane (F; arrows). Mutant epidermis had a normal cornified envelope (G,H) with persistent corneodesmosomes in distal layers of the stratum corneum (G, H, red arrowheads) and the cornified layer had multiple lipid inclusions (G, black arrowheads). Unlike the uniform contents of wild type cornified cells mutant cell layers contained vesicular fibrillar structures (I, red arrows) and frequent inclusion bodies (I, black arrows). EM scale bars in C–F, H, I equal 200 nm and 2 µm in G. C–F and I were stained with ruthenium tetroxide, G and H with osmium tetroxide.</p

Pathology of the Abca12 mutant epidermis.

<p>Abca12 protein is detected in suprabasal keratinocytes in the granular and cornified cell layers of mutant and wild type epidermis (A). Keratinocytes in Abca12 mutant skin undergo premature differentiation highlighted by strong filaggrin expression in cells juxtaposed to K14⁺ basal keratinocytes (B) and increased co-expression of keratins 10 and 14 (white arrows), especially in basal cells (yellow arrows) (C). Increases in co-expression of K10 and K14 are significant at both E18.5 (p = 0.016) and at P1 (p = 4.44×10⁻⁶) (D). Samples are of E18.5 (A) or P1 (B, C) epidermis counterstained with DAPI. Scale bars = 30 µm.</p