Vpr associates with proteasome in fission yeast and mammalian cells.

<p><b>A</b>. Vpr is displaced from the nuclear membrane by overproduction of Uch2. Fission yeast cells carrying or not carrying Uch2 were stained with DAPI 17 hrs after <i>vpr</i> gene induction. Green color, GFP; Blue color, nuclear DNA. <b>B</b>. Vpr is displaced from the nuclear membrane in the <i>cut8</i> mutant. Mts4, a fission yeast homologue of mammalian S2, is a 19S proteasome-associated protein. Cut8 displays normal phenotype at the permissive 25°C, but shows mutant phenotype at non-permissive 37°C. <b>C</b>. Co-migration of Vpr with proteasome in fission yeast cells (<b>i</b>) and HeLa cells (<b>ii</b>) analyzed by glycerol gradient. Extracts from fission yeast cells expressing <i>vpr</i> were fractionated by centrifugation on a 10–40% glycerol gradient. Equal amounts of proteins from each fraction of the gradient were separated on 12% SDS-PAGE and probed with antibodies against Vpr and 19S (Mts4) subunits of the proteasome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011371#pone.0011371-Wilkinson2" target="_blank">[34]</a>. Lanes 1–8 indicates different fractions collected from the top (low molecular weight) to bottom of the gradient (high molecular weight). Note that not all fractions are shown here. <b>D.i</b>. Co-immunoprecipitation shows interaction of Vpr with Mts2 in yeast cells. IP was carried out with anti-HA as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011371#pone.0011371-Huard1" target="_blank">[59]</a>. A HA-tag alone plasmid control was used in this experiment. The recovered proteins were fractionated on SDS-PAGE and immunoblotted with anti-HA, anti-Vpr and anti-Mts2 antibodies. CL, cell lysates; IP:HA, immunoprecipitation with a HA-tagged control plasmid; IP:HA-Vpr, immunoprecipitation with a HA-Vpr carrying plasmid. <b>ii</b>. HeLa cells were transfected with HA-Vpr or HA-Kir2.1 (control). Kir2.1 is an irrelevant protein to Vpr and used here as a control. IP was carried out with anti-HA, recovered proteins were fractionated on SDS-PAGE and immunoblotted with anti-S2 (a mammalian homologue of fission yeast Mts4) and anti-S5a antibodies. <b>iii</b>. HeLa cells were co-transfected with pSG5-ZZ-β1, which codes for a proteasomal β1subunit <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011371#pone.0011371-Klare1" target="_blank">[38]</a>, or control pSG5-ZZ plasmid (Ctr) together with HA-Vpr. The protein A-tagged β1 or control protein were pulled down by anti-protein A antibody, then blotted with anti-HA antibody.</p

Proposed model of Vpr-proteasome interactions.

<p>Details are in the text.</p

Fission yeast strains, human cell lines, plasmids and HIV-1 viral stocks.

<p>Fission yeast strains, human cell lines, plasmids and HIV-1 viral stocks.</p

hHR23A is required for Vpr-mediated stimulation of HIV-1 replication in non-dividing MAGI-CCR5 cells and macrophages.

<p><b>A.i.</b> Cell proliferation of MAGI-CCR5 cells in different FBS concentration. MAGI-CCR5 cells were plated at 7,000 cells/well in DMEM containing 10% or 0.1% of FBS. Cellular proliferation was measured over a period of 5 days. Cells in 0.1% FBS were viable over the experimental period, as they remained adherent to plates. <b>ii.</b> Depletion of hHR23A significantly reduces viral replication in non-dividing MAGI cells in a Vpr-dependent manner. MAGI-CCR5 cells were transfected with hHR23A-targeting (+) or control (−) siRNA, plated in DMEM with 10% or 0.1% FBS, and infected with HIV-1_Ada Vpr(+) or Vpr(−) 24 hrs after hHR23A knockdown. Viral replication was evaluated 48 hrs after infection by staining; blue cells were counted as infected. Results are presented as percent of control, i.e., the number of blue cells in cultures transfected control siRNA and infected with Vpr-positive HIV-1 and show average ± SE of quadruplicate determinations. <b>B.</b> Vpr-dependent HIV-1 viral replication in macrophages is mediated through hHR23A. <b>i.</b> Monocyte-derived macrophages pretreated with hHR23A siRNA or control (Ctr) siRNA were infected with HIV-1_Ada (Vpr+) or (Vpr−) viruses. Cells were collected 72 hrs <i>p.i.</i> and viral replication was determined by measuring p24. Results are presented as inhibition of HIV-1 replication in cells treated with hHR23A siRNA relative to cells treated with control siRNA, and show mean ± SE of three independent experiments with cells from different donors, each performed in triplicate. Statistical analysis was performed using Student's t-test, and <i>p</i> value is shown. <b>ii.</b> Monocyte-derived macrophages were transfected with hHR23A siRNA or control siRNA. Cells were collected 72 hrs <i>p.t.</i> and subjected to Western blot analysis using anti-Rad23A and anti-β-actin antibodies.</p

hHR23A is critical for Vpr-proteasome interaction.

<p><b>A</b>. <i>In vitro</i> and <i>in vivo</i> interactions of Rhp23 with HIV-1 Vpr. (<b>i</b>) Rhp23, a fission yeast homologue of mammalian hHR23A<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011371#pone.0011371-Elder1" target="_blank">[15]</a>, interacts with Vpr in the yeast two-hybrid system. The <i>vpr</i> gene was inserted into the pGBT9 plasmid and <i>rph23</i> was fused to the activation domain in the pGAD-GH plasmid. The interaction was measured by β-galactosidase assay on cell extracts. (<b>ii</b>) <i>In vitro</i> interaction of Vpr with Rhp23. Bacterial cell lysates over-expressing GST and GST-Vpr proteins were immobilized on GST-agarose beads. <i>In vitro</i> translated 35S-labeled Rhp23 (shown by arrow) was incubated with the immobilized GST and GST-Vpr. The Coomassie staining (low panel) shows total proteins, and autoradiography (top panel) shows bound 35S-labeled Rhp23. <b>B</b>. Interaction of Rph23 with proteasome in the presence or absence of Vpr. HA-Rhp23-carrying plasmid was transfected into fission yeast in the presence or absence of Vpr. Following immunoprecipitation with anti-HA antibody, precipitates were tested with anti-Mts4, which recognizes the 19S regulatory subunit of the proteasome. <b>C</b>.<b>i</b>. Depletion of hHR23A abolished the interaction of Vpr with proteasome in HeLa cells. The HA-Vpr or HA-Kir2.1 (control) expressing vectors were transfected into HeLa cells with (lane marked 23A) or without hHR23A depletion by siRNA. The HA-tagged proteins were pulled down by anti-HA antibody, and then blotted with anti-S2 and anti-S5a antibodies, which recognize S2 and S5a, respectively, of the 19S regulatory subunits of the proteasome. <b>ii</b>. 50 µg of supernatants from lanes 2 (None) and 3 (23A) of <b>a</b> were blotted with anti-Rad23A or β-actin antibody. <b>D</b>. Vpr promotes protein poly-ubiquitination <i>via</i> hHR23A. Flag-tagged hHR23A was co-expressed with HA-ubiquitin in the presence or absence of Vpr in HeLa cells. Forty-eight hours after transfection, cells were collected and cell extracts were subject to Western blot analysis using indicated antibodies. hHR23(c57) is a non-functional mutant derivative of hHR23A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011371#pone.0011371-Zhu1" target="_blank">[43]</a>. MG132 was used to inhibit the proteasome activities.</p

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

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

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

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