Mechanisms of anti-HIV activity of CAP treatment.

<p>A1. MDM treated with CAP or helium (control) were analyzed by flow cytometry for expression of CCR5 and CD4. A2. Results are presented as mean ± SEM for analysis performed for MDM from two donors. P values (relative to control) were calculated using Student’s unpaired two-tail <i>t</i>-test. B. Fusion between HIV-1 and MDM was analyzed by fluorescence resonance energy transfer-based fusion assay. Cleavage of CCF2 represents virus-cell fusion. C. MDM treated with CAP or helium (control) were infected in triplicate wells with HIV-1 ADA (R5 virus) or VSV-G-psudotyped HIV-1 NL4-3 (X4 virus), and viral production was measured on day 7 by RT activity. Results are presented as mean ± SD. *p = 0.0037, **p = 0.027 relative to control, calculated using Student’s unpaired two-tail <i>t</i>-test. D. MDM treated with CAP or helium (control) were infected with HIV-1 ADA and incubated for 21 days. Virus was collected, adjusted to the same RT activity by dilution, and used to infect indicator TZM-bl cells. Results are presented as mean ± SD for 5 independent replicates. ****p<0.0001 by Student’s unpaired two-tail <i>t</i>-test.</p

Analysis of CAP cytotoxicity.

<p>A. Morphology of MDM after CAP treatment. Macrophages in the CAP-treated area were imaged 1 h and 24 h after treatment (45 sec at 4.5 kV). B. Cell viability was assessed by MTT assay. MTT conversion to formazan was measured at OD₅₇₀.</p

CAP effects on HIV-1 replication.

<p>A. Monocyte-derived macrophages treated with CAP or helium (control) were infected with HIV-1 ADA and viral replication was monitored for 15 days by RT activity in the culture supernatant. B. Results (mean±SD) are presented for RT analysis on day 15 after infection (performed as in panel A) for 7 different donors. ****p<0.0001 by Student’s unpaired two-tail <i>t</i>-test. C. MDM infected with HIV-1 ADA as in A were analyzed 4 h post infection by qPCR for positive-strand cDNA. Cells treated with AZT (3 μM) and uninfected cells are shown as negative controls. Results are presented as mean±SD for four independent infections with cells from one representative donor. ***p = 0.0004 by Student’s unpaired two-tail <i>t</i>-test. D. HIV-infected MDM were analyzed 48 h post-infection by Alu-GAG qPCR for integrated proviral DNA. Results are presented as mean±SD for four independent infections with cells from one representative donor. *p = 0.0494 by Student’s unpaired two-tail <i>t</i>-test. E. HIV-1 ADA was treated with CAP or helium (control) and used to infect MDM. Virus infection was assessed by measuring reverse transcriptase activity in culture supernatant on day 10–15 post-infection. Results (mean±SD) are presented for 6 experiments with MDM from independent donors. ***p<0.001 by Student’s unpaired two-tail <i>t</i>-test.</p

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