Nef Activates Rac in Resting CD4⁺ T Lymphocytes

<div><p>(A) HIV-1 Nef activates Rac in Jurkat T cells. Jurkat T cells (lane 1) were transduced with a control empty vector (FUGW; lane 2) or the same vector expressing HIV-1 NA7 Nef (FUGWCNA7; lane 3). Rac_GTP was precipitated from cell extracts with recombinant PAK1 PBD–GST. PBD–GST bound Rac_GTP (top), total Rac present in extracts (middle), and Nef (bottom) were detected by immunoblotting.</p> <p>(B) Flow cytometric analysis of Gag and CD4 expression in resting CD4⁺ T lymphocytes transduced with HIV-1 derived vectors in the presence of IL-7. Percentages of cells productively infected with <i>nef</i>-deleted H-Δ vector (boxed area in middle panel) or with HIV-1 NA7 <i>nef</i> containing H-NA7 vector (right panel) are shown. Results obtained with uninfected control CD4⁺ T cells cultured in the presence of IL-7 are also shown (left panel).</p> <p>(C) HIV-1 Nef specifically activates Rac in resting primary CD4⁺ T lymphocytes. Rac_GTP and CDC42_GTP were precipitated with PAK1 PBD–GST from extracts prepared from CD4⁺ T lymphocytes transduced with HIV-1 derived vectors, shown in (B), and analyzed as described in (A).</p></div

HIV-1 Nef Disrupts CCR5-Mediated Migration

<div><p>(A) HIV-1 Nef does not downregulate CCR5. Flow cytometric analysis of CCR5 and GFP in Jurkat T cells transiently expressing GFP alone (panel 1) or CCR5 and GFP in the absence (panel 2) and presence (panel 3) of HIV-1 Nef. Histograms of CCR5 expression for cell populations within a single GFP fluorescence intensity interval indicated by the rectangle in panel 1 are shown in panels 4 to 6, respectively.</p> <p>(B) Percentage of cells migrated to MIP-1β and expressing GFP alone (open circle), GFP and CCR5 (open triangle), or GFP, CCR5 and HIV-1 Nef (filled triangle) is shown as a function of GFP fluorescence intensity.</p></div

DOCK2, ELMO1, and Rac Are Abundant Nef-Associated Proteins in T Cells

<div><p>(A) Schematic representation of epitope-tagged HIV-1 Nef (NA7-hf). The structured regions of Nef are boxed and the disordered regions, as determined by X-ray crystallography and NMR studies, are shown by a thin line. The locations of the N-terminal myristoyl moiety, prolines P72 and P75 in the PP-II helix, arginine R106, leucines L164 and L165 (LL164), and the C-terminal HA-FLAG epitopes are indicated.</p> <p>(B) DOCK2, ELMO1, and Rac2 specifically copurify with HIV-1 Nef from Jurkat T cells. Jurkat T cells (1.8 ×10¹⁰) stably expressing NA7-hf (lane 3) or control Jurkat cells (lane 2) were subjected to the two-step immunopurification procedure described in the text (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020006#s4" target="_blank">Materials and Methods</a>). Polypeptides present in purified immune complexes were resolved by SDS-PAGE and analyzed by LC/MS/MS. We identified 58 DOCK2-specific peptides covering 869 out of 1830 total amino acid residues (47.5% coverage, expectation value 6.0 × 10^–130), 10 ELMO1-specific peptides covering 122 out of 727 total amino acid residues (16.8% coverage, expectation value 1.0 × 10⁻¹⁰), and three Rac-specific (two of which were Rac2-specific) peptides covering 26 out of 192 total amino acid residues (13.5% coverage, expectation value 4.6 × 10⁻⁴). Bands corresponding to DOCK2, ELMO1, Rac2 and their predicted molecular weights, NA7-hf Nef, and the FLAG peptide used for elution are indicated.</p></div

Nef Disrupts T Cell Migration to SDF-1

<div><p>(A) Migration of cell populations shown in (B) expressing GFP (open circle), ectopic CXCR4, and GFP (filled circle), HIV-1 NA7 Nef and GFP (open box), HIV-1 NA7 Nef, ectopic CXCR4 and GFP (filled box) to SDF-1 was measured in transwell assays.</p> <p>(B) Transient expression of ectopic CXCR4 restores CXCR4 levels on the surface of Nef-expressing cells. Flow cytometric analysis of Jurkat T cells transiently expressing GFP (panel 1) or HIV-1 NA7 Nef and GFP (panel 2) and together with ectopic CXCR4 (panels 3 and 4, respectively).</p></div

Nef Potentiates Rac Activation through Association with DOCK2–ELMO1

<div><p>(A and B) Myristoylation signal, P72,P75, and R106 in Nef are required for Rac activation. Rac_GTP and total Rac in the extracts prepared from Jurkat T cells transduced with lentiviral vectors expressing no Nef (−) or the indicated Nef proteins (A) and HEK 293 cells transiently coexpressing the indicated Nef mutants together with DOCK2, ELMO1, and Rac2 (B) were visualized as described in the legend to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020006#pbio-0020006-g003" target="_blank">Figure 3</a> and quantified by direct imaging of chemiluminescent signals. The fraction of total Rac present in the extracts that was PBD–GST bound is shown in the histograms. Data in the histogram shown in (B) are averages of three independent experiments and error bars represent two standard deviations.</p> <p>(C) Myristoylation signal, P72,P75, and R106 in Nef are required for association with DOCK2, ELMO1, and Rac2. The ability of selected Nef mutants to associate with DOCK2, ELMO1, and Rac2 was determined as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020006#pbio-0020006-g002" target="_blank">Figure 2</a>.</p></div

Lentiviral Nef Binds the DOCK2–ELMO1–Rac Complex

<div><p>(A) HIV-1 Nef binds the DOCK2–ELMO1–Rac2 complex. His-DOCK2, Myc-ELMO1, and Myc-Rac2 alone (lanes 1, 3, and 5) or together with NA7-hf Nef (lanes 2, 4, and 6) were transiently expressed in HEK 293 cells as indicated. DOCK2 was precipitated from extracts (lanes 1 and 2) with Ni–NTA resin (lanes 3 and 4). Nef–DOCK2 was then precipitated with anti-FLAG affinity gel (lanes 5 and 6), and the epitope-tagged proteins were detected by immunoblotting and visualized by enhanced chemiluminescence.</p> <p>(B) Rac1 associates with HIV-1 Nef. Nef and associated proteins were isolated from extracts of HEK 293 cells transiently expressing DOCK2, ELMO1, and Rac1 either alone (lanes 1 and 4), with NA7-hf (lanes 2 and 5), or with a Nef variant containing a disrupted myristoylation signal (lanes 3 and 6). Nef and associated proteins were detected in anti-FLAG immunoprecipitates (lanes 1–3) and in extracts (lanes 4–6) by immunoblotting.</p> <p>(C) The interaction with DOCK2, ELMO1, and Rac2 is a conserved function of lentiviral Nef proteins. The ability of selected hf-tagged HIV-1 (lanes 1–3 and 5) and SIV mac239 (lane 4) Nef proteins to bind DOCK2, ELMO1, and Rac2 was determined as described in the legend to (B) above. The protein band in (C) indicated by the asterisk is the heavy chain of anti-FLAG mAb.</p></div

Effect of Nef on IL-2 Expression in HIV-1-Infected CD4⁺ T Lymphocytes Stimulated through CD3 and CD28

<p>CD4⁺ T lymphocytes transduced with H-Δ and H-NA7 HIV-1-derived vectors were not stimulated (unstimulated) or stimulated with immobilized anti-CD3 and anti-CD28 mAbs (anti-CD3, anti-CD28) in the presence of Golgi-Stop for 5 h and stained for intracellular IL-2 and p24 Gag. Percentages of IL-2-positive and IL-2-negative cells in the Gag-negative and Gag-positive populations are shown.</p

ELMO1 and DOCK2 Mediate Rac Activation by HIV-1 Nef

<div><p>(A) ELMO1 is required for Rac activation by Nef in NS1 cells. Rac_GTP and total Rac in the extracts prepared from ELMO1-deficient NS1 cells (lanes 1 and 2) and ELMO1-expressing NS1 cells (lanes 3 and 4) following transduction with a lentiviral vector expressing HIV-1 Nef (lanes 2 and 4) or a control empty vector (lanes 1 and 3) were visualized as described in the legend to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020006#pbio-0020006-g003" target="_blank">Figure 3</a>.</p> <p>(B) Nef activates Rac through DOCK2 and ELMO1 in HEK 293 cells. Rac_GTP and total Rac in the extracts prepared from HEK 293 cells coexpressing the indicated proteins were visualized as described above.</p></div

Nef Disrupts Chemotaxis by Activating Rac through DOCK2–ELMO1

<div><p>(A and B) Jurkat T cells expressing wild-type or mutant HIV-1 Nef proteins and GFP reporter were used in transwell chemotaxis assays with SDF-1. Percentage of migrated cells expressing GFP alone (open circle), or together with HIV-1 NA7 (open square), NA7_(G2_^∇_HA) (open diamond), NA7_(P72A,P75A) (open triangle), NA7_(R106A)(filled circle), and NA7_(LL164AA) (filled triangle) is shown as a function of GFP fluorescence intensity in (A) and in (B) for the single GFP fluorescence intensity interval indicated by the shaded rectangle in (A).</p> <p>(C) Constitutively active Rac GTPases disrupt lymphocyte migration to SDF-1. Migration of Jurkat T cells transiently expressing wild-type (Rac1, Rac2), constitutively active (Rac1_G12V, Rac2_G12V), or as a control HIV-1 Nef were also measured. Data shown are averages of three independent experiments and error bars represent two standard deviations.</p></div

Vpx and Vpr have toggled throughout evolution in their requirement for the N- or the C-terminus of SAMHD1.

<p>(A) 293T cells were transfected with HA-SAMHD1, either WT or ΔC (left panel), or WT or chimeric Human-Rhesus SAMHD1 (consisting of residues 1–114 of human SAMHD1 and 115–626 of rhesus SAMHD1, right panels), plus or minus FLAG-Vpr, and degradation was measured by western blotting as described in the legend for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003496#ppat-1003496-g001" target="_blank">Figure 1</a>. Rhesus SAMHD1 was used as all Vpr tested herein can degrade this SAMHD1. Human SAMHD1 was used in N-terminal chimeras as it contains multiple non-synonymous changes compared to other primate SAMHD1. (B) <i>In vitro</i> ubiquitylation of WT, ΔC, ΔN, and ΔN/ΔC DeBrazza's SAMHD1 (consisting of amino acids 115–595, schematic representation in top panels), in the presence or absence of SIVdeb Vpr, SIVmac Vpx, or SIVrcm Vpx. CRL4 and CRL4-DCAF1 alone were used as controls. Experiment preformed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003496#ppat-1003496-g004" target="_blank">Figure 4B</a> and ubiquitylation of each SAMHD1 construct was analyzed by western blotting. Timepoints shown are 0, 15, 30, and 60 minute incubations. (C) Schematic Vpx/Vpr phylogenetic tree as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003496#ppat-1003496-g001" target="_blank">Figure 1A</a> and SAMHD1 diagrams depicting dependence of Vpx/Vpr on the N- or C-terminus of its autologous SAMHD1. Red indicates strong dependence based on all assays. Light pink indicates slight dependence based on co-IP data. Magenta indicates intermediate dependence.</p