Release of Vpr-Expressing Cells from Thymidine Block Leads to Reentry into the Cell Cycle and Vpr-Induced, G₂-Dependent Apoptosis

<div><p>(A) Thymidine-synchronized HeLa cells transduced with pHR-VPR-G or mock-transduced were released from thymidine block and harvested at specified time points postrelease. Cells from each time point were stained with PI and analyzed for DNA content by flow cytometry.</p><p>(B) pHR-VPR–transduced HeLa cells from the same experiment were monitored by Western blot for PARP cleavage at specified time points. Time points labeled under “Thym + pHR-VPR” indicate hours following release from thymidine block; time points under “pHR-VPR” indicate hours following transduction, as these cells were not synchronized.</p><p>(C) Cells treated as those in (A) and (B) were harvested at 24 and 48 h were analyzed for apoptosis by DAPI staining, and the results are quantified.</p></div

The Apoptotic Effect of Vpr Is Lost in Cells Synchronized in G₁/S

<div><p>(A) HeLa cells were transduced with pHR-VPR-G or mock-transduced and then incubated with 2 mM thymidine. After 24 h of incubation, cells were harvested, stained with PI, and analyzed for DNA content by flow cytometry to determine the percentage of cells in G₁/S and G₂/M. Transduction efficiency of pHR-VPR in both thymidine-treated and cycling cells was 70% to 75% as determined by analysis of GFP expression by flow cytometry (unpublished data).</p><p>(B) Cells from experiments shown in (A) were stained for DAPI at 72 h postinfection, in order to evaluate apoptosis via chromatin morphology</p><p>(C) Quantitation of apoptosis in DAPI-stained samples shown in (B); incubation with 25 μM etoposide for 48 h or 1 μM staurosporine for 8 h was included in both cycling and thymidine-treated cells, for comparative purposes.</p><p>(D) Cycling or thymidine-synchronized HeLa cells were mock-transduced or transduced with either pHR-VPR-R or pHR-VPR(R80A) for apoptosis analysis, using FITC-VAD-FMK. As positive controls for apoptosis, cycling and synchronized cells were treated with etoposide or staurosporine as shown in (C).</p><p>(E) Cycling or thymidine-synchronized HeLa cells expressing Vpr or treated with 25 μM etoposide for 48 h were lysed and analyzed by Western blot for PARP cleavage as a marker of apoptosis/caspase activity. The caspase-cleaved PARP band is observed at 89 kDa.</p><p>(F) Cell lysates from pHR-VPR–transduced cells, with or without thymidine block, were harvested at 12 and 24 h post-transduction and analyzed by Western blot for Vpr expression with antibodies specific to the amino-terminal hemagglutinin tag.</p></div

Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages-3

<p><b>Copyright information:</b></p><p>Taken from "Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages"</p><p>http://www.retrovirology.com/content/4/1/84</p><p>Retrovirology 2007;4():84-84.</p><p>Published online 26 Nov 2007</p><p>PMCID:PMC2211753.</p><p></p>he GFP expression vector. A) G2-arrest activity. The DNA content was analyzed 48 h after transfection by flow cytometry on GFP-positive cells after staining with propidium iodide. Results are expressed as the percentage of the GM/G1 ratio relative to that of the wt HA-Vpr. Values are the means of four independent experiments. Error bars represent 1 standard deviation from the mean. B) Pro-apoptotic activity. Cell surface PS exposure was analyzed 72 h after transfection by flow cytometry on GFP-positive cells after staining with phycoerythrin-labelled Annexin V. Results are expressed as the percentage of GFP-positive cells displaying surface PS exposure relative to that measured with wt HA-Vpr. Values are the means of four independent experiments. Error bars represent 1 standard deviation from the mean. C) Expression of wt and mutated HA-tagged Vpr proteins. Lysates from HPB-ALL transfected cells were analyzed by western-blotting using anti-GFP (upper panels) and anti-HA antibodies (lower panels)

Vpr Induces Histone 3 Phosphorylation in HeLa but Not in SupT1 Cells

<p>HeLa or Supt1 cells were transduced with pHR-VPR-G and at indicated time points, lysed, and analyzed by Western blot with a phospho-specific antibody that recognizes phosphorylation of H3 at serine-10. Nocodazole (250 ng/ml) and doxorubicin (1 μM) were used as positive and negative controls, respectively.</p

Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages-6

<p><b>Copyright information:</b></p><p>Taken from "Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages"</p><p>http://www.retrovirology.com/content/4/1/84</p><p>Retrovirology 2007;4():84-84.</p><p>Published online 26 Nov 2007</p><p>PMCID:PMC2211753.</p><p></p>he wt or mutated (clones 11 and 35, and Vpr-R90K and -W54R single-point mutants) HIV-1 Vpr fused either to LexABD (upper panels) or to the Gal4 DNA binding domain (Gal4BD) (lower panels), in combination with each of the Gal4AD-hybrids indicated on the top was analyzed for histidine auxotrophy and β-Gal activity. Double transformants were patched on selective medium with histidine (+His) and then replica-plated on medium without histidine (-His) and on Whatman filters for β-Gal assay. Growth in the absence of histidine and expression of β-galactosidase indicated an interaction between hybrid proteins. B) Amino acid substitutions found in the hCG1-binding deficient Vpr mutants (clones 11 and 35). Mutants were derived by error prone PCR-mediated mutagenesis from the primary sequence of the VprLai strain that is shown at the top. C) Isolation of single-point Vpr mutants defective for the interaction with hCG1. Single-point mutants derived from Vpr clones 11 and 35 fused to LexABD were expressed in L40 strain in combination with each of the Gal4AD-hybrids indicated on the top. Double transformants were assessed as described in A)

Functional Analysis of Vpr Mutants

<div><p>(A) SupT1 cells were infected with pHR-VPR-R or indicated mutants, at an MOI of 0.5. At 48 h postinfection, cells were stained with hypotonic PI to determine the cell cycle profiles. At 72 h postinfection, cells were incubated with FITC-VAD-FMK and analyzed by flow cytometry to determine the percentage of cells with active caspases.</p><p>(B) Cells from above treatments were lysed at 48 h postinfection, and Western blot was performed to assay for phosphorylation of BRCA1 at Ser1423 by the ATR kinase. To establish the role of ATR in BRCA1 phosphorylation, parallel infections were treated with caffeine (2 mM).</p><p>(C) Induction of apoptosis by Vpr-GFP and Vpr(R80A)-GFP fusion proteins. HPB-ALL cells were transfected with indicated constructs or mock-transfected and 48 after transfection, phosphatidylserine exposure was analyzed by flow cytometry using phycoerythrin-conjugated annexin V.</p></div

Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages-5

<p><b>Copyright information:</b></p><p>Taken from "Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages"</p><p>http://www.retrovirology.com/content/4/1/84</p><p>Retrovirology 2007;4():84-84.</p><p>Published online 26 Nov 2007</p><p>PMCID:PMC2211753.</p><p></p>ted with an HIV-1-based packaging vector lacking the gene in combination with vectors for expression of the wt or mutated HA-tagged Vpr protein. 48 h later, proteins from cell and virion lysates were separated by SDS-PAGE and analyzed by Western blotting with anti-HA and anti-CAp24 antibodies. B and C) The L23F or K27M mutations were introduced into the gene of the HIV-1YU-2 molecular clone. In B) Lysates from transfected 293T cells and virions isolated from cell supernatants were subjected to SDS-PAGE followed by Western blotting, using a rabbit polyclonal anti-Vpr and a mouse anti-CAp24 (provided from the NIH AIDS Research and Reference Reagent Program). In C) Replication of wild type and mutated HIV-1 in monocyte-derived macrophages. The wild type HIV-1YU-2 (WT, open diamonds) and the -defective (ΔVpr, open squares), Vpr-L23F (black circles) and -K27M (black triangles) mutant viruses were produced by transfection of 293T cells with proviral DNAs. Monocyte-derived macrophages from four healthy donors were infected in triplicates with 0.5 ng of CAp24. Virus production was then monitored by measuring the p24 antigen by ELISA 10, 14 and 17 days after infection. Results are expressed as the level of p24 in the supernatants of infected cells. Values are the means of four experiments and error bars represent 1 standard deviation from the mean

Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages-7

<p><b>Copyright information:</b></p><p>Taken from "Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages"</p><p>http://www.retrovirology.com/content/4/1/84</p><p>Retrovirology 2007;4():84-84.</p><p>Published online 26 Nov 2007</p><p>PMCID:PMC2211753.</p><p></p>ted in light blue, yellow and purple, respectively. The L23, K27, A30 and F34 residues are colored in red. The unstructured N- and C-terminal domains are represented in dark blue. B) CPK representation of Vpr. Residues are colored according to their hydrophobicity, except for L23 and K27 which are colored in yellow. The yellow box is enlarged in C), and this region shows a pocket that is organized around the L23 and K27 residues within the first α-helix and may represent a site for hCG1 binding. D) Helical-wheel diagram of the first α-helix of Vpr extending from a.a. D17 to F34. Residues L23, K27, A30 and F34 which have been mutated in the present study are indicated. Hydrophilic residues are in blue, whereas hydrophobic residues are in red

siRNA-Mediated Knockdown of ATR or Bax, but Not of ANT, Suppresses Vpr-Induced Apoptosis

<div><p>(A) HeLa cells were transfected with nonspecific (NS) siRNA or siRNA targeted to ATR, or ATM as indicated. At 48 h post-transfection, cells were either treated with 25 μM etoposide, treated with 25 μM MNNG, mock-transduced, or transduced with pHR-VPR-R. Additionally, cells transfected with siRNAs targeted to Bax or ANT were transduced with pHR-VPR-R or mock-transduced (lower left dot plots). Cells from each treatment were assayed for caspase activity as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020127#ppat-0020127-g002" target="_blank">Figure 2</a>B.</p><p>(B) Cells treated as in (A) were harvested at specified time points post-transduction and assayed for caspase activity.</p><p>(C) Cells treated as in (A) were stained with DAPI, and the results were quantified by microscopy.</p><p>(D) Cells treated with the indicated siRNAs were lysed and analyzed by Western blot to verify knockdown efficiency.</p><p>(E) Primary human CD4⁺ lymphocytes were infected with DHIV3, infected with DHIV3ΔVPR, or mock-infected. At 48 h postinfection, cells in each treatment were lysed and assayed for protein concentration. Equal amounts of protein from each treatment were incubated with Bax6A7 monoclonal antibody. Antibody–protein complexes were precipitated with agarose beads and boiled and then subjected to Western blot analysis with a polyclonal antibody to Bax.</p><p>(F) HeLa cells treated with the indicated siRNAs and transduced with pHR-VPR or mock-transduced were lysed; reactivity to Bax6A7 antibody was assayed as described in (E).</p></div

Vpr-Induced Caspase Activation and Smac Release from the Mitochondria Are Temporally Delayed in Relation to G₂ Arrest

<div><p>(A) pHR-VPR-G and pHR-VPR-R are bicistronic lentiviral vectors that encode HIV-1_NL4–3<i>vpr,</i> an internal ribosome entry site, and either the gene for GFP or that for mRFP, respectively; pHR-VPR(R80A) was derived from pHR-VPR (both the mRFP and the GFP versions) by site-directed mutagenesis; DHIV3 is an envelope-truncated (see gray box) version of HIV-1_NL4–3; DHIV3-ΔVPR was derived from DHIV-3 by introducing a frameshift mutation in <i>vpr</i>.</p><p>(B) SupT1 T lymphocytes were transduced by spin-infection in the presence of 10 μg/ml Polybrene with indicated vectors. Mock-infected cells were subjected to spin-infection in the presence of 10 μg/ml Polybrene without virus. Cells were collected at specified time points post-transduction, stained with propidium iodide, and analyzed for DNA content by flow cytometry to determine cell cycle profiles. The percentage of cells transduced with pHR vectors and DHIV3 viruses ranged between 70% to 80% and between 65% to 70%, respectively, as determined by mRFP or GFP expression (with pHR vectors) or intracellular p24 staining (with DHIV3 vectors).</p><p>(C) Caspase activation was measured as an indication of apoptosis. SupT1 cells were infected with indicated vectors, harvested, and incubated with FITC-VAD-FMK. The percentage of caspase-active cells at each time point was measured by flow cytometry.</p><p>(D) Infected SupT1 cells were lysed, and lysates were fractionated into mitochondrial (m) and cytoplasmic (c) fractions and then assayed by Western blot. Western blots were probed with antibodies specific to Smac to measure release from mitochondria and with anti-VDAC antibodies to measure mitochondrial contamination in the cytoplasmic fractions. As a positive control for apoptosis, SupT1 cells were treated with 0.8 μg/ml doxorubicin (dox) for 48 h.</p><p>(E) Examples of flow cytometric analysis of caspase activation, corresponding to the 72-h time points from (C).</p></div