Hhp2 suppresses <i>PR</i>-induced cell death and apoptosis in HeLa cells.

<p><b>(A)</b> Expression of HIV-1 <i>PR</i> induces apoptosis in HeLa cells. Apoptosis were detected by staining with Annexin V and FACScan analysis as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref064" target="_blank">64</a>]. The percentage of apoptotic cells was measured in HeLa cells 24 hrs after transfection of a control (Ctr) pIRES2-EGFP plasmid (left) or a <i>PR</i>-containing pIRES2-EGFP-<i>PR</i> plasmid (right). The average number ± SD of apoptotic cells are shown in the top right quadrant (<b>a</b>), using data from at least three independent measurements. (<b>b</b>) Expression of HIV-1 PR induced caspase-3 and caspase-8 cleavage in HeLa cells as shown by western blot analyses performed as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref025" target="_blank">25</a>]. Lane 1, HeLa cells transfected with a control plasmid; lane 2, <i>PR</i>-expressing transfected HeLa cells. The analysis was carried out 48 hrs after plasmid transfection. (<b>B</b>) Hhp2 suppresses <i>PR</i>-induced cell death and apoptosis in HeLa cells. Since no specific antibody against Hhp2 was available, to confirm <i>hhp2</i> gene expression in HeLa cells, <i>hhp2</i> gene transcription was measured by RT-PCR and detected on agarose gel as shown in (<b>a</b>). Lane 1, HeLa cells transfected with a control plasmid; lane 2, <i>hhp2</i>-expressing HeLa cells. The gel shows the 1.2-kb <i>hhp2</i> cDNA that was amplified. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The suppressive effect of Hhp2 on HIV-1 PR-induced cell death and apoptosis was also quantified 24 hrs after gene expression using a Trypan blue assay (<b>b</b>) and Annexin V staining (<b>c</b>), respectively. The average numbers ± SD of dead cells are shown, the error bars represent results of at least three independent measurements. The effect of Hhp2 on HIV-1 PR-mediated caspase-3 and caspase-8 cleavage was detected by western blot analysis as shown in (<b>d</b>) and quantified in (<b>e</b>). A statistical t-test was used to determine whether there was a significant difference in the cleavage of caspase-3 and caspase-8 taking place in the control HeLa cells and HIV-1 <i>PR</i>-expressing cells, with or without Hhp2. p-values less than 0.01 were considered statistically significant.</p

A fission yeast protein kinase Hhp2 suppresses PR activities.

<p>(<b>A</b>) Fission yeast genome-wide searches for multicopy suppressors of PR-induced cell death of fission yeast cells revealed six unique cDNA clones out of ten isolates with overlapping open reading frame (ORF) of the <i>hhp2</i> gene. The numbering and relative positions of each nucleotide were derived from the SPAC23C4 cosmid of fission yeast. (<b>B</b>) Overexpression of <i>hhp2</i> restored colony formation in <i>PR</i>-expressing fission yeast cells. Expression of the <i>hhp2</i> gene was induced at lower levels with 1 nM thiamine added to the media. Under this condition, HIV-1 PR still prevented colony formation as shown. Ctr, an empty pYZ1N plasmid control. <i>Gene</i>-off, no HIV-1 PR or Hhp2 protein production; <i>Gene</i>-on, <i>i</i>.<i>e</i>., PR protein production in the presence of Hhp2 or an empty plasmid vector. Agar plates were incubated at 30°C under the indicated conditions for 6 days before pictures were taken. (<b>C</b>) Overexpression of <i>hhp2</i> reduced the percentage of cells showing the GFP pattern over time. The GFP-p6-Vpr fusion product was used here. The Hhp2 effect was measured over time after gene induction as indicated. The percentage of GFP pattern, <i>i</i>.<i>e</i>., the putative protein cleavage of GFP-p6-Vpr fusion construct by HIV-1 PR was quantified and is shown in (<b>b</b>). Error bars shown in (<b>C</b>) represent results of at least three different experiments with an average of 100–200 cells counted at each time point.</p

Expression of the HIV-1 <i>PR</i> gene prevents cellular growth and causes oxidative stress, changes of mitochondrial morphology, and cell death in fission yeast.

<p>(<b>A</b>) Inducible expression of the HIV-1 <i>PR</i> gene in RE294 was detected 24 hrs after gene induction by western blot analysis (<b>a</b>). Lane 1, <i>PR</i>-repressing cells; lane 2, <i>PR</i>-inducing cells. Blot shows a 12-kDa protein band that specifically reacted to an antiserum against HIV-1 PR. Expression of the <i>PR</i> gene induced cellular growth arrest in liquid medium (<b>b</b>) and prevented yeast colony formation on agar plates (<b>c</b>) and in liquid medium (<b>d</b>). <i>PR</i>-off, <i>i</i>.<i>e</i>., no PR protein production. <i>PR</i>-on, <i>i</i>.<i>e</i>., PR protein production. All cells were grown at 30°C, and the cell growth was measured at OD₆₅₀ in the time period shown using a spectrophotometer. Pictures of agar plates were taken 6 days after incubation at 30°C under the indicated conditions. Error bars shown in (<b>b</b>) of the growth assay represent at least three independent experiments. (<b>B</b>) HIV-1 <i>PR</i> expression induced cell death (<b>a</b>), oxidative stress (<b>b</b>), and mitochondrial morphological changes (<b>c</b>) in fission yeast. Twenty-four hours after inducible <i>PR</i> expression, cell viability was measured by the yeast live/dead assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref030" target="_blank">30</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref033" target="_blank">33</a>] (<b>a</b>). In this assay, viable cells are typically shown in orange-red color (left, <i>PR</i>-off); metabolically deceased cells are shown in green-yellow color (right, <i>PR</i>-on). The production of reactive oxygen species (ROS) was measured by an ROS indicator dye DHE (<b>b</b>). Mitochondrial morphologies (<b>c</b>) were visualized by staining fission yeast cells with a mitochondria-specific fluorescent probe, DASPMI, as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref022" target="_blank">22</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref036" target="_blank">36</a>]. Note that normal mitochondria appear like a thread or necklace of multiple small dots concentrated around the edge a cell, at the growing ends of the cell, or as a tubular network extended along the periphery of the cell (Fig 1B-b, left). In contrast, different sizes of mitochondrial aggregates that are situated almost randomly throughout the <i>PR</i>-expressing cells are shown here, indicating changes in mitochondrial morphology (Fig 1B-b, right).</p

HIV-1 Protease in the Fission Yeast <i>Schizosaccharomyces pombe</i>

<div><p>Background</p><p>HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (<i>Schizosaccharomyces pombe</i>) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability.</p><p>Results</p><p>A fission yeast strain RE294 was created that carried a single integrated copy of the <i>PR</i> gene in its chromosome. The <i>PR</i> gene was expressed using an inducible <i>nmt1</i> promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 <i>PR</i> expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8.</p><p>Conclusions</p><p>This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.</p></div

Growth inhibition and cell death induced by HIV-1 PR can be circumvented by IDV in fission yeast.

<p>(<b>A</b>) Treatment of HIV-1 <i>PR</i>-expressing fission yeast cells with protease inhibitor IDV restored colony formation in a dose-dependent manner (<b>a</b>) and cellular growth over time (<b>b</b>). The concentration of IDV that was added in (<b>a</b>) is shown, and 100 μg/mL of IDV was added to the <i>PR</i>-expressing cells in (<b>b</b>). All cells were grown at 30°C and cell growth was measured by OD₆₅₀ at the time point indicating using a spectrophotometer. Error bars shown in (<b>b</b>) of the growth assay represent at least three independent experiments. <b>(B</b>) IDV prevents HIV-1 PR-induced oxidative stress, changes in mitochondrial morphology, and cell death. (<b>a</b>) Fission yeast cell death induced by HIV-1 PR, as shown by the yeast live/dead assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref030" target="_blank">30</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref033" target="_blank">33</a>], was prevented by adding 100 μg/mL of IDV before gene induction. Pictures were taken at 24 hrs after gene induction. ROS production (<b>b</b>) was detected and mitochondrial morphology (<b>c</b>) was observed using DHE and DASPMI as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.g001" target="_blank">Fig 1</a>. Ctr, control, <i>i</i>.<i>e</i>., no IDV added; + IDV, 100 μg/mL of IDV was added prior to HIV-1 <i>PR</i> gene induction.</p

Target-specific enzymatic protein cleavage of indigenous viral proteins by HIV-1 PR and inhibition by IDV in fission yeast.

<p><b>(A</b>) HIV-1 PR specifically cleaves GFP-p6/MA-Vpr fusion protein constructs that contain indigenous cleavage sites of HIV-1 p6 or MA. (<b>a</b>) Schematic drawing to show the rationale for tests of the proteolytic substrate specificity of HIV-1 PR in fission yeast. Green fluorescent protein (GFP) was distributed uniformly throughout fission yeast cells, i.<i>e</i>., the “GFP pattern” [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref039" target="_blank">39</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref065" target="_blank">65</a>]. HIV-1 viral protein R (Vpr) localizes predominantly to the nuclear membrane and appears as a “ring-like” structure, <i>i</i>.<i>e</i>., the “Vpr pattern” [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref039" target="_blank">39</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref065" target="_blank">65</a>]. (<b>b</b>) Expression of HIV-1 <i>PR</i> in fission yeast did not affect subcellular locations of GFP or HIV-1 Vpr-attached GFP. The top panel shows expression of GFP only; the bottom panel shows Vpr-attached GFP. (<b>c</b>) HIV-1 PR cleaves substrate linkers of HIV-1 MA (left) and p6 (middle), but not of LF (right). Cells were examined 20 hrs after <i>PR</i> gene induction. <b>(B</b>) IDV blocked the formation of the “GFP pattern” in a dose-dependent manner in fission yeast. The GFP-p6-Vpr fusion product was used here. The IDV effect was measured 20 hrs after gene induction at increasing concentrations as shown in (<b>a</b>). The percentage of cells exhibiting the GFP pattern, <i>i</i>.<i>e</i>., the putative protein cleavage of GFP-p6-Vpr fusion construct by HIV-1 PR, was quantified and is shown in (<b>b</b>). An average of 100–200 cells was counted for each concentration. Scale bar: 10 μm.</p

Vpr suppresses tumor regression in a neuroblastoma mouse model.

<p>Suppression of neuroblastoma tumor growth by Vpr is demonstrated here either by pre-transduction of Adv-Vpr (<b>A-B</b>) or post-intratumoral injection (<b>C</b>). For pre-transduction, wild type (WT) or DOX-resistant SK-N-SH were grown in DMEM supplemented with 10% FBS at 37°C with a 95%Air/5%CO₂ atmosphere. Fresh cells were first grown in a 12-well plate for 36 hours and adenoviral transduction was carried out 5 hours before cell inoculation with MOI of 2.5, which was determined empirically. Cells were then treated with Trpsin- EDTA, re-suspended in DMEM and washed with PBS 3 times. Final cells were suspended in DMEM for inoculation. About 2×10⁶ cells in the volume of 100 µl were injected <i>s.c.</i> in the left flank of C57-SCID mice. 3–4 mice were injected for each treatment. These treatment groups include an Adv viral control, Adv-Vpr and Adv-F34IVpr. The F34IVpr mutant was used here as a control since a single amino acid change of amino acid 34 from Phenylalanine (F) to Isoleucine (I) renders Vpr unable to cause apoptosis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone-0011466-g003" target="_blank">Figure 3C</a>) but allows for the cell cycle to enter a prolonged G2 phase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Benko1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Chen1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Vodicka1" target="_blank">[28]</a>. The tumor size was measured every 7 days by measuring two perpendicular tumor diameters using calipers. Final tumor measurement was at 26 days post-transduction and mice were then sacrificed for further analysis. For intra-lesional injection of Vpr, the WT and DOX-R SK-N-SH neuroblastoma cells were prepared essentially the same way as described above. 200 µl of the Adv, Adv-Vpr or Adv-F34Ivpr was then injected discretely 3-times into the tumors 2 weeks after cell inoculation with a viral concentration of 1,012/ml. The tumor size was measured every 7 days. Final measurement of tumor size was at 39 days post-injection and mice were then sacrificed for further analysis. Three independent experiments were carried out and results of these experiments with average tumor size with standard deviation (SD) are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone-0011466-t002" target="_blank">Table 2</a>.</p

Vpr induces cell cycle G2 arrest in various cancer cell types.

<p>All cancer cell lines (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone-0011466-t001" target="_blank">Table 1</a> for details; only 3 cell lines are shown here as examples) were grown as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#s4" target="_blank">Materials and Methods</a>. Cells were transduced with Adv or Adv-Vpr with MOI of 1.0. The cells were harvested 48 hrs post-infection (<i>p.i.</i>), Cells were then prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#s4" target="_blank">Materials and Methods</a>. Cellular DNA content was analyzed by FACScan flow cytometry (Becton Dickinson) as we previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Li1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Liang1" target="_blank">[19]</a>. The cell cycle profiles were modeled using ModFit software (Verity Software House, Inc.).</p

Quantitative summary of Vpr-induced G2 arrest in various cancer types.

<p><b>Note:</b> *, PP2A is mutated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Ostrakhovitch1" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Suzuki1" target="_blank">[34]</a>; **, F34IVpr causes cell cycle G2 shift but no cycling arrest <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Benko1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Chen1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Vodicka1" target="_blank">[28]</a>; WT, wild type; DOX-R, doxorubicin-resistant; G2 arrest: (+), strong G2 arrest; (−), no G2 arrest, (+/−), attenuated G2.</p

Summary of Vpr-induced tumor regression of WT and DOX-resistant neuroblastoma in C57-SCID mice.

<p><b>Note:</b> Tumor sizes were measured at 39 days post-intratumoral injection (Week 0). Levels of statistical significance of the t-test results between the Adv control and the testing groups (Ad-Vpr or Ad-F34IVpr): *, p<0.05; **, p<0.001; Weighted average sums of the t-tests <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011466#pone.0011466-Tan1" target="_blank">[31]</a> for both wild type and Dox-R mice showed statistic differences at the level of p<0.05. Note: na, non-applicable.</p