Visualizing Vpr-Induced G2 Arrest and Apoptosis

<div><p>Vpr is an accessory protein of human immunodeficiency virus type 1 (HIV-1) with multiple functions. The induction of G2 arrest by Vpr plays a particularly important role in efficient viral replication because the transcriptional activity of the HIV-1 long terminal repeat is most active in G2 phase. The regulation of apoptosis by Vpr is also important for immune suppression and pathogenesis during HIV infection. However, it is not known whether Vpr-induced apoptosis depends on the ability of Vpr to induce G2 arrest, and the dynamics of Vpr-induced G2 arrest and apoptosis have not been visualized. We performed time-lapse imaging to examine the temporal relationship between Vpr-induced G2 arrest and apoptosis using HeLa cells containing the fluorescent ubiquitination-based cell cycle indicator2 (Fucci2). The dynamics of G2 arrest and subsequent long-term mitotic cell rounding in cells transfected with the Vpr-expression vector were visualized. These cells underwent nuclear mis-segregation after prolonged mitotic processes and then entered G1 phase. Some cells subsequently displayed evidence of apoptosis after prolonged mitotic processes and nuclear mis-segregation. Interestingly, Vpr-induced apoptosis was seldom observed in S or G2 phase. Likewise, visualization of synchronized HeLa/Fucci2 cells infected with an adenoviral vector expressing Vpr clearly showed that Vpr arrests the cell cycle at G2 phase, but does not induce apoptosis at S or G2 phase. Furthermore, time-lapse imaging of HeLa/Fucci2 cells expressing SCAT3.1, a caspase-3-sensitive fusion protein, clearly demonstrated that Vpr induces caspase-3-dependent apoptosis. Finally, to examine whether the effects of Vpr on G2 arrest and apoptosis were reversible, we performed live-cell imaging of a destabilizing domain fusion Vpr, which enabled rapid stabilization and destabilization by Shield1. The effects of Vpr on G2 arrest and subsequent apoptosis were reversible. This study is the first to characterize the dynamics of the morphological changes that occur during Vpr-induced G2 arrest and apoptosis.</p></div

Vpr induces cell cycle arrest in G2 phase and apoptosis in HeLa/Fucci2 cells.

<p>(A) Schema illustrating the cell cycle alteration(s) observed in HeLa/Fucci2 cells. HeLa/Fucci2 cells exhibit red nuclei in G1 and yellow nuclei in S/G2/M phase. (B) Western blot analysis for the detection of Vpr expression and apoptosis. HeLa/Fucci2 cells were transfected with pME18Neo/Flag-Vpr, pME18Neo/Flag- R80A, or pME18Neo. At 72 h after transfection, the cells were lysed and subjected to Western blot analysis with anti-Flag monoclonal antibody (MAb) M2, anti-β-actin MAb, and anti-cleaved caspase-3 (Asp175) antibody. (C) Representative images of Fucci2 fluorescence of cells in G1, S, and G2/M phases of the cell cycle from at least 200 Alexa680-positive cells that were selected automatically using CELAVIEW microscope at 72 h after transfection. HeLa/Fucci2 cells were transfected with either pME18Neo/Flag-Vpr or pME18Neo/Flag-R80A. At 72 h after transfecti on, cells were stained with anti-Flag MAb M2, followed by Alexa680 conjugated anti-mouse IgG MAb for the detection of cells expressing wild-type or R80A Vpr, and then stained with Hoechst 33342 to measure DNA content. The DNA content of Alexa680-positive cells was analyzed by using a CELAVIEW microscope. (D) Quantitation of (C). The percentages of cells are in the G1, S, and G2/M phase were indicated in the <i>bar graph.</i> Data are present as means ± SD of triplicate wells.</p

Vpr induces cleavage of SCAT3.1 at the same time as caspase-3 activation.

<p>HeLa/Fucci2 cells were transfected with pME18Neo/Flag-Vpr-IRES-SCAT3.1 or the control pME18Neo/Flag-IRES-SCAT3.1. At 24 h after transfection, cells expressing SCAT3.1 in the cytoplasm were observed with the LCV110 Imaging System at 15 minute intervals for 96 h. Nuclei with red fluorescence are in G1 phase. Yellow or cyan fluorescence in the cytoplasm identifies inactivated or activated caspase-3, respectively. The ECFP and Venus fluorescence of SCAT3.1 and the mCherry and Venus fluorescence of Fucci2 are shown at 24, 36, 48, 60, 72, 84, and 96 h post-transfection. Five SCAT3.1-positive cells were selected as representative images and are marked as #1 to #5 in the upper part of each cell. The scale bars represent 100 µh. High-magnification images of SCAT3.1-expressing cells #1 to #5 are shown for cells transfected with the control pME18Neo/Flag-IRES-SCAT3.1 (#1 and #2) at 24, 32, 36, 48, 51, 56, 60, 68, 70, 72, 80, 84, or 96 h after transfection, or pME18Neo/Flag-Vpr-IRES-SCAT3.1 (#3 to #5) at 24, 27, 30, 36, 42, 48, 54, 60, 61, 67, 68, or 72 h after transfection.</p

Visualizing Vpr-induced cell cycle arrest and cell death.

<p>HeLa/Fucci2 cells were transfected with pME18Neo/Flag-Vpr-IRES-ECFP or the control pME18Neo/Flag-IRES-ECFP. At 24 h after transfection, cells were monitored by time-lapse imaging using LCV110 Imaging System at 15 minute intervals until 96 h post-transfection. Cells showing red or yellow fluorescence in the nucleus are in the G1 or S/G2/M phases of the cell cycle, respectively. ECFP and Fucci2 fluorescence is shown at 24, 36, 48, 60, 72, 84, and 96 h post-transfection. Eight ECFP-positive cells were selected as representative images, and are marked as *1 to *8 on the right of each cell. The scale bars represent 100 μ. High-magnification images of ECFP-expressing cells *1 to *8 are shown for cells transfected with the control pME18Neo/Flag-IRES-ECFP (*1 and *2) at 24, 27, 28, 36, 42, 43, 48, 60, 67, 72, 80, 88, or 96 h, or pME18Neo/Flag-Vpr-IRES-ECFP (*3 to *8) at 24, 26, 28, 36, 48, 54, 60, 66, 72, 73, 84, 91, or 96 h after transfection.</p

Cell cycle arrest and cell death in cells expressing a destabilizing domain (DD) Vpr fusion in the presence and absence of Shield1.

<p>(A) Schemas illustrating the treatment with Shield1. HeLa cells were transfected with pME18Neo/DD-Vpr-IRES-ZsGreen1 (I-V) or the control pME18Neo/DD-IRES-ZsGreen1 (i and ii) and cultured for 24 h. The cells were then treated in the absence (i and I) or presence (ii and II) of 500 nM Shield1 for 24 h. The cells were washed to remove Shield1 and further cultured in the absence (IV) or presence (V) of 500 nM Shield1 for 24 h. (B) The cells were fixed, permeabilized, stained with anti-DD MAb followed by Alexa594 secondary MAb, and analyzed by confocal laser scanning microscopy. Cells showing red and green fluorescence express DD-Vpr and ZsGreen1, respectively. The scale bar represents 10 µm. (C) Cells were lysed and subjected to Western blot analysis with anti-DD MAb, anti-ZsGreen1 antibody, and anti-β-actin MAb. (D) Cells were stained with PI. ZsGreen1-positive cells were analyzed by flow cytometry using CELL Quest for acquisition and ModFit LT for quantitative analysis of DNA content. The percentage of cells in G2/M phase is indicated in the upper right of each graph. (E) Cells were stained with SR-DEVD-FMK to identify cells with active caspase-3. The percentage of cells with active caspase-3 was measured by flow cytometry in ZsGreen1-positive cells.</p

Proposed model to explain the temporal relationships between various Vpr functions.

<p>(A) Vpr induces apoptosis at G1 (a) and G2 (b) without G2 arrest. Vpr induces G2 arrest and subsequent long-term mitotic cell rounding following apoptosis (c) or nuclear mis-segregation or abnormal cell division (d and e). After nuclear mis-segregation or abnormal cell division, most of the cells undergo apoptosis (d) and other cells survive with micro nuclei (e). Furthermore, some Vpr-expressing cells underwent G2 arrest throughout the observation period (f). All apoptosis was dependent on caspase-3. Bold arrow, G2 arrest; arrow with dotted line, long-term mitotic cell rounding; and gray arrowhead, caspase-3 activation. (B) Interruption of Vpr expression leads to four phenomena: cell division with normal cell cycle progression (a), G2 arrest (b), G2 arrest with apoptosis (c), and G2 arrest with abnormal cell division (d).</p

Expression of DD-Vpr is required for persistent G2 arrest and apoptosis after G2 arrest.

<p>(A) Schemas illustrating the treatment with Shield1. HeLa/Fucci2 cells were transfected with pME18Neo/DD-Vpr-IRES-ECFP. Twenty-four hours after transfection, the cells were cultured in the absence (i) or presence (ii and iii) of 500 nM Shield1 for 23 h (1st cultivation). The cells were washed to remove Shield1 and further cultured in the presence (ii) or absence (iii) of 500 nM Shield1 for 49 h (2nd cultivation). (B) DD-Vpr expression was monitored using Western blot analysis with anti-DD MAb, anti-GFP MAb, and anti-β-actin MAb at 0, 1, 4, 25, and 49 h after the 2nd cultivation. (C-E) ECFP-expressing cells that were in G2 phase 1 h after the 2nd cultivation (marked as ^☆ on the left of each cell) were selected and monitored by time-lapse imaging using LCV110 Imaging System at 15 minute intervals for a further 48 h. (C) Cells showing red or yellow fluorescence in the nucleus are in G1 or S/G2/M phase, respectively. ECFP and Fucci2 fluorescence are shown at 1, 17, 41, and 49 h after the 2nd cultivation. Six ECFP-positive cells that were in G2 phase at 1 h after the 2nd cultivation were selected as representative images and are marked as ^☆1 to ^☆6 on the left of each cell. The scale bars represent 100 µm. (D) The total number of ECFP-expressing cells that had been in G2 phase at 1 h after the 2nd cultivation were counted in particular areas. (E) The effect of continuous DD-Vpr expression in G2 arrested cells was determined by assessing Fucci2 fluorescence and morphology. The proportion of cells undergoing normal cell division (white), persistent G2 arrest (gray), cell death (black), and abnormal cell division (horizontal line) were calculated and plotted.</p

Summary of viral replication kinetics, nuclear localization, nuclear export capacity, and CRM1 binding of the NP-NES3 consensus sequence mutants.

a<p>% virus titer ± SD compared with WT at the 46 h of replication kinetic assay from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g004" target="_blank">Fig. 4C</a>.</p>b<p>No viral rescue by reverse genetics.</p>c<p>% cell count ± SD with nuclear localization of NP from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g005" target="_blank">Fig. 5B</a>.</p>d<p>−, ±, + indicate not occur, partially occur, occur of nuclear export capacity, respectively derived from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g006" target="_blank">Fig. 6</a>.</p>e<p>% intensity of the pull-downed NP band compared with WT from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105081#pone-0105081-g007" target="_blank">Fig. 7C</a>.</p

SNP constructs show that the 23-bp deletion in the upstream region of <i>PRNP</i> and/or a 12-bp deletion in intron1, coupled with the absence of the Sp1 SNP and the presence of exon1, are required for the negative feedback response to PrP overexpression.

<p>(A) Map of the portion of bovine <i>PRNP</i> containing the 5′-flanking region and exons 1 and 2 is shown on the top line. Dotted box = Luciferase gene; black boxes = exon 1 and exon 2, which include numbers denoting the position of the reported transcription start site (+1) of the <i>PRNP</i> promoter region. The 23-bp indel, 12-bp indel, and SNP regions are also indicated above the reporter gene constructs. The absence (−) and presence (+) of each region in the reporter gene constructs are shown in the Table on the right. (B) Graph representing the relative luciferase activities obtained with the above reporter plasmids in the presence of either an empty vector, pEF-BOS (EM, open bars), or pEF-boPrP (PrP, solid bars). The pGL3-Control vector (with the standard SV40 promoter) was used for normalization between different experiments (relative light units (<i>RLU</i>) = (firefly luciferase_construct/<i>total protein</i>_construct)/(firefly luciferase_control/<i>total protein</i>_control)). Relative luciferase activities (Mean ± S.D.) for 3 replicate experiments were compared with that of the pGL3-control plasmid (1%). A significant difference of luciferase activity in pEF-boPrP-transfected cells as compared with corresponding empty vector-transfected cells is shown by one asterisk (*, <0.05) or two asterisks (**, <0.01). NS indicates no significant difference.</p

Intracellular localization of NP-NES3 mutant proteins.

<p>(A) HeLa cells were grown on cover glass and transfected with pCAGGS encoding wild-type NP-NES3 or its mutants (Φ1, Φ2, Φ3, or Φ4) for 48 h before immunofluorescence staining with an anti-NP MAb followed by anti-mouse Alexa Fluor 488 and Hoechst 333342. The cells were then observed under a confocal laser-scanning microscope. The white and yellow arrow heads indicate predominant localization of NP in the cytoplasm (cytoplasmic staining > nuclear staining) and nucleus (nuclear staining > cytoplasmic staining), respectively. (B) Nuclear localization of NP wild-type and NP-NES3 mutants from A. Data are presented as the percentage (± SD) of total cell count with predominant nuclear or cytoplasmic staining of NP from five separate fields.</p