14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

<p>Abstract</p> <p>Background</p> <p>Despite continuing advances in our understanding of AIDS pathogenesis, the mechanism of CD4+ T cell depletion in HIV-1-infected individuals remains unclear. The HIV-1 Vpr accessory protein causes cell death, likely through a mechanism related to its ability to arrest cells in the G₂,M phase. Recent evidence implicated the scaffold protein, 14-3-3, in Vpr cell cycle blockade.</p> <p>Results</p> <p>We found that in human T cells, 14-3-3 plays an active role in mediating Vpr-induced cell cycle arrest and reveal a dramatic increase in the amount of Cdk1, Cdc25C, and CyclinB1 bound to 14-3-3 θ during Vpr_v-induced G₂,M arrest. By contrast, a cell-cycle-arrest-dead Vpr mutant failed to augment 14-3-3 θ association with Cdk1 and CyclinB1. Moreover, G₂,M arrest caused by HIV-1 infection strongly correlated with a disruption in 14-3-3 θ binding to centrosomal proteins, Plk1 and centrin. Finally, Vpr caused elevated levels of CyclinB1, Plk1, and Cdk1 in a complex with the nuclear transport and spindle assembly protein, importin β.</p> <p>Conclusion</p> <p>Thus, our data reveal a new facet of Vpr-induced cell cycle arrest involving previously unrecognized abnormal rearrangements of multiprotein assemblies containing key cell cycle regulatory proteins.</p> <p>Reviewers</p> <p>This article was reviewed by David Kaplan, Nathaniel R. Landau and Yan Zhou.</p

Increased Cdk1, Cdc25C and CyclinB1 association with 14-3-3 θ but stable nucleocytoplasmic distribution during HIV- and Vpr-induced G,M arrest

Jurkat cells were infected as in Fig. 1 with RT- NL4-3virions either with (Vpr) or without (Δ) hVpr supplied in trans or with RT+ NL4-3(HIV; MOI 2). (A) Cell lysates were harvested two days post-infection for immunoprecipitation with 14-3-3 θ and immunoblotting for CyclinB1, Cdc25C-P.S216, Cdc25C, Cdk1-P.Y15, Cdk1, 14-3-3 θ, and Vpr as indicated. The 14-3-3 θ signal in the IP does not reflect poor immunoprecipitation of 14-3-3 θ but rather the result of membrane stripping prior to 14-3-3 θ blotting. (B) DNA content analysis (y-axis) is shown in flow cytometric dot plots against GFP (x-axis) on the right and as a histogram in the inset for the samples in (A). Note that the y-axis of the parent graph becomes the x-axis of the inset graph. The quadrant gate demarcates approximate G1 (lower) and S/G,M (upper) populations and the percentage of cells in each relevant quadrant is indicated. The DNA histogram profile analysis was separated into GFP-positive (+) and negative (-) populations by the x-axis gate for the HIV-infected culture; as expected the infected cells (+) show Garrest, but the uninfected cells (-) are mostly G1. (C) G,M cell cycle arrest caused by Vprand HIV infection does not alter the cytoplasmic and nuclear distribution of 14-3-3 θ, Cdc25C, Cdk1, and CyclinB1. Jurkat T cells shown in (A-B) that were infected with NL4-3RT- Δ Vpr (Δ), RT- wt Vpr (Vpr), or NL4-3RT+ (HIV) for two days were lysed and biochemically separated into cytoplasmic and nuclear fractions. Lysate fractions were blotted as in (A) (the lower panel of Vpr blot represents a longer exposure in which Vpris more apparent), with the addition of probes for HIV-1 Vif, Poly(ADP-ribose) polymerase (PARP) as a nuclear marker, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic loading control. Cell cycle profiles and GFP expression are shown in (B). (D) Viral lysates (20 μg) of RT- NL4-3virions with (+) or without (-) Vpr were western blotted for CyclinB1, Cdk1, p24, and Vpr as indicated.<p><b>Copyright information:</b></p><p>Taken from "14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr"</p><p>http://www.biology-direct.com/content/3/1/17</p><p>Biology Direct 2008;3():17-17.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2390528.</p><p></p

Mutant Vpr fails to stimulate the association of CyclinB1, Cdk1, and Plk1 with 14-3-3 θ but still binds 14-3-3 θ itself

(A) Jurkat cells were infected as in Fig. 2 with RT- NL4-3virions (Vpr) lacking Vpr (Δ) or containing either wild-type (wt), R80A mutant (80A), or I70S mutant (70S) Vpr. Cells lysates were harvested two days post-infection. for immunoprecipitation with 14-3-3 θ antibody (IP: 14-3-3 θ) and the lysates (input) and IP were blotted as indicated with antibodies recognizing Plk1, CyclinB1, Cdk7, Cdk1, 14-3-3 θ, and Vpr. (B) DNA content analysis of the samples in (A), top row (Vpr), and of aphidicolin-synchronized Jurkat cells released from the G1 block for the indicated number of hours (bottom row; sync.). (C) Immunoprecipitation and western blot were performed as in (A) of lysates from the cell cycle synchronized cells shown in (B, bottom row). (D) Jurkat cells were infected with NL4-3(RT+) derivatives containing either wild-type Vpr and Vif (lane 1), Vpr but no Vif (lane 2), neither Vpr nor Vif (lane 3), or R80A mutant Vpr and no Vif (lane 4). Two days post-infection cells were lysed, immunoprecipitated, and immunoblotted as in (A) (top). Flow cytometric DNA content analysis was performed at the time of harvest and shown for the GFP+ (HIV-infected) population of each sample (bottom, numbering corresponds to lane numbers of blots). GFP expression is shown as an inset with the percentage of cells in the GFP-positive gate indicated within the plot.<p><b>Copyright information:</b></p><p>Taken from "14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr"</p><p>http://www.biology-direct.com/content/3/1/17</p><p>Biology Direct 2008;3():17-17.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2390528.</p><p></p

Cell cycle regulatory protein binding to 14-3-3 θ is also enhanced during G,M arrest induced by adriamycin

Jurkat T cells were treated with adriamycin (Adr; 0.2 μg/ml) for 24, 48, or 72 hours (h) or untreated (0) and examined for 14-3-3 θ co-immunoprecipitating (IP) proteins. (A) Western blot analysis of Plk1, CyclinB1, Cdk1, 14-3-3 θ and centrin in whole cell lysates before IP (input) and after 14-3-3 θ IP (IP: 14-3-3 θ) at the time of adriamycin treatment indicated. These data are representative of three independent experiments. Although the amount of 14-3-3 θ appears less in the untreated (0) time point of the IP, this was not a reproducible finding. (B) Viability of the samples used in the IP in (A) determined by flow cytometry detection of propidium iodide (PI) exclusion and large size (high forward scatter). The percentage of viable cells is indicated. Flow cytometric histograms of the DNA content for each sample determined by propidium iodide DNA staining (x-axis) are shown as an inset.<p><b>Copyright information:</b></p><p>Taken from "14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr"</p><p>http://www.biology-direct.com/content/3/1/17</p><p>Biology Direct 2008;3():17-17.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2390528.</p><p></p

Cell cycle regulatory proteins reside in the centrosome during G,M arrest induced by HIV-1 infection and adriamycin

(A) Western blot analysis of centrosomes isolated from Jurkat cells infected with NL4-3(HIV; MOI of 2) for two days. Centrosomes were isolated by discontinuous sucrose gradient and fractions were collected and separated by SDS-PAGE and western blotted for Plk1, CyclinB1, γ-tubulin, Cdk1, 14-3-3 θ, centrin, Vif, and Vpr. Lanes 1–6 represent fractions from the bottom of the gradient upward, with centrosomes most abundant in lane 3. Whole cell lysates were run in lane 8 and volume from the top of the gradient equivalent to that used for each fraction was run in lane 7 to demonstrate sedimentation of centrosomal proteins through the gradient. "L" indicates a light exposure and "D" indicates a darker exposure of the chemilumigraph of the middle part of the gel. (B) Viability (top) and DNA content analysis (bottom) by flow cytometry for the culture in (A; HIV (+)) and untreated Jurkats without (-) HIV infection. The percentage of viable cells was determined by propidium iodide (PI) exclusion and high forward scatter and is indicated in the lower right corner. Infection efficiency was measured by GFP expression (inset; gated population) and the percentage is indicated. DNA content was measured by flow cytometric detection of DNA stained with propidium iodide. The GFP-positive population was analyzed for the HIV-infected culture. (C) Jurkat T cells were treated with adriamycin (adr; 0.2 μg/ml; right panel) for two days or grown asynchronously (left panel) and centrosomes were isolated by discontinuous sucrose gradients as in (A). Centrosomes were most abundant in lanes 2–3 (untreated cells) or lanes 3–4 (adriamycin treated cells). Lanes were as described in panel A. (D) Cell cycle flow cytometric analysis of Jurkat cells in (C).<p><b>Copyright information:</b></p><p>Taken from "14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr"</p><p>http://www.biology-direct.com/content/3/1/17</p><p>Biology Direct 2008;3():17-17.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2390528.</p><p></p

Decreased expression of 14-3-3 θ, β, γ, and Chk1 protects against Vpr- and HIV-1 (-)-induced cell cycle arrest

(A) Diagram of the basic HIV-1 genomes used throughout the study. -deficient NL4-3, NL4-3, (top) also lacks , which was replaced with EGFP. Vpr protein associated with virions (Vpr) was delivered into cells using an RT point mutant, D186N, derivative of NL4-3(bottom). (B) Jurkat T cells were transfected with 500 pmol of nonspecific (ns), 14-3-3 θ, or Chk1 siRNA and four days later mock-infected or infected with either NL4-3(NL4-3), NL4-3Vif- (NL4-3 vif-), or RT- NL4-3(Vpr). Cell cycle arrest by was measured by flow cytometric detection of DRAQ5 DNA staining 44 hours post-infection. DNA analysis of cultures infected with RT+ NL4-3was restricted to actively infected GFP+ cells. Data is representative of six independent experiments. (C) The Dean-Jett-Fox cell cycle model was used for determination of G1 and G,M populations and the ratio is plotted for the FACS data in (B). The transfected siRNA is indicated in the legend. (D) Western blot detection of 14-3-3 θ and Chk1 expression four days post-transfection for the samples in (B) and (C). β-actin was probed as a loading control. (E) Live cell counts were measured for mock-infected and Vpr-infected samples in (B-D) at the indicated times after infection by flow cytometric constant time acquisition. (F) Jurkat cells were transfected as in (B) with the addition of a co-transfected sample that received both 14-3-3 θ and Chk1 siRNA (θ + Chk1). Western blot analysis of 14-3-3 θ, Chk1, and β-actin, as a loading control, is shown. (G) Cell cycle analysis as in (C) for the samples in (F).<p><b>Copyright information:</b></p><p>Taken from "14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr"</p><p>http://www.biology-direct.com/content/3/1/17</p><p>Biology Direct 2008;3():17-17.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2390528.</p><p></p