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

Exposed Hydrophobic Residues in Human Immunodeficiency Virus Type 1 Vpr Helix-1 Are Important for Cell Cycle Arrest and Cell Death

The human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein R (Vpr) is a major determinant for virus-induced G2/M cell cycle arrest and cytopathicity. Vpr is thought to perform these functions through the interaction with partner proteins. The NMR structure of Vpr revealed solvent exposed hydrophobic amino acids along helices 1 and 3 of Vpr, which could be putative protein binding domains. We previously showed that the hydrophobic patch along helix-3 was important for G2/M blockade and cytopathicity. Mutations of the exposed hydrophobic residues along helix-1 were found to reduce Vpr-induced cell cycle arrest and cell death as well. The levels of toxicity during virion delivery of Vpr correlated with G2/M arrest. Thus, the exposed hydrophobic amino acids in the amino-terminal helix-1 are important for the cell cycle arrest and cytopathicity functions of Vpr

Physician privacy concerns when disclosing patient data for public health purposes during a pandemic influenza outbreak

Background: Privacy concerns by providers have been a barrier to disclosing patient information for public health\ud purposes. This is the case even for mandated notifiable disease reporting. In the context of a pandemic it has been\ud argued that the public good should supersede an individual’s right to privacy. The precise nature of these provider\ud privacy concerns, and whether they are diluted in the context of a pandemic are not known. Our objective was to\ud understand the privacy barriers which could potentially influence family physicians’ reporting of patient-level\ud surveillance data to public health agencies during the Fall 2009 pandemic H1N1 influenza outbreak.\ud Methods: Thirty seven family doctors participated in a series of five focus groups between October 29-31 2009.\ud They also completed a survey about the data they were willing to disclose to public health units. Descriptive\ud statistics were used to summarize the amount of patient detail the participants were willing to disclose, factors that\ud would facilitate data disclosure, and the consensus on those factors. The analysis of the qualitative data was based\ud on grounded theory.\ud Results: The family doctors were reluctant to disclose patient data to public health units. This was due to concerns\ud about the extent to which public health agencies are dependable to protect health information (trusting beliefs),\ud and the possibility of loss due to disclosing health information (risk beliefs). We identified six specific actions that\ud public health units can take which would affect these beliefs, and potentially increase the willingness to disclose\ud patient information for public health purposes.\ud Conclusions: The uncertainty surrounding a pandemic of a new strain of influenza has not changed the privacy\ud concerns of physicians about disclosing patient data. It is important to address these concerns to ensure reliable\ud reporting during future outbreaks.University of Ottawa Open Access Author Fun

Cross-border trafficking in human beings: prevention and intervention strategies for reducing sexual exploitation

Over the years, growing attention has been given to the phenomenon of trafficking in human beings (THB). Sexual exploitation was until recently by far the most commonly identified feature of THB, followed by forced labour. Many activities to combat trafficking for the purpose of sexual exploitation have been initiated by numerous supranational, international as well as national organizations. Much is written about these initiatives, but some areas have been neglected. Knowledge on ‘what works’ is in particular limited. The growing attention to THB entails a demand for more information. The severity of the crime and the impact on its victims makes it of utmost importance to gain more insight into the working and effectiveness of anti-trafficking strategies and interventions. The main objective of this review was to assess the presently available evidence on the effects of interventions that aim to prevent and suppress trafficking in human beings

Protein Kinase A Phosphorylation Activates Vpr-Induced Cell Cycle Arrest during Human Immunodeficiency Virus Type 1 Infection▿

Infection with human immunodeficiency virus type 1 (HIV-1) causes an inexorable depletion of CD4+ T cells. The loss of these cells is particularly pronounced in the mucosal immune system during acute infection, and the data suggest that direct viral cytopathicity is a major factor. Cell cycle arrest caused by the HIV-1 accessory protein Vpr is strongly correlated with virus-induced cell death, and phosphorylation of Vpr serine 79 (S79) is required to activate G2/M cell cycle blockade. However, the kinase responsible for phosphorylating Vpr remains unknown. Our bioinformatic analyses revealed that S79 is part of a putative phosphorylation site recognized by protein kinase A (PKA). We show here that PKA interacts with Vpr and directly phosphorylates S79. Inhibition of PKA activity during HIV-1 infection abrogates Vpr cell cycle arrest. These findings provide new insight into the signaling event that activates Vpr cell cycle arrest, ultimately leading to the death of infected T cells

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

Hydrophobic residues on Vpr helix-1 are important for incorporation into virions.

<p>(<b>A</b>) 293T cells were co-transfected with pcDNA3-hVpr plasmids expressing WT or mutant Vpr (or the empty vector control), pLVSV-G, and pNL4-3_{e-n-GFP RTm, VprΔ22-86} to produce virus for virion delivery of Vpr. At day 2 the virus stocks were harvested and the 293T cells were lysed. Western blot indicates protein levels for WT or mutant Vpr (bottom). A western blot for β-actin is used as a loading control (top). (<b>B</b>) Lysates were prepared from the virus stocks in (A). Viral lysates were analyzed for protein levels of WT or mutant Vpr by western blotting as in (A) (bottom). The HIV-1 p24 capsid (p24 CA) is shown as a protein loading control (top). (<b>C</b>) Densitometry of all bands in (B) was performed. The intensity of the Vpr protein band was normalized to p24 CA and plotted as % Vpr incorporation relative to WT Vpr for each mutant. The data are shown as the mean ± the standard deviation of three independent experiments.</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

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