Human Immunodeficiency Virus Type 1 Vif causes dysfunction of Cdk1 and CyclinB1: implications for cell cycle arrest

The two major cytopathic factors in human immunodeficiency virus type 1 (HIV-1), the accessory proteins viral infectivity factor (Vif) and viral protein R (Vpr), inhibit cell-cycle progression at the G2 phase of the cell cycle. Although Vpr-induced blockade and the associated T-cell death have been well studied, the molecular mechanism of G2 arrest by Vif remains undefined. To elucidate how Vif induces arrest, we infected synchronized Jurkat T-cells and examined the effect of Vif on the activation of Cdk1 and CyclinB1, the chief cell-cycle factors for the G2 to M phase transition. We found that the characteristic dephosphorylation of an inhibitory phosphate on Cdk1 did not occur in infected cells expressing Vif. In addition, the nuclear translocation of Cdk1 and CyclinB1 was disregulated. Finally, Vif-induced cell cycle arrest was correlated with proviral expression of Vif. Taken together, our results suggest that Vif impairs mitotic entry by interfering with Cdk1-CyclinB1 activation

The transcription factor BATF operates as an essential differentiation checkpoint in early effector CD8+ T cells

The transcription factor BATF is required for interleukin 17 (IL-17)-producing helper T cell (TH17) and follicular helper T cell (TFH) differentiation. Here, we show that BATF also has a fundamental role in regulating effector CD8+ T cell differentiation. BATF-deficient CD8+ T cells show profound defects in effector expansion and undergo proliferative and metabolic catastrophe early after antigen encounter. BATF, together with IRF4 and Jun proteins, binds to and promotes early expression of genes encoding lineage-specific transcription-factors (T-bet and Blimp-1) and cytokine receptors, while paradoxically repressing genes encoding effector molecules (IFN-γ and granzyme B). Thus, BATF amplifies TCR-dependent transcription factor expression and augments inflammatory signal propagation but restrains effector gene expression. This checkpoint prevents irreversible commitment to an effector fate until a critical threshold of downstream transcriptional activity has been achieved

The histone demethylase UTX regulates the lineage-specific epigenetic program of invariant natural killer T cells

Invariant natural killer T cells (iNKT cells) are innate-like lymphocytes that protect against infection, autoimmune disease and cancer. However, little is known about the epigenetic regulation of iNKT cell development. Here we found that the H3K27me3 histone demethylase UTX was an essential cell-intrinsic factor that controlled an iNKT-cell lineage-specific gene-expression program and epigenetic landscape in a demethylase-Activity-dependent manner. UTX-deficient iNKT cells exhibited impaired expression of iNKT cell signature genes due to a decrease in activation-Associated H3K4me3 marks and an increase in repressive H3K27me3 marks within the promoters occupied by UTX. We found that JunB regulated iNKT cell development and that the expression of genes that were targets of both JunB and the iNKT cell master transcription factor PLZF was UTX dependent. We identified iNKT cell super-enhancers and demonstrated that UTX-mediated regulation of super-enhancer accessibility was a key mechanism for commitment to the iNKT cell lineage. Our findings reveal how UTX regulates the development of iNKT cells through multiple epigenetic mechanisms

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

The epigenetic landscape of T cell exhaustion.

Exhausted T cells in cancer and chronic viral infection express distinctive patterns of genes, including sustained expression of programmed cell death protein 1 (PD-1). However, the regulation of gene expression in exhausted T cells is poorly understood. Here, we define the accessible chromatin landscape in exhausted CD8+ T cells and show that it is distinct from functional memory CD8+ T cells. Exhausted CD8+ T cells in humans and a mouse model of chronic viral infection acquire a state-specific epigenetic landscape organized into functional modules of enhancers. Genome editing shows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that contains essential RAR, T-bet, and Sox3 motifs. Functional enhancer maps may offer targets for genome editing that alter gene expression preferentially in exhausted CD8+ T cells

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

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

Vpr G2/M arrest correlates with cell death induced by virion delivery.

<p>(<b>A</b>) WT or mutant Vpr proteins (denoted by the single letter amino acid changes) were delivered (Vpr_v) into Jurkat cells, and separately Jurkat cells were co-transfected with pcDNA3-hVpr plasmids expressing WT or mutant Vpr (or the empty vector control) and pEGFP-N1 as a transfection marker at a ratio of 5∶1. Infected and transfected cells were analyzed for DNA content of the PI-stained cells by flow cytometry. G1 and G2/M populations were modeled using the Watson Pragmatic cell cycle model, and the G2,M/G1 ratios were plotted on the x-axis. (<b>B</b>) The viability of the Vpr_v-treated cells was monitored over time by flow cytometry (detection of PI-negative, forward-scatter high events), and the percentage of viable cells is plotted over time. These data are representative of three experiments. (<b>C</b>) The G2,M/G1 ratios and viability of Vpr_v-treated cells at 165 hour post-infection from three independent experiments were plotted. Each measurement is color-coded as in (B). Spearman's rank test was used to determine the correlation.</p

Exposed hydrophobic residues on Vpr helix-1 are important for transfected Vpr G2/M arrest.

<p>Jurkat cells were co-transfected with pcDNA3-hVpr plasmids expressing WT or mutant Vpr (or the empty vector control) and pEGFP-N1 as a transfection marker at a ratio of 5∶1. (<b>A</b>) Western blot of the Jurkat cells for WT or mutant Vpr (bottom). A western blot of β-actin was used as a protein loading control (top). (<b>B</b>) Histograms of cell cycle analysis at 61 hr post-transfection show DNA content of GFP+, PI-stained cells by flow cytometry. All samples represent 10,000 cellular events. G1 and G2/M populations were modeled using the Watson Pragmatic cell cycle model, and the G2,M/G1 ratio in each transfection is shown. The data are representative of three experiments.</p