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, 90 (2), 1139-43

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Perturbs the G1-S Cell Cycle Progression via Deregulation of the Cyclin D1 Gene

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Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Perturbs the G1-S Cell Cycle Progression via Deregulation of the Cyclin D1 Gene

Hye-Ra Lee et al. J Virol.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) infection modulates the host cell cycle to create an environment optimal for its viral-DNA replication during the lytic life cycle. We report here that KSHV vIRF4 targets the β-catenin/CBP cofactor and blocks its occupancy on the cyclin D1 promoter, suppressing the G1-S cell cycle progression and enhancing KSHV replication. This shows that KSHV vIRF4 suppresses host G1-S transition, possibly providing an intracellular milieu favorable for its replication.

Figures

FIG 1
FIG 1
vIRF4 suppresses β-catenin-mediated TOPFLASH activity. (A) Effect of vIRF4 on β-catenin-mediated TOPFLASH activity. TOPFLASH (containing a wild-type TCF/LEF binding site) and FOPFLASH (containing a TCF/LEF binding site-defective mutant) reporters were transfected into 293T cells with vIRF4 and β-catenin as indicated. (B) Comparison of the effects of vIRF4 and LANA on β-catenin-mediated TOPFLASH activity. 293T cells were transfected with either β-catenin/vIRF4 or β-catenin/LANA, together with TOPFLASH reporter plasmid. (C) Effects of vIRF4 on the activation of LANA-mediated TOPFLASH activity. BJAB cells were transfected with β-catenin/LANA, along with vIRF4, in a dosage-dependent manner. Equal amounts of total proteins were analyzed by immunoblotting (IB) with an anti-β-catenin antibody, anti-Au, anti-V5, or anti-tubulin antibody. ***, P < 0.00001. The error bars indicate standard deviations.
FIG 2
FIG 2
The DNA-binding domain region of vIRF4 is necessary for modulation of TOPFLASH and cyclin D1 promoter activity. (A and B) Mapping of the region of vIRF4 responsible for β-catenin-mediated TOPFLASH or cyclin D1 activity. 293T cells were cotransfected with the indicated vIRF4 constructs, along with a β-catenin and TOPFLASH (A) or cyclin D1 promoter (B) construct. The cell lysates were used for luciferase assay, followed by IB with the indicated antibodies to determine expression levels of WT vIRF4 and mutants. In the vIRF4 diagram (top), TAD represents the transcriptional activation domain. (C and D) Effects of vIRF4 on the expression of cyclin D1 and other β-catenin/TCF-regulated genes. TRExBCBL1-Vector, TRExBCBL1-vIRF4, and TRExBCBL1-vIRF4ΔDBD cells were mock treated or Doxy treated for 24 h. The cell lysates were used for either quantitative reverse transcription (qRT)-PCR analyses with cyclin D1-, survivin-, SIAH-, axin-, β-catenin-, and GAPDH-specific primers (C) or IB with anti-cyclin D1, anti-Au, or anti-actin antibodies (D). *, P < 0.05; **, P < 0.0001; ***, P < 0.00001. The error bars indicate standard deviations.
FIG 3
FIG 3
vIRF4 inhibits β-catenin/CBP-mediated cyclin D1 expression and G1-S cell cycle transition. (A) Effect of vIRF4 in a TCF/β-catenin/CBP-dependent manner in either TOPFLASH reporter activity (left) or cyclin D1 reporter activity (right). 293T cells were transfected with β-catenin, CBP, and/or vIRF4. (B) TCF/β-catenin/CBP occupancy on the cyclin D1 promoter. TRExBCBL1-vIRF4 or TRExBCBL1-vIRF4ΔDBD cells were mock treated or Doxy treated for 24 h. β-Catenin, TCF4, or CBP antibodies were used for ChIP assay, and ChIP DNAs were subjected to real-time PCR using primers for the cyclin D1 promoter regions. (C) vIRF4-CBP interaction. 293T cells were transiently transfected with V5-tagged vIRF4 and/or hemagglutinin (HA)-tagged CBP, followed by immunoprecipitation (IP) with an anti-HA antibody and IB with an anti-β-catenin and an anti-V5 antibody. (D) Enhancing G1 accumulation of the cells expressing vIRF4. TRExBCBL1-Vector, TRExBCBL1-vIRF4, or TRExBCBL1-vIRF4ΔDBD cells were stimulated with Doxy (1 μg/ml) and then assessed for PI staining. The data represent the means (plus standard deviations) of the combined results from three independent experiments. *, P < 0.05; ***, P < 0.00001. (E) Inhibition of newly synthesized cellular DNA by vIRF4. TRExBCBL1-Vector and TRExBCBL1-vIRF4 cells were treated with Doxy (1 μg/ml) for 24 h, and the cells were incorporated with BrdU, followed by immunostaining with anti-BrdU antibody for confocal analysis.
FIG 4
FIG 4
vIRF4 expression leads to a decrease in cellular-DNA replication and an increase in KSHV replication. (A and B) vIRF4-dependent cellular and KSHV replication at the single-cell level. In order to perform the smDNA FISH analysis, we generated oligonucleotide probes complementary to the noncoding regions of chromosomal MCM7 and the terminal-repeat regions of KSHV and labeled the probes with Alexa 594 and Cy5, respectively. TRExBCBL1-Vector, TRExBCBL1-vIRF4, and TRExBCBL1-vIRF4ΔDBD cells were treated or not with Doxy (1 μg/ml), together with TPA (20 ng/ml) treatment. The images were acquired with a Zeiss Axiovert Illumination optical-sectioning system. Sixty z-stacks were taken automatically at 0.3-μm intervals at an exposure time of 0.5 s. (C) Effects of vIRF4-mediated G1-S transition on KSHV viral-DNA copy numbers. The reactivated KSHV contained supernatants from TRExBCBL1-Vector, TRExBCBL1-vIRF4, and TRExBCBL1-vIRF4ΔDBD mutant cells that were collected and filtered at 72 h after Doxy and TPA treatment. Virion-associated DNA was purified, and KSHV DNA levels were determined by quantitative PCR (qPCR) with specific primers against the KSHV late gene (ORF26). Shown are the relative rates of KSHV DNA copy number/μl for Vector versus vIRF4 (or the vIRF4ΔDBD mutant) expressed in TRExBCBL1 cell lines. (D) Effects of vIRF4-mediated G1-S transition on KSHV viral-protein expression. TREx BCBL1-Vector and -vIRF4 cells were treated with TPA alone or together with doxycycline for 24 h. The cells were lysed, and IB was subsequently performed with the indicated antibodies. The error bars indicate standard deviations.

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