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, 83 (13), 6739-47

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 Targets MDM2 to Deregulate the p53 Tumor Suppressor Pathway

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Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 Targets MDM2 to Deregulate the p53 Tumor Suppressor Pathway

Hye-Ra Lee et al. J Virol.

Abstract

Cells infected by viruses utilize interferon (IFN)-mediated and p53-mediated irreversible cell cycle arrest and apoptosis as part of the overall host surveillance mechanism to ultimately block viral replication and dissemination. Viruses, in turn, have evolved elaborate mechanisms to subvert IFN- and p53-mediated host innate immune responses. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes several viral IFN regulatory factors (vIRF1 to vIRF4) within a cluster of loci, their functions being primarily to inhibit host IFN-mediated innate immunity and deregulate p53-mediated cell growth control. Despite its significant homology and similar genomic location to other vIRFs, vIRF4 is distinctive, as it does not target and antagonize host IFN-mediated signal transduction. Here, we show that KSHV vIRF4 interacts with the murine double minute 2 (MDM2) E3 ubiquitin ligase, leading to the reduction of p53, a tumor suppressor, via proteasome-mediated degradation. The central region of vIRF4 is required for its interaction with MDM2, which led to the suppression of MDM2 autoubiquitination and, thereby, a dramatic increase in MDM2 stability. Consequently, vIRF4 expression markedly enhanced p53 ubiquitination and degradation, effectively suppressing p53-mediated apoptosis. These results indicate that KSHV vIRF4 targets and stabilizes the MDM2 E3 ubiquitin ligase to facilitate the proteasome-mediated degradation of p53, perhaps to circumvent host growth surveillance and facilitate viral replication in infected cells. Taken together, the indications are that the downregulation of p53-mediated cell growth control is a common characteristic of the four KSHV vIRFs and that p53 is indeed a key factor in the host's immune surveillance program against viral infections.

Figures

FIG. 1.
FIG. 1.
The central region of vIRF4 is required for MDM2 interaction. (A) Coimmunoprecipitation (Co-IP) of MDM2 with vIRF4 (WT). 293T cells were transiently transfected with the indicated constructs, followed by immunoprecipitation (IP) with an anti-V5 antibody and immunoblotting (IB) with an anti-MDM2 antibody. (B) Co-IP of MDM2 with vIRF4 (WT) or vIRF1 (WT). 293T cells were transfected MDM2 and the indicated vIRF4 or vIRF1 constructs, followed by IP with an anti-V5 antibody and IB with an anti-MDM2 antibody. One percent of the whole-cell lysates (WCL) was used as the input. (C) Schematic representation of the plasmid constructs. (D) Co-IP of MDM2 with the WT or several vIRF4 mutants. 293T cells were transfected with the indicated vIRF4 constructs along with MDM2, followed by IP with an anti-V5 antibody and IB with an anti-MDM2 antibody. One percent of the WCL was used as the input.
FIG. 2.
FIG. 2.
vIRF4 augments MDM2 stability by suppressing the ubiquitination of MDM2. (A) An increase in the stability of MDM2 is induced by vIRF4. (A, left) After transfection with MDM2, GST, and an empty vector or MDM2, GST, and vIRF4 (WT or mutant), cells were treated with CHX (20 μg/ml) for the indicated periods of time, and then immunoblotted (IB) with anti-MDM2 antibody. (A, middle) At 48 h posttransfection with GST and/or vIRF4, 293T cells were treated with CHX (20 μg/ml) for the indicated periods of time. Whole-cell lysates (WCL) were used for immunoprecipitation (IP) or IB with the indicated antibodies. (A, right) At 48 h posttransfection of 293T cells with vIRF4 or mock transfection, total RNAs were prepared and MDM2 mRNA levels were analyzed by qRT-PCR. (B and C) Effects of vIRF4 on the ubiquitination of MDM2. 293T (B) or HCT116 (C) cells were cotransfected with the indicated vIRF4 constructs along with MDM2 and ubiquitin constructs. The cells were then treated with LLNL (25 μM), followed by IP with an anti-MDM2 antibody and IB with an anti-ubiquitin antibody. One percent of the WCL was used as the input. M, MDM2 (C464A) mutant that no longer possesses E3 ligase activity. (D) MDM2 accumulation is dependent on vIRF4 interaction. HCT116 cells were cotransfected with MDM2, ubiquitin, and the WT or mutant forms of vIRF4. The cells were then treated with LLNL (25 μM), followed by IP with an anti-MDM2 antibody and IB with an anti-ubiquitin antibody. One percent of the WCL was used as the input.
FIG. 3.
FIG. 3.
vIRF4 enhances the ubiquitination of p53 through its interaction with MDM2. (A) The effects of vIRF4 and MDM2 on the ubiquitination of p53. 293T cells were transfected with vIRF4 or MDM2, followed by immunoprecipitation (IP) with an anti-p53 antibody and immunoblotting (IB) with an anti-ubiquitin antibody. One percent of the whole-cell lysates (WCL) was used as the input. (B and C) Effects of vIRF4-MDM2 interaction on the ubiquitination of p53. 293T (B) or HCT116 (C) cells were cotransfected with the vIRF4 constructs along with the MDM2 and ubiquitin constructs. Cells were then treated with LLNL (25 μM), followed by IP with an anti-MDM2 antibody and IB with an anti-ubiquitin antibody. One percent of the WCL was used as the input.
FIG. 4.
FIG. 4.
vIRF4 downregulates p53 protein levels in stable cell lines and interacts with endogenous MDM2 during viral reactivation. (A) p53 expression in Dox-induced TREx/BCBL-1 vIRF4 cells. Upon stimulation with Dox (1 μg/ml), equal amounts of the total proteins were analyzed by immunoblotting (IB) with an anti-p53 antibody at the indicated times. An anti-actin antibody was used to monitor general protein levels, while an anti-Au antibody was used to monitor vIRF4 expression. (B and C) The expression levels of p53 and MDM2 in Dox-induced TREx/BCBL-1 vIRF4 cells and Dox-induced TREx/BCBL-1 vIRF4 (1-605) cells were compared. Twenty-four hours after stimulation with Dox (1 μg/ml), equal amounts of the total proteins were analyzed by IB with anti-p53 and anti-MDM2 antibodies. An anti-tubulin antibody was used to monitor general protein levels, while an anti-Au antibody was used to monitor vIRF4 protein expression. (D, left) At 24 h posttreatment of TREx/BCBL-1 pcDNA5/FRT/TO and TREx/BCBL-1 vIRF4 cells with TPA (20 ng/ml) and/or Dox (1 μg/ml), equal amounts of the total proteins were used for immunoprecipitation (IP) and IB with the indicated antibodies. One percent of the whole-cell lysates was used as the input. An anti-tubulin antibody was used to monitor general protein levels. (D, right) At 24 h posttreatment with TPA (20 ng/ml) and/or Dox (1 μg/ml), total RNAs were isolated from TREx/BCBL-1 pcDNA/FRT/TO and TREx/BCBL-1 vIRF4 cells and the p53 mRNA levels were analyzed by qRT-PCR. Vector, TREx/BCBL-1 pcDNA5/FRT/To cells; vIRF4, TREx/BCBL-1 vIRF4 cells.
FIG. 5.
FIG. 5.
vIRF4 enhances the ubiquitination of p53 in stable cell lines. (A) Effects of vIRF4 expression on the ubiquitination of p53 in stable cell lines. Upon stimulation with Dox (1 μg/ml), cells were also treated with LLNL (25 μM) for 4 h before being harvested at the indicated times. Cells lysates were then used for immunoprecipitation (IP) with an anti-p53 antibody, followed by immunoblotting (IB) with an anti-ubiquitin antibody. An anti-tubulin antibody was used to monitor general protein levels, while an anti-Au antibody was used to monitor vIRF4 protein expression. (B) vIRF4 protects cells from apoptosis induced by two different DNA damage reagents. (B, top) HCT116 cells stably expressing the WT or mutant forms of vIRF4 were treated with 5-FU for 24 h and then assessed for annexin V and PI staining, followed by FACS analysis. (B, bottom) TREx/BCBL-1 cells stably expressing the WT or mutant forms of vIRF4 via stimulation with Dox (1 μg/ml) were treated with etoposide for 24 h and then assessed for annexin V and PI staining, followed by FACS analysis. Data represent the means (±SD) of the combined results from three independent experiments. (C) At the indicated times after stimulation with TPA (20 ng/ml), equal amounts of the total proteins were analyzed by IB with KSHV-specific antibodies. An anti-tubulin antibody was used to monitor general protein levels. V, TREx/BCBL-1 pcDNA5/FRT/To cells; 4, TREx/BCBL-1 vIRF4 cells. Similar results were obtained from three independent experiments.

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