Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Jul;75(13):6193-8.
doi: 10.1128/JVI.75.13.6193-6198.2001.

Viral Interferon Regulatory Factor 1 of Kaposi's Sarcoma-Associated Herpesvirus Binds to p53 and Represses p53-dependent Transcription and Apoptosis

Affiliations
Free PMC article

Viral Interferon Regulatory Factor 1 of Kaposi's Sarcoma-Associated Herpesvirus Binds to p53 and Represses p53-dependent Transcription and Apoptosis

T Seo et al. J Virol. .
Free PMC article

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is related to the development of Kaposi's sarcoma. Open reading frame K9 of KSHV encodes viral interferon regulatory factor 1 (vIRF1), which functions as a repressor of interferon- and IRF1-mediated signal transduction. In addition, vIRF1 acts as an oncogene to induce cellular transformation. Here we show that vIRF1 directly associates with the tumor suppressor p53 and represses its functions. The vIRF1 interaction domains of p53 are the DNA binding domain (amino acids [aa] 100 to 300) and the tetramerization domain (aa 300 to 393). p53 interacts with the central region (aa 152 to 360) of vIRF1. vIRF1 suppresses p53-dependent transcription and deregulates its apoptotic activity. These results suggest that vIRF1 may regulate cellular function by inhibiting p53.

Figures

FIG. 1
FIG. 1
In vivo interaction of vIRF1 with p53. (A) A GST expression plasmid (pEBG) or a GST-vIRF1 expression plasmid (pEBG/vIRF1) was cotransfected with an HA-p53 expression plasmid (pcDNA3/HA-p53) into 293T cells. Whole-cell extracts were prepared 48 h after transfection and precipitated with glutathione-Sepharose 4B beads. The precipitated proteins were washed and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). GST fusion protein and HA-p53 were detected by Western blotting with anti-HA (top and bottom) and anti-GST (middle). Lanes: 1, GST alone; 2, no expression plasmid; 3, GST with HA-p53; 4, GST-vIRF1 with HA-p53. (B) A vIRF1 expression plasmid (pcDNA3/vIRF1) was cotransfected with or without the HA-p53 expression plasmid. Whole-cell extracts were incubated with protein G resin after preincubation with anti-HA. The resulting immunoprecipitates were washed and resolved by SDS-PAGE. vIRF1 and HA-p53 were detected by Western blotting with anti-vIRF1 rabbit polyclonal antibody (top) or with anti-HA (αHA) antibody (bottom). Lanes: 1 and 3, vIRF1 alone; 2 and 4, vIRF1 with HA-p53. IP, immunoprecipitation. Anti-vIRF1 polyclonal antibody was obtained from postimmune sera collected from rabbits immunized with the GST-vIRF1 fusion protein. (C) In vivo interaction of vIRF1 with p53 in KSHV-infected BCBL-1 cells. BCBL-1 cells were treated with TPA as previously described (20), and whole-cell extracts were prepared after the indicated number of hours. vIRF1 expression was detected by Western blotting with anti-vIRF1 antibody (left). Whole-cell extracts from BJAB and BCBL-1 cells were immunoblotted with anti-vIRF1 and anti-p53 (lanes 1 and 2). Direct coimmunoprecipitation (IP) was performed by using BCBL-1 cells after TPA stimulation. BCBL-1 and BJAB cells (1 × 107 cells) were harvested 48 h after TPA stimulation, and whole-cell extracts were incubated with protein G resin after being preincubated with either anti-p53 (αp53) (lanes 3 and 4) or anti-HA (αHA) (lane 5) antibody. vIRF1 was detected by Western blotting with anti-vIRF1 (right top) or anti-p53 (right bottom) antibody.
FIG. 2
FIG. 2
Identification of domains involved in vIRF1-p53 interaction. (A) Top, schematic representation of p53 and its functional domains. The TA domain (aa 1 to 42), DBD (aa 1 to 42), and TD (aa 300 to 393) are shown. Bottom, GST pulldown assays (10) performed with wild-type and mutant GST-p53 fusion proteins by using 35S-labeled, in vitro-translated vIRF1. The input (20%) and GST pulldown mixtures were resolved by SDS-PAGE, and vIRF1 was visualized by autoradiography. (B) Left, schematic representation of vIRF1 and its deletion mutants. Right, an experiment similar to that described for panel A that was performed by using the GST-p53 and 35S-labeled, in vitro-translated vIRF1 and vIRF1 mutant proteins.
FIG. 3
FIG. 3
vIRF1 represses p53-dependent transcription. Luciferase activity was measured with a luminometer. The total amount of transfected DNA in each experiment was kept constant by the addition of a blank vector (pcDNA3). The activity of the reporter alone was normalized to a value of 1, and each luciferase measurement was normalized to the internal control, β-galactosidase activity. Each experiment was carried out at least three times. (A) A synthetic p53 response element fused to a luciferase gene (PG13-Luc) was inhibited by vIRF1 in the presence of p53 in 293T cells. 293T cells were cotransfected with PG13-Luc (1 μg), a β-galactosidase expression plasmid (0.5 μg), a p53 expression plasmid (pcDNA3/HA-p53) (0.5 μg), and increasing amounts of an expression plasmid encoding vIRF1 (pcDNA3/vIRF1). Equal amounts of total cellular extracts were resolved by SDS-PAGE and subjected to HA-specific immunoblotting. (B) PG13-Luc was inhibited by vIRF1 in the presence of p53. Saos2 cells were cotransfected with PG13-Luc (1 μg), a β-galactosidase expression plasmid (RSV/β-gal) (0.5 μg), a p53 expression plasmid (pcDNA3/HA-p53) (0.5 μg), and increasing amounts of an expression plasmid encoding vIRF1 (pcDNA3/vIRF1). (C) The WWP-Luc plasmid was inhibited by vIRF1 in the presence of p53. Saos2 cells were cotransfected with WWP-Luc (1 μg) and the other plasmids listed in panel B. (D) vIRF1 represses p53-dependent transcription in BJAB cells. BJAB cells were cotransfected by electroporation (14) with PG13-Luc (5 μg), an HA-p53 expression plasmid, and a vIRF1 expression plasmid. (E) vIRF1 represses transcriptional activation by p53 and does not influence an unrelated transcriptional activator. 293T cells were cotransfected with a Gal4-Luc reporter plasmid (pFR-Luc) (1 μg), a vIRF1 expression plasmid, and either a Gal4-p53 expression plasmid (0.5 μg) or a Gal4-SP1 expression plasmid (0.5 μg). (F) vIRF1 does not repress transcriptional activation by p53(TA) (aa 1 to 42). 293T cells were cotransfected with pFR-Luc (1 μg), a Gal4-p53(TA) expression plasmid, and a vIRF1 expression plasmid. (G) vIRF1 represses p53-induced p21 expression in 293T cells. A vIRF1 expression plasmid (pcDNA3/vIRF1) and an HA-p53 expression plasmid (pcDNA3/HA-p53) were cotransfected into 293T cells by the calcium phosphate method. Cells were harvested 48 h after transfection. Lanes: 1, no expression plasmid; 2, pcDNA3/HA-p53 (7 μg) plus pcDNA3 (15 μg); 3, pcDNA3/HA-p53 (7 μg) plus pcDNA3/vIRF1 (15 μg); 4, pcDNA3 (7 μg) plus pcDNA3/vIRF1 (15 μg). p21 was detected with a monoclonal antibody to p21 (top), and β-actin was detected with a monoclonal antibody to β-actin (bottom).
FIG. 4
FIG. 4
vIRF1 deregulates p53-induced apoptosis in Saos2 cells. Saos2 cells grown on coverslips in 35-mm-diameter dishes were transfected with Superfect transfection reagent (Qiagen, Hilden, Germany). Forty-eight hours after transfection, cells were fixed and the TUNEL reaction was performed as recommended by the manufacturer (Roche). The analyses were performed with a Zeiss confocal microscope with fluorescein isothiocyanate filter sets. (A) Left top, mock transfection (no expression plasmid); right top, pcDNA3/HA-p53 (1 μg) plus pcDNA3 (1 μg); bottom left, pcDNA3/HA-p53 (1 μg) plus pcDNA3/vIRF1 (1 μg); bottom right, pcDNA3/vIRF1 (1 μg) plus pcDNA3 (1 μg). Magnification, ×100. In the insert at the top right, the magnification of the cell indicated by the arrow is ×400. (B) Schematic representation of TUNEL-positive cell percentages. The bars represent the percentages of transfected cells that showed apoptosis; apoptosis was determined by counting the green dead cells. The values shown are means calculated from two duplicate experiments. A total of 1,000 cells were counted in each experiment.

Similar articles

See all similar articles

Cited by 50 articles

See all "Cited by" articles

Publication types

Feedback