Kaposi's sarcoma-associated herpesvirus open reading frame 50 represses p53-induced transcriptional activity and apoptosis - PubMed (original) (raw)

Kaposi's sarcoma-associated herpesvirus open reading frame 50 represses p53-induced transcriptional activity and apoptosis

Y Gwack et al. J Virol. 2001 Jul.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 50 (ORF50) encodes a viral transcriptional activator which stimulates the transcription of viral early and late genes of KSHV. Here we show that ORF50 represses transcriptional activity of p53 and p53-induced apoptosis through interaction with CREB binding protein (CBP). This inhibitory effect of ORF50 on the transcriptional activity of p53 was relieved by the addition of CBP. ORF50 mutants, which are defective in interaction with CBP, lost the inhibitory effects on p53. Our data provide a framework for delineating the regulatory mechanisms used by KSHV to modulate cellular transcription and the cell cycle.

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Figures

FIG. 1

FIG. 1

ORF50 represses p53-induced transactivation. (A) The domains within ORF50 and the mutated forms of ORF50 are shown. ORF50 contains the basic domain, the leucine zipper motif (LZ), and the transcriptional activation domain (TAD). The LXXLL motif, which interacts with CBP, is located between amino acids 593 and 597 of ORF50. (B) The fold activation of the luciferase activity was determined with the p53-responsive reporters, and the following reporters were used: PG13-Luc (synthetic p53 binding site), MDM2-Luc (the promoter of MDM2 protein), and WWP-Luc (the promoter of p21). All of the ORF50 mutants were introduced into pME18S, a mammalian expression vector containing the SRα promoter and a Flag tag. 293T cells were transfected with the indicated vectors, and extracts were analyzed by blotting with anti-Flag antibody (upper right panel).

FIG. 2

FIG. 2

ORF50 repressed transactivation of p53 through interaction with CBP. (A) To determine whether inhibition by ORF50 could be relieved by the addition of CBP, the fold activation in luciferase activity was determined. (B) ORF50 inhibited the transcriptional activation of Gal4/p53. (C) ORF50 repressed p53 transactivation, while the ORF50 mutant LXXAA, which does not bind to CBP, could not inhibit p53 transactivation in the B-cell lymphoma cell line, BJAB.

FIG. 3

FIG. 3

ORF50 represses p53-induced apoptosis. (A) Apoptosis assays in SAOS2 cells were performed. The hypoploid cell fraction was analyzed by using a fluorescence-activated cell sorter. PI, propidium iodide. (B) To verify the results of fluorescence-activated cell sorting, the level of apoptosis was determined by counting DAPI-stained condensed nuclei in GFP-expressing cells by using a fluorescence microscope.

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