The Kaposi's sarcoma-associated herpesvirus K8 protein interacts with CREB-binding protein (CBP) and represses CBP-mediated transcription

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame K8 encodes a basic region-leucine zipper protein of 237 amino acids that homodimerizes with its bZIP domain. KSHV K8 shows significant homology to the Epstein-Barr virus (EBV) immediate-early protein Zta, a key regulator in the reactivation and replication of EBV. In this study, we report that K8, like its homolog EBV Zta, interacts with cellular CREB-binding protein (CBP) in vivo and in vitro. This interaction requires the C/H3 domain of CBP and the basic region of K8. K8 represses CBP-mediated transcription by competing with limited amounts of cellular CBP, exemplified by the reduced expression from the AP-1 and human immunodeficiency virus long terminal repeat promoters.

FIG. 1

In vivo interaction of K8 and CBP. (A) The coimmunoprecipitation of in vivo-synthesized K8 and CBP was analyzed by Western blotting. 293T cells were transfected with an HA-tagged CBP expression vector and either a blank (pEBG) vector or an expression vector carrying GST-fused K8 (pEBG-K8). The cells were harvested, lysed, and precipitated with either anti-HA antibody (α-HA) or anti-GST antibody (α-GST), and protein G resin. The proteins were analyzed by SDS-PAGE and immunoblotted with anti-HA or anti-GST antibodies. (B) Coimmunoprecipitation assay in the KSHV-positive BCBL-1 cell lines. The BJAB cell lines were used as a KSHV-negative control. BCBL-1 cell lines, with or without TPA induction (48 h), were harvested, and the appropriate lysates were precipitated and immunoblotted with either CBP-specific monoclonal antibody (α-CBP) or rabbit polyclonal anti-K8 antibody (α-K8), respectively. (C) K8 colocalizes with CBP in 293T cells. The GFP-K8 and HA-CBP expression vectors were transfected into 293T cells. Cells were fixed and immunostained 48 h after transfection. HA-CBP was detected using a rhodamine-conjugated secondary antibody against a mouse monoclonal HA antibody. (D) Colocalization of K8 and CBP in BCBL-1 cells. For the expression of lytic gene product K8, BCBL-1 cells were treated with TPA for 48 h. K8 was detected by indirect immunofluorescence using a rabbit anti-K8 antibody as a primary antibody and FITC-conjugated goat anti-rabbit IgG as a secondary antibody. CBP was detected with a mouse CBP-specific monoclonal antibody and TRITC-conjugated goat anti-mouse IgG. The nucleus of the cell was stained with DAPI.

FIG. 2

In vitro interaction and the interacting domains of K8 with CBP. (A) The domains of CBP and the GST-fused CBP fragments are indicated. KIX, kinase-induced domain interacting domain; ZF, zinc finger; CTAD, carboxyl-terminal transcriptional activation domain. (B) GST pulldown assays with in vitro-translated, ³⁵S-labeled K8 and deletion mutants of CBP fused to GST. (C) Domains within K8 and the deletion mutants. The K8 fragments were introduced into pGEX4T-1. (D) GST pulldown assays were performed with in vitro-translated, ³⁵S-labeled CBP2 and GST-fused K8 fragments. (E) GST pulldown assays of in vitro-translated K8 and its mutant K8(1–189) with GST-fused K8 and CBP2. (F) Localization of K8 deletion mutants was investigated by immunostaining 293T cells transfected with each Flag-tagged K8 deletion mutant. (G) Localization of K8 and NLS-deleted mutants K8(190–237) and K8(1–115) cloned into an NLS-containing CMV2N3T vector.

FIG. 3

K8 represses the transcription of AP-1 in a CBP-dependent manner. (A) Transient reporter assays were performed in which 293T cells were transiently cotransfected with a reporter gene construct (AP-1 Luc) and K8 expression plasmids CMV2N3T K8, K8(190–237), and K8(1–115). The amounts of expression vectors are shown. In all assays, the fold activation was determined by luciferase activity derived from the reporter after normalizing it to β-Gal activity from a cotransfected RSV β-Gal control plasmid. All experiments were performed at least in triplicate, and the total amount of each expression vector was kept constant. The luciferase activity of AP-1 in the absence of the other expression vectors and TPA induction is normalized to a value of 1. (B) AP-1 reporter assays with induction by c-Fos expression vector instead of TPA. (C) CBP relieves the repression of AP-1 by K8 in a dose-dependent manner, but does not activate the AP-1 promoter. (D) Transient reporter assay with the same reporter and expression vectors in BJAB cell lines.

FIG. 4

K8 represses the transcription of HIV LTR in a CBP-dependent manner. (A) Transient reporter assays in 293T cells with a reporter gene, HIV LTR luciferase, and the same expression vectors. The luciferase activity of HIV LTR in the absence of the other expression vectors is normalized to a value of 1. (B) CBP relieves the repression of HIV LTR by K8 in a dose-dependent manner but does not much activate the HIV LTR promoter alone. (C) Transient reporter assays with the same reporter and expression vectors in BJAB cell lines. (D) The equivalent expression of each plasmid was verified by Western blotting.