The Epstein-Barr virus BZLF1 protein interacts physically and functionally with the histone acetylase CREB-binding protein

Abstract

The Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) is a key regulator of the EBV latent-to-lytic switch. Z is a transcriptional activator which induces EBV early gene expression. We demonstrate here that Z interacts with CREB-binding protein (CBP), a histone acetylase and transcriptional coactivator. This interaction requires the amino-terminal region of CBP as well as the transactivation and leucine zipper domains of Z. We show that CBP enhances Z-mediated transactivation of EBV early promoters, in reporter gene assays and in the context of the endogenous genome. We also demonstrate that Z decreases CREB transactivation function and that this inhibitory effect is reversed by overexpression of CBP. We show that Z also interacts directly with CREB. However, mutational analysis indicates that Z inhibition of CREB activity requires the direct interaction between Z and CBP but not the direct interaction between Z and CREB. We propose that Z interacts with CBP to enhance viral early gene transcription. In addition, the Z-CBP interaction may control host cellular transcription factor activity through competition for limiting amounts of cellular CBP.

FIG. 1

Z and CBP associate in vivo. (A) Anti-CBP antibody and a control antibody (Ab.) (rabbit serum) were used to coimmunoprecipitate Z from mock-, adenovirus-LacZ-, or adenovirus-Z-infected HeLa cell extracts (100 μg) (lanes 1 to 6). Direct loads (10 μg) of each extract were used to confirm the presence of Z (lanes 7 to 9). Western blot analysis was performed with an anti-Z antibody. (B) Anti-CBP antibody and a control antibody (rabbit serum) were used to coimmunoprecipitate Rb from adenovirus-LacZ- or adenovirus-Z-infected HeLa cell extracts (100 μg) (lanes 1 to 4). Direct loads (20 μg) of each extract were used to confirm the presence of Rb (lanes 5 and 6). Western blot analysis was performed with an anti-Rb antibody.

FIG. 2

Z binds to the amino-terminal portion of CBP. (A) Schematic of the CBP protein. The regions previously reported to bind various proteins are indicated. Below the CBP map are the five segments of CBP that have been fused to GST. (B) Affinity chromatography experiments were performed in which 10 μl of in vitro-translated Z was incubated with GST alone, GST-Z, or the GST-CBP segments (amino acids 1 to 721, 706 to 1009, 1069 to 1459, 1459 to 1891, and 1892 to 2441) bound to glutathione-agarose beads. Lane 1 shows the direct load of in vitro-translated Z protein (10 μl).

FIG. 3

The Z-CBP association requires several regions of the Z protein. (A) Schematics of the wild-type Z protein (Z wt) and the various Z mutants used in this study. The transactivator (TA), dimerization (DIM), and DNA binding domains are indicated. (B) Aliquots of 5 μl of in vitro-translated wild-type Z protein (lanes 2 to 4), 10 μl of in vitro-translated ZΔLZ (lanes 6 to 8), 5 μl of in vitro-translated ZΔ214/218 (lanes 10 to 12), 5 μl of in vitro-translated Z-CT (lanes 14 to 16), 5 μl of in vitro-translated ZΔter (lanes 18 to 20), or 5 μl of in vitro-translated Z-NT (lanes 22 to 24) were incubated with GST alone, GST-Z, or GST-CBP(1-721). Lanes 1, 5, 9, 13, 17, and 21 contain the direct loads for each protein (3 μl of Z, 10 μl of ZΔLZ, 5 μl of ZΔ214/218, 5 μl of Z-CT, 5 μl of ZΔter, and 3 μl of Z-NT).

FIG. 4

CBP enhances Z-mediated activation of EBV early gene promoters. (A) Transient reporter assays were performed in which DG75 cells were transfected with 5 μg of each promoter construct (EApBS-CAT or BHRF1-CAT) and 1 μg of Z expression plasmid with or without 2 μg of CBP expression plasmid or CBP-HAT(−) expression plasmid (for a total of 8 μg DNA). CAT assays were performed as described in the text. The results are a combination of at least two separate experiments. (B) Extracts (containing either vector alone, Z, or Z plus CBP) from one of the CAT assays above were analyzed for Z protein levels. Western blot analysis was performed with an anti-Z antibody. (C and D) Transient reporter assays were performed in which DG75 cells were transfected with 5 μg of each promoter construct (EApBS-CAT [C] or BHRF1-CAT [D]) and 1 μg of Z, Z311, or ZE₂ter expression plasmid with or without 2 μg of CBP expression plasmid (for a total of 8 μg DNA). CAT assays were performed as described in the text. The results are a combination of two separate experiments.

FIG. 5

CBP enhances Z-mediated transactivation of BMRF1 from the endogenous viral genome. D98/HE-R-1 cells were transfected with either 2 μg of vector (vec.; lanes 1 to 4) or 2 μg of Z expression plasmid (lanes 5 to 8), along with increasing amounts (0, 2, 4, and 6 μg) of CBP expression plasmid. Cells were harvested 14 h posttransfection. Western blot analysis was performed with an anti-EAD antibody.

FIG. 6

Z inhibits CREB activity. (A) Transient reporter assays were performed in which DG75 cells were transfected with 5 μg of GAL4-E1B-CAT, 1 μg each of SG424 (GAL4 DNA-binding domain alone) or GAL4-CREB, and 1 μg each of control vector or Z expression plasmid with or without 1 μg of CBP expression plasmid (8 μg of total DNA). CAT assays were performed as described in the text. The results are a combination of two separate experiments. (B) DG75 cells were transfected with 5 μg of Zp-CRE-CAT and 1 μg each (or a combination) of control vector, CREB, PKA, Z, or CBP expression plasmid (9 μg in total). CAT assays were performed as described in the text. The results are a combination of two separate experiments.

FIG. 7

Z interacts directly with CREB. (A) Anti-CBP antibody (Ab.) and a control antibody (rabbit serum) were used to coimmunoprecipitate (Ip) CREB from adenovirus-LacZ- or adenovirus-Z-infected HeLa cell extracts (10 μg). Western blot analysis was performed with an anti-CREB antibody. (B) Anti-CREB antibody was used to coimmunoprecipitate Z from mock-, adenovirus-LacZ-, or adenovirus-Z-infected HeLa cell extracts (100 μg) (lanes 1 to 3). Direct loads (10 μg) of each extract were used to confirm the presence of Z (lanes 4 to 6). Western blot analysis was performed with an anti-Z antibody. The results from the control antibody used in this experiment are presented in Fig. 1. (C) Aliquots of 4 μl of in vitro-translated wild-type Z protein (lanes 2 to 5), 5 μl of in vitro-translated Z-CT protein (lanes 7 to 10), or 5 μl of in vitro-translated ZΔ200 protein (lanes 12 to 15) were incubated with GST alone, GST-Z, GST-CREB, or GST-CBP(1-721). The direct loads (lanes 1, 6, and 11) contain 4 μl of Z, 5 μl of Z-CT, or 5 μl of ZΔ200. (D) DG75 cells were transfected with 5 μg of GAL4-E1B-CAT, 1 μg each of SG424 (GAL4 DNA-binding domain alone) or GAL4-CREB, and 1 μg each of vector, Z, Z311, Z-CT, or ZΔ200 expression plasmid. CAT assays were performed as described in the text. The results are a combination of two separate experiments. Fold activation was calculated by taking the ratio of GAL4-CREB activity to SG424 activity in the presence of each Z construct.