Epstein-Barr virus immediate-early protein BRLF1 interacts with CBP, promoting enhanced BRLF1 transactivation

Abstract

The Epstein-Barr virus (EBV) immediate-early protein BRLF1 is a transcriptional activator that mediates the switch from latent to lytic viral replication. Many transcriptional activators function, in part, due to an interaction with histone acetylases, such as CREB-binding protein (CBP). Here we demonstrate that BRLF1 interacts with the amino and carboxy termini of CBP and that multiple domains of the BRLF1 protein are necessary for this interaction. Furthermore, we show that the interaction between BRLF1 and CBP is important for BRLF1-induced activation of the early lytic EBV gene SM in Raji cells.

FIG. 1

BRLF1 interacts with CBP in vivo. (A) HeLa cells were mock infected or infected with adenovirus vectors expressing beta-galactosidase (AdLacZ) or BRLF1 (AdBRLF1). Anti-CBP antibody (Ab) or a control rabbit antibody were used to coimmunoprecipitate BRLF1 (300 μg; lanes 4 to 9). Immunocomplexes were electrophoresed on a 7.5% polyacrylamide gel, transferred to nitrocellulose, and immunoblotted for BRLF1. Proteins were visualized by chemiluminescence and autoradiography. Direct loads (30 μg; lanes 1 to 3) confirmed the presence of BRLF1 in the HeLa cell extracts. (B) Coimmunoprecipitation experiments (as described for panel A) were performed using extracts from Akata cells with or without anti-immunoglobulin G (anti-IgG) treatment (100 μg/ml; Sigma) for 4 h, which induces lytic EBV infection. The direct load lane is lysate from Akata cells treated with an anti-IgG antibody. (C) The same CBP and control rabbit antibodies used for panels A and B as well as a BRLF1-reactive antibody were used to immunoprecipitate GST or GST-BRLF1 fusion protein (GST-R), followed by immunoblot analysis with a BRLF1 antibody.

FIG. 1

BRLF1 interacts with CBP in vivo. (A) HeLa cells were mock infected or infected with adenovirus vectors expressing beta-galactosidase (AdLacZ) or BRLF1 (AdBRLF1). Anti-CBP antibody (Ab) or a control rabbit antibody were used to coimmunoprecipitate BRLF1 (300 μg; lanes 4 to 9). Immunocomplexes were electrophoresed on a 7.5% polyacrylamide gel, transferred to nitrocellulose, and immunoblotted for BRLF1. Proteins were visualized by chemiluminescence and autoradiography. Direct loads (30 μg; lanes 1 to 3) confirmed the presence of BRLF1 in the HeLa cell extracts. (B) Coimmunoprecipitation experiments (as described for panel A) were performed using extracts from Akata cells with or without anti-immunoglobulin G (anti-IgG) treatment (100 μg/ml; Sigma) for 4 h, which induces lytic EBV infection. The direct load lane is lysate from Akata cells treated with an anti-IgG antibody. (C) The same CBP and control rabbit antibodies used for panels A and B as well as a BRLF1-reactive antibody were used to immunoprecipitate GST or GST-BRLF1 fusion protein (GST-R), followed by immunoblot analysis with a BRLF1 antibody.

FIG. 1

BRLF1 interacts with CBP in vivo. (A) HeLa cells were mock infected or infected with adenovirus vectors expressing beta-galactosidase (AdLacZ) or BRLF1 (AdBRLF1). Anti-CBP antibody (Ab) or a control rabbit antibody were used to coimmunoprecipitate BRLF1 (300 μg; lanes 4 to 9). Immunocomplexes were electrophoresed on a 7.5% polyacrylamide gel, transferred to nitrocellulose, and immunoblotted for BRLF1. Proteins were visualized by chemiluminescence and autoradiography. Direct loads (30 μg; lanes 1 to 3) confirmed the presence of BRLF1 in the HeLa cell extracts. (B) Coimmunoprecipitation experiments (as described for panel A) were performed using extracts from Akata cells with or without anti-immunoglobulin G (anti-IgG) treatment (100 μg/ml; Sigma) for 4 h, which induces lytic EBV infection. The direct load lane is lysate from Akata cells treated with an anti-IgG antibody. (C) The same CBP and control rabbit antibodies used for panels A and B as well as a BRLF1-reactive antibody were used to immunoprecipitate GST or GST-BRLF1 fusion protein (GST-R), followed by immunoblot analysis with a BRLF1 antibody.

FIG. 2

BRLF1 interacts with the amino- and carboxy-terminal regions of CBP in vitro. (A) Schematic diagram of GST-CBP fusion constructs. Regions implicated in CBP-protein interactions are indicated. The five CBP fragments fused in frame with GST are indicated below the GST-CBP construct (29). HAT, histone acetyltransferase. (B) Five microliters of [³⁵S]methionine-labeled in vitro-translated BRLF1 was incubated with GST alone (lane 2), GST-BRLF1 (lane 3), or GST-CBP constructs (lanes 4 to 8) bound to glutathione beads. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. A direct load of in vitro-translated BRLF1 (2.5 μl) is shown in lane 1.

FIG. 2

BRLF1 interacts with the amino- and carboxy-terminal regions of CBP in vitro. (A) Schematic diagram of GST-CBP fusion constructs. Regions implicated in CBP-protein interactions are indicated. The five CBP fragments fused in frame with GST are indicated below the GST-CBP construct (29). HAT, histone acetyltransferase. (B) Five microliters of [³⁵S]methionine-labeled in vitro-translated BRLF1 was incubated with GST alone (lane 2), GST-BRLF1 (lane 3), or GST-CBP constructs (lanes 4 to 8) bound to glutathione beads. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. A direct load of in vitro-translated BRLF1 (2.5 μl) is shown in lane 1.

FIG. 3

Multiple domains in the BRLF1 protein are required for efficient interaction with CBP. (A) Schematic diagram of BRLF1 and BRLF1 mutants used in the mapping study. The DNA binding, dimerization, and transactivation domains are indicated. (B) Five microliters of [³⁵S] methionine-labeled in vitro-translated BRLF1 or BRLF1 mutants was incubated with GST alone (lanes 5 to 8) or with GST-CBP 1-721 (lanes 9 to 12) bound to glutathione beads. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. Direct loads of in vitro-translated protein (2.5 μl) were also included (lanes 1 to 4). (C) Five microliters of in vitro-translated BRLF1 or BRLF1 mutants was incubated with GST alone (lanes 5 to 8) or with GST-CBP 1892-2441 (lanes 9 to 12) bound to glutathione beads. Direct loads of in vitro-translated protein (2.5 μl) were also included (lanes 1 to 4).

FIG. 4

BRLF1-CBP interaction affects BRLF1 transactivator function. (A) Transient reporter assays were performed in which HeLa cells were transfected with 4 μg of promoter construct (S-CAT, containing SM gene promoter sequences), 0.3 μg of BRLF1 expression vector (CMV-RIE) (26), or control vector (pHD1013), with or without 1 μg of the CMV-CBP expression vector (29). CAT activity was determined as previously described (20). The results represent data from two separate experiments. (B) The S-CAT plasmid (1 μg) was transfected into HeLa cells with either vector DNA (8.5 μg) (SG5), wild-type E1A vector (8 μg), an E1A mutant (Δ2-36) which is unable to bind CBP (8 μg), the BRLF1 expression vector (pRTS15; a gift from Diane Hayward) (0.5 μg) and control vector DNA (8 μg), the BRLF1 expression vector (0.5 μg) and wild-type E1A (8 μg), or the BRLF1 expression vector (0.5 μg) and the E1A mutant (Δ2-36) (8 μg). The E1A vectors were gifts from David Livingston (8). (C) Immunoblot analysis was performed using extracts from the experiment shown in panel 4B to assess the level of transfected wild-type versus mutant E1A proteins and of BRLF1.

FIG. 4

BRLF1-CBP interaction affects BRLF1 transactivator function. (A) Transient reporter assays were performed in which HeLa cells were transfected with 4 μg of promoter construct (S-CAT, containing SM gene promoter sequences), 0.3 μg of BRLF1 expression vector (CMV-RIE) (26), or control vector (pHD1013), with or without 1 μg of the CMV-CBP expression vector (29). CAT activity was determined as previously described (20). The results represent data from two separate experiments. (B) The S-CAT plasmid (1 μg) was transfected into HeLa cells with either vector DNA (8.5 μg) (SG5), wild-type E1A vector (8 μg), an E1A mutant (Δ2-36) which is unable to bind CBP (8 μg), the BRLF1 expression vector (pRTS15; a gift from Diane Hayward) (0.5 μg) and control vector DNA (8 μg), the BRLF1 expression vector (0.5 μg) and wild-type E1A (8 μg), or the BRLF1 expression vector (0.5 μg) and the E1A mutant (Δ2-36) (8 μg). The E1A vectors were gifts from David Livingston (8). (C) Immunoblot analysis was performed using extracts from the experiment shown in panel 4B to assess the level of transfected wild-type versus mutant E1A proteins and of BRLF1.

FIG. 4

BRLF1-CBP interaction affects BRLF1 transactivator function. (A) Transient reporter assays were performed in which HeLa cells were transfected with 4 μg of promoter construct (S-CAT, containing SM gene promoter sequences), 0.3 μg of BRLF1 expression vector (CMV-RIE) (26), or control vector (pHD1013), with or without 1 μg of the CMV-CBP expression vector (29). CAT activity was determined as previously described (20). The results represent data from two separate experiments. (B) The S-CAT plasmid (1 μg) was transfected into HeLa cells with either vector DNA (8.5 μg) (SG5), wild-type E1A vector (8 μg), an E1A mutant (Δ2-36) which is unable to bind CBP (8 μg), the BRLF1 expression vector (pRTS15; a gift from Diane Hayward) (0.5 μg) and control vector DNA (8 μg), the BRLF1 expression vector (0.5 μg) and wild-type E1A (8 μg), or the BRLF1 expression vector (0.5 μg) and the E1A mutant (Δ2-36) (8 μg). The E1A vectors were gifts from David Livingston (8). (C) Immunoblot analysis was performed using extracts from the experiment shown in panel 4B to assess the level of transfected wild-type versus mutant E1A proteins and of BRLF1.

FIG. 5

BRLF1 interaction with CBP is important for activation of the early SM gene in Raji cells. (A) Latently infected, EBV-positive Raji cells were transfected with a control vector or expression vectors for BZLF1 or BRLF1. Cell extracts were harvested 1 day later and immunoblotted with antibodies directed against the BMRF1 or SM early EBV proteins. BMRF1 protein was detected using a monoclonal antibody (Capricorn; 1:100 dilution) and SM protein was detected using a rabbit polyclonal antibody (1:400 dilution) provided by Sankar Swaminathan. (B) Raji cells were transfected with 2 μg of the BRLF1 expression vector (pRTS15) or control vector (SG5) in the presence or absence of the CBP expression vector (5 μg) (keeping the total DNA level constant). Cell extracts were harvested at 24 h posttransfection and immunoblotted for SM early protein. (C) Raji cells were transfected with the BRLF1 expression vector (2 μg), control vector (2 μg), wild-type E1A (10 μg), or mutant E1A (Δ2-36) (10 μg), keeping the total DNA level constant. Cell extracts were harvested at 24 h posttransfection and immunoblotted for SM early protein. (D) The same extracts shown in panel C were immunoblotted using an antibody which recognizes BRLF1.