The acidic activation domain of the Epstein-Barr virus transcription factor R interacts in vitro with both TBP and TFIIB and is cell-specifically potentiated by a proline-rich region

Abstract

In cells latently infected with Epstein-Barr virus (EBV), the expression of two viral transactivators, EB1 and R, is responsible for the switch from latency to a productive cycle. R contains a DNA-binding/dimerization domain localized at the N-terminus. The domain required for transcriptional activation is localized at the C-terminus and contains two regions of very different amino acid composition. The first is very rich in prolines, whereas the second is rich in acidic residues and contains two potential alpha-helices. We investigated the activation potential of these subregions when linked to the heterologous Gal4 DNA-binding domain. We found that the acidic region--more precisely, the second putative alpha-helix--is an activating domain. In contrast, the proline-rich region is insufficient by itself for activation but collaborates with the acidic region in a cell-specific manner to make transactivation more efficient. We demonstrated that R interacts in vitro with the basal transcription factors TBP and TFIIB, and that the acidic domain of R mediates these interactions.

Figure 1

The activity of the C-terminal acidic domain is increased by a contiguous proline-rich domain. A. Schematic representation of the wild-type R protein. B. Schematic representation of the chimeric proteins containing the Gal4 DNA-binding domain fused to various regions of the R protein. The limits of the R regions fused to Gal4 are indicated on the right. C. Schematic representation of the reporter gene pβGG4, comprising the β-globin ORF, a TATA box, and a single Gal4 binding site. D and E. HeLa cells were transfected with 3 μg of reporter gene plasmid together with increasing amounts of Gal4 fusion-expressing plasmids: 0.4, 2, and 10 ng (D) or 2, 10, and 50 ng (E). Transcriptional activation was determined by quantitative SI analysis using total cellular RNA isolated from transfected cells. The specific β-globin bands are indicated in the margin by β. Plasmid pCMVβ was cotransfected as an internal control. The specific bands are indicated by C (control) in the margin. The level of activation was quantified by cutting out the bands and counting the amount of radioactivity in each band. The results are expressed as relative activities.

Figure 2

The effect of the proline-rich domain is cell-specific. A. Schematic representation of fusion proteins between the Gal4 DNA-binding domain and subdomains of the R protein. B. Schematic representation of the reporter gene pβGG4. C. HepG2 cells were transfected with 3 μg of reporter gene plasmid together with increasing amounts of Gal4 fusion-expressing plasmids: 2, 10, and 50 ng. Transcriptional activation was determined as described in Figure 1.

Figure 3

The A1 subregion of R potentiates transcrip-tional activation by domain A2 in HepG2 cells. A. Schematic representation of fusion proteins between the Gal4 DNA-binding domain and subdomains of the R protein. B. Schematic representation of the reporter gene pβGG4. C. HepG2 cells were transfected with 3 μg of reporter gene plasmid together with increasing amounts (10, 50, or 250 ng) of expression plasmids Gal4-A, Gal4-Al, and Gal4-A2. As a control (lane 1), the cells were cotransfected with the reporter gene plasmid together with 250 ng of plasmid expressing the DNA-binding domain of Gal4.

Figure 4

Unbound Gal4-VP16 represses R-induced activation. A. Schematic representation of the reporter gene pβGR, comprising the β-globin ORF, a TATA box, and two binding sites for R. B. HeLa cells were transfected with 3 μg of the reporter gene together with plasmids expressing the proteins indicated in the figure. The fusions are described in Figure 1. Transcriptional activation was determined as for Figure 1.

Figure 5

The acidic domain of R contacts both TBP and TFIID in vitro. A. Schematic representation of the R protein. B. Schematic representation of the R mutant proteins tested for their capacity to bind TBP or TFIIB. Each variant has been assigned a recognition number (1–5). C. The R mutant proteins presented in B were translated in vitro in the presence of [³⁵S] methionine. Aliquots of the lysate extracts were either loaded directly onto SDS PAGE (lanes 1–5) or loaded onto minicolumns of GST- or GSThllB-agarose beads (lanes 6–15), nickel-NTA-agarose, or HisIID-nickel-NTA-agarose beads (lanes 16–25). The eluates were loaded onto SDS-PAGE. The gels were then autoradiographed. The mutant proteins used in each lane are indicated by their corresponding number. M indicates the lanes that correspond to the molecular weight markers.