AR1 is an integral part of the adenovirus type 2 E1A-CR3 transactivation domain

Abstract

We have previously shown that the nonconserved carboxy-terminal exon of the adenovirus type 2 E1A-289R protein contains two interchangeable sequence elements, auxiliary region (AR) 1 and AR2, that are required for efficient CR3-mediated transcriptional activation of the viral E4 promoter (M. Bondesson, C. Svensson, S. Linder, and G. Akusjärvi, EMBO J. 11:3347-3354, 1992). Here we show that CR3-mediated transactivation of all adenovirus early promoters and the HSP70 promoter requires the AR1 element. We further show that AR2 can substitute for AR1 only when artificially juxtaposed to CR3. AR1 consists of six tandem glutamic acid-proline (EP) repeats and is positioned immediately downstream of CR3. Genetic dissection of AR1 showed that the number of EP repeats in AR1 is critical for CR3 function. Thus, reducing or increasing the number of EP repeats reduces the CR3 transactivation capacity. Furthermore, the introduction of amino acid substitutions into AR1 suggested that the net negative charge in AR1 is of critical importance for its function as an enhancer of CR3-mediated transcriptional activation. Using an in vitro binding approach, we showed that the AR1 element is not part of the CR3 promoter localization signal mediating contact with the Sp1, ATF-2, or c-Jun upstream-binding transcription factors. Previous studies have suggested that the 49-amino-acid sequence constituting CR3 represents the minimal domain required for E1A-induced activation of viral early promoters. Since AR1 was required for efficient CR3-mediated transcriptional activation of all tested promoters, we suggest that the carboxy-terminal boundary for the CR3 transactivation domain should be extended to include the AR1 element.

FIG. 1

AR1 is essential for E1A transactivation of early viral promoters. (A) A schematic representation of the E1A deletions mutants. (B) Relative CAT and luciferase (HSP70) activities, based on the mean values of data from at least three independent experiments, are shown as percentages of wtE1A activation (set at 100%) with the respective reporter plasmids. Error bars show the standard derivations. The fold activation values relative to the basal reporter activity are marked to the right of each panel. (C) Western blot showing E1A protein expression in transfected HeLa cells. The positions of protein molecular size standards are marked on the left side (in kilodaltons).

FIG. 1

AR1 is essential for E1A transactivation of early viral promoters. (A) A schematic representation of the E1A deletions mutants. (B) Relative CAT and luciferase (HSP70) activities, based on the mean values of data from at least three independent experiments, are shown as percentages of wtE1A activation (set at 100%) with the respective reporter plasmids. Error bars show the standard derivations. The fold activation values relative to the basal reporter activity are marked to the right of each panel. (C) Western blot showing E1A protein expression in transfected HeLa cells. The positions of protein molecular size standards are marked on the left side (in kilodaltons).

FIG. 1

AR1 is essential for E1A transactivation of early viral promoters. (A) A schematic representation of the E1A deletions mutants. (B) Relative CAT and luciferase (HSP70) activities, based on the mean values of data from at least three independent experiments, are shown as percentages of wtE1A activation (set at 100%) with the respective reporter plasmids. Error bars show the standard derivations. The fold activation values relative to the basal reporter activity are marked to the right of each panel. (C) Western blot showing E1A protein expression in transfected HeLa cells. The positions of protein molecular size standards are marked on the left side (in kilodaltons).

FIG. 2

The number of EP repeats in AR1 is critical for function. HeLa cells were cotransfected with an Ad5 E4CAT reporter plasmid and either wtE1A or plasmids encoding E1A mutant proteins (schematically illustrated to the right). Relative CAT activities, based on the mean values of data from at least three independent experiments, are shown as percentages of wtE1A activation (set at 100%). Error bars show the standard derivations. The fold activation relative to basal reporter activity is marked to the right of each E1A protein.

FIG. 3

The negative charge of AR1 is important for its function. Levels of CAT activity in HeLa cells cotransfected with the E4CAT reporter and E1A activator plasmids were determined. Relative CAT activities, based on the mean values of data from at least three independent experiments, are shown as percentages of wtE1A activation (set at 100%). Error bars show the standard derivations. The fold activation relative to the basal reporter activity is marked to the right of each E1A mutant protein. The various conservative or nonconservative amino acid substitutions in the six EP repeats are schematically shown in boldface.

FIG. 4

AR1 is not part of the CR3 promoter-targeting signal. (A) A schematic representation of the N-terminally truncated E1A mutant proteins used for in vitro binding. (B) In vitro-transcribed and -translated, ³⁵S-labelled deletion mutants of E1A, illustrated in panel A, were tested for binding to GST, GST/c-Jun, GST/ATF-2, and GST/Sp1(DBD). The positions of protein molecular size standards are marked on the right side (in kilodaltons). The first panel (in vitro translation products [IVT]) was run with 1/10 of the E1A protein input used in the GST fusion protein binding assay.

FIG. 4

AR1 is not part of the CR3 promoter-targeting signal. (A) A schematic representation of the N-terminally truncated E1A mutant proteins used for in vitro binding. (B) In vitro-transcribed and -translated, ³⁵S-labelled deletion mutants of E1A, illustrated in panel A, were tested for binding to GST, GST/c-Jun, GST/ATF-2, and GST/Sp1(DBD). The positions of protein molecular size standards are marked on the right side (in kilodaltons). The first panel (in vitro translation products [IVT]) was run with 1/10 of the E1A protein input used in the GST fusion protein binding assay.

FIG. 5

E1A proteins from six adenovirus serotypes have an excess of negatively charged amino acid residues at the hypothetical AR1 position. The boxed region indicates the highly conserved CR3 promoter-targeting domain. Negatively charged residues are indicated in boldface type.