The Transcriptional Repressor BS69 is a Conserved Target of the E1A Proteins from Several Human Adenovirus Species

Abstract

Early region 1A (E1A) is the first viral protein produced upon human adenovirus (HAdV) infection. This multifunctional protein transcriptionally activates other HAdV early genes and reprograms gene expression in host cells to support productive infection. E1A functions by interacting with key cellular regulatory proteins through short linear motifs (SLiMs). In this study, the molecular determinants of interaction between E1A and BS69, a cellular repressor that negatively regulates E1A transactivation, were systematically defined by mutagenesis experiments. We found that a minimal sequence comprised of MPNLVPEV, which contains a conserved PXLXP motif and spans residues 112⁻119 in HAdV-C5 E1A, was necessary and sufficient in binding to the myeloid, Nervy, and DEAF-1 (MYND) domain of BS69. Our study also identified residues P113 and L115 as critical for this interaction. Furthermore, the HAdV-C5 and -A12 E1A proteins from species C and A bound BS69, but those of HAdV-B3, -E4, -D9, -F40, and -G52 from species B, E, D, F, and G, respectively, did not. In addition, BS69 functioned as a repressor of E1A-mediated transactivation, but only for HAdV-C5 and HAdV-A12 E1A. Thus, the PXLXP motif present in a subset of HAdV E1A proteins confers interaction with BS69, which serves as a negative regulator of E1A mediated transcriptional activation.

Keywords: BS69; E1A; ZMYND11; human adenovirus; short linear motifs; transcriptional regulation.

Figure 1

The interaction between early region 1A (E1A) and BS69 is present in only a subset of human adenovirus (HAdV) species. (A) Yeast two-hybrid analysis of the ability of the E1A CR2 portions from representative HAdV types to bind BS69. Results are shown as mean ± SD, n = 3. Significance markers are assigned in comparison to the no myeloid, Nervy, and DEAF-1 (MYND) control for each respective E1A type (* p ≤ 0.05). (B) Western blot detection of expression of the LexA-DBD E1A CR2 fusions and the BS69 MYND construct in yeast. G6PD was used as a loading control. (C) Multiple sequence alignment of E1A CR2 proximal regions of HAdV-B3, -E4, -C5, -D9, -A12, -F40, and -G52. Residues with high conservation are shaded in black, with darker shading indicating higher levels of conservation. The previously identified PXLXP motif contributing to HAdV-C5 E1A interaction with BS69 and the corresponding motif in HAdV-A12 E1A are indicated in red. A second N-terminal PXLXP motif present in the E1A proteins of HAdV-D9 and -A12 is indicated in blue. (D) Coimmunoprecipitation analysis of the ability of E1A proteins from representative HAdV types to bind BS69 in mammalian cells. Vectors expressing GFP-tagged full-length E1A and HA-tagged BS69 MYND were co-transfected into human HT1080 cells. Cell lysates were subjected to immunoprecipitation using an anti-GFP antibody. Western blots were probed with anti-GFP and anti-HA antibodies.

Figure 2

Mapping the minimal BS69 interacting region in HAdV-C5 E1A CR2. (A) Vectors expressing the indicated E1A truncation mutants and BS69 MYND domain were transformed as bait and prey respectively in yeast. Sequences of the E1A truncation mutants, T1–T8, are shown in the legend beside the graph. V = vector control, where HAdV-C5 E1A CR2 was co-transformed with an empty prey vector. Results are shown as mean ± SD of percent activity normalized to E1A CR2, n = 4. Significance markers are assigned in comparison to the CR2 positive control (* p ≤ 0.05). (B) Western blot of yeast cell lysates to confirm protein expression. Two blots were run concurrently in parallel to include all the samples. Bait and prey proteins were visualized using anti-LexA DBD and anti-HA antibodies respectively. G6PD was used as a loading control. V = vector control.

Figure 3

Determination of specific residues of HAdV-C5 E1A CR2 required to interact with BS69. (A) Sequences of the E1A CR2 point mutants, M1–M10, used to determine the importance of specific residues within and adjacent to the HAdV-C5 E1A PXLXP motif for interaction with BS69. (B) Vectors expressing E1A CR2 point mutants and BS69 MYND were transformed as bait and prey respectively in yeast. Results are shown as mean ± SD of percent activity normalized to HAdV-C5 CR2, n = 3. Significance markers are assigned in comparison to the CR2 positive control (* p ≤ 0.05). (C) Western blot of yeast cell lysates to confirm protein expression. Bait and prey proteins were visualized using anti-LexA DBD and anti-HA antibodies respectively. G6PD was used as a loading control.

Figure 4

Analysis of BS69 interaction with the two putative PXLXP motifs present in the HAdV-A12 E1A protein. (A) Sequences of the motifs corresponding to the wildtype (WT) and mutant N-terminal and C-terminal PXLXP motifs in HAdV-A12 E1A tested. Putative PXLXP motifs are indicated in red and mutations in the motifs are indicated in blue. (B) Vectors expressing the motifs depicted in (A) and BS69 MYND were tested for interaction with BS69 by yeast two-hybrid analyses. Results are shown as mean ± SD of percent activity normalized to the HAdV-A12 N-terminal WT positive control, n = 3. Significance markers are assigned in comparison to the LexA negative control (* p ≤ 0.05). (C) Western blot of yeast cell lysates to confirm protein expression. Bait and prey proteins were visualized using anti-LexA DBD and anti-HA antibodies respectively. G6PD was used as a loading control.

Figure 5

Analysis of BS69 interaction with the E1A proteins from HAdV-A18 and -A31. (A) Sequence alignment of the relevant portions of HAdV-A12, -A18, and -A31. PXLXP like sequences are indicated in red, with divergent residues indicated in blue. (B) The indicated fragments corresponding to the N-terminal (N), middle (M), and C-terminal (C) PXLXP motifs in HAdV-A18 and -A31 E1A were tested for interaction with BS69 MYND by yeast two-hybrid analyses. Results are shown as mean ± SD of percent activity normalized to HAdV-C5 CR2 positive control, n = 3. Significance markers are assigned in comparison to the LexA negative control (* p ≤ 0.05). (C) Western blot analysis of expression of the motifs tested.

Figure 6

The PXLXP motif in HAdV-D9 E1A does not bind BS69, but can be functionalized by a single amino acid change. (A) Sequences of the WT PXLXP motif from HAdV-D9 E1A and mutants containing single amino acid changes to more closely resemble the HAdV-A12 E1A N-terminal BS69-binding motif. (B) Vectors expressing the motifs depicted in (A) and BS69 MYND were tested for interaction with BS69 by yeast two-hybrid analyses. The HAdV-A12 E1A N-terminal PXLXP motif was used as a positive control. Results are shown as mean ± SD of percent activity normalized to HAdV-A12 CR2 positive control, n = 3. Significance markers are assigned in comparison to the LexA negative control (* p ≤ 0.05). (C) Western blot analysis of expression of the fragments tested.

Figure 7

Binding by the HAdV-A12 N-terminal PXLXP motif is influenced by a proximal proline residue. (A) Sequences corresponding to the WT N-terminal PXLXP motif from HAdV-A12 E1A and a mutant containing a single amino acid change of proline to threonine. (B) Vectors expressing the motifs depicted in (A) and BS69 MYND were tested for interaction with BS69 by yeast two-hybrid analyses. Results are shown as mean ± SD of percent activity normalized to HAdV-A12 N-terminal WT positive control, n = 3. Significance markers are assigned in comparison to the LexA negative control (* p ≤ 0.05). (C) Western blot analysis of expression of the fragments tested.

Figure 8

The MYND domain of BS69 inhibits E1A-mediated transcriptional activation in a dose-dependent manner dependent on the PXLXP motif. (A) Depiction of the domain structure of BS69 and the truncation mutants used in this study. (B) Luciferase assay showing repression of E1A transactivation by BS69 MYND. HT1080 cells were co-transfected with the pGL2-(GAL4)₆-Luc reporter, either pM 13S HAdV-C5 E1A or pM 13S HAdV-C5 E1A L115A, and increasing amounts of HA BS69 WT vector. Luciferase activity was normalized by protein concentration and reported as percent activation compared to WT E1A alone. Results are shown as mean ± SD of activity normalized to WT E1A with no BS69, n = 2. Significance markers are assigned in comparison to the samples not transfected with a BS69 vector (* p ≤ 0.05). (C) The MYND domain of BS69 is necessary and sufficient to repress E1A transactivation. HT1080 cells were co-transfected with the pGL2-(GAL4)₆-Luc reporter and pM 13S HAdV-C5 E1A. Cells were also co-transfected with increasing amounts (0, 0.05, and 0.25 μg) of the indicated BS69 constructs (WT BS69, MYND, or ∆MYND). Luciferase activity was normalized by protein concentration and reported as percent activation over that of an empty vector. Results are shown as mean ± SD of activity normalized to E1A with no BS69, n = 2. Significance markers are assigned in comparison to the samples not transfected with a BS69 vector (* p ≤ 0.05).

Figure 9

BS69-mediated repression of transactivation by HAdV-C5 and -A12 E1A. The effect of expressing BS69 on E1A-mediated transactivation was measured using luciferase assays. HT1080 cells were co-transfected with pGL2-(GAL4)₆-Luc reporter, pM HAdV-C5, -D9, -A12, or -F40 E1A, and increasing amounts (0, 0.05, and 0.25 μg) of HA BS69 WT. Luciferase activity was normalized by protein concentration and reported as fold activation over that of an empty vector. Results are shown as mean ± SD of percent activity normalized to the E1A of each respective species with no BS69, n = 2. Significance markers are assigned in comparison to the samples not transfected with a BS69 vector in each respective adenovirus type (* p ≤ 0.05).