The adenovirus E4orf4 protein targets PP2A to the ACF chromatin-remodeling factor and induces cell death through regulation of SNF2h-containing complexes

Abstract

The adenovirus E4 open-reading-frame 4 (E4orf4) protein regulates the progression of viral infection and when expressed individually it induces non-classical apoptosis in transformed cells. Here we show that E4orf4 associates with the ATP-dependent chromatin-remodeling factor ACF that consists of a sucrose non fermenting-2h (SNF2h) ATPase and an Acf1 regulatory subunit. Furthermore, E4orf4 targets protein phosphatase 2A (PP2A) to this complex and to chromatin. Obstruction of SNF2h activity inhibits E4orf4-induced cell death, whereas knockdown of Acf1 results in enhanced E4orf4-induced toxicity in both mammalian and yeast cells, and Acf1 overexpression inhibits E4orf4's ability to downregulate early adenovirus gene expression in the context of viral infection. Knockdown of the Acf1 homolog, WSTF, inhibits E4orf4-induced cell death. Based on these results we suggest that the E4orf4-PP2A complex inhibits ACF and facilitates enhanced chromatin-remodeling activities of other SNF2h-containing complexes, such as WSTF-SNF2h. The resulting switch in chromatin remodeling determines life versus death decisions and contributes to E4orf4 functions during adenovirus infection.

Figure 1.

E4orf4 associates with Acf1 and SNF2h in mammalian cells. (A) HEK293T cells were transfected with a plasmid expressing E4orf4 or with an empty vector. Nuclear extracts were prepared 24 h later and subjected to immunoprecipitation (IP) with Acf1-specific antibodies or with pre-immune serum. The western blot shows the presence of Acf1, the PP2A-C subunit and E4orf4 in the immune complexes and in input lysates. The amount of proteins in the input represents 10% of the amount of proteins used for IP. (B) HEK293T cells were transfected with a plasmid expressing Acf1-GFP, together with a plasmid expressing E4orf4 or the empty vector. Nuclear extracts were prepared as in (A) and subjected to IP with E4orf4-specific antibodies. The western blot showing input lysates and immune complexes (IP: αE4orf4) was stained sequentially with antibodies to E4orf4, GFP and SNF2h. The amount of proteins in the input represents 10% of the amount of proteins used for IP. (C) HEK293T cells were transfected with a plasmid expressing E4orf4 or an empty vector and were processed as in (B). Western blots were stained sequentially with antibodies to Acf1, SNF2h and E4orf4. (D) HEK293T cells were transfected with plasmids expressing Acf1-GFP or the corresponding empty vector, together with a plasmid expressing E4orf4 from a doxycycline-inducible promoter and a plasmid expressing GFP, which served as a transfection efficiency and loading control. Nuclear extracts were prepared at the indicated times after E4orf4 induction and subjected to IP with GFP-specific antibodies. The western blot showing input lysates and immune complexes (IP: α-GFP) was probed sequentially with antibodies to E4orf4 and GFP. The amount of proteins in the input represents 10% of the amount of proteins used for IP. (E) HEK293T cells were transfected with an empty vector or a plasmid expressing Acf1-GFP and with a plasmid expressing GFP, which served as a transfection efficiency and loading control. The cells were subsequently infected with dl366* or dl366*+E4orf4 adenoviruses. Cells were harvested 24 h after infection and equal amounts of nuclear extracts were subjected to IP with GFP-specific antibodies. The western blot showing input lysates and immune complexes (IP: αGFP) was probed sequentially with antibodies to E4orf4, Acf1 and GFP.

Figure 2.

E4orf4 targets PP2A to an Acf1-containing complex. (A) HEK293 cells were transfected with a plasmid expressing Acf1-Flag together with a plasmid expressing WT E4orf4 (wt), the E4orf4 R81F84A mutant that cannot bind PP2A (mut) or the corresponding empty vector (−). Nuclear extracts were prepared 24 h later and were subjected to immunoprecipitation (IP) with Flag-specific antibodies or with anti-HA antibodies, serving as control IgG. The western blot showing input lysates and immune complexes (IP-αFlag or control IgG) was stained sequentially with antibodies to E4orf4, SNF2h and the Flag tag. (B) HEK293 cells were transfected with plasmids expressing WT E4orf4 (wt) or the E4orf4 R81F84A mutant (mut). Nuclear extracts were prepared as in (A) and subjected to IP with Acf1-specific antibodies or with pre-immune serum. A western blot showing input lysates and immune complexes was stained sequentially with antibodies to the PP2A-C subunit, SNF2h and Acf1. The amount of proteins in the input represents 10% of the amount of proteins used for IP in both (A) and (B).

Figure 3.

A subpopulation of PP2A binds more tightly to chromatin in the presence of E4orf4. HEK293T cells were transfected with an empty vector (−) or a plasmid expressing E4orf4 (+) (A), or were transfected with plasmids expressing either WT E4orf4 (wt) or the R81F84A mutant (mut) (B). Twenty-four hours later a chromatin fraction was prepared from nuclei and subjected to extraction with increasing salt concentrations. Proteins were chromatographed on SDS–PAGE and western blots were sequentially stained with antibodies to Acf1, SNF2h, PP2A-B55, PP2A-C and E4orf4 in A or stained with antibodies to SNF2h, PP2A-B55 and E4orf4 in (B). Arrowheads mark the relevant protein bands and the asterisk marks a non-specific band. The stained western blot was subjected to densitometry. The relative band intensity for each individual extraction step out of the sum of intensities of all extraction steps for each sample was calculated and the ratio between control (defined as 1) and E4orf4 values is shown below the bands. The western blots shown here represent three independent experiments with similar results.

Figure 4.

Expression of an Acf1 mutant which cannot interact with SNF2h interferes with E4orf4-induced cell death. HEK293 cells were transfected with plasmids expressing Acf1-GFP, Acf1-DN-GFP or GFP alone together with a plasmid expressing E4orf4 or its corresponding empty vector. (A) A representative picture of cells containing the indicated plasmids was taken 24 h after transfection, using a Zeiss Axioskop at a 400-fold magnification. (B) Cells in duplicate plates were fixed 24 h after transfection and stained with E4orf4-specific antibodies and with DAPI. The number of nuclei with abnormal morphology (condensed or fragmented) was counted in cells expressing both E4orf4 and GFP, and the percentage of cells with abnormal nuclei was calculated. The average of two independent experiments with two replicates each is shown. Error bars represent pooled standard deviation and statistical significance was determined using a paired Student’s t-test. (C) Proteins were extracted from parallel plates as in (B) and a western blot was stained sequentially with antibodies to E4orf4 and to GFP. (D) Nuclear extracts were prepared from parallel plates as in (B) and were subjected to immunoprecipitation with antibodies to GFP. A western blot was stained sequentially with antibodies to GFP, SNF2h and E4orf4. Input lysates represent 10% of the extracts used for the immunoprecipitation.

Figure 5.

An active SNF2h ATPase is required for E4orf4-induced cell death. (A, B) HEK293 cells were transfected with plasmids expressing WT SNF2h (wt), a catalytically inactive SNF2h mutant (mut) or an empty vector (−) together with a plasmid expressing E4orf4 or its corresponding empty vector. The SNF2h proteins were GFP tagged. Twenty-four hours later, the cells were either stained with antibodies to E4orf4 and with DAPI or harvested for western blot analysis. Induction of cell death was measured by a DAPI assay (A), as described in the legend to Figure 4. The average of three experiments, each containing two replicates is shown. Error bars represent pooled standard deviation and statistical significance was determined using a paired Student’s t-test. Proteins extracted from parallel plates were subjected to western blot analysis (B) using antibodies to GFP and E4orf4. (C, D) Cells from a HEK293-derived cell line expressing SNF2h–shRNA from a doxycycline-inducible promoter were induced with doxycycline (Dox) or with the solvent control (EtOH). After 3 days the cells were transfected with plasmids expressing E4orf4 or an empty vector together with a vector control or a plasmid expressing Flag-tagged SNF2h, which was rendered resistant to the shRNA by the introduction of silent mutations. Twenty-four hours after transfection the cells were either fixed and stained with antibodies to E4orf4 and the Flag tag and with DAPI or were extracted for western blot analysis. Induction of cell death (C) was measured by the DAPI assay described above. The average of two experiments with two replicates each is shown. Error bars represent pooled standard deviation and statistical significance was determined using a paired Student’s t-test. A western blot of total lysates (D) was stained sequentially with antibodies to SNF2h, E4orf4 and α-Tubulin.

Figure 6.

Acf1 knockdown enhances E4orf4-induced toxicity. (A, B) Cells from a HEK293-derived cell line expressing Acf1-shRNA from a doxycycline-inducible promoter were induced with doxycycline (Dox) or with the solvent control (EtOH). After 3 days the cells were transfected with plasmids expressing E4orf4 or an empty vector together with a plasmid expressing GFP-tagged Acf1, which was rendered resistant to the shRNA by the introduction of silent mutations or its corresponding empty vector. Twenty-four hours after transfection the cells were either fixed and stained with antibodies to E4orf4 and with DAPI, or were extracted for western blot analysis. Induction of cell death (A) was measured by the DAPI assay described above. The average of two experiments with two replicates each is shown. Error bars represent pooled standard deviation and statistical significance was determined using a paired Student’s t-test. A western blot of total lysates (B) was stained sequentially with antibodies to Acf1, E4orf4 and α-Tubulin. (C) WT300 and itc1Δ yeast cells were transformed with the plasmid pDAD2-E4orf4, expressing E4orf4 from a weak galactose-inducible promoter or with the empty vector. The yeast cells were serially diluted (1:5) and plated on glucose and galactose plates.

Figure 7.

WSTF knockdown inhibits E4orf4-induced cell death. (A, B) Cells from a HEK293-derived cell line expressing WSTF-shRNA from a doxycycline-inducible promoter were induced with doxycycline (Dox) or with the solvent control (EtOH). After 3 days the cells were transfected with plasmids expressing E4orf4 or an empty vector together with a plasmid expressing Flag-tagged WSTF, which was rendered resistant to the shRNA by the introduction of silent mutations, or its corresponding empty vector. Twenty-four hours after transfection the cells were either fixed and stained with antibodies to the Flag tag and E4orf4 and with DAPI, or were extracted for western blot analysis. Induction of cell death (A) was measured by the DAPI assay described above. The average of two independent experiments with two replicates each is shown. Error bars represent pooled standard deviation and statistical significance was determined using a Student’s t-test. A western blot of total lysates (B) was stained sequentially with antibodies to WSTF, E4orf4 and α-Tubulin.

Figure 8.

Acf1 overexpression inhibits downregulation of the early adenovirus E2A-72 kDa protein by E4orf4. HEK293 cells were transfected with plasmids expressing Acf1-GFP or GFP alone and were subsequently either mock-infected (M) or infected with the adenovirus mutants dl366* or dl366*+E4orf4. The cells were harvested 24 h post-infection and protein extracts were prepared and chromatographed on SDS–PAGE. A western blot was stained sequentially with antibodies to GFP, E2A-72 kDa, α-Tubulin and E4orf4. This blot represents three independent experiments with similar results.

Figure 9.

A working model: E4orf4 functions by shifting the balance between different SNF2h-containing chromatin-remodeling complexes. Various SNF2h-containing complexes participate in chromatin remodeling and affect transcription, DNA replication, DNA repair, etc. E4orf4 targets PP2A to Acf1-containing chromatin remodelers and inhibits them. This inhibition leads to a shift in the balance between various SNF2h remodeling complexes, allowing, for example, more activity of a WSTF–SNF2h complex. The variation in chromatin remodeler activity alters chromatin remodeling and produces changes in transcription, DNA replication, DNA repair or other processes that require remodeling. These events contribute to E4orf4 functions during virus infection and lead to cell death when E4orf4 is expressed individually.