Adenovirus E4orf4 protein downregulates MYC expression through interaction with the PP2A-B55 subunit

Abstract

The adenovirus E4 open reading frame 4 (E4orf4) protein is a multifunctional viral regulator that is involved in the temporal regulation of viral gene expression by modulating cellular and viral genes at the transcription and translation levels and by controlling alternative splicing of adenoviral late mRNAs. When expressed individually, E4orf4 induces apoptosis in transformed cells. Using oligonucleotide microarray analysis, validated by quantitative real time PCR, we found that MYC (also known as c-Myc) is downregulated early after the induction of E4orf4 expression. As a result, Myc protein levels are reduced in E4orf4-expressing cells. MYC downregulation is observed both when E4orf4 is expressed individually and within the context of viral infection. E4orf4 reduces MYC transcription but does not affect transcriptional elongation or RNA stability. An interaction with the PP2A-B55 subunit is required for the downregulation of MYC by E4orf4. Since Myc overexpression was previously shown to inhibit adenovirus replication, the downregulation of Myc by E4orf4 would contribute to efficient virus infection.

FIG. 1.

Induction of E4orf4 expression in clone 13 cells. E4orf4 expression was induced in clone 13 cells by 1 μg/ml doxycycline. At the indicated times postinduction, cells were either harvested and subjected to Western blot analysis with E4orf4-specific antibodies (A) or fixed and stained with E4orf4-specific antibodies and with DAPI (4′,6′-diamidino-2-phenylindole) (B).

FIG. 2.

Quantitative real time PCR analysis of MYC RNA levels following induction of E4orf4 expression. Clone 13 and control T-REX cells were treated with 1 μg/ml doxycycline (Dox) or with the ethanol vehicle. Two plates of cells were harvested at each of the indicated times postinduction, and RNA was prepared. The various RNA samples were subjected to quantitative real time PCR analysis using primers for MYC and GAPDH. MYC RNA levels were normalized to those of the housekeeping GAPDH RNAs, and relative quantification was obtained by further normalizing these results to MYC levels in clone 13 cells treated with ethanol for 24 h. Relative MYC RNA levels representing log₁₀(relative quantification) are shown.

FIG. 3.

Downregulation of Myc protein upon induction of E4orf4 expression. (A) Clone 13 and T-REX cells were subjected to treatment with 1 μg/ml doxycycline (Dox) for the indicated times in hours (Hrs). Cells were then harvested, and proteins were subjected to Western blot analysis with antibodies to Myc, α-tubulin, and E4orf4. (B) Results of the Western blot stained with Myc-specific antibodies were quantified by densitometry. Values at time zero of induction in both clone 13 and T-REX cells were defined as 100%, and relative Myc protein levels are plotted for each time point. (C) E4orf4 expression was induced by a 24-h treatment of various cell clones derived from H1299 cells and the 293-derived clone 13 cells with 1 μg/ml doxycycline (Dox). Proteins were then extracted and subjected to Western blot analysis with Myc- and α-tubulin-specific antibodies.

FIG. 4.

E4orf4-induced downregulation of MYC RNA levels in the context of virus infection. H1299 cells were mock infected or infected with dl366* and dl366*+orf4 viruses at 10 PFU/cell. RNAs were prepared from these cells 6 h postinfection and subjected to quantitative real time PCR with primers to MYC and to GAPDH. MYC RNA levels were normalized to GAPDH levels, and the resulting relative quantification is shown as a percentage of MYC levels in mock-infected cells. The graph shows the average of two independent experiments, and error bars represent standard deviations.

FIG. 5.

Investigation of the mechanisms underlying E4orf4-induced MYC downregulation. (A) T-REX and clone 13 cells were treated with 1 μg/ml doxycycline (Dox) or with ethanol (EtOH) vehicle for 5 h. Actinomycin D was then added (5 μg/ml), and cells from two plates per each point were harvested at the indicated times after the addition of the drug. RNA was prepared and subjected to quantitative real time PCR analysis with primers to MYC and the housekeeping GAPDH gene. Relative quantification was obtained by normalizing MYC results to GAPDH levels. The values at the time of addition of actinomycin D (time zero) were defined as 100% for each group of treated cells. Broken line with diamonds, T-REX with EtOH treatment; broken line with squares, T-REX with doxycycline treatment; solid line with triangles, clone 13 with EtOH treatment; solid line with circles, clone 13 with doxycycline treatment. (B) RNAs were extracted from clone 13 cells treated with ethanol or 1 μg/ml doxycycline for 6 h and were subjected to semiquantitative RT-PCR analysis with primers to MYC exon 1 or 3 and to GAPDH. Amplified cDNAs were taken for agarose gel analysis after 21 (lanes 1 and 4), 24 (lanes 2 and 5), and 27 (lanes 3 and 6) amplification cycles for the MYC reactions and after 17 (lanes 1 and 4), 22 (lanes 2 and 5), and 25 (lanes 3 and 6) amplification cycles for the GAPDH reaction. (C) Clone 13 cells were treated with ethanol or 1 μg/ml doxycycline for 6 h, and nuclei were prepared and subjected to nuclear run-on analysis. Hybridization of the ³²P-labeled RNA products to a filter carrying DNA sequences of E4orf4, MYC, and β-ACTIN was visualized by use of a PhosphorImager. (D) Results of C were quantified using TINA software. The alteration shown here using a logarithmic scale is the ratio between intensities of hybridization for doxycycline- and EtOH-treated cells normalized to the ratio for the control β-ACTIN gene.

FIG. 6.

The PP2A-B55 subunit is required for E4orf4-induced downregulation of Myc expression. (A) 293 cells were transfected with an empty vector, WT E4orf4, or the E4orf4 R4 (R73/74/75A) or R81/F84A mutant. Cells were harvested 24 h later, and proteins were subjected to Western blot analysis sequentially stained with antibodies to Myc, E4orf4, and α-tubulin. Densitometry was used to quantify Myc and α-tubulin levels, and Myc levels were normalized to α-tubulin levels. Myc expression in vector-transfected cells was defined as 1, and relative Myc levels are shown below the blot. (B) NTO (lanes 1 and 2) and IR-B55 (lanes 3 and 4) cells were transfected with WT E4orf4 (lanes 2 and 4) or an empty vector (lanes 1 and 3) and with a plasmid expressing green fluorescent protein. Cells were harvested 24 h later, and green fluorescent protein-transfected cells were collected using a FACSAria cell sorter. Proteins were extracted and subjected to West-ern blot analysis and sequentially stained with antibodies to Myc, PP2A-B55, E4orf4, and α-tubulin. The asterisk marks a nonspecific protein band. (C) IR-B55 cells were transfected with plasmids expressing a PP2A-B55-HA mutant resistant to the B55 shRNA (lanes 3 and 4), E4orf4 (lanes 2 and 4), or empty vectors (lane 1). Cells were harvested 24 h later, and proteins were subjected to Western blot analysis and sequentially stained with antibodies to Myc, the ΗΑ tag, Ε4orf4, and α-tubulin.