The adenovirus E4orf6 protein can promote E1A/E1B-induced focus formation by interfering with p53 tumor suppressor function - PubMed (original) (raw)

The adenovirus E4orf6 protein can promote E1A/E1B-induced focus formation by interfering with p53 tumor suppressor function

M Nevels et al. Proc Natl Acad Sci U S A. 1997.

Abstract

We have recently shown that the adenovirus type 5 E4orf6 protein interacts with the cellular tumor suppressor protein p53 and blocks p53 transcriptional functions. Here we report that the E4orf6 protein can promote focus formation of primary rodent epithelial cells in cooperation with adenovirus E1A and E1A plus E1B proteins. The E4orf6 protein can also inhibit p53-mediated suppression of E1A plus E1B-19kDa-induced focus formation. Mutant analysis of the E4orf6 protein demonstrates that these activities correlate with the ability of the adenovirus protein to relieve transcriptional repression mediated by the carboxyl-terminal region of p53 in transient transfection assays. We further demonstrate that expression of wild-type E4orf6 correlates with a dramatic reduction of p53 steady-state levels in transformed rat cells. Our data demonstrate that adenovirus type 5 encodes two different proteins, E1B-55kDa and E4orf6, that bind to p53 and contribute to transformation by modulating p53 transcriptional functions.

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Figures

Figure 1

Figure 1

Effects of E4orf6, E4orf6/7, and E4orf6 deletion mutants on p53 transcriptional functions. (A) Inhibition of Gal4-p53 transactivation. Subconfluent H1299 cells were transfected with the indicated amounts of reporter and effector plasmids (μg of DNA) tabulated below each bar in the graphs, and luciferase activity was determined. The mean and standard deviation are presented for four experiments, each performed in duplicate. (B) Relief of Gal4-p53C-mediated repression. Three micrograms of reporter plasmid pGalTK-LUC and the indicated amount of pCMV-E4 effector plasmids were cotransfected with 1 μg of pGal4-p53C into H1299 cells and luciferase activity was determined. Bar 1 (counting from the left) represents luciferase activity produced by transfecting 3 μg of the reporter plasmid in the absence of any effector plasmids. Bar 2 represents luciferase activity produced by cotransfecting 3 μg of pGalTK-LUC and 1 μg of pGal4-p53C. The mean and standard deviation are presented for three experiments, each performed in duplicate. (C) Expression of wild-type E4orf6 and E4orf6 mutant proteins in transfected H1299 cells. Luciferase assay samples were immunoblotted by normalizing the amount of extract used according to β-galactosidase activity and probing the immunoblot with anti-E4orf6 monoclonal antibody RSA3 (lanes 1–4) and anti-HA monoclonal antibody 12CA5 (lanes 5 and 6). The position of molecular mass markers is indicated at left. (D) In vivo interaction of p53 with E4orf6, E4orf6/7, and E4orf6 deletion mutants in H1299 cells. The same extracts were subjected to immunoprecipitation using anti-p53 monoclonal antibody DO-1 (34). The precipitates were resolved on the same SDS/15% polyacrylamide gel, and coprecipitated E4 proteins were visualized by immunoblotting using RSA3 (lanes 1–4) or 12CA5 (lanes 5 and 6) monoclonal antibodies on two separate filters. The coimmunoprecipitation reaction with E4orf6/7 (lane 4) was resolved on a separate SDS/15% polyacrylamide gel. The bands representing the IgG proteins are indicated at left.

Figure 2

Figure 2

Wild-type E4orf6 and E4ΔN efficiently interfere with p53-mediated suppression of pAd5 _dl_338_Xho_I-C-induced transformation. Primary BRK cells were transfected with the indicated amounts of plasmids (μg of DNA per 3 × 106 cells). Focus-forming activity is represented as a percentage of E1A plus E1B-19kDa activity. The average number of foci for pAd5 _dl_338_Xho_I-C was 21 in four independent experiments.

Figure 3

Figure 3

Transformation experiments. (A) E4orf6 cooperates with E1A to promote focus formation. Primary BRK cells were transfected with the indicated amounts of plasmids, and morphologically transformed colonies were scored 3 weeks after transfection. The mean number of dense foci from three independent experiments is presented. (B) E4orf6 cooperates with E1A and E1B proteins to enhance the number of cell transformants. Focus-forming activity is represented as a percentage of pAd5 _Xho_I-C activity. The average number of foci for pAd5 _Xho_I-C was 33 in four independent experiments.

Figure 4

Figure 4

Protein and DNA analysis. (A) Immunoblot showing the steady-state levels of E1B-55kDa, E4orf6, E4ΔN, and p53 proteins in cell pools originating from cotransfections with pAd5 _Xho_I-C. The same amount of total protein form each pool was resolved on SDS/12% polyacrylamide gels and subjected to immunoblot analysis with monoclonal antibodies 2A6 (E1B-55kDa), RSA3 (E4orf6), 12CA5 (E4ΔN), and PAb421 (p53). The bands representing the viral proteins and p53 are indicated at right. (B) Analysis of viral cDNAs in ABS, ABSΔC, and ABSΔN pool cells. The viral cDNAs were PCR amplified as described in the text. The following primers were used: orf6fw and 861rev (lanes 2 and 5); orf6fw and 461rev (lanes 3 and 6); and 331fw and flufix (lanes 4 and 7). Genomic DNA isolated from BRK cells was used in the control reactions. The size of the PCR products (bp) is indicated at right.

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