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. 1998 Sep;72(9):7144-53.
doi: 10.1128/JVI.72.9.7144-7153.1998.

The early region 4 orf4 protein of human adenovirus type 5 induces p53-independent cell death by apoptosis

R C Marcellus  1 , J N Lavoie, D Boivin, G C Shore, G Ketner, P E Branton
Affiliations

The early region 4 orf4 protein of human adenovirus type 5 induces p53-independent cell death by apoptosis

R C Marcellus et al. J Virol. 1998 Sep.

Abstract

Previous studies by our group showed that infection of human and rodent cells by human adenovirus type 5 (Ad5) results in the induction of p53-independent apoptosis and cell death that are dependent upon transactivation of early region 4 (E4). To identify which E4 products are involved, studies were conducted with p53-deficient human SAOS-2 cells infected with various Ad5 E4 mutants. An E4orf6-deficient mutant was defective in cell killing, whereas another that expressed only E4orf6 and E4orf4 killed like wild-type virus, suggesting that E4orf6 may be responsible for cytotoxicity; however, a mutant expressing only E4orf4 induced high levels of cell death, indicating that this E4 product may also be able to induce cytotoxicity. To define the E4 cell death-inducing functions more precisely, cDNAs encoding individual E4 products were introduced into cells by DNA transfection in the absence of other Ad5 proteins. In cotransfections with a cDNA encoding firefly luciferase, enzymatic activity was high in all cases except with E4orf4, where luciferase levels were less than 20% of those in controls. In addition, drug selection of several cell types following transfection with retroviral vector DNA encoding individual E4 products as well as puromycin resistance yielded a large number of cell colonies except when E4orf4 was expressed. These data demonstrated that E4orf4 is the only E4 product capable of independent cell killing. Cell death induced by E4orf4 was due to apoptosis, as evidenced by 4',6-diamidino-2-phenylindole (DAPI) staining of cell nuclei in E4orf4-expressing cells. Thus, although E4orf6 may play some role, these results suggested that E4orf4 may be the major E4 product responsible for induction of p53-independent apoptosis.

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Figures

FIG. 1
FIG. 1
Ad5 E4 and E4 mutants. At the top is right end of the Ad5 genome including positions in base pairs and map units. The locations of two SmaI restriction enzyme sites used in the preparation of E4 mutants are also presented. Below are the positions of several open reading frames for E4 proteins that are encoded from right (amino terminus) to left (carboxy terminus). At the bottom are the structures of several E4 deletion mutants. The E4 proteins expressed by these mutants are summarized at the right.
FIG. 2
FIG. 2
Viability of SAOS-2 cells infected with mutant and wild-type (wt) Ad5. p53-deficient human SAOS-2 cells were mock infected or infected with wild-type Ad5 or a series of mutants which contain defects in E4, and at various times following infection they were tested for viability by a trypan blue exclusion assay, as described in Materials and Methods. The results are presented as the logarithm of the percentage of viable cells. Panels A and B show two separate representative studies involving two sets of E4 mutants.
FIG. 3
FIG. 3
Analysis of cell killing by measurement of luciferase activity. 1A.A6 cells were cotransfected with pcDNA3 plasmids expressing firefly luciferase and individual Ad5 E4 proteins. After 48 h the cells were harvested and the extracts were analyzed for luciferase activity as described in Materials and Methods. The data represent the average of five separate experiments, each involving duplicate samples (standard errors are indicated by lines). The luciferase activity obtained with the pcDNA3 alone control was set at 100%.
FIG. 4
FIG. 4
Analysis of cell killing by an Ad vector expressing E4orf6. SAOS-2 cells were infected either with wild-type (wt) Ad5 or with Ad vector AdE4orf6 that expresses only E4orf6. Cell killing was measured by trypan blue exclusion as in Fig. 2.
FIG. 5
FIG. 5
Analysis of E4 killing by colony inhibition. E1A-expressing, p53-minus 1A.A6 cells (A), SAOS-2 cells (B), or HeLa cells (C) were transfected with the puromycin resistance-containing pBABE retroviral plasmid expressing individual E4 products. The cells were plated and then grown in the presence of puromycin for 14 days, as described in Materials and Methods, after which time the number of colonies was tabulated. The results are the mean of four (A) or two (B and C) experiments involving two plates of each E4orf per experiment (standard error indicated by bars). Colony formation in the presence of pcDNA3, which varied between about 80 and 120 per plate, was set at 100%.
FIG. 6
FIG. 6
CHO (A) or 293 (B) cells were transfected with plasmid pcDNA3 expressing an HA-tagged version of E4orf4 together with the pHOOK plasmid to facilitate magnetic sorting of transfected cells (see Materials and Methods). After sorting, the cells were analyzed either by immunofluorescence with an antibody directed against HA or by staining for DNA with DAPI. A single representative field for each cell line is presented.
FIG. 7
FIG. 7
Analysis of viral protein expression with wild-type and E4 mutant Ad5. HeLa cells were infected by wild-type Ad5 or mutants dl1010, dl1013, or dl1015 and harvested at 16 h p.i. Cell extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted. (A) Anti-E1A M73 monoclonal antibody. (B) Anti-E4orf6 serum. (C) Anti-E4orf4 serum.
FIG. 8
FIG. 8
Analysis of cell killing and E4 expression by wild-type (wt) Ad5 and mutant dl359. SAOS-2 cells were infected with wild-type Ad5 or mutant dl359 that is defective for E4orf4 expression, and at various times after infection cell viability was assessed by trypan blue exclusion as in Fig. 2. (A) Cell viability by trypan blue staining. Data are expressed as percent cell viability. (B) Expression of E4orf6 and E4orf6/7. Portions of the cultures were harvested at 16 h p.i. and analyzed by immunoblotting as in Fig. 7 with an antiserum that recognizes the amino terminus of E4orf6 and E4orf6/7.
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