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We have reported that an 11,600-Da nuclear membrane glycoprotein named adenovirus death protein (ADP), encoded by the E3 region, is required for the efficient death (lysis) of adenovirus (Ad)-infected cells. We postulated that ADP mediates the release of virions from cells at the conclusion of replication. Here we provide further characterization of cells infected by adp+ and adp- Ads. Using virus mutants with deletions in the individual E3 genes, we show that only mutants that lack ADP have small plaques that are slow to develop. Mutants in the adp gene replicated as well as wild-type Ad, but the cells lysed much more slowly. Cell lysis and viability were determined by plaque size, cell morphology, trypan blue exclusion, the release of lactate dehydrogenase, and the MTT assay for mitochondrial activity. ADP is required for efficient lysis of human A549, KB, 293, and MCF-7 cells. A549 cells infected with adp+ Ads began to die at 2-3 days postinfection and were dead by 6 days. With adp mutants, > 80% of cells remained viable for 5-6 days; when the medium was changed, > 80% of cells were viable after 7 days and 10-20% after 14 days. When the MTT assay was used, there was an increase in mitochondrial activity, suggesting that Ad infection stimulates respiratory metabolism. Nearly all nuclei from wild-type Adinfected cells lacked DAPI-stained DNA by 7 days, whereas with an adp mutant nearly all nuclei stained brightly after 15 days. Nuclei from adp mutant-infected cells were extremely swollen and full of virus, and appeared to have an intact nuclear membrane. Cells infected with wild-type Ad had many vacuoles and perhaps a disrupted nuclear membrane; they did not display features typical of apoptosis.

Adenovirus (Ad) infection is concluded by assembly of virions in the cell nucleus followed by lysis of cells by an unknown mechanism. We have described an Ad nuclear membrane glycoprotein of 11,600 kDa (E3-11.6K) which is encoded by the E3 transcription unit and which is synthesized in small amounts from the E3 promoter at early stages of infection but in large amounts from the major late promoter at very late stages of infection. We now report that E3-11.6K is required for the efficient lysis (death) of Ad-infected cells, and we propose that the function of E3-11.6K is to mediate the release of Ad progeny from infected cells. We have renamed E3-11.6K the Ad death protein (ADP). Virus mutants that lack ADP replicated as well as adp+ Ad, but the cells lysed more slowly, virus release from the cell was retarded, and the plaques were small and developed slowly. Cells infected with adp+ viruses began to lyse at 2 or 3 days postinfection (p.i.) and were completely lysed by 5 or 6 days p.i. In contrast, cells infected with adp mutants did not begin significant lysis until 5 or 6 days p.i. Cell lysis and viability were determined by plaque size, extracellular virus, cell morphology, release of lactate dehydrogenase, trypan blue exclusion, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay for mitochondrial activity, RNA degradation, and DNA degradation as determined by agarose gel electrophoresis and the terminal deoxynucleotidyltransferase end labeling assay. Protein synthesis was almost nonexistent at 3 days p.i. in cells infected with adp+ Ads, but it was still increasing in cells infected with adp mutants. Host cell protein synthesis was undetectable at 1 day p.i. in cells infected with adp+ Ads or adp mutants. Cells infected with adp mutants showed Ad cytopathic effect at 1 or 2 days p.i. in that they rounded up and detached, but the cells remained metabolically active and intact for >5 days p.i. When examined by electron microscopy, the nuclei were extremely swollen and full of virus, and the nuclear membrane appeared to be intact. ADP is unrelated in sequence to other known cell death-promoting proteins.