Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Apr;70(4):2296–2306. doi: 10.1128/jvi.70.4.2296-2306.1996

The adenovirus death protein (E3-11.6K) is required at very late stages of infection for efficient cell lysis and release of adenovirus from infected cells.

A E Tollefson 1, A Scaria 1, T W Hermiston 1, J S Ryerse 1, L J Wold 1, W S Wold 1
PMCID: PMC190071  PMID: 8642656

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Banda N. K., Bernier J., Kurahara D. K., Kurrle R., Haigwood N., Sekaly R. P., Finkel T. H. Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis. J Exp Med. 1992 Oct 1;176(4):1099–1106. doi: 10.1084/jem.176.4.1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bett A. J., Krougliak V., Graham F. L. DNA sequence of the deletion/insertion in early region 3 of Ad5 dl309. Virus Res. 1995 Nov;39(1):75–82. [PubMed] [Google Scholar]
  3. Boyd J. M., Malstrom S., Subramanian T., Venkatesh L. K., Schaeper U., Elangovan B., D'Sa-Eipper C., Chinnadurai G. Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins. Cell. 1994 Oct 21;79(2):341–351. doi: 10.1016/0092-8674(94)90202-x. [DOI] [PubMed] [Google Scholar]
  4. Challberg S. S., Ketner G. Deletion mutants of adenovirus 2: isolation and initial characterization of virus carrying mutations near the right end of the viral genome. Virology. 1981 Oct 15;114(1):196–209. doi: 10.1016/0042-6822(81)90265-8. [DOI] [PubMed] [Google Scholar]
  5. Chen M. J., Holskin B., Strickler J., Gorniak J., Clark M. A., Johnson P. J., Mitcho M., Shalloway D. Induction by E1A oncogene expression of cellular susceptibility to lysis by TNF. Nature. 1987 Dec 10;330(6148):581–583. doi: 10.1038/330581a0. [DOI] [PubMed] [Google Scholar]
  6. Chen P. H., Ornelles D. A., Shenk T. The adenovirus L3 23-kilodalton proteinase cleaves the amino-terminal head domain from cytokeratin 18 and disrupts the cytokeratin network of HeLa cells. J Virol. 1993 Jun;67(6):3507–3514. doi: 10.1128/jvi.67.6.3507-3514.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chiou S. K., Tseng C. C., Rao L., White E. Functional complementation of the adenovirus E1B 19-kilodalton protein with Bcl-2 in the inhibition of apoptosis in infected cells. J Virol. 1994 Oct;68(10):6553–6566. doi: 10.1128/jvi.68.10.6553-6566.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coulombe P. A., Hutton M. E., Vassar R., Fuchs E. A function for keratins and a common thread among different types of epidermolysis bullosa simplex diseases. J Cell Biol. 1991 Dec;115(6):1661–1674. doi: 10.1083/jcb.115.6.1661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. D'Halluin J. C. Virus assembly. Curr Top Microbiol Immunol. 1995;199(Pt 1):47–66. [PubMed] [Google Scholar]
  10. Debbas M., White E. Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B. Genes Dev. 1993 Apr;7(4):546–554. doi: 10.1101/gad.7.4.546. [DOI] [PubMed] [Google Scholar]
  11. Deutscher S. L., Bhat B. M., Pursley M. H., Cladaras C., Wold W. S. Novel deletion mutants that enhance a distant upstream 5' splice in the E3 transcription unit of adenovirus 2. Nucleic Acids Res. 1985 Aug 26;13(16):5771–5788. doi: 10.1093/nar/13.16.5771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Duerksen-Hughes P. J., Hermiston T. W., Wold W. S., Gooding L. R. The amino-terminal portion of CD1 of the adenovirus E1A proteins is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse cells. J Virol. 1991 Mar;65(3):1236–1244. doi: 10.1128/jvi.65.3.1236-1244.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feinstein E., Kimchi A., Wallach D., Boldin M., Varfolomeev E. The death domain: a module shared by proteins with diverse cellular functions. Trends Biochem Sci. 1995 Sep;20(9):342–344. doi: 10.1016/s0968-0004(00)89070-2. [DOI] [PubMed] [Google Scholar]
  14. Givol I., Tsarfaty I., Resau J., Rulong S., da Silva P. P., Nasioulas G., DuHadaway J., Hughes S. H., Ewert D. L. Bcl-2 expressed using a retroviral vector is localized primarily in the nuclear membrane and the endoplasmic reticulum of chicken embryo fibroblasts. Cell Growth Differ. 1994 Apr;5(4):419–429. [PubMed] [Google Scholar]
  15. Gooding L. R., Elmore L. W., Tollefson A. E., Brady H. A., Wold W. S. A 14,700 MW protein from the E3 region of adenovirus inhibits cytolysis by tumor necrosis factor. Cell. 1988 May 6;53(3):341–346. doi: 10.1016/0092-8674(88)90154-7. [DOI] [PubMed] [Google Scholar]
  16. Gooding L. R., Ranheim T. S., Tollefson A. E., Aquino L., Duerksen-Hughes P., Horton T. M., Wold W. S. The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus function together to protect many but not all mouse cell lines against lysis by tumor necrosis factor. J Virol. 1991 Aug;65(8):4114–4123. doi: 10.1128/jvi.65.8.4114-4123.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gooding L. R., Sofola I. O., Tollefson A. E., Duerksen-Hughes P., Wold W. S. The adenovirus E3-14.7K protein is a general inhibitor of tumor necrosis factor-mediated cytolysis. J Immunol. 1990 Nov 1;145(9):3080–3086. [PubMed] [Google Scholar]
  18. Greber U. F., Willetts M., Webster P., Helenius A. Stepwise dismantling of adenovirus 2 during entry into cells. Cell. 1993 Nov 5;75(3):477–486. doi: 10.1016/0092-8674(93)90382-z. [DOI] [PubMed] [Google Scholar]
  19. Green M., Wold W. S. Human adenoviruses: growth, purification, and transfection assay. Methods Enzymol. 1979;58:425–435. doi: 10.1016/s0076-6879(79)58157-9. [DOI] [PubMed] [Google Scholar]
  20. Groux H., Torpier G., Monté D., Mouton Y., Capron A., Ameisen J. C. Activation-induced death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals. J Exp Med. 1992 Feb 1;175(2):331–340. doi: 10.1084/jem.175.2.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hawkins L. K., Wold W. S. A 12,500 MW protein is coded by region E3 of adenovirus. Virology. 1992 Jun;188(2):486–494. doi: 10.1016/0042-6822(92)90502-g. [DOI] [PubMed] [Google Scholar]
  22. Hinshaw V. S., Olsen C. W., Dybdahl-Sissoko N., Evans D. Apoptosis: a mechanism of cell killing by influenza A and B viruses. J Virol. 1994 Jun;68(6):3667–3673. doi: 10.1128/jvi.68.6.3667-3673.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Horton T. M., Ranheim T. S., Aquino L., Kusher D. I., Saha S. K., Ware C. F., Wold W. S., Gooding L. R. Adenovirus E3 14.7K protein functions in the absence of other adenovirus proteins to protect transfected cells from tumor necrosis factor cytolysis. J Virol. 1991 May;65(5):2629–2639. doi: 10.1128/jvi.65.5.2629-2639.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jones N., Shenk T. Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells. Cell. 1979 Jul;17(3):683–689. doi: 10.1016/0092-8674(79)90275-7. [DOI] [PubMed] [Google Scholar]
  25. Kerr J. F., Wyllie A. H., Currie A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972 Aug;26(4):239–257. doi: 10.1038/bjc.1972.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kumar S. ICE-like proteases in apoptosis. Trends Biochem Sci. 1995 May;20(5):198–202. doi: 10.1016/s0968-0004(00)89007-6. [DOI] [PubMed] [Google Scholar]
  27. Lithgow T., van Driel R., Bertram J. F., Strasser A. The protein product of the oncogene bcl-2 is a component of the nuclear envelope, the endoplasmic reticulum, and the outer mitochondrial membrane. Cell Growth Differ. 1994 Apr;5(4):411–417. [PubMed] [Google Scholar]
  28. Lowe S. W., Ruley H. E. Stabilization of the p53 tumor suppressor is induced by adenovirus 5 E1A and accompanies apoptosis. Genes Dev. 1993 Apr;7(4):535–545. doi: 10.1101/gad.7.4.535. [DOI] [PubMed] [Google Scholar]
  29. Martin S. J., Green D. R. Protease activation during apoptosis: death by a thousand cuts? Cell. 1995 Aug 11;82(3):349–352. doi: 10.1016/0092-8674(95)90422-0. [DOI] [PubMed] [Google Scholar]
  30. Noteborn M. H., Todd D., Verschueren C. A., de Gauw H. W., Curran W. L., Veldkamp S., Douglas A. J., McNulty M. S., van der EB A. J., Koch G. A single chicken anemia virus protein induces apoptosis. J Virol. 1994 Jan;68(1):346–351. doi: 10.1128/jvi.68.1.346-351.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Oltvai Z. N., Korsmeyer S. J. Checkpoints of dueling dimers foil death wishes. Cell. 1994 Oct 21;79(2):189–192. doi: 10.1016/0092-8674(94)90188-0. [DOI] [PubMed] [Google Scholar]
  32. Ranheim T. S., Shisler J., Horton T. M., Wold L. J., Gooding L. R., Wold W. S. Characterization of mutants within the gene for the adenovirus E3 14.7-kilodalton protein which prevents cytolysis by tumor necrosis factor. J Virol. 1993 Apr;67(4):2159–2167. doi: 10.1128/jvi.67.4.2159-2167.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rao L., Debbas M., Sabbatini P., Hockenbery D., Korsmeyer S., White E. The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7742–7746. doi: 10.1073/pnas.89.16.7742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reed J. C. Bcl-2 and the regulation of programmed cell death. J Cell Biol. 1994 Jan;124(1-2):1–6. doi: 10.1083/jcb.124.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sabbatini P., Chiou S. K., Rao L., White E. Modulation of p53-mediated transcriptional repression and apoptosis by the adenovirus E1B 19K protein. Mol Cell Biol. 1995 Feb;15(2):1060–1070. doi: 10.1128/mcb.15.2.1060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Scaria A., Tollefson A. E., Saha S. K., Wold W. S. The E3-11.6K protein of adenovirus is an Asn-glycosylated integral membrane protein that localizes to the nuclear membrane. Virology. 1992 Dec;191(2):743–753. doi: 10.1016/0042-6822(92)90250-s. [DOI] [PubMed] [Google Scholar]
  37. Shen Y., Shenk T. E. Viruses and apoptosis. Curr Opin Genet Dev. 1995 Feb;5(1):105–111. doi: 10.1016/s0959-437x(95)90061-6. [DOI] [PubMed] [Google Scholar]
  38. Shisler J., Duerksen-Hughes P., Hermiston T. M., Wold W. S., Gooding L. R. Induction of susceptibility to tumor necrosis factor by E1A is dependent on binding to either p300 or p105-Rb and induction of DNA synthesis. J Virol. 1996 Jan;70(1):68–77. doi: 10.1128/jvi.70.1.68-77.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Steller H. Mechanisms and genes of cellular suicide. Science. 1995 Mar 10;267(5203):1445–1449. doi: 10.1126/science.7878463. [DOI] [PubMed] [Google Scholar]
  40. Stewart P. L., Burnett R. M. Adenovirus structure by X-ray crystallography and electron microscopy. Curr Top Microbiol Immunol. 1995;199(Pt 1):25–38. doi: 10.1007/978-3-642-79496-4_2. [DOI] [PubMed] [Google Scholar]
  41. Subramanian T., Boyd J. M., Chinnadurai G. Functional substitution identifies a cell survival promoting domain common to adenovirus E1B 19 kDa and Bcl-2 proteins. Oncogene. 1995 Dec 7;11(11):2403–2409. [PubMed] [Google Scholar]
  42. Subramanian T., Tarodi B., Govindarajan R., Boyd J. M., Yoshida K., Chinnadurai G. Mutational analysis of the transforming and apoptosis suppression activities of the adenovirus E1B 175R protein. Gene. 1993 Feb 28;124(2):173–181. doi: 10.1016/0378-1119(93)90391-f. [DOI] [PubMed] [Google Scholar]
  43. Takizawa T., Matsukawa S., Higuchi Y., Nakamura S., Nakanishi Y., Fukuda R. Induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells. J Gen Virol. 1993 Nov;74(Pt 11):2347–2355. doi: 10.1099/0022-1317-74-11-2347. [DOI] [PubMed] [Google Scholar]
  44. Thimmappaya B., Weinberger C., Schneider R. J., Shenk T. Adenovirus VAI RNA is required for efficient translation of viral mRNAs at late times after infection. Cell. 1982 Dec;31(3 Pt 2):543–551. doi: 10.1016/0092-8674(82)90310-5. [DOI] [PubMed] [Google Scholar]
  45. Thompson C. B. Apoptosis in the pathogenesis and treatment of disease. Science. 1995 Mar 10;267(5203):1456–1462. doi: 10.1126/science.7878464. [DOI] [PubMed] [Google Scholar]
  46. Tollefson A. E., Scaria A., Saha S. K., Wold W. S. The 11,600-MW protein encoded by region E3 of adenovirus is expressed early but is greatly amplified at late stages of infection. J Virol. 1992 Jun;66(6):3633–3642. doi: 10.1128/jvi.66.6.3633-3642.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ubol S., Tucker P. C., Griffin D. E., Hardwick J. M. Neurovirulent strains of Alphavirus induce apoptosis in bcl-2-expressing cells: role of a single amino acid change in the E2 glycoprotein. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5202–5206. doi: 10.1073/pnas.91.11.5202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Vassar R., Coulombe P. A., Degenstein L., Albers K., Fuchs E. Mutant keratin expression in transgenic mice causes marked abnormalities resembling a human genetic skin disease. Cell. 1991 Jan 25;64(2):365–380. doi: 10.1016/0092-8674(91)90645-f. [DOI] [PubMed] [Google Scholar]
  49. Voelkel-Johnson C., Entingh A. J., Wold W. S., Gooding L. R., Laster S. M. Activation of intracellular proteases is an early event in TNF-induced apoptosis. J Immunol. 1995 Feb 15;154(4):1707–1716. [PubMed] [Google Scholar]
  50. Wang E. W., Scott M. O., Ricciardi R. P. An adenovirus mRNA which encodes a 14,700-Mr protein that maps to the last open reading frame of region E3 is expressed during infection. J Virol. 1988 Apr;62(4):1456–1459. doi: 10.1128/jvi.62.4.1456-1459.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. White E., Cipriani R., Sabbatini P., Denton A. Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins. J Virol. 1991 Jun;65(6):2968–2978. doi: 10.1128/jvi.65.6.2968-2978.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. White E., Grodzicker T., Stillman B. W. Mutations in the gene encoding the adenovirus early region 1B 19,000-molecular-weight tumor antigen cause the degradation of chromosomal DNA. J Virol. 1984 Nov;52(2):410–419. doi: 10.1128/jvi.52.2.410-419.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. White E., Sabbatini P., Debbas M., Wold W. S., Kusher D. I., Gooding L. R. The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha. Mol Cell Biol. 1992 Jun;12(6):2570–2580. doi: 10.1128/mcb.12.6.2570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wickham T. J., Mathias P., Cheresh D. A., Nemerow G. R. Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. Cell. 1993 Apr 23;73(2):309–319. doi: 10.1016/0092-8674(93)90231-e. [DOI] [PubMed] [Google Scholar]
  55. Wold W. S., Cladaras C., Magie S. C., Yacoub N. Mapping a new gene that encodes an 11,600-molecular-weight protein in the E3 transcription unit of adenovirus 2. J Virol. 1984 Nov;52(2):307–313. doi: 10.1128/jvi.52.2.307-313.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wold W. S., Deutscher S. L., Takemori N., Bhat B. M., Magie S. C. Evidence that AGUAUAUGA and CCAAGAUGA initiate translation in the same mRNA region E3 of adenovirus. Virology. 1986 Jan 15;148(1):168–180. doi: 10.1016/0042-6822(86)90412-5. [DOI] [PubMed] [Google Scholar]
  57. Wold W. S., Gooding L. R. Region E3 of adenovirus: a cassette of genes involved in host immunosurveillance and virus-cell interactions. Virology. 1991 Sep;184(1):1–8. doi: 10.1016/0042-6822(91)90815-s. [DOI] [PubMed] [Google Scholar]
  58. Wold W. S., Hermiston T. W., Tollefson A. E. Adenovirus proteins that subvert host defenses. Trends Microbiol. 1994 Nov;2(11):437–443. doi: 10.1016/0966-842x(94)90801-x. [DOI] [PubMed] [Google Scholar]
  59. Yew P. R., Berk A. J. Inhibition of p53 transactivation required for transformation by adenovirus early 1B protein. Nature. 1992 May 7;357(6373):82–85. doi: 10.1038/357082a0. [DOI] [PubMed] [Google Scholar]
  60. Zhang Y., Schneider R. J. Adenovirus inhibition of cell translation facilitates release of virus particles and enhances degradation of the cytokeratin network. J Virol. 1994 Apr;68(4):2544–2555. doi: 10.1128/jvi.68.4.2544-2555.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES