Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Feb;71(2):1115–1123. doi: 10.1128/jvi.71.2.1115-1123.1997

Structural analysis of the adenovirus type 5 E1B 55-kilodalton-E4orf6 protein complex.

S Rubenwolf 1, H Schütt 1, M Nevels 1, H Wolf 1, T Dobner 1
PMCID: PMC191163  PMID: 8995632

Abstract

The adenovirus type 5 (Ad5) early 1B (E1B) 55-kDa (E1B-55kDa)-E4orf6 protein complex has been implicated in the selective modulation of nucleocytoplasmic mRNA transport at late times after infection. Using a combined immunoprecipitation-immunoblotting assay, we mapped the domains in E1B-55kDa required for the interaction with the E4orf6 protein in lytically infected A549 cells. Several domains in the 496-residue 55-kDa polypeptide contributed to a stable association with the E4orf6 protein in E1B mutant virus-infected cells. Linker insertion mutations at amino acids 180 and 224 caused reduced binding of the E4orf6 protein, whereas linker insertion mutations at amino acid 143 and in the central domain of E1B-55kDa eliminated the binding of the E4orf6 protein. Earlier work showing that the central domain of E1B-55kDa is required for binding to p53 and the recent observation that the E4orf6 protein also interacts with the tumor suppressor protein led us to suspect that p53 might play a role in the E1B-E4 protein interaction. However, coimmunoprecipitation assays with extracts prepared from infected p53-negative H1299 cells established that p53 is not needed for the E1B-E4 protein interaction in adenovirus-infected cells. Using two different protein-protein interaction assays, we also mapped the region in the E4orf6 protein required for E1B-55kDa interaction to the amino-terminal 55 amino acid residues. Interestingly, both binding assays established that the same region in the E4orf6/7 protein can potentially interact with E1B-55kDa. Our results demonstrate that two distinct segments in the 55-kDa protein encoding the transformation and late lytic functions independently interact with p53 and the E4orf6 protein in vivo and provide further insight by which the multifunctional 55-kDa EIB protein can exert its multiple activities in lytically infected cells and in adenovirus transformation.

Full Text

The Full Text of this article is available as a PDF (474.8 KB).

Selected References

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

  1. Babiss L. E., Ginsberg H. S. Adenovirus type 5 early region 1b gene product is required for efficient shutoff of host protein synthesis. J Virol. 1984 Apr;50(1):202–212. doi: 10.1128/jvi.50.1.202-212.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Babiss L. E., Ginsberg H. S., Darnell J. E., Jr Adenovirus E1B proteins are required for accumulation of late viral mRNA and for effects on cellular mRNA translation and transport. Mol Cell Biol. 1985 Oct;5(10):2552–2558. doi: 10.1128/mcb.5.10.2552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barker D. D., Berk A. J. Adenovirus proteins from both E1B reading frames are required for transformation of rodent cells by viral infection and DNA transfection. Virology. 1987 Jan;156(1):107–121. doi: 10.1016/0042-6822(87)90441-7. [DOI] [PubMed] [Google Scholar]
  4. Blair Zajdel M. E., Blair G. E. The intracellular distribution of the transformation-associated protein p53 in adenovirus-transformed rodent cells. Oncogene. 1988 Jun;2(6):579–584. [PubMed] [Google Scholar]
  5. Boulanger P. A., Blair G. E. Expression and interactions of human adenovirus oncoproteins. Biochem J. 1991 Apr 15;275(Pt 2):281–299. doi: 10.1042/bj2750281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Braithwaite A. W., Jenkins J. R. Ability of p53 and the adenovirus E1b 58-kilodalton protein to form a complex is determined by p53. J Virol. 1989 Apr;63(4):1792–1799. doi: 10.1128/jvi.63.4.1792-1799.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bridge E., Ketner G. Interaction of adenoviral E4 and E1b products in late gene expression. Virology. 1990 Feb;174(2):345–353. doi: 10.1016/0042-6822(90)90088-9. [DOI] [PubMed] [Google Scholar]
  8. Brown C. R., Doxsey S. J., White E., Welch W. J. Both viral (adenovirus E1B) and cellular (hsp 70, p53) components interact with centrosomes. J Cell Physiol. 1994 Jul;160(1):47–60. doi: 10.1002/jcp.1041600107. [DOI] [PubMed] [Google Scholar]
  9. Cutt J. R., Shenk T., Hearing P. Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells. J Virol. 1987 Feb;61(2):543–552. doi: 10.1128/jvi.61.2.543-552.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dobner T., Horikoshi N., Rubenwolf S., Shenk T. Blockage by adenovirus E4orf6 of transcriptional activation by the p53 tumor suppressor. Science. 1996 Jun 7;272(5267):1470–1473. doi: 10.1126/science.272.5267.1470. [DOI] [PubMed] [Google Scholar]
  11. Gaynor R. B., Hillman D., Berk A. J. Adenovirus early region 1A protein activates transcription of a nonviral gene introduced into mammalian cells by infection or transfection. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1193–1197. doi: 10.1073/pnas.81.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Giard D. J., Aaronson S. A., Todaro G. J., Arnstein P., Kersey J. H., Dosik H., Parks W. P. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst. 1973 Nov;51(5):1417–1423. doi: 10.1093/jnci/51.5.1417. [DOI] [PubMed] [Google Scholar]
  13. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  14. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  15. Grand R. J., Gallimore P. H. Adenovirus type 12 early region 1 proteins: a study of their subcellular localization and protein-protein interactions. J Gen Virol. 1984 Dec;65(Pt 12):2149–2166. doi: 10.1099/0022-1317-65-12-2149. [DOI] [PubMed] [Google Scholar]
  16. Grand R. J., Grant M. L., Gallimore P. H. Enhanced expression of p53 in human cells infected with mutant adenoviruses. Virology. 1994 Sep;203(2):229–240. doi: 10.1006/viro.1994.1480. [DOI] [PubMed] [Google Scholar]
  17. Halbert D. N., Cutt J. R., Shenk T. Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff. J Virol. 1985 Oct;56(1):250–257. doi: 10.1128/jvi.56.1.250-257.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hearing P., Shenk T. Sequence-independent autoregulation of the adenovirus type 5 E1A transcription unit. Mol Cell Biol. 1985 Nov;5(11):3214–3221. doi: 10.1128/mcb.5.11.3214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Huang M. M., Hearing P. Adenovirus early region 4 encodes two gene products with redundant effects in lytic infection. J Virol. 1989 Jun;63(6):2605–2615. doi: 10.1128/jvi.63.6.2605-2615.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kaelin W. G., Jr, Krek W., Sellers W. R., DeCaprio J. A., Ajchenbaum F., Fuchs C. S., Chittenden T., Li Y., Farnham P. J., Blanar M. A. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell. 1992 Jul 24;70(2):351–364. doi: 10.1016/0092-8674(92)90108-o. [DOI] [PubMed] [Google Scholar]
  21. Kao C. C., Yew P. R., Berk A. J. Domains required for in vitro association between the cellular p53 and the adenovirus 2 E1B 55K proteins. Virology. 1990 Dec;179(2):806–814. doi: 10.1016/0042-6822(90)90148-k. [DOI] [PubMed] [Google Scholar]
  22. Karasuyama H., Tohyama N., Tada T. Autocrine growth and tumorigenicity of interleukin 2-dependent helper T cells transfected with IL-2 gene. J Exp Med. 1989 Jan 1;169(1):13–25. doi: 10.1084/jem.169.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Leppard K. N., Shenk T. The adenovirus E1B 55 kd protein influences mRNA transport via an intranuclear effect on RNA metabolism. EMBO J. 1989 Aug;8(8):2329–2336. doi: 10.1002/j.1460-2075.1989.tb08360.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Liang S., Hitomi M., Tartakoff A. M. Adenoviral E1B-55kDa protein inhibits yeast mRNA export and perturbs nuclear structure. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7372–7375. doi: 10.1073/pnas.92.16.7372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Malette P., Yee S. P., Branton P. E. Studies on the phosphorylation of the 58000 dalton early region 1B protein of human adenovirus type 5. J Gen Virol. 1983 May;64(Pt 5):1069–1078. doi: 10.1099/0022-1317-64-5-1069. [DOI] [PubMed] [Google Scholar]
  26. Marton M. J., Baim S. B., Ornelles D. A., Shenk T. The adenovirus E4 17-kilodalton protein complexes with the cellular transcription factor E2F, altering its DNA-binding properties and stimulating E1A-independent accumulation of E2 mRNA. J Virol. 1990 May;64(5):2345–2359. doi: 10.1128/jvi.64.5.2345-2359.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Matlashewski G., Banks L., Pim D., Crawford L. Analysis of human p53 proteins and mRNA levels in normal and transformed cells. Eur J Biochem. 1986 Feb 3;154(3):665–672. doi: 10.1111/j.1432-1033.1986.tb09449.x. [DOI] [PubMed] [Google Scholar]
  28. Mitsudomi T., Steinberg S. M., Nau M. M., Carbone D., D'Amico D., Bodner S., Oie H. K., Linnoila R. I., Mulshine J. L., Minna J. D. p53 gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features. Oncogene. 1992 Jan;7(1):171–180. [PubMed] [Google Scholar]
  29. Neill S. D., Hemstrom C., Virtanen A., Nevins J. R. An adenovirus E4 gene product trans-activates E2 transcription and stimulates stable E2F binding through a direct association with E2F. Proc Natl Acad Sci U S A. 1990 Mar;87(5):2008–2012. doi: 10.1073/pnas.87.5.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Niman H. L., Houghten R. A., Walker L. E., Reisfeld R. A., Wilson I. A., Hogle J. M., Lerner R. A. Generation of protein-reactive antibodies by short peptides is an event of high frequency: implications for the structural basis of immune recognition. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4949–4953. doi: 10.1073/pnas.80.16.4949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nordqvist K., Akusjärvi G. Adenovirus early region 4 stimulates mRNA accumulation via 5' introns. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9543–9547. doi: 10.1073/pnas.87.24.9543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nordqvist K., Ohman K., Akusjärvi G. Human adenovirus encodes two proteins which have opposite effects on accumulation of alternatively spliced mRNAs. Mol Cell Biol. 1994 Jan;14(1):437–445. doi: 10.1128/mcb.14.1.437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Obert S., O'Connor R. J., Schmid S., Hearing P. The adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex. Mol Cell Biol. 1994 Feb;14(2):1333–1346. doi: 10.1128/mcb.14.2.1333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ohman K., Nordqvist K., Akusjärvi G. Two adenovirus proteins with redundant activities in virus growth facilitates tripartite leader mRNA accumulation. Virology. 1993 May;194(1):50–58. doi: 10.1006/viro.1993.1234. [DOI] [PubMed] [Google Scholar]
  35. Ornelles D. A., Shenk T. Localization of the adenovirus early region 1B 55-kilodalton protein during lytic infection: association with nuclear viral inclusions requires the early region 4 34-kilodalton protein. J Virol. 1991 Jan;65(1):424–429. doi: 10.1128/jvi.65.1.424-429.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pilder S., Moore M., Logan J., Shenk T. The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs. Mol Cell Biol. 1986 Feb;6(2):470–476. doi: 10.1128/mcb.6.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rowe D. T., Branton P. E., Graham F. L. The kinetics of synthesis of early viral proteins in KB cells infected with wild-type and transformation-defective host-range mutants of human adenovirus type 5. J Gen Virol. 1984 Mar;65(Pt 3):585–597. doi: 10.1099/0022-1317-65-3-585. [DOI] [PubMed] [Google Scholar]
  38. Rowe D. T., Graham F. L., Branton P. E. Intracellular localization of adenovirus type 5 tumor antigens in productively infected cells. Virology. 1983 Sep;129(2):456–468. doi: 10.1016/0042-6822(83)90183-6. [DOI] [PubMed] [Google Scholar]
  39. Sandler A. B., Ketner G. Adenovirus early region 4 is essential for normal stability of late nuclear RNAs. J Virol. 1989 Feb;63(2):624–630. doi: 10.1128/jvi.63.2.624-630.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sarnow P., Hearing P., Anderson C. W., Halbert D. N., Shenk T., Levine A. J. Adenovirus early region 1B 58,000-dalton tumor antigen is physically associated with an early region 4 25,000-dalton protein in productively infected cells. J Virol. 1984 Mar;49(3):692–700. doi: 10.1128/jvi.49.3.692-700.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sarnow P., Sullivan C. A., Levine A. J. A monoclonal antibody detecting the adenovirus type 5-E1b-58Kd tumor antigen: characterization of the E1b-58Kd tumor antigen in adenovirus-infected and -transformed cells. Virology. 1982 Jul 30;120(2):510–517. doi: 10.1016/0042-6822(82)90054-x. [DOI] [PubMed] [Google Scholar]
  42. Smiley J. K., Young M. A., Flint S. J. Intranuclear location of the adenovirus type 5 E1B 55-kilodalton protein. J Virol. 1990 Sep;64(9):4558–4564. doi: 10.1128/jvi.64.9.4558-4564.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stillman B. Functions of the adenovirus E1B tumour antigens. Cancer Surv. 1986;5(2):389–404. [PubMed] [Google Scholar]
  44. Teodoro J. G., Halliday T., Whalen S. G., Takayesu D., Graham F. L., Branton P. E. Phosphorylation at the carboxy terminus of the 55-kilodalton adenovirus type 5 E1B protein regulates transforming activity. J Virol. 1994 Feb;68(2):776–786. doi: 10.1128/jvi.68.2.776-786.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Triezenberg S. J., Kingsbury R. C., McKnight S. L. Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev. 1988 Jun;2(6):718–729. doi: 10.1101/gad.2.6.718. [DOI] [PubMed] [Google Scholar]
  46. Weinberg D. H., Ketner G. A cell line that supports the growth of a defective early region 4 deletion mutant of human adenovirus type 2. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5383–5386. doi: 10.1073/pnas.80.17.5383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Weinberg D. H., Ketner G. Adenoviral early region 4 is required for efficient viral DNA replication and for late gene expression. J Virol. 1986 Mar;57(3):833–838. doi: 10.1128/jvi.57.3.833-838.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Yew P. R., Kao C. C., Berk A. J. Dissection of functional domains in the adenovirus 2 early 1B 55K polypeptide by suppressor-linker insertional mutagenesis. Virology. 1990 Dec;179(2):795–805. doi: 10.1016/0042-6822(90)90147-j. [DOI] [PubMed] [Google Scholar]
  50. Yew P. R., Liu X., Berk A. J. Adenovirus E1B oncoprotein tethers a transcriptional repression domain to p53. Genes Dev. 1994 Jan;8(2):190–202. doi: 10.1101/gad.8.2.190. [DOI] [PubMed] [Google Scholar]
  51. Zantema A., Fransen J. A., Davis-Olivier A., Ramaekers F. C., Vooijs G. P., DeLeys B., Van der Eb A. J. Localization of the E1B proteins of adenovirus 5 in transformed cells, as revealed by interaction with monoclonal antibodies. Virology. 1985 Apr 15;142(1):44–58. doi: 10.1016/0042-6822(85)90421-0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES