Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Journal of Virology logoLink to Journal of Virology
. 1988 Nov;62(11):4153–4166. doi: 10.1128/jvi.62.11.4153-4166.1988

Differential distribution of the adenovirus E1A proteins and colocalization of E1A with the 70-kilodalton cellular heat shock protein in infected cells.

E White 1, D Spector 1, W Welch 1
PMCID: PMC253847  PMID: 2971821

Abstract

Five distinct localization patterns were observed for the adenovirus E1A proteins in the nuclei of infected HeLa cells: diffuse, reticular, nucleolar, punctate, and peripheral. The variable distribution of E1A was correlated with the time postinfection and the cell cycle stage of the host cell at the time of infection. All staining patterns, with the exception of peripheral E1A localization, were associated with the early phase of infection since only the diffuse, reticular, nucleolar, and punctate staining patterns were observed in the presence of hydroxyurea. Because the E1A proteins (12S and 13S) stimulate the expression of the cellular heat shock 70-kilodalton protein (hsp70), we examined the intracellular distribution of hsp70 in the adenovirus-infected cells. Whereas hsp70 was predominantly cytoplasmic in the cells before infection, after adenovirus infection most of the protein was now found within the nucleus. Specifically, hsp70 was found within the nucleoli as well as exhibiting reticular, diffuse, and punctate nuclear staining patterns, analogous to those observed for the E1A proteins. Double-label indirect immunofluorescence of E1A and hsp70 in infected cells demonstrated a colocalization of these proteins in the nucleus. Translocation of hsp70 to the nucleus was dependent upon both adenovirus infection and expression of the E1A proteins. The localization of hsp70 was unaltered by infection with an E1A 9S cDNA virus which does not synthesize a functional E1A gene product. Moreover, the discrete nuclear localization patterns of E1A and the colocalization of E1A with hsp70 were not observed in adenovirus-transformed 293 cells which constitutively express E1A and E1B. E1A displayed exclusively diffuse nuclear staining in 293 cells; however, localization of E1A into the discrete nuclear patterns occurred after adenovirus infection of 293 cells. Immunoprecipitation of labeled infected-cell extracts with a monoclonal antibody directed against the E1A proteins resulted in precipitation of small amounts of hsp70 along with E1A. These data indicate that the adenovirus E1A proteins colocalize with, and possibly form a physical complex with, cellular hsp70 in infected cells. The relevance of this association, with respect to the function of these proteins during infection and the association of other oncoproteins with hsp70, is discussed.

Full text

PDF
4153

Images in this article

Selected References

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

  1. Bardwell J. C., Craig E. A. Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous. Proc Natl Acad Sci U S A. 1984 Feb;81(3):848–852. doi: 10.1073/pnas.81.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berk A. J. Adenovirus promoters and E1A transactivation. Annu Rev Genet. 1986;20:45–79. doi: 10.1146/annurev.ge.20.120186.000401. [DOI] [PubMed] [Google Scholar]
  3. Branton P. E., Bayley S. T., Graham F. L. Transformation by human adenoviruses. Biochim Biophys Acta. 1985;780(1):67–94. doi: 10.1016/0304-419x(84)90007-6. [DOI] [PubMed] [Google Scholar]
  4. Chatterjee P. K., Bruner M., Flint S. J., Harter M. L. DNA-binding properties of an adenovirus 289R E1A protein. EMBO J. 1988 Mar;7(3):835–841. doi: 10.1002/j.1460-2075.1988.tb02882.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clarke C. F., Cheng K., Frey A. B., Stein R., Hinds P. W., Levine A. J. Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP. Mol Cell Biol. 1988 Mar;8(3):1206–1215. doi: 10.1128/mcb.8.3.1206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dodson M., Echols H., Wickner S., Alfano C., Mensa-Wilmot K., Gomes B., LeBowitz J., Roberts J. D., McMacken R. Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7638–7642. doi: 10.1073/pnas.83.20.7638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feldman L. T., Nevins J. R. Localization of the adenovirus E1Aa protein, a positive-acting transcriptional factor, in infected cells infected cells. Mol Cell Biol. 1983 May;3(5):829–838. doi: 10.1128/mcb.3.5.829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ferguson B., Jones N., Richter J., Rosenberg M. Adenovirus E1a gene product expressed at high levels in Escherichia coli is functional. Science. 1984 Jun 22;224(4655):1343–1346. doi: 10.1126/science.6374895. [DOI] [PubMed] [Google Scholar]
  9. Flint S. J. Cellular transformation by adenoviruses. Pharmacol Ther. 1984;26(1):59–88. doi: 10.1016/0163-7258(84)90051-2. [DOI] [PubMed] [Google Scholar]
  10. Gaynor R. B., Tsukamoto A., Montell C., Berk A. J. Enhanced expression of adenovirus transforming proteins. J Virol. 1982 Oct;44(1):276–285. doi: 10.1128/jvi.44.1.276-285.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Haley K. P., Overhauser J., Babiss L. E., Ginsberg H. S., Jones N. C. Transformation properties of type 5 adenovirus mutants that differentially express the E1A gene products. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5734–5738. doi: 10.1073/pnas.81.18.5734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harlow E., Crawford L. V., Pim D. C., Williamson N. M. Monoclonal antibodies specific for simian virus 40 tumor antigens. J Virol. 1981 Sep;39(3):861–869. doi: 10.1128/jvi.39.3.861-869.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harlow E., Franza B. R., Jr, Schley C. Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products. J Virol. 1985 Sep;55(3):533–546. doi: 10.1128/jvi.55.3.533-546.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harlow E., Whyte P., Franza B. R., Jr, Schley C. Association of adenovirus early-region 1A proteins with cellular polypeptides. Mol Cell Biol. 1986 May;6(5):1579–1589. doi: 10.1128/mcb.6.5.1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hinds P. W., Finlay C. A., Frey A. B., Levine A. J. Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines. Mol Cell Biol. 1987 Aug;7(8):2863–2869. doi: 10.1128/mcb.7.8.2863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Houweling A., van den Elsen P. J., van der Eb A. J. Partial transformation of primary rat cells by the leftmost 4.5% fragment of adenovirus 5 DNA. Virology. 1980 Sep;105(2):537–550. doi: 10.1016/0042-6822(80)90054-9. [DOI] [PubMed] [Google Scholar]
  18. Imperiale M. J., Kao H. T., Feldman L. T., Nevins J. R., Strickland S. Common control of the heat shock gene and early adenovirus genes: evidence for a cellular E1A-like activity. Mol Cell Biol. 1984 May;4(5):867–874. doi: 10.1128/mcb.4.5.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Itikawa H., Ryu J. Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B. J Bacteriol. 1979 May;138(2):339–344. doi: 10.1128/jb.138.2.339-344.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kaczmarek L., Ferguson B., Rosenberg M., Baserga R. Induction of cellular DNA synthesis by purified adenovirus E1A proteins. Virology. 1986 Jul 15;152(1):1–10. doi: 10.1016/0042-6822(86)90366-1. [DOI] [PubMed] [Google Scholar]
  21. Kao H. T., Capasso O., Heintz N., Nevins J. R. Cell cycle control of the human HSP70 gene: implications for the role of a cellular E1A-like function. Mol Cell Biol. 1985 Apr;5(4):628–633. doi: 10.1128/mcb.5.4.628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kao H. T., Nevins J. R. Transcriptional activation and subsequent control of the human heat shock gene during adenovirus infection. Mol Cell Biol. 1983 Nov;3(11):2058–2065. doi: 10.1128/mcb.3.11.2058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Khandjian E. W., Türler H. Simian virus 40 and polyoma virus induce synthesis of heat shock proteins in permissive cells. Mol Cell Biol. 1983 Jan;3(1):1–8. doi: 10.1128/mcb.3.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kingston R. E., Baldwin A. S., Jr, Sharp P. A. Regulation of heat shock protein 70 gene expression by c-myc. Nature. 1984 Nov 15;312(5991):280–282. doi: 10.1038/312280a0. [DOI] [PubMed] [Google Scholar]
  25. Kingston R. E., Cowie A., Morimoto R. I., Gwinn K. A. Binding of polyomavirus large T antigen to the human hsp70 promoter is not required for trans activation. Mol Cell Biol. 1986 Sep;6(9):3180–3190. doi: 10.1128/mcb.6.9.3180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Krippl B., Ferguson B., Jones N., Rosenberg M., Westphal H. Mapping of functional domains in adenovirus E1A proteins. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7480–7484. doi: 10.1073/pnas.82.22.7480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Leong K., Berk A. J. Adenovirus early region 1A protein increases the number of template molecules transcribed in cell-free extracts. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5844–5848. doi: 10.1073/pnas.83.16.5844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lonberg-Holm K., Philipson L. Early events of virus-cell interaction in an adenovirus system. J Virol. 1969 Oct;4(4):323–338. doi: 10.1128/jvi.4.4.323-338.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lucher L. A., Loewenstein P. M., Green M. Phosphorylation in vitro of Escherichia coli-produced 235R and 266R tumor antigens encoded by human adenovirus type 12 early transformation region 1A. J Virol. 1985 Oct;56(1):183–193. doi: 10.1128/jvi.56.1.183-193.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Milarski K. L., Morimoto R. I. Expression of human HSP70 during the synthetic phase of the cell cycle. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9517–9521. doi: 10.1073/pnas.83.24.9517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Moran E., Grodzicker T., Roberts R. J., Mathews M. B., Zerler B. Lytic and transforming functions of individual products of the adenovirus E1A gene. J Virol. 1986 Mar;57(3):765–775. doi: 10.1128/jvi.57.3.765-775.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Moran E., Mathews M. B. Multiple functional domains in the adenovirus E1A gene. Cell. 1987 Jan 30;48(2):177–178. doi: 10.1016/0092-8674(87)90418-1. [DOI] [PubMed] [Google Scholar]
  34. Nevins J. R. Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product. Cell. 1982 Jul;29(3):913–919. doi: 10.1016/0092-8674(82)90453-6. [DOI] [PubMed] [Google Scholar]
  35. Pinhasi-Kimhi O., Michalovitz D., Ben-Zeev A., Oren M. Specific interaction between the p53 cellular tumour antigen and major heat shock proteins. Nature. 1986 Mar 13;320(6058):182–184. doi: 10.1038/320182a0. [DOI] [PubMed] [Google Scholar]
  36. Ruley H. E. Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature. 1983 Aug 18;304(5927):602–606. doi: 10.1038/304602a0. [DOI] [PubMed] [Google Scholar]
  37. Schmitt R. C., Fahnestock M. L., Lewis J. B. Differential nuclear localization of the major adenovirus type 2 E1a proteins. J Virol. 1987 Feb;61(2):247–255. doi: 10.1128/jvi.61.2.247-255.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simon M. C., Kitchener K., Kao H. T., Hickey E., Weber L., Voellmy R., Heintz N., Nevins J. R. Selective induction of human heat shock gene transcription by the adenovirus E1A gene products, including the 12S E1A product. Mol Cell Biol. 1987 Aug;7(8):2884–2890. doi: 10.1128/mcb.7.8.2884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Spindler K. R., Rosser D. S., Berk A. J. Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli. J Virol. 1984 Jan;49(1):132–141. doi: 10.1128/jvi.49.1.132-141.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stabel S., Argos P., Philipson L. The release of growth arrest by microinjection of adenovirus E1A DNA. EMBO J. 1985 Sep;4(9):2329–2336. doi: 10.1002/j.1460-2075.1985.tb03934.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stürzbecher H. W., Chumakov P., Welch W. J., Jenkins J. R. Mutant p53 proteins bind hsp 72/73 cellular heat shock-related proteins in SV40-transformed monkey cells. Oncogene. 1987 May;1(2):201–211. [PubMed] [Google Scholar]
  42. Thomas G. P., Mathews M. B. DNA replication and the early to late transition in adenovirus infection. Cell. 1980 Nov;22(2 Pt 2):523–533. doi: 10.1016/0092-8674(80)90362-1. [DOI] [PubMed] [Google Scholar]
  43. Tsukamoto A. S., Ponticelli A., Berk A. J., Gaynor R. B. Genetic mapping of a major site of phosphorylation in adenovirus type 2 E1A proteins. J Virol. 1986 Jul;59(1):14–22. doi: 10.1128/jvi.59.1.14-22.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Walter G., Carbone A., Welch W. J. Medium tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with 73-kilodalton heat shock protein. J Virol. 1987 Feb;61(2):405–410. doi: 10.1128/jvi.61.2.405-410.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Welch W. J., Feramisco J. R. Nuclear and nucleolar localization of the 72,000-dalton heat shock protein in heat-shocked mammalian cells. J Biol Chem. 1984 Apr 10;259(7):4501–4513. [PubMed] [Google Scholar]
  46. Welch W. J., Mizzen L. A. Characterization of the thermotolerant cell. II. Effects on the intracellular distribution of heat-shock protein 70, intermediate filaments, and small nuclear ribonucleoprotein complexes. J Cell Biol. 1988 Apr;106(4):1117–1130. doi: 10.1083/jcb.106.4.1117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Welch W. J., Suhan J. P. Cellular and biochemical events in mammalian cells during and after recovery from physiological stress. J Cell Biol. 1986 Nov;103(5):2035–2052. doi: 10.1083/jcb.103.5.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. White E., Blose S. H., Stillman B. W. Nuclear envelope localization of an adenovirus tumor antigen maintains the integrity of cellular DNA. Mol Cell Biol. 1984 Dec;4(12):2865–2875. doi: 10.1128/mcb.4.12.2865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. White E., Denton A., Stillman B. Role of the adenovirus E1B 19,000-dalton tumor antigen in regulating early gene expression. J Virol. 1988 Sep;62(9):3445–3454. doi: 10.1128/jvi.62.9.3445-3454.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. White E., Stillman B. Expression of adenovirus E1B mutant phenotypes is dependent on the host cell and on synthesis of E1A proteins. J Virol. 1987 Feb;61(2):426–435. doi: 10.1128/jvi.61.2.426-435.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wu B. J., Hurst H. C., Jones N. C., Morimoto R. I. The E1A 13S product of adenovirus 5 activates transcription of the cellular human HSP70 gene. Mol Cell Biol. 1986 Aug;6(8):2994–2999. doi: 10.1128/mcb.6.8.2994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yamamoto T., McIntyre J., Sell S. M., Georgopoulos C., Skowyra D., Zylicz M. Enzymology of the pre-priming steps in lambda dv DNA replication in vitro. J Biol Chem. 1987 Jun 15;262(17):7996–7999. [PubMed] [Google Scholar]
  54. Yee S. P., Branton P. E. Detection of cellular proteins associated with human adenovirus type 5 early region 1A polypeptides. Virology. 1985 Nov;147(1):142–153. doi: 10.1016/0042-6822(85)90234-x. [DOI] [PubMed] [Google Scholar]
  55. Yee S. P., Rowe D. T., Tremblay M. L., McDermott M., Branton P. E. Identification of human adenovirus early region 1 products by using antisera against synthetic peptides corresponding to the predicted carboxy termini. J Virol. 1983 Jun;46(3):1003–1013. doi: 10.1128/jvi.46.3.1003-1013.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yoshinaga S., Dean N., Han M., Berk A. J. Adenovirus stimulation of transcription by RNA polymerase III: evidence for an E1A-dependent increase in transcription factor IIIC concentration. EMBO J. 1986 Feb;5(2):343–354. doi: 10.1002/j.1460-2075.1986.tb04218.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zerler B., Moran B., Maruyama K., Moomaw J., Grodzicker T., Ruley H. E. Adenovirus E1A coding sequences that enable ras and pmt oncogenes to transform cultured primary cells. Mol Cell Biol. 1986 Mar;6(3):887–899. doi: 10.1128/mcb.6.3.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zhai Z. H., Nickerson J. A., Krochmalnic G., Penman S. Alterations in nuclear matrix structure after adenovirus infection. J Virol. 1987 Apr;61(4):1007–1018. doi: 10.1128/jvi.61.4.1007-1018.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES