Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1987 Mar;7(3):1004–1011. doi: 10.1128/mcb.7.3.1004

An adenovirus type 5 E1A protein with a single amino acid substitution blocks wild-type E1A transactivation.

G M Glenn, R P Ricciardi
PMCID: PMC365170  PMID: 2951587

Abstract

The E1A gene of adenovirus type 5 encodes a 289-amino-acid (289R) protein that transactivates early adenovirus promoters. We showed that the 289R protein of the E1A missense mutant gene hr5 is novel in that it inhibits the wild-type (wt) E1A protein from stimulating transcription from each of the early viral promoters E2, E3, and E4. Since both the hr5 and wt genes produced similar levels of E1A proteins, the ability of hr5 E1A to block transactivation was attributed to the replacement of serine by asparagine as position 185. We confirmed that this single amino acid substitution was responsible for blocking transactivation by showing equal inhibition with an hr5-wt hybrid E1A gene containing this missense mutation as the only alteration. The smaller 243R E1A protein of hr5 was not necessary for inhibition. Transcriptional activity from each early promoter was inhibited by at least 50% when the hr5 and wt E1A genes were present in equimolar amounts; complete inhibition occurred with a fivefold molar excess of the hr5 gene. Two other E1A missense mutant genes (hr3 and hr4) with amino acid substitutions in close proximity to that of hr5 failed to block wt E1A-induced transcription when similarly tested. Also, the hr5 E1A gene failed to impede the pseudorabies immediate early gene from transactivating the adenovirus E3 promoter, demonstrating that hr5 E1A inhibits wt E1A activation at the transcriptional, rather than the posttranscriptional, level. Although several possibilities were considered to account for this inhibition, the most likely is that the nonfunctional hr5 E1A protein competes with the wt 289R protein for a cellular transcription factor required for transactivation.

Full text

PDF
1004

Images in this article

1007Image
on p.1007
1007Image
on p.1007
1008Image
on p.1008
1009Image
on p.1009

Selected References

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

  1. Berk A. J., Lee F., Harrison T., Williams J., Sharp P. A. Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs. Cell. 1979 Aug;17(4):935–944. doi: 10.1016/0092-8674(79)90333-7. [DOI] [PubMed] [Google Scholar]
  2. Berk A. J., Sharp P. A. Structure of the adenovirus 2 early mRNAs. Cell. 1978 Jul;14(3):695–711. doi: 10.1016/0092-8674(78)90252-0. [DOI] [PubMed] [Google Scholar]
  3. Bernards R., Schrier P. I., Houweling A., Bos J. L., van der Eb A. J., Zijlstra M., Melief C. J. Tumorigenicity of cells transformed by adenovirus type 12 by evasion of T-cell immunity. 1983 Oct 27-Nov 2Nature. 305(5937):776–779. doi: 10.1038/305776a0. [DOI] [PubMed] [Google Scholar]
  4. Borrelli E., Hen R., Chambon P. Adenovirus-2 E1A products repress enhancer-induced stimulation of transcription. Nature. 1984 Dec 13;312(5995):608–612. doi: 10.1038/312608a0. [DOI] [PubMed] [Google Scholar]
  5. Carthew R. W., Chodosh L. A., Sharp P. A. An RNA polymerase II transcription factor binds to an upstream element in the adenovirus major late promoter. Cell. 1985 Dec;43(2 Pt 1):439–448. doi: 10.1016/0092-8674(85)90174-6. [DOI] [PubMed] [Google Scholar]
  6. Chen I. S., Cann A. J., Shah N. P., Gaynor R. B. Functional relation between HTLV-II x and adenovirus E1A proteins in transcriptional activation. Science. 1985 Nov 1;230(4725):570–573. doi: 10.1126/science.2996140. [DOI] [PubMed] [Google Scholar]
  7. Davison B. L., Egly J. M., Mulvihill E. R., Chambon P. Formation of stable preinitiation complexes between eukaryotic class B transcription factors and promoter sequences. Nature. 1983 Feb 24;301(5902):680–686. doi: 10.1038/301680a0. [DOI] [PubMed] [Google Scholar]
  8. Eager K. B., Williams J., Breiding D., Pan S., Knowles B., Appella E., Ricciardi R. P. Expression of histocompatibility antigens H-2K, -D, and -L is reduced in adenovirus-12-transformed mouse cells and is restored by interferon gamma. Proc Natl Acad Sci U S A. 1985 Aug;82(16):5525–5529. doi: 10.1073/pnas.82.16.5525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elkaim R., Goding C., Kédinger C. The adenovirus-2 EIIa early gene promoter: sequences required for efficient in vitro and in vivo transcription. Nucleic Acids Res. 1983 Oct 25;11(20):7105–7117. doi: 10.1093/nar/11.20.7105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Feldman L. T., Imperiale M. J., Nevins J. R. Activation of early adenovirus transcription by the herpesvirus immediate early gene: evidence for a common cellular control factor. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4952–4956. doi: 10.1073/pnas.79.16.4952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fire A., Samuels M., Sharp P. A. Interactions between RNA polymerase II, factors, and template leading to accurate transcription. J Biol Chem. 1984 Feb 25;259(4):2509–2516. [PubMed] [Google Scholar]
  12. Gaynor R. B., Feldman L. T., Berk A. J. Transcription of class III genes activated by viral immediate early proteins. Science. 1985 Oct 25;230(4724):447–450. doi: 10.1126/science.2996135. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Glenn G. M., Ricciardi R. P. Adenovirus 5 early region 1A host range mutants hr3, hr4, and hr5 contain point mutations which generate single amino acid substitutions. J Virol. 1985 Oct;56(1):66–74. doi: 10.1128/jvi.56.1.66-74.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Green M. R., Treisman R., Maniatis T. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products. Cell. 1983 Nov;35(1):137–148. doi: 10.1016/0092-8674(83)90216-7. [DOI] [PubMed] [Google Scholar]
  20. Harrison T., Graham F., Williams J. Host-range mutants of adenovirus type 5 defective for growth in HeLa cells. Virology. 1977 Mar;77(1):319–329. doi: 10.1016/0042-6822(77)90428-7. [DOI] [PubMed] [Google Scholar]
  21. Hen R., Borrelli E., Chambon P. Repression of the immunoglobulin heavy chain enhancer by the adenovirus-2 E1A products. Science. 1985 Dec 20;230(4732):1391–1394. doi: 10.1126/science.2999984. [DOI] [PubMed] [Google Scholar]
  22. Hoeffler W. K., Roeder R. G. Enhancement of RNA polymerase III transcription by the E1A gene product of adenovirus. Cell. 1985 Jul;41(3):955–963. doi: 10.1016/s0092-8674(85)80076-3. [DOI] [PubMed] [Google Scholar]
  23. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  24. Ihara S., Feldman L., Watanabe S., Ben-Porat T. Characterization of the immediate-early functions of pseudorabies virus. Virology. 1983 Dec;131(2):437–454. doi: 10.1016/0042-6822(83)90510-x. [DOI] [PubMed] [Google Scholar]
  25. Imperiale M. J., Feldman L. T., Nevins J. R. Activation of gene expression by adenovirus and herpesvirus regulatory genes acting in trans and by a cis-acting adenovirus enhancer element. Cell. 1983 Nov;35(1):127–136. doi: 10.1016/0092-8674(83)90215-5. [DOI] [PubMed] [Google Scholar]
  26. Imperiale M. J., Hart R. P., Nevins J. R. An enhancer-like element in the adenovirus E2 promoter contains sequences essential for uninduced and E1A-induced transcription. Proc Natl Acad Sci U S A. 1985 Jan;82(2):381–385. doi: 10.1073/pnas.82.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jones N., Shenk T. An adenovirus type 5 early gene function regulates expression of other early viral genes. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3665–3669. doi: 10.1073/pnas.76.8.3665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Keegan L., Gill G., Ptashne M. Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. Science. 1986 Feb 14;231(4739):699–704. doi: 10.1126/science.3080805. [DOI] [PubMed] [Google Scholar]
  30. Kingston R. E., Kaufman R. J., Sharp P. A. Regulation of transcription of the adenovirus EII promoter by EIa gene products: absence of sequence specificity. Mol Cell Biol. 1984 Oct;4(10):1970–1977. doi: 10.1128/mcb.4.10.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kovesdi I., Reichel R., Nevins J. R. E1A transcription induction: enhanced binding of a factor to upstream promoter sequences. Science. 1986 Feb 14;231(4739):719–722. doi: 10.1126/science.2935935. [DOI] [PubMed] [Google Scholar]
  32. Kovesdi I., Reichel R., Nevins J. R. Identification of a cellular transcription factor involved in E1A trans-activation. Cell. 1986 Apr 25;45(2):219–228. doi: 10.1016/0092-8674(86)90386-7. [DOI] [PubMed] [Google Scholar]
  33. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  34. 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]
  35. Leff T., Corden J., Elkaim R., Sassone-Corsi P. Transcriptional analysis of the adenovirus-5 EIII promoter: absence of sequence specificity for stimulation by EIa gene products. Nucleic Acids Res. 1985 Feb 25;13(4):1209–1221. doi: 10.1093/nar/13.4.1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lusky M., Botchan M. Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences. Nature. 1981 Sep 3;293(5827):79–81. doi: 10.1038/293079a0. [DOI] [PubMed] [Google Scholar]
  37. Montell C., Fisher E. F., Caruthers M. H., Berk A. J. Resolving the functions of overlapping viral genes by site-specific mutagenesis at a mRNA splice site. Nature. 1982 Feb 4;295(5848):380–384. doi: 10.1038/295380a0. [DOI] [PubMed] [Google Scholar]
  38. Murthy S. C., Bhat G. P., Thimmappaya B. Adenovirus EIIA early promoter: transcriptional control elements and induction by the viral pre-early EIA gene, which appears to be sequence independent. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2230–2234. doi: 10.1073/pnas.82.8.2230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Perricaudet M., Akusjärvi G., Virtanen A., Pettersson U. Structure of two spliced mRNAs from the transforming region of human subgroup C adenoviruses. Nature. 1979 Oct 25;281(5733):694–696. doi: 10.1038/281694a0. [DOI] [PubMed] [Google Scholar]
  40. Ricciardi R. P., Jones R. L., Cepko C. L., Sharp P. A., Roberts B. E. Expression of early adenovirus genes requires a viral encoded acidic polypeptide. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6121–6125. doi: 10.1073/pnas.78.10.6121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rowe D. T., Yee S. P., Otis J., Graham F. L., Branton P. E. Characterization of human adenovirus type 5 early region 1A polypeptides using antitumor sera and an antiserum specific for the carboxy terminus. Virology. 1983 Jun;127(2):253–271. doi: 10.1016/0042-6822(83)90142-3. [DOI] [PubMed] [Google Scholar]
  42. Sawadogo M., Roeder R. G. Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4394–4398. doi: 10.1073/pnas.82.13.4394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sawadogo M., Roeder R. G. Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region. Cell. 1985 Nov;43(1):165–175. doi: 10.1016/0092-8674(85)90021-2. [DOI] [PubMed] [Google Scholar]
  44. Scott M. O., Kimelman D., Norris D., Ricciardi R. P. Production of a monospecific antiserum against the early region 1A proteins of adenovirus 12 and adenovirus 5 by an adenovirus 12 early region 1A-beta-galactosidase fusion protein antigen expressed in bacteria. J Virol. 1984 Jun;50(3):895–903. doi: 10.1128/jvi.50.3.895-903.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Smith D. H., Kegler D. M., Ziff E. B. Vector expression of adenovirus type 5 E1a proteins: evidence for E1a autoregulation. Mol Cell Biol. 1985 Oct;5(10):2684–2696. doi: 10.1128/mcb.5.10.2684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Solnick D., Anderson M. A. Transformation-deficient adenovirus mutant defective in expression of region 1A but not region 1B. J Virol. 1982 Apr;42(1):106–113. doi: 10.1128/jvi.42.1.106-113.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Spector D. J., McGrogan M., Raskas H. J. Regulation of the appearance of cytoplasmic RNAs from region 1 of the adenovirus 2 genome. J Mol Biol. 1978 Dec 15;126(3):395–414. doi: 10.1016/0022-2836(78)90048-7. [DOI] [PubMed] [Google Scholar]
  48. Stein R., Ziff E. B. HeLa cell beta-tubulin gene transcription is stimulated by adenovirus 5 in parallel with viral early genes by an E1a-dependent mechanism. Mol Cell Biol. 1984 Dec;4(12):2792–2801. doi: 10.1128/mcb.4.12.2792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Svensson C., Akusjärvi G. Adenovirus 2 early region 1A stimulates expression of both viral and cellular genes. EMBO J. 1984 Apr;3(4):789–794. doi: 10.1002/j.1460-2075.1984.tb01886.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tibbetts C., Larsen P. L., Jones S. N. Autoregulation of adenovirus E1A gene expression. J Virol. 1986 Mar;57(3):1055–1064. doi: 10.1128/jvi.57.3.1055-1064.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Vasavada R., Eager K. B., Barbanti-Brodano G., Caputo A., Ricciardi R. P. Adenovirus type 12 early region 1A proteins repress class I HLA expression in transformed human cells. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5257–5261. doi: 10.1073/pnas.83.14.5257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Velcich A., Ziff E. Adenovirus E1a proteins repress transcription from the SV40 early promoter. Cell. 1985 Mar;40(3):705–716. doi: 10.1016/0092-8674(85)90219-3. [DOI] [PubMed] [Google Scholar]
  53. Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Weeks D. L., Jones N. C. E1A control of gene expression is mediated by sequences 5' to the transcriptional starts of the early viral genes. Mol Cell Biol. 1983 Jul;3(7):1222–1234. doi: 10.1128/mcb.3.7.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES