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. 1982 May;42(2):488–501. doi: 10.1128/jvi.42.2.488-501.1982

Expression of the gene encoding the adenovirus DNA terminal protein precursor in productively infected and transformed cells.

M H Binger, S J Flint, D M Rekosh
PMCID: PMC256875  PMID: 7086968

Abstract

The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed.

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  1. Akusjärvi G., Persson H. Controls of RNA splicing and termination in the major late adenovirus transcription unit. Nature. 1981 Jul 30;292(5822):420–426. doi: 10.1038/292420a0. [DOI] [PubMed] [Google Scholar]
  2. Amin M., Mirza A., Weber J. Genetic analysis of adenovirus type 2. VII. Cleavage-modified affinity for DNA of internal virion proteins. Virology. 1977 Jul 1;80(1):83–97. doi: 10.1016/0042-6822(77)90382-8. [DOI] [PubMed] [Google Scholar]
  3. Anderson C. W., Baum P. R., Gesteland R. F. Processing of adenovirus 2-induced proteins. J Virol. 1973 Aug;12(2):241–252. doi: 10.1128/jvi.12.2.241-252.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baker C. C., Ziff E. B. Promoters and heterogeneous 5' termini of the messenger RNAs of adenovirus serotype 2. J Mol Biol. 1981 Jun 25;149(2):189–221. doi: 10.1016/0022-2836(81)90298-9. [DOI] [PubMed] [Google Scholar]
  5. Berget S. M., Flint S. J., Williams J. F., Sharp P. A. Adenovirus transcription. IV. Synthesis of viral-specific RNA in human cells infected with temperature-sensitive mutants of adenovirus 5. J Virol. 1976 Sep;19(3):879–889. doi: 10.1128/jvi.19.3.879-889.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Bhatti A. R., Weber J. Protease of adenovirus type 2: partial characterization. Virology. 1979 Jul 30;96(2):478–485. doi: 10.1016/0042-6822(79)90105-3. [DOI] [PubMed] [Google Scholar]
  8. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  9. Carter T. H., Blanton R. A. Autoregulation of adenovirus type 5 early gene expression II. Effect of temperature-sensitive early mutations on virus RNA accumulation. J Virol. 1978 Nov;28(2):450–456. doi: 10.1128/jvi.28.2.450-456.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carter T. H., Blanton R. A. Possible role of the 72,000 dalton DNA-binding protein in regulation of adenovirus type 5 early gene expression. J Virol. 1978 Feb;25(2):664–674. doi: 10.1128/jvi.25.2.664-674.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Carusi E. A. Evidence for blocked 5'-termini in human adenovirus DNA. Virology. 1977 Jan;76(1):380–394. doi: 10.1016/0042-6822(77)90310-5. [DOI] [PubMed] [Google Scholar]
  12. Challberg M. D., Desiderio S. V., Kelly T. J., Jr Adenovirus DNA replication in vitro: characterization of a protein covalently linked to nascent DNA strands. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5105–5109. doi: 10.1073/pnas.77.9.5105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Challberg M. D., Kelly T. J., Jr Adenovirus DNA replication in vitro. Proc Natl Acad Sci U S A. 1979 Feb;76(2):655–659. doi: 10.1073/pnas.76.2.655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Challberg M. D., Kelly T. J., Jr Processing of the adenovirus terminal protein. J Virol. 1981 Apr;38(1):272–277. doi: 10.1128/jvi.38.1.272-277.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Chow L. T., Broker T. R., Lewis J. B. Complex splicing patterns of RNAs from the early regions of adenovirus-2. J Mol Biol. 1979 Oct 25;134(2):265–303. doi: 10.1016/0022-2836(79)90036-6. [DOI] [PubMed] [Google Scholar]
  16. Chow L. T., Broker T. R. The spliced structures of adenovirus 2 fiber message and the other late mRNAs. Cell. 1978 Oct;15(2):497–510. doi: 10.1016/0092-8674(78)90019-3. [DOI] [PubMed] [Google Scholar]
  17. Chow L. T., Lewis J. B., Broker T. R. RNA transcription and splicing at early and intermediate times after adenovirus-2 infection. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):401–414. doi: 10.1101/sqb.1980.044.01.044. [DOI] [PubMed] [Google Scholar]
  18. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  19. Cleveland D. W., Lopata M. A., MacDonald R. J., Cowan N. J., Rutter W. J., Kirschner M. W. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes. Cell. 1980 May;20(1):95–105. doi: 10.1016/0092-8674(80)90238-x. [DOI] [PubMed] [Google Scholar]
  20. Craig E. A., Raskas H. J. Effect of cycloheximide on RNA metabolism early in productive infection with adenovirus 2. J Virol. 1974 Jul;14(1):26–32. doi: 10.1128/jvi.14.1.26-32.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ensinger M. J., Ginsberg H. S. Selection and preliminary characterization of temperature-sensitive mutants of type 5 adenovirus. J Virol. 1972 Sep;10(3):328–339. doi: 10.1128/jvi.10.3.328-339.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Esche H., Mathews M. B., Lewis J. B. Proteins and messenger RNAs of the transforming region of wild-type and mutant adenoviruses. J Mol Biol. 1980 Sep 25;142(3):399–417. doi: 10.1016/0022-2836(80)90279-x. [DOI] [PubMed] [Google Scholar]
  23. Flint S. J., Sambrook J., Williams J. F., Sharp P. A. Viral nucleic acid sequences in transformed cells. IV. A study of the sequences of adenovirus 5 DNA and RNA in four lines of adenovirus 5-transformed rodent cells using specific fragments of the viral genome. Virology. 1976 Jul 15;72(2):456–470. doi: 10.1016/0042-6822(76)90174-4. [DOI] [PubMed] [Google Scholar]
  24. Flint S. J., Sharp P. A. Adenovirus transcription. V. Quantitation of viral RNA sequences in adenovirus 2-infected and transformed cells. J Mol Biol. 1976 Sep 25;106(3):749–774. doi: 10.1016/0022-2836(76)90263-1. [DOI] [PubMed] [Google Scholar]
  25. Flint S. J. Splicing and the regulation of viral gene expression. Curr Top Microbiol Immunol. 1981;93:47–79. doi: 10.1007/978-3-642-68123-3_4. [DOI] [PubMed] [Google Scholar]
  26. Flint S. J., Wewerka-Lutz Y., Levine A. S., Sambrook J., Sharp P. A. Adenovirus transcription. II. RNA sequences complementary to simian virus 40 and adenovirus 2DNA in AD2+ND1- and AD2+ND3-infected cells. J Virol. 1975 Sep;16(3):662–673. doi: 10.1128/jvi.16.3.662-673.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Gallimore P. H. Viral DNA in transformed cells. II. A study of the sequences of adenovirus 2 DNA IN NINE LINES OF TRANSFORMED RAT CELLS USING SPECIFIC FRAGMENTS OF THE VIRAL GENOME;. J Mol Biol. 1974 Oct 15;89(1):49–72. doi: 10.1016/0022-2836(74)90162-4. [DOI] [PubMed] [Google Scholar]
  28. Galos R. S., Williams J., Binger M. H., Flint S. J. Location of additional early gene sequences in the adenoviral chromosome. Cell. 1979 Aug;17(4):945–956. doi: 10.1016/0092-8674(79)90334-9. [DOI] [PubMed] [Google Scholar]
  29. Grodzicker T., Anderson C., Sambrook J., Mathews M. B. The physical locations of structural genes in adenovirus DNA. Virology. 1977 Jul 1;80(1):111–126. doi: 10.1016/0042-6822(77)90384-1. [DOI] [PubMed] [Google Scholar]
  30. Halbert D. N., Spector D. J., Raskas H. J. In vitro translation products specified by the transforming region of adenovirus type 2. J Virol. 1979 Sep;31(3):621–629. doi: 10.1128/jvi.31.3.621-629.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Harter M. L., Lewis J. B., Anderson C. W. Adenovirus type 2 terminal protein: purification and comparison of tryptic peptides with known adenovirus-coded proteins. J Virol. 1979 Sep;31(3):823–835. doi: 10.1128/jvi.31.3.823-835.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Horwitz M. S., Ariga H. Multiple rounds of adenovirus DNA synthesis in vitro. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1476–1480. doi: 10.1073/pnas.78.3.1476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Horwitz M. S. Temperature-sensitive replication of H5ts125 adenovirus DNA in vitro. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4291–4295. doi: 10.1073/pnas.75.9.4291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Johansson K., Persson H., Lewis A. M., Pettersson U., Tibbetts C., Philipson L. Viral DNA sequences and gene products in hamster cells transformed by adenovirus type 2. J Virol. 1978 Sep;27(3):628–639. doi: 10.1128/jvi.27.3.628-639.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Kedinger C., Brison O., Perrin F., Wilhelm J. Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei. J Virol. 1978 May;26(2):364–379. doi: 10.1128/jvi.26.2.364-379.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  40. Lewis J. B., Anderson C. W., Atkins J. F. Further mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments. Cell. 1977 Sep;12(1):37–44. doi: 10.1016/0092-8674(77)90183-0. [DOI] [PubMed] [Google Scholar]
  41. Lewis J. B., Atkins J. F., Baum P. R., Solem R., Gesteland R. F., Anderson C. W. Location and identification of the genes for adenovirus type 2 early polypeptides. Cell. 1976 Jan;7(1):141–151. doi: 10.1016/0092-8674(76)90264-6. [DOI] [PubMed] [Google Scholar]
  42. Lewis J. B., Esche H., Smart J. E., Stillman B. W., Harter M. L., Mathews M. B. Organization and expression of the left third of the genome of adenovirus. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):493–508. doi: 10.1101/sqb.1980.044.01.052. [DOI] [PubMed] [Google Scholar]
  43. Lewis J. B., Mathews M. B. Control of adenovirus early gene expression: a class of immediate early products. Cell. 1980 Aug;21(1):303–313. doi: 10.1016/0092-8674(80)90138-5. [DOI] [PubMed] [Google Scholar]
  44. Lewis J. B., Mathews M. B. Viral messenger RNAs in six lines of adenovirus-transformed cells. Virology. 1981 Dec;115(2):345–360. doi: 10.1016/0042-6822(81)90116-1. [DOI] [PubMed] [Google Scholar]
  45. Lichy J. H., Horwitz M. S., Hurwitz J. Formation of a covalent complex between the 80,000-dalton adenovirus terminal protein and 5'-dCMP in vitro. Proc Natl Acad Sci U S A. 1981 May;78(5):2678–2682. doi: 10.1073/pnas.78.5.2678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Miller J. S., Ricciardi R. P., Roberts B. E., Paterson B. M., Mathews M. B. Arrangement of messenger RNAs and protein coding sequences in the major late transcription unit of adenovirus 2. J Mol Biol. 1980 Oct 5;142(4):455–488. doi: 10.1016/0022-2836(80)90258-2. [DOI] [PubMed] [Google Scholar]
  47. Nevins J. R., Wilson M. C. Regulation of adenovirus-2 gene expression at the level of transcriptional termination and RNA processing. Nature. 1981 Mar 12;290(5802):113–118. doi: 10.1038/290113a0. [DOI] [PubMed] [Google Scholar]
  48. Nevins J. R., Winkler J. J. Regulation of early adenovirus transcription: a protein product of early region 2 specifically represses region 4 transcription. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1893–1897. doi: 10.1073/pnas.77.4.1893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  50. Pincus S., Robertson W., Rekosh D. Characterization of the effect of aphidicolin on adenovirus DNA replication: evidence in support of a protein primer model of initiation. Nucleic Acids Res. 1981 Oct 10;9(19):4919–4938. doi: 10.1093/nar/9.19.4919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Rekosh D. M., Russell W. C., Bellet A. J., Robinson A. J. Identification of a protein linked to the ends of adenovirus DNA. Cell. 1977 Jun;11(2):283–295. doi: 10.1016/0092-8674(77)90045-9. [DOI] [PubMed] [Google Scholar]
  52. Rekosh D. Analysis of the DNA-terminal protein from different serotypes of human adenovirus. J Virol. 1981 Oct;40(1):329–333. doi: 10.1128/jvi.40.1.329-333.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Ricciardi R. P., Miller J. S., Roberts B. E. Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4927–4931. doi: 10.1073/pnas.76.10.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Robinson A. J., Bellett J. D. A circular DNA-protein complex adenoviruses and its possible role in DNA replication. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):523–531. doi: 10.1101/sqb.1974.039.01.064. [DOI] [PubMed] [Google Scholar]
  55. Robinson A. J., Younghusband H. B., Bellett A. J. A circula DNA-protein complex from adenoviruses. Virology. 1973 Nov;56(1):54–69. doi: 10.1016/0042-6822(73)90287-0. [DOI] [PubMed] [Google Scholar]
  56. Russell W. C., Hayashi K., Sanderson P. J., Pereira H. G. Adenovirus antigens--a study of their properties and sequential development in infection. J Gen Virol. 1967 Oct;1(4):495–507. doi: 10.1099/0022-1317-1-4-495. [DOI] [PubMed] [Google Scholar]
  57. Sambrook J., Botchan M., Gallimore P., Ozanne B., Pettersson U., Williams J., Sharp P. A. Viral DNA sequences in cells transformed by simian virus 40, adenovirus type 2 and adenovirus type 5. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):615–632. doi: 10.1101/sqb.1974.039.01.075. [DOI] [PubMed] [Google Scholar]
  58. Shaw A. R., Ziff E. B. Transcripts from the adenovirus-2 major late promoter yield a single early family of 3' coterminal mRNAs and five late families. Cell. 1980 Dec;22(3):905–916. doi: 10.1016/0092-8674(80)90568-1. [DOI] [PubMed] [Google Scholar]
  59. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  60. 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]
  61. Van Der Vliet P. C., Levine A. J., Ensinger M. J., Ginsberg H. S. Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovrius defective in viral DNA synthesis. J Virol. 1975 Feb;15(2):348–354. doi: 10.1128/jvi.15.2.348-354.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Van der Vliet P. C., Zandberg J., Jansz H. S. Evidence for a function of the adenovirus DNA-binding protein in initiation in DNA synthesis as well as in elongation of nascent DNA chains. Virology. 1977 Jul 1;80(1):98–110. doi: 10.1016/0042-6822(77)90383-x. [DOI] [PubMed] [Google Scholar]
  63. Visser L., van Maarschalkerweerd M. W., Rozijn T. H., Wassenaar A. D., Reemst A. M., Sussenbach J. S. Viral DNA sequences in adenovirus-transformed cells. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):541–550. doi: 10.1101/sqb.1980.044.01.056. [DOI] [PubMed] [Google Scholar]
  64. Vliet P. C., Sussenbach J. S. An adenovirus type 5 gene function required for initiation of viral DNA replication. Virology. 1975 Oct;67(2):415–426. doi: 10.1016/0042-6822(75)90443-2. [DOI] [PubMed] [Google Scholar]
  65. Weber J., Begin M., Khittoo G. Genetic Analysis of Adenovirus Type 2 II. Preliminary Phenotypic Characterization of Temperature-Sensitive Mutants. J Virol. 1975 May;15(5):1049–1056. doi: 10.1128/jvi.15.5.1049-1056.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Weber J. Genetic analysis of adenovirus type 2 III. Temperature sensitivity of processing viral proteins. J Virol. 1976 Feb;17(2):462–471. doi: 10.1128/jvi.17.2.462-471.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Wilkie N. M., Ustacelebi S., Williams J. F. Characterization of temperature-sensitive mutants of adenovirus type 5: nucleic acid synthesis. Virology. 1973 Feb;51(2):499–503. doi: 10.1016/0042-6822(73)90450-9. [DOI] [PubMed] [Google Scholar]
  68. Williams J. F. Enhancement of adenovirus plaque formation on HeLa cells by magnesium chloride. J Gen Virol. 1970 Dec;9(3):251–255. doi: 10.1099/0022-1317-9-3-251. [DOI] [PubMed] [Google Scholar]
  69. Williams J. F. Oncogenic transformation of hamster embryo cells in vitro by adenovirus type 5. Nature. 1973 May 18;243(5403):162–163. doi: 10.1038/243162a0. [DOI] [PubMed] [Google Scholar]
  70. Williams J., Galos R. S., Binger M. H., Flint S. J. Location of additional early regions within the left quarter of the adenoviral genome. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):353–365. doi: 10.1101/sqb.1980.044.01.040. [DOI] [PubMed] [Google Scholar]
  71. Willians J. F., Young C. S., Austin P. E. Genetic analysis of human adenovirus type 5 in permissive and nonpermissive cells. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):427–437. doi: 10.1101/sqb.1974.039.01.055. [DOI] [PubMed] [Google Scholar]
  72. Ziff E. B., Evans R. M. Coincidence of the promoter and capped 5' terminus of RNA from the adenovirus 2 major late transcription unit. Cell. 1978 Dec;15(4):1463–1475. doi: 10.1016/0092-8674(78)90070-3. [DOI] [PubMed] [Google Scholar]

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