Characterization of an Early Temperature-sensitive and Cytocidal Double Mutant of Adenovirus Type 2 Free

Abstract

SUMMARY

Human adenovirus type 2 mutant, H2 111, presented a double phenotype: temperature-sensitive () for initiation and elongation of DNA synthesis, and cytocidal () by its large-plaque formation and the nucleolytic cleavage of both viral and cellular DNAs. Both characters were recessive since they were efficiently complemented by wild-type or other DNA-negative mutants. H2 111 DNA-terminal protein complex formed at 33 °C and chased at 39.5 °C showed a decreased affinity for glass fibre filters, concurrently with the loss of protein-linked DNA ends. H2 111 DNA breakdown occurring upon shift-up to 39.5 °C therefore appeared to start in close proximity to the genome extremities. Marker rescue experiments showed that the character was abolished by co-infection with plasmid recombinants containing whole or part of the E2A region, encoding for the 72K DNA-binding protein. The N-terminal domain of this 72K protein has been assigned between 0 and 200 amino acids, and is supposed to have a function in late transcription control. Since H2 111 mapped between 0 and 300 residues (63.6 to 65.9 map units), its mutation was most likely located between 200 and 300 amino acids, namely in the C-terminal domain of the protein, which is involved in DNA replication. Recombination between H2 111 and H5 313 mutant revealed that the function was localized in the E1B zone, between 3.8 and 11.3 map units. The nucleolytic and cytocidal effects were complemented by HEK 293 cells, an H5-transformed cell line expressing the left-most 12.5% of the viral genome. H2 111 appeared, therefore, as a double - mutant. The gene product rendered temperature-sensitive by the H2 111 mutation was found to act stoichiometrically, and not catalytically, a result compatible with a lesion in the 72K protein. Although inactive at the non-permissive temperature, the early 72K protein was normally synthesized and stable in H2 111-infected cells at 39.5 °C. Assymetric complementation obtained with the DNA-defective H5 36 implied a certain degree of type specificity in the DNA-binding protein function.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-65-8-1305
1984-08-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/65/8/JV0650081305.html?itemId=/content/journal/jgv/10.1099/0022-1317-65-8-1305&mimeType=html&fmt=ahah

References

  1. Aiello L., Guilfoyle R., Huebner K., Weinman R. 1979; Adenovirus 5 DNA sequences present and RNA sequences transcribed in transformed human embryo kidney cells (HEK-Ad-5 or 293). Virology 94:460–469
    [Google Scholar]
  2. Asselbergs F. A., Mathews M. B., Smart J. E. 1983; Structural characterization of the proteins encoded by adenovirus early region 2A. Journal of Molecular Biology 163:177–207
    [Google Scholar]
  3. Berk A. J., Lee F., Harisson T., Williams J., Sharp P. A. 1979; Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs. Cell 17:944–955
    [Google Scholar]
  4. Boulanger P., Lemay P., Blair G. E., Russell W. C. 1979; Characterization of adenovirus protein IX. Virology 44:783–800
    [Google Scholar]
  5. Chinnadurai G. 1983; Adenovirus 2 lp+ codes for a 19 Kd tumor antigen that plays an essential role in cell transformation. Cell 33:759–766
    [Google Scholar]
  6. Coombs D. H., Pearson G. D. 1978; Filter-binding assay for covalent DNA-protein complexes: adenovirus DNA-terminal protein complex. Proceedings of the National Academy of Sciences, U,. S,. A 75:5291–5295
    [Google Scholar]
  7. D’Halluin J. C., Allart C., Cousin C., Boulanger P. A., Martin G. R. 1979; Adenovirus early function required for protection of viral and cellular DNA. Journal of Virology 32:61–91
    [Google Scholar]
  8. D’Halluin J. C., Milleville M., Boulanger P. 1980; Effects of novobiocin on adenovirus DNA synthesis and encapsidation. Nucleic Acids Research 8:1625–1641
    [Google Scholar]
  9. D’Halluin J. C., Cousin C., Boulanger P. 1982; Physical mapping of adenovirus type 2 ts mutants by restriction nuclease analysis of interserotypic recombinants. Journal of Virology 41:401–413
    [Google Scholar]
  10. D’Halluin J. C., Milleville M., Boulanger P. 1983; Restriction maps of human adenovirus types 2, 5 and 3 for Bcl I, Cla I, Pvu I and Sph I endonucleases. Gene 21:167–171
    [Google Scholar]
  11. Ensinger M. J., Ginsberg H. S. 1972; Selection and preliminary characterization of temperature-sensitive mutants of type 5 adenovirus. Journal of Virology 10:328–339
    [Google Scholar]
  12. Fatt R. B. L., Mak S. 1982; Mapping of an adenovirus function involved in the inhibition of DNA degradation. Journal of Virology 42:969–977
    [Google Scholar]
  13. Flint S. I., Sambrook J., Williams J. F., Sharp P. A. 1976; 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 72:456–470
    [Google Scholar]
  14. Galos R. S., Williams J., Binger M. H., Flint S. J. 1979; Location of additional early gene sequences in the adenoviral chromosome. Cell 17:945–956
    [Google Scholar]
  15. Graham F. L., Abrahams P. J., Mulder C., Heijneker H. L., Warnaar S. O., De Vries F. A. J., van Der Eb A. I. 1974; Studies on in vitro transformation by DNA and DNA fragments of human adenovirus and SV40. Cold Spring Harbor Symposia on Quantitative Biology 39:637–650
    [Google Scholar]
  16. Graham F. L., Smiley J., Russell W. C., Nairn R. 1977; Characteristics of a human cell line transformed by DNA from human adenovirus type 5. Journal of General Virology 36:59–72
    [Google Scholar]
  17. Jones N., Shenk T. 1978; Isolation of deletion and substitution mutants of adenovirus type 5. Cell 13:181–188
    [Google Scholar]
  18. Jones N., Shenk T. 1979; An adenovirus type 5 early gene function regulates expression of other early viral genes. Proceedings of the National Academy of Sciences, U,. S,. A 76:3665–3669
    [Google Scholar]
  19. Kessler S. W. 1975; Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. Journal of Immunology 15:1617–1624
    [Google Scholar]
  20. Klessig D. F., Grodzicker T. 1979; Mutations that allow human Ad2 and Ad5 to express late genes in monkey cells map in the viral gene encoding the 72 K DNA binding protein. Cell 17:957–966
    [Google Scholar]
  21. Kruijer W., van Schaik F. M. A., Sussenbach J. S. 1981; Structure and organization of the gene coding for the DNA-binding protein of adenovirus type 5. Nucleic Acids Research 9:4439–4457
    [Google Scholar]
  22. Kruijer W., van Schaik F. M. A., Sussenbach J. S. 1982; Nucleotide sequence of the gene encoding adenovirus type 2 DNA binding protein. Nucleic Acids Research 10:4493–4500
    [Google Scholar]
  23. Martin G. R., Warocquier R., Cousin C., D’Halluin J. C., Boulanger P. A. 1978; Isolation and phenotypic characterization of human adenovirus type 2 temperature-sensitive mutants. Journal of General Virology 41:303–314
    [Google Scholar]
  24. Reiter T., Futterer J., Weingartner B., Winnacker E. L. 1977; Initiation of adenovirus DNA replication. Journal of Virology 35:662–671
    [Google Scholar]
  25. Rekosh D. M. K., Russell W. C., Bellett A. J. D., Robinson A. J. 1977; Identification of a protein linked to the ends of adenovirus DNA. Cell 11:283–295
    [Google Scholar]
  26. Rice S. A., Klessig D. F. 1984; The function(s) provided by the adenovirus-specified, DNA-binding protein required for viral late gene expression is independent of the role of the protein in viral DNA replication. Journal of Virology 49:35–49
    [Google Scholar]
  27. Robinson A. J., Bellett A. J. D. 1974; A circular DNA-protein complex from adenoviruses and its possible role in DNA replication. Cold Spring Harbor Symposia on Quantitative Biology 39:523–531
    [Google Scholar]
  28. Ross S. R., Flint S. J., Levine A. J. 1980; Identification of the adenovirus early proteins and their genomic map positions. Virology 100:419–432
    [Google Scholar]
  29. Russell W. C., Hayashi K., Sanderson P. J., Pereira H. G. 1967; Adenovirus antigens. A study of their properties and sequential development in infection. Journal of General Virology 1:495–507
    [Google Scholar]
  30. Sambrook J., Botchan M., Gallimore P., Ozanne B., Pettersson U., Williams J., Sharp P. A. 1974; Viral DNA sequences in cells transformed by simian virus 40, adenovirus type 2 and adenovirus type 5. Cold Spring Harbor Symposia on Quantitative Biology 39:615–632
    [Google Scholar]
  31. Sharp P. A., Sugden B., Sambrook J. 1973; Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 12:3055–3063
    [Google Scholar]
  32. Snustad D. P. 1968; Dominance interaction in E. coli cells mixedly infected with bacteriophage T4D wild-type and amber mutants and their possible implications as to type of gene product function: catalytic vs. stoichiometric. Virology 35:550–563
    [Google Scholar]
  33. Spector D. J., McGrogan M., Raskas H. J. 1978; Regulation of the appearance of cytoplasmic RNAs from region 1 of the adenovirus 2 genome. Journal of Molecular Biology 126:395–414
    [Google Scholar]
  34. Tooze J. (editor) 1980; DNA tumor viruses. In Molecular Biology of Tumor Viruses 2nd edn., part 2 pp 931–939 New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  35. Torpier G., D’Halluin J. C., Boulanger P. 1971; Electron microscopic observations on KB cells infected with adeno-associated satellite virus. Journal of Microscopy 11:259–264
    [Google Scholar]
  36. van Der Vliet P. C., Levine A. J., Ensinger M. J., Ginsberg H. S. 1975; Thermolabile DNA-binding proteins from cells infected with a temperature-sensitive mutant of adenovirus defective in viral DNA synthesis. Journal of Virology 15:348–354
    [Google Scholar]
  37. Weber J., Begin M., Carstens E. B. 1977; Genetic analysis of adenovirus type 2. IV. Coordinate regulation of polypeptides 80 K, Ilia and V. Virology 76:709–724
    [Google Scholar]
  38. Williams J. F. 1973; Oncogenic transformation of hamster embryo cells in vitro by adenovirus type 5. Nature, London 243:162–163
    [Google Scholar]
  39. Winnacker E. L. 1978; Adenovirus DNA: structure and function of a novel replicon. Cell 14:761–773
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-65-8-1305
Loading
/content/journal/jgv/10.1099/0022-1317-65-8-1305
Loading

Data & Media loading...

Most cited Most Cited RSS feed