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. 1997 May;71(5):3375–3384. doi: 10.1128/jvi.71.5.3375-3384.1997

Bipartite structure and functional independence of adenovirus type 5 packaging elements.

S I Schmid 1, P Hearing 1
PMCID: PMC191481  PMID: 9094606

Abstract

Selectivity and polarity of adenovirus type 5 DNA packaging are believed to be directed by an interaction of putative packaging factors with the cis-acting adenovirus packaging domain located within the genomic left end (nucleotides 194 to 380). In previous studies, this packaging domain was mutationally dissected into at least seven functional elements called A repeats. These elements, albeit redundant in function, exhibit differences in the ability to support viral packaging, with elements I, II, V, and VI as the most critical repeats. Viral packaging was shown to be sensitive to spatial changes between individual A repeats. To study the importance of spatial constraints in more detail, we performed site-directed mutagenesis of the 21-bp linker regions separating A repeats I and II, as well as A repeats V and VI. The results of our mutational analysis reveal previously unrecognized sequences that are critical for DNA encapsidation in vivo. On the basis of these results, we present a more complex consensus motif for the adenovirus packaging elements which is bipartite in structure. DNA encapsidation is compromised by changes in spacing between the two conserved parts of the consensus motif, rather than between different A repeats. Genetic evidence implicating packaging elements as independent units in viral DNA packaging is derived from the selection of revertants from a packaging-deficient adenovirus: multimerization of packaging repeats is sufficient for the evolution of packaging-competent viruses. Finally, we identify minimally sized segments of the adenovirus packaging domain that can confer viability and packaging activity to viruses carrying gross truncations within their left-end sequences. Coinfection experiments using the revertant as well as the minimal-packaging-domain mutant viruses strengthen existing arguments for the involvement of limiting, trans-acting components in viral DNA packaging.

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Selected References

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  1. Bjornsti M. A., Reilly B. E., Anderson D. L. Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: oriented and quantized in vitro packaging of DNA protein gp3. J Virol. 1983 Jan;45(1):383–396. doi: 10.1128/jvi.45.1.383-396.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolwig G. M., Bruder J. T., Hearing P. Different binding site requirements for binding and activation for the bipartite enhancer factor EF-1A. Nucleic Acids Res. 1992 Dec 25;20(24):6555–6564. doi: 10.1093/nar/20.24.6555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brough D. E., Lizonova A., Hsu C., Kulesa V. A., Kovesdi I. A gene transfer vector-cell line system for complete functional complementation of adenovirus early regions E1 and E4. J Virol. 1996 Sep;70(9):6497–6501. doi: 10.1128/jvi.70.9.6497-6501.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bruder J. T., Hearing P. Nuclear factor EF-1A binds to the adenovirus E1A core enhancer element and to other transcriptional control regions. Mol Cell Biol. 1989 Nov;9(11):5143–5153. doi: 10.1128/mcb.9.11.5143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. D'Halluin J. C., Martin G. R., Torpier G., Boulanger P. A. Adenovirus type 2 assembly analyzed by reversible cross-linking of labile intermediates. J Virol. 1978 May;26(2):357–363. doi: 10.1128/jvi.26.2.357-363.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. D'Halluin J. C., Milleville M., Boulanger P. A., Martin G. R. Temperature-sensitive mutant of adenovirus type 2 blocked in virion assembly: accumulation of light intermediate particles. J Virol. 1978 May;26(2):344–356. doi: 10.1128/jvi.26.2.344-356.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. D'Halluin J. C., Milleville M., Martin G. R., Boulanger P. Morphogenesis of human adenovirus type 2 studied with fiber- and fiber and penton base-defective temperature-sensitive mutants. J Virol. 1980 Jan;33(1):88–99. doi: 10.1128/jvi.33.1.88-99.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Daniell E. Genome structure of incomplete particles of adenovirus. J Virol. 1976 Aug;19(2):685–708. doi: 10.1128/jvi.19.2.685-708.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Earnshaw W. C., Casjens S. R. DNA packaging by the double-stranded DNA bacteriophages. Cell. 1980 Sep;21(2):319–331. doi: 10.1016/0092-8674(80)90468-7. [DOI] [PubMed] [Google Scholar]
  10. Edvardsson B., Everitt E., Jörnvall H., Prage L., Philipson L. Intermediates in adenovirus assembly. J Virol. 1976 Aug;19(2):533–547. doi: 10.1128/jvi.19.2.533-547.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Edvardsson B., Ustacelebi S., Williams J., Philipson L. Assembly intermediates among adenovirus type 5 temperature-sensitive mutants. J Virol. 1978 Feb;25(2):641–651. doi: 10.1128/jvi.25.2.641-651.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Engelhardt J. F., Ye X., Doranz B., Wilson J. M. Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6196–6200. doi: 10.1073/pnas.91.13.6196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  14. Fredman J. N., Pettit S. C., Horwitz M. S., Engler J. A. Linker insertion mutations in the adenovirus preterminal protein that affect DNA replication activity in vivo and in vitro. J Virol. 1991 Sep;65(9):4591–4597. doi: 10.1128/jvi.65.9.4591-4597.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Freimuth P. I., Ginsberg H. S. Codon insertion mutants of the adenovirus terminal protein. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7816–7820. doi: 10.1073/pnas.83.20.7816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Gräble M., Hearing P. Adenovirus type 5 packaging domain is composed of a repeated element that is functionally redundant. J Virol. 1990 May;64(5):2047–2056. doi: 10.1128/jvi.64.5.2047-2056.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gräble M., Hearing P. cis and trans requirements for the selective packaging of adenovirus type 5 DNA. J Virol. 1992 Feb;66(2):723–731. doi: 10.1128/jvi.66.2.723-731.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hammarskjöld M. L., Winberg G. Encapsidation of adenovirus 16 DNA is directed by a small DNA sequence at the left end of the genome. Cell. 1980 Jul;20(3):787–795. doi: 10.1016/0092-8674(80)90325-6. [DOI] [PubMed] [Google Scholar]
  20. Hearing P., Samulski R. J., Wishart W. L., Shenk T. Identification of a repeated sequence element required for efficient encapsidation of the adenovirus type 5 chromosome. J Virol. 1987 Aug;61(8):2555–2558. doi: 10.1128/jvi.61.8.2555-2558.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hearing P., Shenk T. The adenovirus type 5 E1A enhancer contains two functionally distinct domains: one is specific for E1A and the other modulates all early units in cis. Cell. 1986 Apr 25;45(2):229–236. doi: 10.1016/0092-8674(86)90387-9. [DOI] [PubMed] [Google Scholar]
  22. Hearing P., Shenk T. The adenovirus type 5 E1A transcriptional control region contains a duplicated enhancer element. Cell. 1983 Jul;33(3):695–703. doi: 10.1016/0092-8674(83)90012-0. [DOI] [PubMed] [Google Scholar]
  23. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Kosturko L. D., Sharnick S. V., Tibbetts C. Polar encapsidation of adenovirus DNA: cloning and DNA sequence of the left end of adenovirus type 3. J Virol. 1982 Sep;43(3):1132–1137. doi: 10.1128/jvi.43.3.1132-1137.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pettit S. C., Horwitz M. S., Engler J. A. Mutations of the precursor to the terminal protein of adenovirus serotypes 2 and 5. J Virol. 1989 Dec;63(12):5244–5250. doi: 10.1128/jvi.63.12.5244-5250.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Robinson C. C., Tibbetts C. Polar encapsidation of adenovirus DNA: evolutionary variants reveal dispensable sequences near the left ends of Ad3 genomes. Virology. 1984 Sep;137(2):276–286. doi: 10.1016/0042-6822(84)90219-8. [DOI] [PubMed] [Google Scholar]
  29. Stow N. D. Cloning of a DNA fragment from the left-hand terminus of the adenovirus type 2 genome and its use in site-directed mutagenesis. J Virol. 1981 Jan;37(1):171–180. doi: 10.1128/jvi.37.1.171-180.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tibbetts C. Viral DNA sequences from incomplete particles of human adenovirus type 7. Cell. 1977 Sep;12(1):243–249. doi: 10.1016/0092-8674(77)90202-1. [DOI] [PubMed] [Google Scholar]
  31. Yang Y., Nunes F. A., Berencsi K., Furth E. E., Gönczöl E., Wilson J. M. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4407–4411. doi: 10.1073/pnas.91.10.4407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yang Y., Nunes F. A., Berencsi K., Gönczöl E., Engelhardt J. F., Wilson J. M. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat Genet. 1994 Jul;7(3):362–369. doi: 10.1038/ng0794-362. [DOI] [PubMed] [Google Scholar]

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