Skip to main content
SpringerLink
Log in

Manipulation of Early Region 4

  • Protocol
Adenovirus Methods and Protocols

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 21))

  • 511 Accesses

Abstract

Early region 4 (E4; Fig. 1A) occupies the right-hand 3000 bp of the human adenovirus genome. The sequences of E4 and E4 cDNAs indicate that E4 encodes seven polypeptides (1–4), most of which have been detected in infected cells. Analysis of E4 mutants has implicated E4 products in a variety of processes that occur in infected cells, including viral early and late gene expression, DNA replication, the shutoff of host-cell protein synthesis, transformation, and the ability to stimulate replication of adenovirus-associated virus (AAV). Specific E4 products have been implicated in many of those processes and, in some cases, information on the molecular mechanisms of E4 function is emerging. Current knowledge of the functions of E4 products is summarized below.

A) The organization of adenovirus early region 4 E4 open reading frames (ORFs) are indicated by open boxes above a scale that indicates position in map units (0 to 100; 1 map unit is approx 360 bp) and nucleotide numbers. ORFs 1,2,3,4, and 6 are colinear with the viral DNA; ORFs 3/4 and 6/7 are the result of inframe splicing of the E4 mRNA precursor E4 is transcribed from right to left. (B) Reconstruction of an intact viral genome by ligation in vitro (C) Reconstruction of an intact viral genome by overlap recombination. (D) Construction of an E4 mutant by recombination between a plasmid and an E4 deletion mutant In C and D, the X indicates a potential homologous recombination event. In B, C, and D, the position of a hypothetical E4 mutation is noted by a V

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 79.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Fryer, G. A., Katoh, Y., and Roberts, R. J (1984) Characterization of the major mRNAs from adenovirus 2 early region 4 by cDNA cloning and sequencing Nucleic Acids Res 12, 3503–3519

    Article  Google Scholar 

  2. Roberts, R. J., Akusjarvi, G., Alestrom, P., Gelinas, R E., Gingeras, T R., Sciaky, D., and Pettersson, U. (1986) A consensus sequence for the adenovirus-2 genome, in Adenovirus DNA The Viral Genome and Its Expression (Doerfler, W., ed), Martinus Nijhoff, Boston, pp. 1–51

    Google Scholar 

  3. Virtanen, A., Gilardi, P., Naslund, A., Le Moullec, J M., Pettersson, U., and Perricaudet, M. (1984) mRNAs from human adenovirus 2 early region 4 J Virol 51, 822–831

    PubMed  CAS  Google Scholar 

  4. Dix, I and Leppard, K N (1992) Open reading frames 1 and 2 of adenovirus region E4 are conserved between human serotypes 2 and 5. J. Gen. Virol 73, 2975–2976

    Article  PubMed  CAS  Google Scholar 

  5. Javier, R. T. (1994) Adenovirus type 9 E4 open reading frame 1 encodes a transforming protein required for the production of mammary tumors in rats. J Virol 68, 3917–3924

    PubMed  CAS  Google Scholar 

  6. Bridge, E and Ketner, G. (1989) Redundant control of adenovirus late gene expression by early region 4. J Virol 63, 631–638.

    PubMed  CAS  Google Scholar 

  7. Huang, M. and Hearing, P. (1989) Adenovirus early region 4 encodes two products with redundant activities in lytic infection J Virol 63, 2605–2615

    PubMed  CAS  Google Scholar 

  8. Halbert, D N., Cutt, J R., and Shenk, T (1985) Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff J Virol 56, 250–257

    PubMed  CAS  Google Scholar 

  9. Weinberg, D. H. and Ketner, D. (1983) A cell line that supports the growth of a defective early region 4 deletion mutant of human adenovirus type 2. Proc. Natl Acad Sci USA 80, 5383–5386

    Article  PubMed  CAS  Google Scholar 

  10. Ohman, K., Nordqvist, K., and Akusjarvi, G (1993) Two adenovirus proteins with redundant activities in virus growth facilitate tripartite leader mRNA accumulation. Virology 194, 50–58.

    Article  PubMed  CAS  Google Scholar 

  11. Nordqvist, K., Ohman, K., and Akusjarvi, G (1994) Human adenovirus encodes two proteins which have opposite effects on accumulation of alternatively spliced mRNAs. Mol Cell Biol 14, 437–445.

    PubMed  CAS  Google Scholar 

  12. Weitzman, M D., Fisher, K. J., and Wilson, J M (1996) Recruitment of wildtype and recombinant adeno-associated virus into adenovirus replication centers J Virol 70, 1845–1854.

    PubMed  CAS  Google Scholar 

  13. Cutt, J. R., Shenk, T., and Hearing, P (1987) Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively-infected cells J Virol 61, 543–552

    PubMed  CAS  Google Scholar 

  14. Sarnow, P., Hearing, P., Anderson, C. W., Halbert, D. N., Shenk, T., and Levine, A J. (1984) Adenovirus early region 1b 58,000-dalton tumor antigen is physically associated with an early region 4 25,000-dalton protein in productively infected cells J Virol 49, 692–700

    PubMed  CAS  Google Scholar 

  15. Bridge, E and Ketner, G (1990) Interaction of adenoviral E4 and E1b proteins in late gene expression Virology 174, 345–353

    Article  PubMed  CAS  Google Scholar 

  16. Dobner, T., Horikoshi, N., Rubenwolf, S., and Shenk, T. (1996) Blockage by adenovirus E4orf6 of transcriptional activation by the p53 tumor suppressor Science 272, 1470–1473

    Article  PubMed  CAS  Google Scholar 

  17. Ornelles, D A. and Shenk, T. (1991) Localization of the adenovirus early region 1B 55-kilodalton protein during lytic infection: association with nuclear viral inclusions requires the early region 4 34-kilodalton protein. J Virol. 65, 424–439.

    PubMed  CAS  Google Scholar 

  18. Carvalho, T., Seeler, J S., Ohman, K., Jordan, P., Pettersson, U., Akusjarvi, G., Carmo-Fonseca, M., and Dejean, A (1995) Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix-associated PML bodies J. Cell Biol 131, 45–56

    Article  PubMed  CAS  Google Scholar 

  19. Muller, U., Kleinberger, T., and Shenk, T. (1992) Adenovirus E4orf4 protein reduces phosphorylation of c-Fos and E1a proteins while simultaneously reducing the level of AP-1. J. Virol. 66, 5867–5878.

    PubMed  CAS  Google Scholar 

  20. Bondesson, M., Ohman, K., Mannervik, M., Fan, S., and Akusjarvi, G. (1996) Adenovirus E4 open reading frame 4 protein autoregulates E4 transcription by inhibiting E1A transactivation of the E4 promoter. J Virol. 70, 3844–3851

    PubMed  CAS  Google Scholar 

  21. Medghalchi, S., Padmanabhan, R., and Ketner, G (1997) Early region 4 modulates adenovirus DNA replication by two genetically separable mechanisms Virology 236, 8–17.

    Article  PubMed  CAS  Google Scholar 

  22. Kleinberger, T and Shenk, T. (1993) Adenovirus E4 ORF4 protein binds to protein phosphatase 2A, and the complex down regulates E1A-enhanced junB transcription. J Virol 67, 7556–7560.

    PubMed  CAS  Google Scholar 

  23. Bridge, E., Medghalchi, S., Ubol, S., Leesong, M., and Ketner, G. (1993) Adenovirus early region 4 and viral DNA synthesis Virology 193, 794–801

    Article  PubMed  CAS  Google Scholar 

  24. Huang, M. and Hearing, P (1989) The adenovirus early region 4 open reading frame 6/7 protein regulates the DNA binding activity of cellular transcription factor E2F through a direct complex. Genes Dev. 3, 1699–1710

    Article  PubMed  CAS  Google Scholar 

  25. Neill, S. D., Hemstrom, C., Virtanen, A., and Nevins, J. R (1990) An adenovirus E4 gene product trans-activates E2 transcription and stimulates stable E2F binding through a direct association with E2F. Proc Natl. Acad Sci USA 87, 2008–2012.

    Article  PubMed  CAS  Google Scholar 

  26. Swaminathan, S. and Thimmapaya, B. (1996) Transactivation of the E2 early promoter by E1A and E4 6/7 in the context of the viral chromosome. J. Mol Biol 258, 736–746

    Article  PubMed  CAS  Google Scholar 

  27. Raychaudhuri, P., Bagchi, S. D., Neill, S., and Nevins, J. R. (1990) Activation of the E2F transcription factor in adenovirus-infected cells involves the E1A-dependent stimulation of DNA binding activity and induction of cooperative binding mediated by an E4 gene product. J Virol. 64, 2702–2710

    PubMed  CAS  Google Scholar 

  28. Berkner, K. L. and Sharp, P. A. (1983) Generation of adenovirus by transfection of plasmids. Nucleic Acids Res. 11, 6003–6020.

    Article  PubMed  CAS  Google Scholar 

  29. Challberg, S. S. and Ketner, G (1981) Deletion mutants of adenovirus 2. isolation and initial characterization of virus carrying mutations near the right end of the viral genome Virology 114, 196–209.

    Article  PubMed  CAS  Google Scholar 

  30. Robinson, A J. and Bellett, A. D. J (1974) A circular DNA-protein complex from adenoviruses and its possible role in DNA replication. Cold Spring Harbor Symp Quant Biol 39, 523–531.

    Google Scholar 

  31. Graham, F. L and van der Eb, A J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. VwoEogy 52, 456–467.

    CAS  Google Scholar 

  32. Cameron, I R., Wilkie, N. M., and McNab, J C. M. (1983) The infectivity of herpes simples DNA in rat embryo cells is enhanced synergistically by DMSO and glucose. J Virological Methods 6, 183–191

    Article  CAS  Google Scholar 

  33. Kroughliak, V. and Graham, F (1995) Development of cell lines capable of complementing E1, E4, and protein IX defective adenovirus type 5 mutants Human Gene Ther 6, 1575–1586

    Article  Google Scholar 

  34. Yeh, P., Didieu, J-F., Orsini, C., Vigne, E., Denefle, P., and Perricaudet, M (1996) Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit J Virol 70, 559–565.

    PubMed  CAS  Google Scholar 

  35. Brough, D E., Lizonova, A., Hsu, C., Kulesa, V. A., and Kovesdi, I (1996) A gene transfer vector-cell line system for complete functional complementation of adenovirus early regions E1 and E4 J. Virol. 70, 6497–6501.

    PubMed  CAS  Google Scholar 

  36. Wang, Q., Jia, X.-C., and Finer, M H. (1995) A packaging cell line for propagation of recombinant adenovirus vectors containing two lethal gene region deletions Gene Ther 2, 775–783

    PubMed  CAS  Google Scholar 

  37. Marton, M., Baim, S. B., Ornelles, D. A., and Shenk, T (1990) The adenovirus E4 17 kilodalton protein complexes with the cellular transcription factor E2F, altering its DNA-binding properties and stimulating E1A-independent accumulation of E2 mRNA. J Virol 64, 2345–2359.

    PubMed  CAS  Google Scholar 

  38. Hemstrom, K., Nordqvist, K., Pettersson, U., and Virtanen, A (1988) Gene product of region E4 of adenovirus type 5 modulates accumulation of certain viral polypeptides. J Virol 62, 3258–3264.

    PubMed  CAS  Google Scholar 

  39. Nordqvist, K and Akusjarvi, G. (1990) Adenovirus early region 4 stimulates mRNA accumulation via 5′ introns. Proc Natl Acad Sci USA 87, 9543–9547

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular Microbiology and Immunology, School 0f Hygiene and Public Health, Johns Hopkins University, Baltimore, MD

    Julie Boyer

  2. Immunology and Infectious Diseases Department, School of Public Health, Johns Hopkins University, Baltimore, MD

    Gary Ketner

Authors
  1. Julie Boyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gary Ketner
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. St. Louis University Health Science Center, St. Louis, MO

    William S. M. Wold

Rights and permissions

Reprints and permissions

Copyright information

© 1999 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Boyer, J., Ketner, G. (1999). Manipulation of Early Region 4. In: Wold, W.S.M. (eds) Adenovirus Methods and Protocols. Methods in Molecular Medicine™, vol 21. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-551-4:33

Download citation

  • DOI: https://doi.org/10.1385/0-89603-551-4:33

  • Publisher Name: Springer, Totowa, NJ

  • Print ISBN: 978-0-89603-551-5

  • Online ISBN: 978-1-59259-603-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation