Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1986 May;58(2):324–330. doi: 10.1128/jvi.58.2.324-330.1986

Deletion mutants that affect expression of Epstein-Barr virus nuclear antigen in COS-1 cells after gene transfer with simian virus 40 vectors containing portions of the BamHI K fragment.

M Polvino-Bodnar, D Shedd, G Miller
PMCID: PMC252916  PMID: 3009849

Abstract

We have identified sequences that affect the efficient expression of Epstein-Barr virus nuclear antigen (EBNA 1) when the structural portion of its gene, found within the 2.9-kilobase-pair BamHI/HindIII fragment called Ilf, is expressed from a simian virus 40 vector. A set of nested deletions at the BamHI end of the fragment was constructed by using BAL 31 digestion, the addition of linkers, and ligation into pSVOd. The mutants were tested for their ability to express antigen in COS-1 monkey cells by using indirect immunofluorescence and immunoblotting. Deletion endpoints were determined by DNA sequencing of the 5' ends of the mutants. The deletion mutants could be subclassified into four groups based on their ability to express EBNA polypeptide. Mutants that retain more than 106 base pairs upstream from the start of the open reading frame in Ilf exhibit antigen expression indistinguishable from that of wild type. Mutants that invade the structural gene by 1,115 or more bases destroy antigen expression. Mutants that alter the splice acceptor site or invade the open reading frame by a short distance make antigen at a markedly lower frequency. There are three mutants, whose deletions map at -78, -70, and -44 base pairs upstream of the open reading frame, that make reduced levels of EBNA. Since these three mutants differ in the extent to which EBNA expression is impaired, the data suggest that there are several critical regions upstream of the open reading frame that regulate EBNA expression in COS-1 cells. It is not known whether these regulatory sequences, which would be located in an intron in the intact genome, play any role in the expression of EBNA in infected lymphocytes.

Full text

PDF
324

Images in this article

327Image
on p.327
327Image
on p.327
328Image
on p.328

Selected References

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

  1. Arnott S., Selsing E. Letter: The structure of polydeoxyguanylic acid with polydeoxycytidylic acid. J Mol Biol. 1974 Sep 15;88(2):551–552. doi: 10.1016/0022-2836(74)90502-6. [DOI] [PubMed] [Google Scholar]
  2. Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Séguin C. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984 Jul 19;310(5974):207–211. doi: 10.1038/310207a0. [DOI] [PubMed] [Google Scholar]
  3. Bhat B. M., Brady H. A., Wold W. S. Virus deletion mutants that affect a 3' splice site in the E3 transcription unit of adenovirus 2. Mol Cell Biol. 1985 Sep;5(9):2405–2413. doi: 10.1128/mcb.5.9.2405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Dillner J., Sternås L., Kallin B., Alexander H., Ehlin-Henriksson B., Jörnvall H., Klein G., Lerner R. Antibodies against a synthetic peptide identify the Epstein-Barr virus-determined nuclear antigen. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4652–4656. doi: 10.1073/pnas.81.15.4652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fischer D. K., Robert M. F., Shedd D., Summers W. P., Robinson J. E., Wolak J., Stefano J. E., Miller G. Identification of Epstein-Barr nuclear antigen polypeptide in mouse and monkey cells after gene transfer with a cloned 2.9-kilobase-pair subfragment of the genome. Proc Natl Acad Sci U S A. 1984 Jan;81(1):43–47. doi: 10.1073/pnas.81.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  8. Grogan E. A., Summers W. P., Dowling S., Shedd D., Gradoville L., Miller G. Two Epstein-Barr viral nuclear neoantigens distinguished by gene transfer, serology, and chromosome binding. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7650–7653. doi: 10.1073/pnas.80.24.7650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hearing J. C., Nicolas J. C., Levine A. J. Identification of Epstein-Barr virus sequences that encode a nuclear antigen expressed in latently infected lymphocytes. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4373–4377. doi: 10.1073/pnas.81.14.4373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Heller M., van Santen V., Kieff E. Simple repeat sequence in Epstein-Barr virus DNA is transcribed in latent and productive infections. J Virol. 1982 Oct;44(1):311–320. doi: 10.1128/jvi.44.1.311-320.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hennessy K., Heller M., van Santen V., Kieff E. Simple repeat array in Epstein-Barr virus DNA encodes part of the Epstein-Barr nuclear antigen. Science. 1983 Jun 24;220(4604):1396–1398. doi: 10.1126/science.6304878. [DOI] [PubMed] [Google Scholar]
  12. Hennessy K., Kieff E. One of two Epstein-Barr virus nuclear antigens contains a glycine-alanine copolymer domain. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5665–5669. doi: 10.1073/pnas.80.18.5665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jeffreys A. J., Wilson V., Thein S. L. Hypervariable 'minisatellite' regions in human DNA. Nature. 1985 Mar 7;314(6006):67–73. doi: 10.1038/314067a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Larsen A., Weintraub H. An altered DNA conformation detected by S1 nuclease occurs at specific regions in active chick globin chromatin. Cell. 1982 Jun;29(2):609–622. doi: 10.1016/0092-8674(82)90177-5. [DOI] [PubMed] [Google Scholar]
  16. Luthman H., Magnusson G. High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res. 1983 Mar 11;11(5):1295–1308. doi: 10.1093/nar/11.5.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mellon P., Parker V., Gluzman Y., Maniatis T. Identification of DNA sequences required for transcription of the human alpha 1-globin gene in a new SV40 host-vector system. Cell. 1981 Dec;27(2 Pt 1):279–288. doi: 10.1016/0092-8674(81)90411-6. [DOI] [PubMed] [Google Scholar]
  18. Robert M. F., Shedd D., Weigel R. J., Fischer D. K., Miller G. Expression in COS-1 cells of Epstein-Barr virus nuclear antigen from a complete gene and a deleted gene. J Virol. 1984 Jun;50(3):822–831. doi: 10.1128/jvi.50.3.822-831.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rowe D., Heston L., Metlay J., Miller G. Identification and expression of a nuclear antigen from the genomic region of the Jijoye strain of Epstein-Barr virus that is missing in its nonimmortalizing deletion mutant, P3HR-1. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7429–7433. doi: 10.1073/pnas.82.21.7429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Simpson R. T., Künzler P. Cromatin and core particles formed from the inner histones and synthetic polydeoxyribonucleotides of defined sequence. Nucleic Acids Res. 1979 Apr;6(4):1387–1415. doi: 10.1093/nar/6.4.1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Summers W. P., Grogan E. A., Shedd D., Robert M., Liu C. R., Miller G. Stable expression in mouse cells of nuclear neoantigen after transfer of a 3.4-megadalton cloned fragment of Epstein-Barr virus DNA. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5688–5692. doi: 10.1073/pnas.79.18.5688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Weigel R., Fischer D. K., Heston L., Miller G. Constitutive expression of Epstein-Barr virus-encoded RNAs and nuclear antigen during latency and after induction of Epstein-Barr virus replication. J Virol. 1985 Jan;53(1):254–259. doi: 10.1128/jvi.53.1.254-259.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Weigel R., Miller G. Latent and viral replicative transcription in vivo from the BamHI K fragment of Epstein-Barr virus DNA. J Virol. 1985 May;54(2):501–508. doi: 10.1128/jvi.54.2.501-508.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weisbrod S. Active chromatin. Nature. 1982 May 27;297(5864):289–295. doi: 10.1038/297289a0. [DOI] [PubMed] [Google Scholar]
  28. Yates J. L., Warren N., Sugden B. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. 1985 Feb 28-Mar 6Nature. 313(6005):812–815. doi: 10.1038/313812a0. [DOI] [PubMed] [Google Scholar]
  29. van Santen V. L., Spritz R. A. mRNA precursor splicing in vivo: sequence requirements determined by deletion analysis of an intervening sequence. Proc Natl Acad Sci U S A. 1985 May;82(9):2885–2889. doi: 10.1073/pnas.82.9.2885. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES