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. 1988 Apr;7(4):1197–1204. doi: 10.1002/j.1460-2075.1988.tb02931.x

Bovine papillomavirus mutant temperature sensitive for transformation, replication and transactivation.

D DiMaio 1, J Settleman 1
PMCID: PMC454456  PMID: 2841117

Abstract

The genetic analysis of the papillomaviruses has been hampered by the lack of mutants conditionally defective for viral biological activities. We report here the construction and characterization of a temperature-sensitive papillomavirus mutant. The mutation is predicted to insert the sequence Pro-Arg-Ser-Arg into the N-terminal half of the bovine papillomavirus type 1 (BPV1) ORF E2 protein, the major viral regulatory protein. The cloned mutant viral DNA displays temperature-sensitive defects in the induction of focus formation in mouse C127 cells, in its establishment as an extrachromosomal plasmid and in transactivation of a BPV1 enhancer. Genetic experiments confirm that this pleiotropic phenotype is caused by the insertion mutation in ORF E2 and that the transformation and replication defects of the mutant at 37 degrees C are corrected in trans by wild-type E2 gene activity. Most cell lines stably transformed by the mutant at 32.5 degrees C display a reduced ability to overgrow a monolayer of normal cells following temperature shift to 37 degrees C and the mutant viral DNA after temperature shift is present in decreased copy number and/or in an integrated state. These results provide strong genetic evidence that continued ORF E2 activity is required for maintenance of BPV1-induced transformation and for normal viral DNA replication.

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Selected References

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

  1. Amtmann E., Müller K., Knapp A., Sauer G. Reversion of bovine papillomavirus-induced transformation and immortalization by a xanthate compound. Exp Cell Res. 1985 Dec;161(2):541–550. doi: 10.1016/0014-4827(85)90109-0. [DOI] [PubMed] [Google Scholar]
  2. Androphy E. J., Lowy D. R., Schiller J. T. Bovine papillomavirus E2 trans-activating gene product binds to specific sites in papillomavirus DNA. Nature. 1987 Jan 1;325(6099):70–73. doi: 10.1038/325070a0. [DOI] [PubMed] [Google Scholar]
  3. Androphy E. J., Schiller J. T., Lowy D. R. Identification of the protein encoded by the E6 transforming gene of bovine papillomavirus. Science. 1985 Oct 25;230(4724):442–445. doi: 10.1126/science.2996134. [DOI] [PubMed] [Google Scholar]
  4. Barany F. Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4202–4206. doi: 10.1073/pnas.82.12.4202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berg L. J., Singh K., Botchan M. Complementation of a bovine papilloma virus low-copy-number mutant: evidence for a temporal requirement of the complementing gene. Mol Cell Biol. 1986 Mar;6(3):859–869. doi: 10.1128/mcb.6.3.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen E. Y., Howley P. M., Levinson A. D., Seeburg P. H. The primary structure and genetic organization of the bovine papillomavirus type 1 genome. Nature. 1982 Oct 7;299(5883):529–534. doi: 10.1038/299529a0. [DOI] [PubMed] [Google Scholar]
  7. Colicelli J., Lobel L. I., Goff S. P. A temperature-sensitive mutation constructed by "linker insertion" mutagenesis. Mol Gen Genet. 1985;199(3):537–539. doi: 10.1007/BF00330771. [DOI] [PubMed] [Google Scholar]
  8. DiMaio D., Guralski D., Schiller J. T. Translation of open reading frame E5 of bovine papillomavirus is required for its transforming activity. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1797–1801. doi: 10.1073/pnas.83.6.1797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DiMaio D. Nonsense mutation in open reading frame E2 of bovine papillomavirus DNA. J Virol. 1986 Feb;57(2):475–480. doi: 10.1128/jvi.57.2.475-480.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DiMaio D., Treisman R., Maniatis T. Bovine papillomavirus vector that propagates as a plasmid in both mouse and bacterial cells. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4030–4034. doi: 10.1073/pnas.79.13.4030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dvoretzky I., Shober R., Chattopadhyay S. K., Lowy D. R. A quantitative in vitro focus assay for bovine papilloma virus. Virology. 1980 Jun;103(2):369–375. doi: 10.1016/0042-6822(80)90195-6. [DOI] [PubMed] [Google Scholar]
  12. Gorman C. M., Howard B. H., Reeves R. Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nucleic Acids Res. 1983 Nov 11;11(21):7631–7648. doi: 10.1093/nar/11.21.7631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Groff D. E., Lancaster W. D. Genetic analysis of the 3' early region transformation and replication functions of bovine papillomavirus type 1. Virology. 1986 Apr 15;150(1):221–230. doi: 10.1016/0042-6822(86)90281-3. [DOI] [PubMed] [Google Scholar]
  15. Haugen T. H., Cripe T. P., Ginder G. D., Karin M., Turek L. P. Trans-activation of an upstream early gene promoter of bovine papilloma virus-1 by a product of the viral E2 gene. EMBO J. 1987 Jan;6(1):145–152. doi: 10.1002/j.1460-2075.1987.tb04732.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hermonat P. L., Howley P. M. Mutational analysis of the 3' open reading frames and the splice junction at nucleotide 3225 of bovine papillomavirus type 1. J Virol. 1987 Dec;61(12):3889–3895. doi: 10.1128/jvi.61.12.3889-3895.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kipreos E. T., Lee G. J., Wang J. Y. Isolation of temperature-sensitive tyrosine kinase mutants of v-abl oncogene by screening with antibodies for phosphotyrosine. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1345–1349. doi: 10.1073/pnas.84.5.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lancaster W. D. Apparent lack of integration of bovine papillomavirus DNA in virus-induced equine and bovine tumor cells and virus-transformed mouse cells. Virology. 1981 Jan 30;108(2):251–255. doi: 10.1016/0042-6822(81)90433-5. [DOI] [PubMed] [Google Scholar]
  19. Law M. F., Lowy D. R., Dvoretzky I., Howley P. M. Mouse cells transformed by bovine papillomavirus contain only extrachromosomal viral DNA sequences. Proc Natl Acad Sci U S A. 1981 May;78(5):2727–2731. doi: 10.1073/pnas.78.5.2727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lowy D. R., Dvoretzky I., Shober R., Law M. F., Engel L., Howley P. M. In vitro tumorigenic transformation by a defined sub-genomic fragment of bovine papilloma virus DNA. Nature. 1980 Sep 4;287(5777):72–74. doi: 10.1038/287072a0. [DOI] [PubMed] [Google Scholar]
  21. Lusky M., Botchan M. R. Genetic analysis of bovine papillomavirus type 1 trans-acting replication factors. J Virol. 1985 Mar;53(3):955–965. doi: 10.1128/jvi.53.3.955-965.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lusky M., Botchan M. R. Transient replication of bovine papilloma virus type 1 plasmids: cis and trans requirements. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3609–3613. doi: 10.1073/pnas.83.11.3609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Moskaluk C., Bastia D. The E2 "gene" of bovine papillomavirus encodes an enhancer-binding protein. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1215–1218. doi: 10.1073/pnas.84.5.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Neary K., Horwitz B. H., DiMaio D. Mutational analysis of open reading frame E4 of bovine papillomavirus type 1. J Virol. 1987 Apr;61(4):1248–1252. doi: 10.1128/jvi.61.4.1248-1252.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rabson M. S., Yee C., Yang Y. C., Howley P. M. Bovine papillomavirus type 1 3' early region transformation and plasmid maintenance functions. J Virol. 1986 Nov;60(2):626–634. doi: 10.1128/jvi.60.2.626-634.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sarver N., Byrne J. C., Howley P. M. Transformation and replication in mouse cells of a bovine papillomavirus--pML2 plasmid vector that can be rescued in bacteria. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7147–7151. doi: 10.1073/pnas.79.23.7147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sarver N., Rabson M. S., Yang Y. C., Byrne J. C., Howley P. M. Localization and analysis of bovine papillomavirus type 1 transforming functions. J Virol. 1984 Nov;52(2):377–388. doi: 10.1128/jvi.52.2.377-388.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schiller J. T., Vass W. C., Lowy D. R. Identification of a second transforming region in bovine papillomavirus DNA. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7880–7884. doi: 10.1073/pnas.81.24.7880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schiller J. T., Vass W. C., Vousden K. H., Lowy D. R. E5 open reading frame of bovine papillomavirus type 1 encodes a transforming gene. J Virol. 1986 Jan;57(1):1–6. doi: 10.1128/jvi.57.1.1-6.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schlegel R., Wade-Glass M., Rabson M. S., Yang Y. C. The E5 transforming gene of bovine papillomavirus encodes a small, hydrophobic polypeptide. Science. 1986 Jul 25;233(4762):464–467. doi: 10.1126/science.3014660. [DOI] [PubMed] [Google Scholar]
  31. Schwarz E., Dürst M., Demankowski C., Lattermann O., Zech R., Wolfsperger E., Suhai S., zur Hausen H. DNA sequence and genome organization of genital human papillomavirus type 6b. EMBO J. 1983;2(12):2341–2348. doi: 10.1002/j.1460-2075.1983.tb01744.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Spalholz B. A., Lambert P. F., Yee C. L., Howley P. M. Bovine papillomavirus transcriptional regulation: localization of the E2-responsive elements of the long control region. J Virol. 1987 Jul;61(7):2128–2137. doi: 10.1128/jvi.61.7.2128-2137.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Spalholz B. A., Yang Y. C., Howley P. M. Transactivation of a bovine papilloma virus transcriptional regulatory element by the E2 gene product. Cell. 1985 Aug;42(1):183–191. doi: 10.1016/s0092-8674(85)80114-8. [DOI] [PubMed] [Google Scholar]
  35. Turek L. P., Byrne J. C., Lowy D. R., Dvoretzky I., Friedman R. M., Howley P. M. Interferon induces morphologic reversion with elimination of extrachromosomal viral genomes in bovine papillomavirus-transformed mouse cells. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7914–7918. doi: 10.1073/pnas.79.24.7914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wallace R. B., Johnson M. J., Suggs S. V., Miyoshi K., Bhatt R., Itakura K. A set of synthetic oligodeoxyribonucleotide primers for DNA sequencing in the plasmid vector pBR322. Gene. 1981 Dec;16(1-3):21–26. doi: 10.1016/0378-1119(81)90057-3. [DOI] [PubMed] [Google Scholar]
  38. Yang Y. C., Okayama H., Howley P. M. Bovine papillomavirus contains multiple transforming genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1030–1034. doi: 10.1073/pnas.82.4.1030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yang Y. C., Spalholz B. A., Rabson M. S., Howley P. M. Dissociation of transforming and trans-activation functions for bovine papillomavirus type 1. Nature. 1985 Dec 12;318(6046):575–577. doi: 10.1038/318575a0. [DOI] [PubMed] [Google Scholar]
  40. van der Eb A. J., Graham F. L. Assay of transforming activity of tumor virus DNA. Methods Enzymol. 1980;65(1):826–839. doi: 10.1016/s0076-6879(80)65077-0. [DOI] [PubMed] [Google Scholar]

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