Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1988 Feb;62(2):417–426. doi: 10.1128/jvi.62.2.417-426.1988

Genomic clones of bovine parvovirus: construction and effect of deletions and terminal sequence inversions on infectivity.

B C Shull 1, K C Chen 1, M Lederman 1, E R Stout 1, R C Bates 1
PMCID: PMC250551  PMID: 3275788

Abstract

Genomic clones of the autonomous parvovirus bovine parvovirus (BPV) were constructed by blunt-end ligation of reannealed virion plus and minus DNA strands into the plasmid pUC8. These clones were stable during propagation in Escherichia coli JM107. All clones tested were found to be infectious by the criteria of plaque titer and progressive cytopathic effect after transfection into bovine fetal lung cells. Sequencing of the recombinant plasmids demonstrated that all of the BPV inserts had left-end (3')-terminal deletions of up to 34 bases. DNA isolated from progeny virions arising from transfected infectious clones was found to be indistinguishable from wild-type DNA by restriction enzyme analysis. Defective genomes could also be detected in the progeny DNA even though the infection was initiated with homogenous, cloned DNA. Full-length genomic clones with 3' flip and 3' flop conformations were constructed and were found to have equal infectivity. Analysis of low-molecular-weight DNA isolated from lysates of cells transfected with these clones demonstrated that rescue and replication of BPV DNA could be detected 3 to 8 days after transfection. Expression of capsid proteins from transfected genomes was demonstrated by hemagglutination, indirect immunofluorescence, and immunoprecipitation of [35S]methionine-labeled cell lysates. Use of appropriate antiserum for immunoprecipitation showed the synthesis of BPV capsid and noncapsid proteins after transfection. Independently, a series of genomic clones with increasingly larger 3'-terminal deletions was prepared from separately subcloned 3'-terminal fragments. Transfection of these clones into bovine fetal lung cells revealed that deletions of up to 34 bases at the 3' end lowered but did not abolish infectivity, while deletions of greater than 52 bases were lethal. End-label analysis showed that the 34-base deletion was repaired to wild-type length in the progeny virus.

Full text

PDF

Images in this article

419Image
on p.419
421Image
on p.421
422Image
on p.422
423Image
on p.423
423Image
on p.423
424Image
on p.424

Selected References

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

  1. Astell C. R., Chow M. B., Ward D. C. Sequence analysis of the termini of virion and replicative forms of minute virus of mice DNA suggests a modified rolling hairpin model for autonomous parvovirus DNA replication. J Virol. 1985 Apr;54(1):171–177. doi: 10.1128/jvi.54.1.171-177.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bates R. C., Snyder C. E., Banerjee P. T., Mitra S. Autonomous parvovirus LuIII encapsidates equal amounts of plus and minus DNA strands. J Virol. 1984 Feb;49(2):319–324. doi: 10.1128/jvi.49.2.319-324.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bates R. C., Storz J., Reed D. E. Isolation and comparison of bovine parvoviruses. J Infect Dis. 1972 Nov;126(5):531–536. doi: 10.1093/infdis/126.5.531. [DOI] [PubMed] [Google Scholar]
  4. Berns K. I., Bohenzky R. A. Adeno-associated viruses: an update. Adv Virus Res. 1987;32:243–306. doi: 10.1016/s0065-3527(08)60479-0. [DOI] [PubMed] [Google Scholar]
  5. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bloom M. E., Race R. E., Wolfinbarger J. B. Identification of a nonvirion protein of Aleutian disease virus: mink with Aleutian disease have antibody to both virion and nonvirion proteins. J Virol. 1982 Aug;43(2):608–616. doi: 10.1128/jvi.43.2.608-616.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boissy R., Astell C. R. An Escherichia coli recBCsbcBrecF host permits the deletion-resistant propagation of plasmid clones containing the 5'-terminal palindrome of minute virus of mice. Gene. 1985;35(1-2):179–185. doi: 10.1016/0378-1119(85)90170-2. [DOI] [PubMed] [Google Scholar]
  8. Chen K. C., Shull B. C., Moses E. A., Lederman M., Stout E. R., Bates R. C. Complete nucleotide sequence and genome organization of bovine parvovirus. J Virol. 1986 Dec;60(3):1085–1097. doi: 10.1128/jvi.60.3.1085-1097.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chow M., Bodnar J. W., Polvino-Bodnar M., Ward D. C. Identification and characterization of a protein covalently bound to DNA of minute virus of mice. J Virol. 1986 Mar;57(3):1094–1104. doi: 10.1128/jvi.57.3.1094-1104.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cotmore S. F., Tattersall P. Characterization and molecular cloning of a human parvovirus genome. Science. 1984 Dec 7;226(4679):1161–1165. doi: 10.1126/science.6095448. [DOI] [PubMed] [Google Scholar]
  11. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  12. Hermonat P. L., Labow M. A., Wright R., Berns K. I., Muzyczka N. Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants. J Virol. 1984 Aug;51(2):329–339. doi: 10.1128/jvi.51.2.329-339.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Laughlin C. A., Tratschin J. D., Coon H., Carter B. J. Cloning of infectious adeno-associated virus genomes in bacterial plasmids. Gene. 1983 Jul;23(1):65–73. doi: 10.1016/0378-1119(83)90217-2. [DOI] [PubMed] [Google Scholar]
  14. Lederman M., Cotmore S. F., Stout E. R., Bates R. C. Detection of bovine parvovirus proteins homologous to the nonstructural NS-1 proteins of other autonomous parvoviruses. J Virol. 1987 Nov;61(11):3612–3616. doi: 10.1128/jvi.61.11.3612-3616.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lopata M. A., Cleveland D. W., Sollner-Webb B. High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res. 1984 Jul 25;12(14):5707–5717. doi: 10.1093/nar/12.14.5707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lusby E., Fife K. H., Berns K. I. Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA. J Virol. 1980 May;34(2):402–409. doi: 10.1128/jvi.34.2.402-409.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Merchlinsky M. J., Tattersall P. J., Leary J. J., Cotmore S. F., Gardiner E. M., Ward D. C. Construction of an infectious molecular clone of the autonomous parvovirus minute virus of mice. J Virol. 1983 Jul;47(1):227–232. doi: 10.1128/jvi.47.1.227-232.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Parris D. S., Bates R. C. Effect of bovine parvovirus replication on DNA, RNA, and protein synthesis in S phase cells. Virology. 1976 Aug;73(1):72–78. doi: 10.1016/0042-6822(76)90061-1. [DOI] [PubMed] [Google Scholar]
  19. Reed K. C., Mann D. A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. doi: 10.1093/nar/13.20.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Revie D., Tseng B. Y., Grafstrom R. H., Goulian M. Covalent association of protein with replicative form DNA of parvovirus H-1. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5539–5543. doi: 10.1073/pnas.76.11.5539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rhode S. L., 3rd, Klaassen B. DNA sequence of the 5' terminus containing the replication origin of parvovirus replicative form DNA. J Virol. 1982 Mar;41(3):990–999. doi: 10.1128/jvi.41.3.990-999.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rhode S. L., 3rd trans-Activation of parvovirus P38 promoter by the 76K noncapsid protein. J Virol. 1985 Sep;55(3):886–889. doi: 10.1128/jvi.55.3.886-889.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sahli R., McMaster G. K., Hirt B. DNA sequence comparison between two tissue-specific variants of the autonomous parvovirus, minute virus of mice. Nucleic Acids Res. 1985 May 24;13(10):3617–3633. doi: 10.1093/nar/13.10.3617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Samulski R. J., Berns K. I., Tan M., Muzyczka N. Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. Proc Natl Acad Sci U S A. 1982 Mar;79(6):2077–2081. doi: 10.1073/pnas.79.6.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Samulski R. J., Srivastava A., Berns K. I., Muzyczka N. Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV. Cell. 1983 May;33(1):135–143. doi: 10.1016/0092-8674(83)90342-2. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Senapathy P., Carter B. J. Molecular cloning of adeno-associated virus variant genomes and generation of infectious virus by recombination in mammalian cells. J Biol Chem. 1984 Apr 10;259(7):4661–4666. [PubMed] [Google Scholar]
  28. Senapathy P., Tratschin J. D., Carter B. J. Replication of adeno-associated virus DNA. Complementation of naturally occurring rep- mutants by a wild-type genome or an ori- mutant and correction of terminal palindrome deletions. J Mol Biol. 1984 Oct 15;179(1):1–20. doi: 10.1016/0022-2836(84)90303-6. [DOI] [PubMed] [Google Scholar]
  29. Summers J., Jones S. E., Anderson M. J. Characterization of the genome of the agent of erythrocyte aplasia permits its classification as a human parvovirus. J Gen Virol. 1983 Nov;64(Pt 11):2527–2532. doi: 10.1099/0022-1317-64-11-2527. [DOI] [PubMed] [Google Scholar]
  30. Sussman D. J., Milman G. Short-term, high-efficiency expression of transfected DNA. Mol Cell Biol. 1984 Aug;4(8):1641–1643. doi: 10.1128/mcb.4.8.1641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tratschin J. D., Miller I. L., Carter B. J. Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function. J Virol. 1984 Sep;51(3):611–619. doi: 10.1128/jvi.51.3.611-619.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES