Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1984 Oct;4(10):2072–2081. doi: 10.1128/mcb.4.10.2072

A human parvovirus, adeno-associated virus, as a eucaryotic vector: transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase.

J D Tratschin, M H West, T Sandbank, B J Carter
PMCID: PMC369024  PMID: 6095038

Abstract

We have used the defective human parvovirus adeno-associated virus (AAV) as a novel eucaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p40 (pAVHiCAT) and p19 (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p19 is increased by E1A, whereas p40 yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

Full text

PDF
2072

Images in this article

Selected References

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

  1. Alton N. K., Vapnek D. Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature. 1979 Dec 20;282(5741):864–869. doi: 10.1038/282864a0. [DOI] [PubMed] [Google Scholar]
  2. Berk A. J., Lee F., Harrison T., Williams J., Sharp P. A. Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs. Cell. 1979 Aug;17(4):935–944. doi: 10.1016/0092-8674(79)90333-7. [DOI] [PubMed] [Google Scholar]
  3. Carlock L. R., Jones N. C. Transformation-defective mutant of adenovirus type 5 containing a single altered E1a mRNA species. J Virol. 1981 Dec;40(3):657–664. doi: 10.1128/jvi.40.3.657-664.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carter B. J., Laughlin C. A., de la Maza L. M., Myers M. Adeno-associated virus autointerference. Virology. 1979 Jan 30;92(2):449–462. doi: 10.1016/0042-6822(79)90149-1. [DOI] [PubMed] [Google Scholar]
  5. Carter B. J., Marcus-Sekura C. J., Laughlin C. A., Ketner G. Properties of an adenovirus type 2 mutant, Ad2dl807, having a deletion near the right-hand genome terminus: failure to help AAV replication. Virology. 1983 Apr 30;126(2):505–516. doi: 10.1016/s0042-6822(83)80008-7. [DOI] [PubMed] [Google Scholar]
  6. Cheung A. K., Hoggan M. D., Hauswirth W. W., Berns K. I. Integration of the adeno-associated virus genome into cellular DNA in latently infected human Detroit 6 cells. J Virol. 1980 Feb;33(2):739–748. doi: 10.1128/jvi.33.2.739-748.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Garrett J. M., Young R. Lethal action of bacteriophage lambda S gene. J Virol. 1982 Dec;44(3):886–892. doi: 10.1128/jvi.44.3.886-892.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gaynor R. B., Berk A. J. Cis-acting induction of adenovirus transcription. Cell. 1983 Jul;33(3):683–693. doi: 10.1016/0092-8674(83)90011-9. [DOI] [PubMed] [Google Scholar]
  9. Gaynor R. B., Hillman D., Berk A. J. Adenovirus early region 1A protein activates transcription of a nonviral gene introduced into mammalian cells by infection or transfection. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1193–1197. doi: 10.1073/pnas.81.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Green M. R., Roeder R. G. Definition of a novel promoter for the major adenovirus-associated virus mRNA. Cell. 1980 Nov;22(1 Pt 1):231–242. doi: 10.1016/0092-8674(80)90171-3. [DOI] [PubMed] [Google Scholar]
  13. Green M. R., Treisman R., Maniatis T. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products. Cell. 1983 Nov;35(1):137–148. doi: 10.1016/0092-8674(83)90216-7. [DOI] [PubMed] [Google Scholar]
  14. Handa H., Shiroki K., Shimojo H. Establishment and characterization of KB cell lines latently infected with adeno-associated virus type 1. Virology. 1977 Oct 1;82(1):84–92. doi: 10.1016/0042-6822(77)90034-4. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  17. Humphreys G. O., Willshaw G. A., Anderson E. S. A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta. 1975 Apr 2;383(4):457–463. doi: 10.1016/0005-2787(75)90318-4. [DOI] [PubMed] [Google Scholar]
  18. Imperiale M. J., Feldman L. T., Nevins J. R. Activation of gene expression by adenovirus and herpesvirus regulatory genes acting in trans and by a cis-acting adenovirus enhancer element. Cell. 1983 Nov;35(1):127–136. doi: 10.1016/0092-8674(83)90215-5. [DOI] [PubMed] [Google Scholar]
  19. Janik J. E., Huston M. M., Rose J. A. Locations of adenovirus genes required for the replication of adenovirus-associated virus. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1925–1929. doi: 10.1073/pnas.78.3.1925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jay F. T., Laughlin C. A., Carter B. J. Eukaryotic translational control: adeno-associated virus protein synthesis is affected by a mutation in the adenovirus DNA-binding protein. Proc Natl Acad Sci U S A. 1981 May;78(5):2927–2931. doi: 10.1073/pnas.78.5.2927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jones N. C., Richter J. D., Weeks D. L., Smith L. D. Regulation of adenovirus transcription by an E1a gene in microinjected Xenopus laevis oocytes. Mol Cell Biol. 1983 Dec;3(12):2131–2142. doi: 10.1128/mcb.3.12.2131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jones N., Shenk T. An adenovirus type 5 early gene function regulates expression of other early viral genes. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3665–3669. doi: 10.1073/pnas.76.8.3665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kozak M. Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo. Nature. 1984 Mar 15;308(5956):241–246. doi: 10.1038/308241a0. [DOI] [PubMed] [Google Scholar]
  24. Langridge J., Langridge P., Bergquist P. L. Extraction of nucleic acids from agarose gels. Anal Biochem. 1980 Apr;103(2):264–271. doi: 10.1016/0003-2697(80)90266-3. [DOI] [PubMed] [Google Scholar]
  25. Laughlin C. A., Jones N., Carter B. J. Effect of deletions in adenovirus early region 1 genes upon replication of adeno-associated virus. J Virol. 1982 Mar;41(3):868–876. doi: 10.1128/jvi.41.3.868-876.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Laughlin C. A., Westphal H., Carter B. J. Spliced adenovirus-associated virus RNA. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5567–5571. doi: 10.1073/pnas.76.11.5567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Liu C. C., Simonsen C. C., Levinson A. D. Initiation of translation at internal AUG codons in mammalian cells. Nature. 1984 May 3;309(5963):82–85. doi: 10.1038/309082a0. [DOI] [PubMed] [Google Scholar]
  29. Lusby E. W., Berns K. I. Mapping of the 5' termini of two adeno-associated virus 2 RNAs in the left half of the genome. J Virol. 1982 Feb;41(2):518–526. doi: 10.1128/jvi.41.2.518-526.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mackett M., Smith G. L., Moss B. Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7415–7419. doi: 10.1073/pnas.79.23.7415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Marcus-Sekura C. J., Carter B. J. Chromatin-like structure of adeno-associated virus DNA in infected cells. J Virol. 1983 Oct;48(1):79–87. doi: 10.1128/jvi.48.1.79-87.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Marcus C. J., Laughlin C. A., Carter B. J. Adeno-associated virus RNA transcription in vivo. Eur J Biochem. 1981 Dec;121(1):147–154. doi: 10.1111/j.1432-1033.1981.tb06443.x. [DOI] [PubMed] [Google Scholar]
  33. McCutchan J. H., Pagano J. S. Enchancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl Cancer Inst. 1968 Aug;41(2):351–357. [PubMed] [Google Scholar]
  34. Montell C., Fisher E. F., Caruthers M. H., Berk A. J. Resolving the functions of overlapping viral genes by site-specific mutagenesis at a mRNA splice site. Nature. 1982 Feb 4;295(5848):380–384. doi: 10.1038/295380a0. [DOI] [PubMed] [Google Scholar]
  35. Moore D. D., Denniston-Thompson K., Furth M. E., Williams B. G., Blattner F. R. Construction of chimeric phages and plasmids containing the origin of replication of bacteriophage lambda. Science. 1977 Dec 9;198(4321):1041–1046. doi: 10.1126/science.929185. [DOI] [PubMed] [Google Scholar]
  36. Muzyczka N. Construction of an SV40-derived cloning vector. Gene. 1980 Oct;11(1-2):63–77. doi: 10.1016/0378-1119(80)90087-6. [DOI] [PubMed] [Google Scholar]
  37. Myers M. W., Carter B. J. Adeno-associated virus replication. The effect of L-canavanine or a helper virus mutation on accumulation of viral capsids and progeny single-stranded DNA. J Biol Chem. 1981 Jan 25;256(2):567–570. [PubMed] [Google Scholar]
  38. Nevins J. R. Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product. Cell. 1982 Jul;29(3):913–919. doi: 10.1016/0092-8674(82)90453-6. [DOI] [PubMed] [Google Scholar]
  39. Nevins J. R. Mechanism of activation of early viral transcription by the adenovirus E1A gene product. Cell. 1981 Oct;26(2 Pt 2):213–220. doi: 10.1016/0092-8674(81)90304-4. [DOI] [PubMed] [Google Scholar]
  40. Panicali D., Paoletti E. Construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4927–4931. doi: 10.1073/pnas.79.16.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ricciardi R. P., Jones R. L., Cepko C. L., Sharp P. A., Roberts B. E. Expression of early adenovirus genes requires a viral encoded acidic polypeptide. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6121–6125. doi: 10.1073/pnas.78.10.6121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Richardson W. D., Westphal H. A cascade of adenovirus early functions is required for expression of adeno-associated virus. Cell. 1981 Nov;27(1 Pt 2):133–141. doi: 10.1016/0092-8674(81)90367-6. [DOI] [PubMed] [Google Scholar]
  43. Rigby P. W. Cloning vectors derived from animal viruses. J Gen Virol. 1983 Feb;64(Pt 2):255–266. doi: 10.1099/0022-1317-64-2-255. [DOI] [PubMed] [Google Scholar]
  44. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. Shenk T., Jones N., Colby W., Fowlkes D. Functional analysis of adenovirus-5 host-range deletion mutants defective for transformation of rat embryo cells. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):367–375. doi: 10.1101/sqb.1980.044.01.041. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Srivastava A., Lusby E. W., Berns K. I. Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol. 1983 Feb;45(2):555–564. doi: 10.1128/jvi.45.2.555-564.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Straus S. E., Sebring E. D., Rose J. A. Concatemers of alternating plus and minus strands are intermediates in adenovirus-associated virus DNA synthesis. Proc Natl Acad Sci U S A. 1976 Mar;73(3):742–746. doi: 10.1073/pnas.73.3.742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Treisman R., Green M. R., Maniatis T. cis and trans activation of globin gene transcription in transient assays. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7428–7432. doi: 10.1073/pnas.80.24.7428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Weeks D. L., Jones N. C. E1A control of gene expression is mediated by sequences 5' to the transcriptional starts of the early viral genes. Mol Cell Biol. 1983 Jul;3(7):1222–1234. doi: 10.1128/mcb.3.7.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wieslander L. A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal Biochem. 1979 Oct 1;98(2):305–309. doi: 10.1016/0003-2697(79)90145-3. [DOI] [PubMed] [Google Scholar]
  55. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES