Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1988 Sep;62(9):3356–3363. doi: 10.1128/jvi.62.9.3356-3363.1988

Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein.

J P Trempe 1, B J Carter 1
PMCID: PMC253458  PMID: 2841488

Abstract

Fine-structure mapping of the capsid-specific mRNAs from adeno-associated virus (AAV) revealed an alternate splicing pattern in these RNAs. S1 nuclease and primer extension analyses showed that splicing of these mRNAs occurs at acceptor sites at nucleotide 2228 (major splice) or 2201 (minor splice). Both splice acceptors were ligated to the same 55-nucleotide leader in mature mRNAs. Both species were present in equal amounts in mRNA derived from AAV plasmid-transfected cells. However, when adenovirus infection accompanied the DNA transfection, the major splice predominated over the minor splice. Using cDNA clones of both the major and minor spliced mRNAs, we demonstrated that the largest AAV capsid protein, VP1, was derived from the minor spliced mRNA. The other capsid proteins, VP2 and VP3, came predominantly from the major spliced mRNA. These results, which describe the previously undetected minor splice, provide a mechanism for the production of all three AAV virion proteins.

Full text

PDF
3356

Images in this article

3358Image
on p.3358
3359Image
on p.3359
3359Image
on p.3359
3360Image
on p.3360
3360Image
on p.3360
3361Image
on p.3361

Selected References

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

  1. Becerra S. P., Rose J. A., Hardy M., Baroudy B. M., Anderson C. W. Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7919–7923. doi: 10.1073/pnas.82.23.7919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Cotmore S. F., Tattersall P. The autonomously replicating parvoviruses of vertebrates. Adv Virus Res. 1987;33:91–174. doi: 10.1016/s0065-3527(08)60317-6. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Green M. R., Roeder R. G. Transcripts of the adeno-associated virus genome: mapping of the major RNAs. J Virol. 1980 Oct;36(1):79–92. doi: 10.1128/jvi.36.1.79-92.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Janik J. E., Huston M. M., Rose J. A. Adeno-associated virus proteins: origin of the capsid components. J Virol. 1984 Nov;52(2):591–597. doi: 10.1128/jvi.52.2.591-597.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Jongeneel C. V., Sahli R., McMaster G. K., Hirt B. A precise map of splice junctions in the mRNAs of minute virus of mice, an autonomous parvovirus. J Virol. 1986 Sep;59(3):564–573. doi: 10.1128/jvi.59.3.564-573.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  13. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  14. Labieniec-Pintel L., Pintel D. The minute virus of mice P39 transcription unit can encode both capsid proteins. J Virol. 1986 Mar;57(3):1163–1167. doi: 10.1128/jvi.57.3.1163-1167.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Labow M. A., Hermonat P. L., Berns K. I. Positive and negative autoregulation of the adeno-associated virus type 2 genome. J Virol. 1986 Oct;60(1):251–258. doi: 10.1128/jvi.60.1.251-258.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. Mendelson E., Trempe J. P., Carter B. J. Identification of the trans-acting Rep proteins of adeno-associated virus by antibodies to a synthetic oligopeptide. J Virol. 1986 Dec;60(3):823–832. doi: 10.1128/jvi.60.3.823-832.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Morgan W. R., Ward D. C. Three splicing patterns are used to excise the small intron common to all minute virus of mice RNAs. J Virol. 1986 Dec;60(3):1170–1174. doi: 10.1128/jvi.60.3.1170-1174.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Myers M. W., Laughlin C. A., Jay F. T., Carter B. J. Adenovirus helper function for growth of adeno-associated virus: effect of temperature-sensitive mutations in adenovirus early gene region 2. J Virol. 1980 Jul;35(1):65–75. doi: 10.1128/jvi.35.1.65-75.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rose J. A., Maizel J. V., Jr, Inman J. K., Shatkin A. J. Structural proteins of adenovirus-associated viruses. J Virol. 1971 Nov;8(5):766–770. doi: 10.1128/jvi.8.5.766-770.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. Tratschin J. D., Tal J., Carter B. J. Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product. Mol Cell Biol. 1986 Aug;6(8):2884–2894. doi: 10.1128/mcb.6.8.2884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tratschin J. D., West M. H., Sandbank T., Carter B. J. A human parvovirus, adeno-associated virus, as a eucaryotic vector: transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase. Mol Cell Biol. 1984 Oct;4(10):2072–2081. doi: 10.1128/mcb.4.10.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Trempe J. P., Carter B. J. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation. J Virol. 1988 Jan;62(1):68–74. doi: 10.1128/jvi.62.1.68-74.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Trempe J. P., Mendelson E., Carter B. J. Characterization of adeno-associated virus rep proteins in human cells by antibodies raised against rep expressed in Escherichia coli. Virology. 1987 Nov;161(1):18–28. doi: 10.1016/0042-6822(87)90166-8. [DOI] [PubMed] [Google Scholar]
  32. West M. H., Trempe J. P., Tratschin J. D., Carter B. J. Gene expression in adeno-associated virus vectors: the effects of chimeric mRNA structure, helper virus, and adenovirus VA1 RNA. Virology. 1987 Sep;160(1):38–47. doi: 10.1016/0042-6822(87)90041-9. [DOI] [PubMed] [Google Scholar]

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

RESOURCES