1887

Abstract

Using site-directed mutagenesis, we tested whether a potential integrin-binding site, (composed of the amino acids RGD) which is predicted in the adeno-associated virus 2 (AAV-2) capsid open reading frame (ORF), plays a role in the infectivity of AAV-2. Nucleotide sequencing of wild-type and mutant capsid protein-coding sequences, however, revealed discrepancies with the published sequence data at several positions, including a frameshift in the carboxy terminus which cancels the RGD motif and extends the capsid ORF by 27 amino acis. This sequence was confirmed by protein sequencing of proteolytic fragments of VP3. Thus, the virus mutant (pTAV-p), in which the intention was to exchange D of the putative RGD motif for E, resulted in replacing I by S in the newly established ORF. A second virus mutant (pTAV-d), in which the intention was to delete the RGD peptide, in fact gave a shift into the ORF of the originally published sequence. The pTAV-p mutant showed a strongly reduced infectivity compared to wild- type AAV-2, whereas pTAV-d was not infectious at all. Neither mutant accumulated viral ssDNA as detected by Hirt extraction. Analysis of virus particle formation and subcellular localization of the capsid proteins revealed a defect of the mutant capsid proteins in capsid assembly. This shows that the newly established C-terminal sequence of the AAV capsid proteins plays an important role in viral assembly.

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1994-12-01
2021-10-16
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References

  1. Becerra S. P., Rose J. A., Hardy M., Baroudy B. M., Anderson C. W. 1985; Direct mapping of adeno-associated virus proteins B and C: a possible ACG initiation codon. Proceedings of the National Academy of Sciences U.S.A: 827919–7923
    [Google Scholar]
  2. Becerra S. P., Koczot F., Fabish P., Rose J. A. 1988; Synthesis of adeno-associated virus structural proteins requires both alternative mRNA splicing and alternative initiations from a single transcript. Journal of Virology 62:2745–2754
    [Google Scholar]
  3. Berns K. I., Bohenzky R. A. 1987; Adeno-associated virus: an update. Advances in Virus Research 32:243–306
    [Google Scholar]
  4. Berns K. I. 1990; Parvovirus replication. Microbiological Reviews 54:316–329
    [Google Scholar]
  5. Cassinotti P., Weitz M., Tratschin J.-D. 1988; Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced mRNA coding for virus capsid protein 1. Virology 167:176–184
    [Google Scholar]
  6. Chen C., Okayama H. 1987; High-efficiency transformation of mammalian cells by plasmid DNA. Molecular and Cellular Biology 7:2745–2752
    [Google Scholar]
  7. Chejanovsky N., Carter B. 1989; Mutagenesis of an AUG codon in the adeno-associated rep gene: effects on viral DNA replication. Virology 173:120–128
    [Google Scholar]
  8. Fox G., Parry N. R., Barnett P. V., Mcginn B., Rowlands D. J., Brown F. 1989; The cell attachment site on foot-and- mouth disease virus includes the amino acid sequence RGD (arginine- glycine-aspartic acid). Journal of General Virology 70:625–637
    [Google Scholar]
  9. Harlow E., Lane D. 1988; Immunoblotting. In Antibodies.A Laboratory Manual pp 471–511 Harlow E., Lane D. Edited by New York: Cold Spring Harbor;
    [Google Scholar]
  10. Heilbronn R., Bürkle A., Stephan S., Zur Hausen H. 1990; The adeno-associated rep gene suppresses herpes simplex virus- induced DNA amplification. Journal of Virology 64:3012–3018
    [Google Scholar]
  11. Hermonat P. L., Labow M. A., Wright R., Berns K. I., Muzyczka N. 1984; Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants. Journal of Virology 51:329–339
    [Google Scholar]
  12. Hynes R. O. 1992; Integrins: versatility, modulations, and signaling in cell adhesion. Cell 69:11–25
    [Google Scholar]
  13. Janik J. E., Huston M. M., Rose J. A. 1984; Adeno-associated virus proteins: origin of the capsid components. Journal of Virology 52:591–597
    [Google Scholar]
  14. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
    [Google Scholar]
  15. Laughlin C. A., Tratschin J., Coon H., Carter B. J. 1983; Cloning of infectious adeno-associated virus genomes in bacterial plasmids. Gene 23:65–73
    [Google Scholar]
  16. Mendelson E., Trempe J. P., Carter B. J. 1986; Identification of trans-active Rep proteins of adeno-associated virus by antibodies to a synthetic oligopeptide. Journal of Virology 60:823–832
    [Google Scholar]
  17. Muzyczka N. 1992; Use of adeno-associated virus as a general transduction vector for mammalian cells. Current Topics in Microbiology and Immunology 158:97–129
    [Google Scholar]
  18. Muralidhar S., Becerra S. P., Rose J. A. 1994; Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity. Journal of Virology 68:170–176
    [Google Scholar]
  19. Myers M. W., Carter B. J. 1980; Assembly of adeno-associated virus. Virology 102:71–82
    [Google Scholar]
  20. Myers M. W., Carter B. J. 1981; Adeno-associated virus replication. Journal of Biological Chemistry 265:567–570
    [Google Scholar]
  21. Redemann B. E., Mendelson E., Carter B. J. 1989; Adeno- associated virus Rep protein synthesis during productive infection. Journal of Virology 63:873–882
    [Google Scholar]
  22. Roivainen M., Hyypia T., Piirainen L., Kalkkinen N., Stanway G., Hovi T. 1991; RGD-dependent entry of coxsackievirus A9 into host cells and its bypass after cleavage of VP1 protein by intestinal proteases. Journal of Virology 65:4735–4740
    [Google Scholar]
  23. Ruffing M., Zentgraf H., Kleinschmidt J. A. 1992; Assembly of viruslike particles by recombinant structural proteins of adeno- associated virus type 2 in insect cells. Journal of Virology 66:6922–6930
    [Google Scholar]
  24. Ruoslahti E., Pierschbacher M. D. 1986; Arg-Gly-Asp: a versatile cell recognition signal. Cell 44:517–518
    [Google Scholar]
  25. Ruoslahti E., Pierschbacher M. D. 1987; New perspectives in cell adhesion: RGD and integrins. Science 238:491–497
    [Google Scholar]
  26. Samulski R. J., Berns K. I., Tan M., Muzyczka N. 1982; Cloning of adeno-associated virus into pBR322: rescue of intact virus from recombinant plasmid in human cells. Proceedings of the National Academy of Sciences U.S.A.: 792077–2081
    [Google Scholar]
  27. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences U.S.A: 745463–5467
    [Google Scholar]
  28. Smuda J. W., Carter B. J. 1991; Adeno-associated viruses having nonsense mutations in the capsid genes: growth in mammalian cells containing an inducible amber suppressor. Virology 184:310–318
    [Google Scholar]
  29. Srivastava A., Lusby E.W., Berns K. I. 1983; Nucleotide sequence and organization of adeno-associated virus 2 genome. Journal of Virology 45:555–564
    [Google Scholar]
  30. Taylor J. W., Ott J., Eckstein F. 1985; The rapid generation of oligonucleotide-directed mutations at high frequency using phospho- thioate-modified DNA. Nucleic Acids Research 13:8765–8785
    [Google Scholar]
  31. Thomas J. O., Kornberg R. D. 1975; An octamer of histones in chromatin and free in solution. Proceedings of the National Academy of Sciences U.S.A: 724350–4354
    [Google Scholar]
  32. Tratschin J. -D., Miller I. L., Carter B. J. 1984; Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function. Journal of Virology 51:611–619
    [Google Scholar]
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