1887

Abstract

Adeno-associated virus (AAV) capsid assembly occurs in the nucleus. Newly synthesized capsid proteins VP1, VP2 and VP3 contain several basic regions (BRs), which may act as nuclear localization signals (NLSs). Mutation of BR2 and BR3, located at the VP1 and VP2 N termini, marginally reduced nuclear uptake of VP1 or VP2, but not of VP3, when expressed in the context of the whole AAV type 2 (AAV2) genome. Combined mutation of BR1, BR2 and BR3 resulted in capsids with slightly reduced amounts of VP1. Expression of isolated VP1/2 N termini revealed an influence of BR3 on nuclear transport, whilst BR1 or BR2 had no effect. However, deletion of an N-terminal fragment in front of the BR elements strongly reduced nuclear uptake of VP1/2 N termini. Mutation of BR4, present in all three capsid proteins, led to their retention in the cytoplasm and to the formation of speckles, resulting in a lack of capsid formation and a significant reduction in VP levels. In a VP fragment comprising BR2, BR3 and BR4, the BR4 element was not necessary for nuclear localization. Mutation of BR5 in the C-terminal part of the VPs resulted in a speckled protein distribution in the nucleus, strongly reduced capsid assembly, and low VP1 and VP2 levels. Taken together, these results showed that BR2 and BR3 have a weak influence on nuclear transport of VP1 and VP2, whilst combined mutation of BR1, BR2 and BR3 influences the stoichiometry of VPs in assembled capsids. BR4 and BR5 play a crucial role in capsid assembly but have no NLS activity.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.043232-0
2012-09-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jgv/93/9/1887.html?itemId=/content/journal/jgv/10.1099/vir.0.043232-0&mimeType=html&fmt=ahah

References

  1. Becerra S. P., Rose J. A., Hardy M., Baroudy B. M., Anderson C. W. 1985; Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon. Proc Natl Acad Sci U S A 82:7919–7923 [View Article][PubMed]
    [Google Scholar]
  2. Becerra S. P., Koczot F., Fabisch P., Rose J. A. 1988; Synthesis of adeno-associated virus structural proteins requires both alternative mRNA splicing and alternative initiations from a single transcript. J Virol 62:2745–2754[PubMed]
    [Google Scholar]
  3. Buller R. M., Rose J. A. 1978; Characterization of adenovirus-associated virus-induced polypeptides in KB cells. J Virol 25:331–338[PubMed]
    [Google Scholar]
  4. Cassinotti P., Weitzand 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. Virology 167:176–184 [View Article]
    [Google Scholar]
  5. Cokol M., Nair R., Rost B. 2000; Finding nuclear localization signals. EMBO Rep 1:411–415 [View Article][PubMed]
    [Google Scholar]
  6. Dingwall C., Laskey R. A. 1991; Nuclear targeting sequences – a consensus?. Trends Biochem Sci 16:478–481 [View Article][PubMed]
    [Google Scholar]
  7. Fried H., Kutay U. 2003; Nucleocytoplasmic transport: taking an inventory. Cell Mol Life Sci 60:1659–1688 [View Article][PubMed]
    [Google Scholar]
  8. Garcia-Bustos J., Heitman J., Hall M. N. 1991; Nuclear protein localization. Biochim Biophys Acta 1071:83–101[PubMed] [CrossRef]
    [Google Scholar]
  9. Grieger J. C., Snowdy S., Samulski R. J. 2006; Separate basic region motifs within the adeno-associated virus capsid proteins are essential for infectivity and assembly. J Virol 80:5199–5210 [View Article][PubMed]
    [Google Scholar]
  10. Grimm D., Kern A., Pawlita M., Ferrari F., Samulski R., Kleinschmidt J. 1999; Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2. Gene Ther 6:1322–1330 [View Article][PubMed]
    [Google Scholar]
  11. Heilbronn R., Bürkle A., Stephan S., zur Hausen H. 1990; The adeno-associated virus rep gene suppresses herpes simplex virus-induced DNA amplification. J Virol 64:3012–3018[PubMed]
    [Google Scholar]
  12. Hoque M., Ishizu K., Matsumoto A., Han S.-I., Arisaka F., Takayama M., Suzuki K., Kato K., Kanda T. other authors 1999; Nuclear transport of the major capsid protein is essential for adeno-associated virus capsid formation. J Virol 73:7912–7915[PubMed]
    [Google Scholar]
  13. Kerr J. R., Cotmore S. F., Bloom M. E., Linden R. M., Parrish C. R. editors 2006 Parvoviruses London: Hodder Arnold;
    [Google Scholar]
  14. Kleinschmidt J. A., King J. A. 2006; Molecular interactions involved in assembling the viral particle and packaging the genome. In Parvoviruses pp. 305–319 Edited by Kerr J. R., Cotmore S. F., Bloom M. E., Linden R. M., Parrish C. R. London: Hodder Arnold;
    [Google Scholar]
  15. Kronenberg S., Kleinschmidt J. A., Böttcher B. 2001; Electron cryo-microscopy and image reconstruction of adeno-associated virus type 2 empty capsids. EMBO Rep 2:997–1002 [View Article][PubMed]
    [Google Scholar]
  16. Lee B. J., Cansizoglu A. E., Süel K. E., Louis T. H., Zhang Z., Chook Y. M. 2006; Rules for nuclear localization sequence recognition by karyopherin beta 2. Cell 126:543–558 [View Article][PubMed]
    [Google Scholar]
  17. Lombardo E., Ramírez J. C., Agbandje-McKenna M., Almendral J. M. 2000; A β-stranded motif drives capsid protein oligomers of the parvovirus minute virus of mice into the nucleus for viral assembly. J Virol 74:3804–3814 [View Article][PubMed]
    [Google Scholar]
  18. Lombardo E., Ramírez J. C., Garcia J., Almendral J. M. 2002; Complementary roles of multiple nuclear targeting signals in the capsid proteins of the parvovirus minute virus of mice during assembly and onset of infection. J Virol 76:7049–7059 [View Article][PubMed]
    [Google Scholar]
  19. 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. J Virol 68:170–176[PubMed]
    [Google Scholar]
  20. Nigg E. A. 1997; Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386:779–787 [View Article][PubMed]
    [Google Scholar]
  21. 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. J Virol 66:6922–6930[PubMed]
    [Google Scholar]
  22. Sayle R. A., Milner-White E. J. 1995; rasmol: Biomolecular graphics for all. Trends Biochem Sci 20:374–376 [View Article][PubMed]
    [Google Scholar]
  23. Sonntag F., Bleker S., Leuchs B., Fischer R., Kleinschmidt J. A. 2006; Adeno-associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus. J Virol 80:11040–11054 [View Article][PubMed]
    [Google Scholar]
  24. Sonntag F., Schmidt K., Kleinschmidt J. A. 2010; A viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proc Natl Acad Sci U S A 107:10220–10225 [View Article][PubMed]
    [Google Scholar]
  25. Tran E. J., Wente S. R. 2006; Dynamic nuclear pore complexes: life on the edge. Cell 125:1041–1053 [View Article][PubMed]
    [Google Scholar]
  26. Trempe J. P., Carter B. J. 1988; Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein. J Virol 62:3356–3363[PubMed]
    [Google Scholar]
  27. Valle N., Riolobos L., Almendral J. M. 2006; Synthesis, post-translational modification and trafficking of the parvovirus structural polypeptides. In Parvoviruses pp. 344–358 Edited by Kerr J. R., Cotmore S. F., Bloom M. E., Linden R. M., Parrish C. R. London: Hodder Arnold;
    [Google Scholar]
  28. Vihinen-Ranta M., Kakkola L., Kalela A., Vilja P., Vuento M. 1997; Characterization of a nuclear localization signal of canine parvovirus capsid proteins. Eur J Biochem 250:389–394 [View Article][PubMed]
    [Google Scholar]
  29. Wistuba A., Weger S., Kern A., Kleinschmidt J. A. 1995; Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins. J Virol 69:5311–5319[PubMed]
    [Google Scholar]
  30. Wistuba A., Kern A., Weger S., Grimm D., Kleinschmidt J. A. 1997; Subcellular compartmentalization of adeno-associated virus type 2 assembly. J Virol 71:1341–1352[PubMed]
    [Google Scholar]
  31. Wobus C. E., Hügle-Dörr B., Girod A., Petersen G., Hallek M., Kleinschmidt J. A. 2000; Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2–cell interaction and neutralization of AAV-2 infection. J Virol 74:9281–9293 [View Article][PubMed]
    [Google Scholar]
  32. Wu P., Xiao W., Conlon T., Hughes J., Agbandje-McKenna M., Ferkol T., Flotte T., Muzyczka N. 2000; Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism. J Virol 74:8635–8647 [View Article][PubMed]
    [Google Scholar]
  33. Xie Q., Bu W., Bhatia S., Hare J., Somasundaram T., Azzi A., Chapman M. S. 2002; The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci U S A 99:10405–10410 [View Article][PubMed]
    [Google Scholar]
  34. Yuan W., Parrish C. R. 2001; Canine parvovirus capsid assembly and differences in mammalian and insect cells. Virology 279:546–557 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.043232-0
Loading
/content/journal/jgv/10.1099/vir.0.043232-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error