1887

Abstract

The simian immunodeficiency virus (SIV) Nef protein contains a consensus Src-homology 3 (SH3) binding motif. However, no SH3-domain proteins showing strong binding to SIV Nef have yet been found, and its potential capacity for high-affinity SH3 binding has therefore remained unproven. Here we have used phage-display-assisted protein engineering to develop artificial SH3 domains that bind tightly to SIV strain mac (SIVmac) Nef. Substitution of six amino acids in the RT loop region of Hck-SH3 with the sequence E/DGWWG resulted in SH3 domains that bound to SIVmac Nef much better than the natural Hck- or Fyn-SH3 domains. These novel SH3 domains also efficiently associated with SIVmac Nef when co-expressed in 293T cells and displayed a strikingly differential specificity when compared with SH3 domains similarly targeted for binding to human immunodeficiency virus type 1 (HIV-1) Nef. Thus, SIVmac Nef is competent for high-affinity SH3 binding, but its natural SH3 protein partners are likely to be different from those of HIV-1 Nef.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-83-12-3147
2002-12-01
2024-04-25
Loading full text...

Full text loading...

/deliver/fulltext/jgv/83/12/0833147a.html?itemId=/content/journal/jgv/10.1099/0022-1317-83-12-3147&mimeType=html&fmt=ahah

References

  1. Carl S., Iafrate A. J., Lang S. M., Stolte N., Stahl-Hennig C., Matz-Rensing K., Fuchs D., Skowronski J., Kirchhoff F. 2000; Simian immunodeficiency virus containing mutations in N-terminal tyrosine residues and in the PxxP motif in Nef replicates efficiently in rhesus macaques. Journal of Virology 74:4155–4164
    [Google Scholar]
  2. Collette Y., Arold S., Picard C., Janvier K., Benichou S., Benarous R., Olive D., Dumas C. 2000; HIV-2 and SIV Nef proteins target different Src family SH3 domains than does HIV-1 Nef because of a triple amino acid substitution. Journal of Biological Chemistry 275:4171–4176
    [Google Scholar]
  3. Cullen B. R. 1999; HIV-1 Nef protein: an invitation to a kill. Nature Medicine 5:985–986
    [Google Scholar]
  4. Dalgarno D. C., Botfield M. C., Rickles R. J. 1997; SH3 domains and drug design: ligands, structure, and biological function. Biopolymers 43:383–400
    [Google Scholar]
  5. Geyer M., Fackler O. T., Peterlin B. M. 2001; Structure–function relationships in HIV-1 Nef. EMBO Reports 2:580–585
    [Google Scholar]
  6. Greenway A. L., Dutartre H., Allen K., McPhee D. A., Olive D., Collette Y. 1999; Simian immunodeficiency virus and human immunodeficiency virus type 1 Nef proteins show distinct patterns and mechanisms of Src kinase activation. Journal of Virology 73:6152–6158
    [Google Scholar]
  7. Hanna Z., Kay D. G., Rebai N., Guimond A., Jothy S., Jolicoeur P. 1998; Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 95:163–175
    [Google Scholar]
  8. Hanna Z., Weng X., Kay D. G., Poudrier J., Lowell C., Jolicoeur P. 2001; The pathogenicity of human immunodeficiency virus (HIV) type 1 Nef in CD4C/HIV transgenic mice is abolished by mutation of its SH3-binding domain, and disease development is delayed in the absence of Hck. Journal of Virology 75:9378–9392
    [Google Scholar]
  9. Hiipakka M., Poikonen K., Saksela K. 1999; SH3 domains with high affinity and engineered ligand specificities targeted to HIV-1 Nef. Journal of Molecular Biology 293:1097–1106
    [Google Scholar]
  10. Hiipakka M., Huotari P., Manninen A., Renkema G. H., Saksela K. 2001; Inhibition of cellular functions of HIV-1 Nef by artificial SH3 domains. Virology 286:152–159
    [Google Scholar]
  11. Jacobsson K., Frykberg L. 1996; Phage display shot-gun cloning of ligand-binding domains of prokaryotic receptors approaches 100% correct clones. Biotechniques 20:1070–1081
    [Google Scholar]
  12. Khan I. H., Sawai E. T., Antonio E., Weber C. J., Mandell C. P., Montbriand P., Luciw P. A. 1998; Role of the SH3-ligand domain of simian immunodeficiency virus Nef in interaction with Nef-associated kinase and simian AIDS in rhesus macaques. Journal of Virology 72:5820–5830
    [Google Scholar]
  13. Lang S. M., Iafrate A. J., Stahl-Hennig C., Kuhn E. M., Nisslein T., Kaup F. J., Haupt M., Hunsmann G., Skowronski J., Kirchhoff F. 1997; Association of simian immunodeficiency virus Nef with cellular serine/threonine kinases is dispensable for the development of AIDS in rhesus macaques. Nature Medicine 3:860–865
    [Google Scholar]
  14. Lee C. H., Leung B., Lemmon M. A., Zheng J., Cowburn D., Kuriyan J., Saksela K. 1995; A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein. EMBO Journal 14:5006–5015
    [Google Scholar]
  15. Lee C. H., Saksela K., Mirza U. A., Chait B. T., Kuriyan J. 1996; Crystal structure of the conserved core of HIV-1 Nef complexed with a Src family SH3 domain. Cell 85:931–942
    [Google Scholar]
  16. Manninen A., Hiipakka M., Vihinen M., Lu W., Mayer B. J., Saksela K. 1998; Domain binding function of HIV-1 Nef is required for association with a PAK-related kinase. Virology 250:273–282
    [Google Scholar]
  17. Manninen A., Renkema G. H., Saksela K. 2000; Synergistic activation of NFAT by HIV-1 Nef and the Ras/MAPK pathway. Journal of Biological Chemistry 275:16513–16517
    [Google Scholar]
  18. Manninen A., Huotari P., Hiipakka M., Renkema G. H., Saksela K. 2001; Activation of NFAT-dependent gene expression by Nef: conservation among divergent Nef alleles, dependence on SH3 binding and membrane association, and cooperation with protein kinase C-theta. Journal of Virology 75:3034–3037
    [Google Scholar]
  19. Mayer B. J. 2001; SH3 domains: complexity in moderation. Journal of Cell Science 114:1253–1263
    [Google Scholar]
  20. Mizushima S., Nagata S. 1990; pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Research 18:5322
    [Google Scholar]
  21. Picard C., Greenway A., Holloway G., Olive D., Collette Y. 2002; Interaction with simian Hck tyrosine kinase reveals convergent evolution of the Nef protein from simian and human immunodeficiency viruses despite differential molecular surface usage. Virology 295:320–327
    [Google Scholar]
  22. Piguet V., Schwartz O., Le Gall S., Trono D. 1999; The downregulation of CD4 and MHC-I by primate lentiviruses: a paradigm for the modulation of cell surface receptors. Immunological Reviews 168:51–63
    [Google Scholar]
  23. Renkema G. H., Saksela K. 2000; Interactions of HIV-1 Nef with cellular signal transducing proteins. Frontiers in Biosciences 5:D268–D283
    [Google Scholar]
  24. Saksela K., Cheng G., Baltimore D. 1995; Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO Journal 14:484–491
    [Google Scholar]
  25. Shugars D. C., Smith M. S., Glueck D. H., Nantermet P. V., Seillier-Moiseiwitsch F., Swanstrom R. 1993; Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo. Journal of Virology 67:4639–4650
    [Google Scholar]
  26. Swigut T., Iafrate A. J., Muench J., Kirchhoff F., Skowronski J. 2000; Simian and human immunodeficiency virus Nef proteins use different surfaces to downregulate class I major histocompatibility complex antigen expression. Journal of Virology 74:5691–5701
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-83-12-3147
Loading
/content/journal/jgv/10.1099/0022-1317-83-12-3147
Loading

Data & Media loading...

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