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Structural and functional studies of ALIX interactions with YPXnL late domains of HIV-1 and EIAV

Nature Structural & Molecular Biology volume 15, pages 4349 (2008) | Download Citation

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Abstract

Retrovirus budding requires short peptide motifs (late domains) located within the viral Gag protein that function by recruiting cellular factors. The YPXnL late domains of HIV and other lentiviruses recruit the protein ALIX (also known as AIP1), which also functions in vesicle formation at the multivesicular body and in the abscission stage of cytokinesis. Here, we report the crystal structures of ALIX in complex with the YPXnL late domains from HIV-1 and EIAV. The two distinct late domains bind at the same site on the ALIX V domain but adopt different conformations that allow them to make equivalent contacts. Binding studies and functional assays verified the importance of key interface residues and revealed that binding affinities are tuned by context-dependent effects. These results reveal how YPXnL late domains recruit ALIX to facilitate virus budding and how ALIX can bind YPXnL sequences with both n = 1 and n = 3.

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References

  1. 1.

    & Retrovirus budding. Virus Res. 106, 87–102 (2004).

  2. 2.

    Late budding domains and host proteins in enveloped virus release. Virology 344, 55–63 (2006).

  3. 3.

    & Retrovirus budding. Annu. Rev. Cell Dev. Biol. 20, 395–425 (2004).

  4. 4.

    et al. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 107, 55–65 (2001).

  5. 5.

    , , & Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release. Proc. Natl. Acad. Sci. USA 88, 3195–3199 (1991).

  6. 6.

    , , & p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease. J. Virol. 69, 6810–6818 (1995).

  7. 7.

    , , & Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function. Proc. Natl. Acad. Sci. USA 99, 955–960 (2002).

  8. 8.

    , & HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nat. Med. 7, 1313–1319 (2001).

  9. 9.

    et al. Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). Proc. Natl. Acad. Sci. USA 98, 7724–7729 (2001).

  10. 10.

    , , , & AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 114, 689–699 (2003).

  11. 11.

    et al. The protein network of HIV budding. Cell 114, 701–713 (2003).

  12. 12.

    , , , & YPXL/I is a protein interaction motif recognized by Aspergillus PalA and its human homologue, AIP1/Alix. Mol. Cell. Biol. 23, 1647–1655 (2003).

  13. 13.

    , , & Equine infectious anemia virus utilizes a YXXL motif within the late assembly domain of the Gag p9 protein. J. Virol. 71, 6541–6546 (1997).

  14. 14.

    , , , & Functions of early (AP-2) and late (AIP1/ALIX) endocytic proteins in equine infectious anemia virus budding. J. Biol. Chem. 280, 40474–40480 (2005).

  15. 15.

    , , & Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins. Proc. Natl. Acad. Sci. USA 100, 12414–12419 (2003).

  16. 16.

    & The ESCRT complexes: structure and mechanism of a membrane-trafficking network. Annu. Rev. Biophys. Biomol. Struct. 35, 277–298 (2006).

  17. 17.

    The multiple personalities of Alix. J. Cell Sci. 119, 3025–3032 (2006).

  18. 18.

    & The emerging shape of the ESCRT machinery. Nat. Rev. Mol. Cell Biol. 8, 355–368 (2007).

  19. 19.

    & Parallels between cytokinesis and retroviral budding- a role for the ESCRT machinery. Science 316, 1908–1912 (2007).

  20. 20.

    et al. Structural basis for budding by the ESCRT-III factor CHMP3. Dev. Cell 10, 821–830 (2006).

  21. 21.

    et al. Human ESCRT-II complex and its role in human immunodeficiency virus type 1 release. J. Virol. 80, 9465–9480 (2006).

  22. 22.

    et al. Structural basis for endosomal targeting by the Bro1 domain. Dev. Cell 8, 937–947 (2005).

  23. 23.

    et al. Protein-protein interactions of ESCRT complexes in the yeast Saccharomyces cerevisiae. Traffic 5, 194–210 (2004).

  24. 24.

    , , & Structure of the Tsg101 UEV domain in complex with the PTAP motif of the HIV-1 p6 protein. Nat. Struct. Biol. 9, 812–817 (2002).

  25. 25.

    et al. Structure and functional interactions of the Tsg101 UEV domain. EMBO J. 21, 2397–2406 (2002).

  26. 26.

    et al. Structural and biochemical studies of ALIX/AIP1 and its role in retrovirus budding. Cell 128, 841–852 (2007).

  27. 27.

    , & Potent rescue of human immunodeficiency virus type 1 late domain mutants by ALIX/AIP1 that depends on its CHMP4 binding site. J. Virol. 81, 6614–6622 (2007).

  28. 28.

    et al. New simian immunodeficiency virus infecting De Brazza's monkeys (Cercopithecus neglectus): evidence for a cercopithecus monkey virus clade. J. Virol. 78, 7748–7762 (2004).

  29. 29.

    , , , & Structural basis for viral late-domain binding to Alix. Nat. Struct. Mol. Biol. 14, 194–199 (2007).

  30. 30.

    , , , & Bro1 is an endosome-associated protein that functions in the MVB pathway in Saccharomyces cerevisiae. J. Cell Sci. 116, 1893–1903 (2003).

  31. 31.

    , , & Cloning of AIP1, a novel protein that associates with the apoptosis-linked gene ALG-2 in a Ca2+-dependent reaction. J. Biol. Chem. 274, 1533–1540 (1999).

  32. 32.

    , , & Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein. Cell Death Differ. 6, 124–129 (1999).

  33. 33.

    , , , & The glioma-associated protein SETA interacts with AIP1/Alix and ALG-2 and modulates apoptosis in astrocytes. J. Biol. Chem. 275, 19275–19281 (2000).

  34. 34.

    et al. Alix (ALG-2-interacting protein X), a protein involved in apoptosis, binds to endophilins and induces cytoplasmic vacuolization. J. Biol. Chem. 277, 29108–29115 (2002).

  35. 35.

    , & POSH, a scaffold protein for JNK signaling, binds to ALG-2 and ALIX in Drosophila. FEBS Lett. 580, 3296–3300 (2006).

  36. 36.

    , , , & An Alix fragment potently inhibits HIV-1 budding: characterization of binding to retroviral YPXL late domains. J. Biol. Chem. 282, 3847–3855 (2007).

  37. 37.

    , & Context-dependent effects of L domains and ubiquitination on viral budding. J. Virol. 78, 5554–5563 (2004).

  38. 38.

    , , & Degradation of AP2 during reticulocyte maturation enhances binding of hsc70 and Alix to a common site on TFR for sorting into exosomes. Traffic 5, 181–193 (2004).

  39. 39.

    et al. Interactions between Nef and AIP1 proliferate multivesicular bodies and facilitate egress of HIV-1. Retrovirology 3, 33 (2006).

  40. 40.

    & Processing of X-ray diffraction data collected in oscillation mode. In Methods in Enzymology Vol. 276 (eds. Carter, C.W., Jr. & Sweet, R.M.) 307–326 (Academic Press, New York, 1997).

  41. 41.

    , & Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).

  42. 42.

    & TLSMD web server for the generation of multi-group TLS models. J. Appl. Cryst. 39, 109–111 (2006).

  43. 43.

    & Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D Biol. Crystallogr. 62, 439–450 (2006).

  44. 44.

    , , , & Efficient anisotropic refinement of macromolecular structures using FFT. Acta Crystallogr. D 55, 247–255 (1999).

  45. 45.

    , , , & TLSANL: TLS parameter analysis program for segmented anisotropic refinement of macromolecular structures. J. Appl. Cryst. 26, 622–624 (1993).

  46. 46.

    CCP4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  47. 47.

    , , & Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

  48. 48.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

  49. 49.

    , & Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 231, 1049–1067 (1993).

  50. 50.

    , & Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal. Biochem. 198, 268–277 (1991).

  51. 51.

    Improving biosensor analysis. J. Mol. Recognit. 12, 279–284 (1999).

  52. 52.

    The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, California, USA, 2002).

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Acknowledgements

We thank P. Bieniasz (Aaron Diamond AIDS Research Center) for kindly providing various HIV-1 proviral plasmids and H. Göttlinger (University of Massachusettes) for helpful discussions. This work was funded by US National Institutes of Health (NIH) grants GM082534 (C.P.H., W.I.S.) and AI051174 (W.I.S.). Portions of this research were performed at the Stanford Synchrotron Radiation Laboratory (SSRL), a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the NIH, National Center for Research Resources, Biomedical Technology Program and National Institute of General Medical Sciences. The Center for Biomolecular Interactions Analysis at the University of Utah is funded in part by NIH grant 1S10RR016787-01 (D.G.M.).

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Author notes

    • Qianting Zhai
    •  & Robert D Fisher

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650, USA.

    • Qianting Zhai
    • , Robert D Fisher
    • , Hyo-Young Chung
    • , Wesley I Sundquist
    •  & Christopher P Hill
  2. Center for Biomolecular Interactions Analysis, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650, USA.

    • David G Myszka

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Correspondence to Wesley I Sundquist or Christopher P Hill.

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https://doi.org/10.1038/nsmb1319

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