Abstract
The modular protein Alix is a central node in endosomal-lysosomal trafficking and the budding of human immunodeficiency virus (HIV)-1. The Gag p6 protein of HIV-1 contains a LYPxnLxxL motif that is required for Alix-mediated budding and binds a region of Alix spanning residues 360–702. The structure of this fragment of Alix has the shape of the letter 'V' and is termed the V domain. The V domain has a topologically complex arrangement of 11 α-helices, with connecting loops that cross three times between the two arms of the V. The conserved residue Phe676 is at the center of a large hydrophobic pocket and is crucial for binding to a peptide model of HIV-1 p6. Overexpression of the V domain inhibits HIV-1 release from cells. This inhibition of release is reversed by mutations that block binding of the Alix V domain to p6.
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References
Freed, E.O. HIV-1 Gag proteins: diverse functions in the virus life cycle. Virology 251, 1–15 (1998).
Bieniasz, P.D. Late budding domains and host proteins in enveloped virus release. Virology 344, 55–63 (2006).
Demirov, D.G. & Freed, E.O. Retrovirus budding. Virus Res. 106, 87–102 (2004).
Morita, E. & Sundquist, W.I. Retrovirus budding. Annu. Rev. Cell Dev. Biol. 20, 395–425 (2004).
Demirov, D.G., Ono, A., Orenstein, J.M. & Freed, E.O. 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).
Garrus, J.E. et al. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 107, 55–65 (2001).
Goila-Gaur, R., Demirov, D.G., Orenstein, J.M., Ono, A. & Freed, E.O. Defects in human immunodeficiency virus budding and endosomal sorting induced by TSG101 overexpression. J. Virol. 77, 6507–6519 (2003).
Martin-Serrano, J., Zang, T. & Bieniasz, P.D. HIV-I and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nat. Med. 7, 1313–1319 (2001).
Martin-Serrano, J., Zang, T. & Bieniasz, P.D. Role of ESCRT-I in retroviral budding. J. Virol. 77, 4794–4804 (2003).
VerPlank, L. 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).
Yuan, B., Li, X.Q. & Goff, S.P. Mutations altering the Moloney murine leukemia virus p12 Gag protein affect virion production and early events of the virus life cycle. EMBO J. 18, 4700–4710 (1999).
Kikonyogo, A. et al. Proteins related to the Nedd4 family of ubiquitin protein ligases interact with the L domain of Rous sarcoma virus and are required for gag budding from cells. Proc. Natl. Acad. Sci. USA 98, 11199–11204 (2001).
Martin-Serrano, J., Eastman, S.W., Chung, W. & Bieniasz, P.D. HECT ubiquitin ligases link viral and cellular PPXY motifs to the vacuolar protein-sorting pathway. J. Cell Biol. 168, 89–101 (2005).
Strack, B., Calistri, A., Accola, M.A., Palu, G. & Gottlinger, H.G. A role for ubiquitin ligase recruitment in retrovirus release. Proc. Natl. Acad. Sci. USA 97, 13063–13068 (2000).
Vito, P., Pellegrini, L., Guiet, C. & D'Adamio, L. 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).
Missotten, M., Nichols, A., Rieger, K. & Sadoul, R. 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).
Chen, C., Vincent, O., Jin, J., Weisz, O.A. & Montelaro, R.C. Functions of early (AP-2) and late (AIP1/ALIX) endocytic proteins in equine infectious anemia virus budding. J. Biol. Chem. 280, 40474–40480 (2005).
Strack, B., Calistri, A., Craig, S., Popova, E. & Gottlinger, H.G. AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 114, 689–699 (2003).
von Schwedler, U.K. et al. The protein network of HIV budding. Cell 114, 701–713 (2003).
Odorizzi, G. The multiple personalities of Alix. J. Cell Sci. 119, 3025–3032 (2006).
Sadoul, R. Do Alix and ALg-2 really control endosomes for better or for worse? Biol. Cell 98, 69–77 (2006).
Pan, S. et al. Involvement of the adaptor protein alix in actin cytoskeleton assembly. J. Biol. Chem. 281, 34640–34650 (2006).
Kim, J. et al. Structural basis for endosomal targeting by the Bro1 domain. Dev. Cell 8, 937–947 (2005).
Munshi, U., Kim, J., Nagashima, K., Hurley, J.H. & Freed, E.O. An Alix fragment potently inhibits HIV-1 budding: characterization of binding to retroviral YPXL late domains. J. Biol. Chem. published online 8 December 2006 (doi:10.1074/jbc.M607489200).
Bowers, K. & Stevens, T.H. Protein transport from the late Golgi to the vacuole in the yeast Saccharomyces cerevisiae. Biochim. Biophys. Acta 1744, 438–454 (2005).
Hurley, J.H. & Emr, S.D. The ESCRT complexes: structure and mechanism of a membrane-trafficking network. Annu. Rev. Biophys. Biomol. Struct. 35, 277–298 (2006).
Babst, M. Protein's final ESCRT. Traffic 6, 2–9 (2005).
van der Goot, F.G. & Gruenberg, J. Intra-endosomal membrane traffic. Trends Cell Biol. 16, 514–521 (2006).
Slagsvold, T., Pattni, K., Malerod, L. & Stenmark, H. Endosomal and non-endosomal functions of ESCRT proteins. Trends Cell Biol. 16, 317–326 (2006).
Chen, B., Borinstein, S.C., Gillis, J., Sykes, V.W. & Bogler, O. The glioma-associated protein SETA interacts with AIP1/Alix and AZIG-2 and modulates apoptosis in astrocytes. J. Biol. Chem. 275, 19275–19281 (2000).
Chatellard-Causse, C. 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).
Matsuo, H. et al. Role of LBPA and Alix in multivesicular liposome formation and endosome organization. Science 303, 531–534 (2004).
Fossen, T. et al. Solution structure of the human immunodeficiency virus type 1 p6 protein. J. Biol. Chem. 280, 42515–42527 (2005).
Hurley, J.H. Leucine in the sky with diamonds. Structure 11, 1192–1193 (2003).
Hoellerer, M.K. et al. Molecular recognition of paxillin LD motifs by the focal adhesion targeting domain. Structure 11, 1207–1217 (2003).
Sheffield, P., Garrard, S. & Derewenda, Z. Overcoming expression and purification problems of RhoGDI using a family of “parallel” expression vectors. Protein Expr. Purif. 15, 34–39 (1999).
Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999).
Terwilliger, T.C. Maximum-likelihood density modification. Acta Crystallogr. D Biol. Crystallogr. 56, 965–972 (2000).
Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. 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).
Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).
Winn, M.D., Isupov, M.N. & Murshudov, G.N. Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D Biol. Crystallogr. 57, 122–133 (2001).
: DeLano, W.L. The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, California, USA, 2002).
Freed, E.O. & Martin, M.A. Evidence for a functional interaction between the V1/V2 and 4 domains of human immunodeficiency virus type 1 envelope glycoprotein gp120. J. Virol. 68, 2503–2512 (1994).
Willey, R.L., Bonifacino, J.S., Potts, B.J., Martin, M.A. & Klausner, R.D. Biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160. Proc. Natl. Acad. Sci. USA 85, 9580–9584 (1988).
Freed, E.O., Orenstein, J.M., Buckler-White, A.J. & Martin, M.A. Single amino acid changes in the human immunodeficiency virus type 1 matrix protein block virus particle production. J. Virol. 68, 5311–5320 (1994).
Acknowledgements
We thank J. Kim, G. Miller and U. Munshi for many contributions to the early stages of this project, F. Soheilian for EM support, D.E. Anderson for mass spectrometry support, B. Beach and T. Leonard for advice and discussions and J. Bonifacino for the use of his calorimeter. C. Hill and W. Sundquist independently determined this structure and suggested the term 'V domain', which we have adopted. Use of the National Synchrotron Light Source at Brookhaven National Laboratory was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no. DE-AC02-98CH10886. HIV-Ig was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH. This research was supported by the Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases (J.H.H.) and National Cancer Institute, Center for Cancer Research (E.O.F.). Funds were also obtained from the Intramural AIDS Targeted Antiviral Program (J.H.H. and E.O.F.) and from the National Cancer Institute under contract N01-CO-12400 (K.N.).
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Lee, S., Joshi, A., Nagashima, K. et al. Structural basis for viral late-domain binding to Alix. Nat Struct Mol Biol 14, 194–199 (2007). https://doi.org/10.1038/nsmb1203
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DOI: https://doi.org/10.1038/nsmb1203
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