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Live-cell visualization of dynamics of HIV budding site interactions with an ESCRT component

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Abstract

HIV (human immunodeficiency virus) diverts the cellular ESCRT (endosomal sorting complex required for transport) machinery to promote virion release from infected cells. The ESCRT consists of four heteromeric complexes (ESCRT-0 to ESCRT-III), which mediate different membrane abscission processes, most importantly formation of intralumenal vesicles at multivesicular bodies. The ATPase VPS4 (vacuolar protein sorting 4) acts at a late stage of ESCRT function, providing energy for ESCRT dissociation. Recruitment of ESCRT by late-domain motifs in the viral Gag polyprotein and a role of ESCRT in HIV release are firmly established, but the order of events, their kinetics and the mechanism of action of individual ESCRT components in HIV budding are unclear at present. Using live-cell imaging, we show late-domain-dependent recruitment of VPS4A to nascent HIV particles at the host cell plasma membrane. Recruitment of VPS4A was transient, resulting in a single or a few bursts of at least two to five VPS4 dodecamers assembling at HIV budding sites. Bursts lasted for 35 s and appeared with variable delay before particle release. These results indicate that VPS4A has a direct role in membrane scission leading to HIV-1 release.

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Figure 1: Recruitment of VPS4A to HIV assembly sites.
Figure 2: Duration of eGFP–VPS4A bursts.
Figure 3: Number of eGFP–VPS4A molecules per burst.
Figure 4: Correlation of VPS4A wild-type bursts with Gag assembly phases.

Change history

  • 15 March 2011

    In the version of this letter initially published online, the first sentence was erroneously truncated and eGFP–VPS4A was misspelled in the second paragraph.

References

  1. Jouvenet, N., Bieniasz, P. D. & Simon, S. M. Imaging the biogenesis of individual HIV-1 virions in live cells. Nature 454, 236–240 (2008).

    CAS  Article  Google Scholar 

  2. Ivanchenko, S. et al. Dynamics of HIV-1 assembly and release. PLoS Pathog. 5, e1000652 (2009).

    Article  Google Scholar 

  3. Lampe, M. et al. Double-labelled HIV-1 particles for study of virus-cell interaction. Virology 360, 92–104 (2007).

    CAS  Article  Google Scholar 

  4. Muller, B. et al. Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative. J. Virol. 78, 10803–10813 (2004).

    Article  Google Scholar 

  5. Scheuring, S. et al. Mammalian cells express two VPS4 proteins both of which are involved in intracellular protein trafficking. J. Mol. Biol. 312, 469–480 (2001).

    CAS  Article  Google Scholar 

  6. Babst, M. A protein’s final ESCRT. Traffic 6, 2–9 (2005).

    CAS  Article  Google Scholar 

  7. Carlton, J. G. & Martin-Serrano, J. The ESCRT machinery: new functions in viral and cellular biology. Biochem. Soc. Trans. 37, 195–199 (2009).

    CAS  Article  Google Scholar 

  8. Hurley, J. H. ESCRT complexes and the biogenesis of multivesicular bodies. Curr. Opin. Cell Biol. 20, 4–11 (2008).

    CAS  Article  Google Scholar 

  9. 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).

    CAS  Article  Google Scholar 

  10. Hanson, P. I., Shim, S. & Merrill, S. A. Cell biology of the ESCRT machinery. Curr. Opin. Cell Biol. 21, 568–574 (2009).

    CAS  Article  Google Scholar 

  11. Bishop, N. & Woodman, P. ATPase-defective mammalian VPS4 localizes to aberrant endosomes and impairs cholesterol trafficking. Mol. Biol. Cell 11, 227–239 (2000).

    CAS  Article  Google Scholar 

  12. Stuchell-Brereton, M. D. et al. ESCRT-III recognition by VPS4 ATPases. Nature 449, 740–744 (2007).

    CAS  Article  Google Scholar 

  13. Neil, S. J., Eastman, S. W., Jouvenet, N. & Bieniasz, P. D. HIV-1 Vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane. PLoS Pathog. 2, e39 (2006).

    Article  Google Scholar 

  14. Babst, M., Wendland, B., Estepa, E. J. & Emr, S. D. The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function. EMBO J. 17, 2982–2993 (1998).

    CAS  Article  Google Scholar 

  15. Scott, A. et al. Structural and mechanistic studies of VPS4 proteins. EMBO J. 24, 3658–3669 (2005).

    CAS  Article  Google Scholar 

  16. Merrill, S. A. & Hanson, P. I. Activation of human VPS4A by ESCRT-III proteins reveals ability of substrates to relieve enzyme autoinhibition. J. Biol. Chem. 285, 35428–35438 (2010).

    CAS  Article  Google Scholar 

  17. Azmi, I. et al. Recycling of ESCRTs by the AAA-ATPase Vps4 is regulated by a conserved VSL region in Vta1. J. Cell Biol. 172, 705–717 (2006).

    CAS  Article  Google Scholar 

  18. Shestakova, A. et al. Assembly of the AAA ATPase Vps4 on ESCRT-III. Mol. Biol. Cell 21, 1059–1071 (2010).

    CAS  Article  Google Scholar 

  19. Ward, D. M. et al. The role of LIP5 and CHMP5 in multivesicular bodyformation and HIV-1 budding in mammalian cells. J. Biol. Chem. 280, 10548–10555 (2005).

    CAS  Article  Google Scholar 

  20. Gonciarz, M. D. et al. Biochemical and structural studies of yeast Vps4 oligomerization. J. Mol. Biol. 384, 878–895 (2008).

    CAS  Article  Google Scholar 

  21. Landsberg, M. J., Vajjhala, P. R., Rothnagel, R., Munn, A. L. & Hankamer, B. Three-dimensional structure of AAA ATPase Vps4: advancing structural insights into the mechanisms of endosomal sorting and enveloped virus budding. Structure 17, 427–437 (2009).

    CAS  Article  Google Scholar 

  22. Yu, Z., Gonciarz, M. D., Sundquist, W. I., Hill, C. P. & Jensen, G. J. Cryo-EM structure of dodecameric Vps4p and its 2:1 complex with Vta1p. J. Mol. Biol. 377, 364–377 (2008).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  24. von Schwedler, U. K. et al. The protein network of HIV budding. Cell 114, 701–713 (2003).

    CAS  Article  Google Scholar 

  25. Fujita, H. et al. Mammalian class E Vps proteins, SBP1 and mVps2/CHMP2A, interact with and regulate the function of an AAA-ATPase SKD1/Vps4B. J. Cell Sci. 117, 2997–3009 (2004).

    CAS  Article  Google Scholar 

  26. Petersen, N. O., Hoddelius, P. L., Wiseman, P. W., Seger, O. & Magnusson, K. E. Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application. Biophys. J. 65, 1135–1146 (1993).

    CAS  Article  Google Scholar 

  27. Wiseman, P. W. & Petersen, N. O. Image correlation spectroscopy. II. Optimization for ultrasensitive detection of preexisting platelet-derived growth factor-beta receptor oligomers on intact cells. Biophys. J. 76, 963–977 (1999).

    CAS  Article  Google Scholar 

  28. Sergeev, M., Costantino, S. & Wiseman, P. W. Measurement of monomer–oligomer distributions via fluorescence moment image analysis. Biophys. J. 91, 3884–3896 (2006).

    CAS  Article  Google Scholar 

  29. Yang, D. & Hurley, J. H. Structural role of the Vps4–Vta1 interface in ESCRT-III recycling. Structure 18, 976–984 (2010).

    Article  Google Scholar 

  30. Morita, E. et al. Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis. EMBO J. 26, 4215–4227 (2007).

    CAS  Article  Google Scholar 

  31. Saksena, S., Wahlman, J., Teis, D., Johnson, A. E. & Emr, S. D. Functional reconstitution of ESCRT-III assembly and disassembly. Cell 136, 97–109 (2009).

    CAS  Article  Google Scholar 

  32. Hanson, P. I., Roth, R., Lin, Y. & Heuser, J. E. Plasma membrane deformation by circular arrays of ESCRT-III protein filaments. J. Cell Biol. 180, 389–402 (2008).

    CAS  Article  Google Scholar 

  33. Wollert, T. et al. The ESCRT machinery at a glance. J. Cell Sci. 122, 2163–2166 (2009).

    CAS  Article  Google Scholar 

  34. Wollert, T., Wunder, C., Lippincott-Schwartz, J. & Hurley, J. H. Membrane scission by the ESCRT-III complex. Nature 458, 172–177 (2009).

    CAS  Article  Google Scholar 

  35. Wollert, T. & Hurley, J. H. Molecular mechanism of multivesicular body biogenesis by ESCRT complexes. Nature 464, 864–869 (2010).

    CAS  Article  Google Scholar 

  36. Fabrikant, G. et al. Computational model of membrane fission catalyzed by ESCRT-III. PLoS Comput. Biol. 5, e1000575 (2009).

    Article  Google Scholar 

  37. Lange, S. et al. Simultaneous transport of different localized mRNA species revealed by live-cell imaging. Traffic 9, 1256–1267 (2008).

    CAS  Article  Google Scholar 

  38. Godinez, W. J. et al. Deterministic and probabilistic approaches for tracking virus particles in time-lapse fluorescence microscopy image sequences. Med. Image Anal. 13, 325–342 (2009).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank W. Sundquist (University of Utah, Salt Lake City) and R. Tsien (University of California, San Diego) for plasmids. We appreciate the assistance of M. Franke, M. Anders-Ößwein, A. M. Heuser, W. Schrimpf, D. Schupp and M. Eckhardt and tracking support from M. Preiner, F. Kohler and S. A. Rahman. We thank J. Hurley for helpful comments, A. Godin for Matlab masking algorithms and W. Godinez, J. P. Bergeestand, K. Rohr for support with the single-particle tracking algorithm. We gratefully acknowledge financial support of the DFG through SPP1175 (D.C.L., C.B., H-G.K.), SFB638 (H-G.K.) and grant MU885/4-2 (B.M.), the German Excellence Initiative through ‘Nanosystems Initiative Munich (NIM)’ (D.C.L., C.B.) and ‘CellNetworks’ (H-G.K.), the Ludwig-Maximilians-University Munich (LMUInnovativ BioImaging Network, D.C.L., C.B.), the EU (HIV-ACE network HEALTH-F3-2008-201095; B.M., H-G.K.), the Natural Sciences and Engineering Research Council of Canada (P.W.W.) and the Canadian Institutes of Health Research (P.W.W.).

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Contributions

V.B. and S.I. carried out experiments. M.S. and P.W.W. carried out the ICS analysis. V.B., S.I. and A.D. analysed data. B.M., D.C.L., H-G.K., S.I., A.D., M.S., P.W.W., C.B. and V.B. wrote the manuscript. B.M., D.C.L., H-G.K., C.B. designed and guided the project.

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Correspondence to Barbara Müller or Don C. Lamb.

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The authors declare no competing financial interests.

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Baumgärtel, V., Ivanchenko, S., Dupont, A. et al. Live-cell visualization of dynamics of HIV budding site interactions with an ESCRT component. Nat Cell Biol 13, 469–474 (2011). https://doi.org/10.1038/ncb2215

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