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Posted: 6 Oct 2011
30 years in 30 weeks, 2003

Since the discovery of HIV, researchers have uncovered, in unprecedented detail, the molecular processes involved in different stages of the viral life cycle, from reverse transcription and integration to viral particle assembly and protease-mediated maturation. One of these processes is budding – a late-stage step in virus infection, during which newly formed viral particles leave the infected cell. In his commentary, Dr. Sundquist covers the history of research in this area and highlights one of the important papers his laboratory published in 2003, which identified a number of cellular proteins that play important role in viral budding.

The protein network of HIV budding.
Cell. 2003 Sep 19;114(6):701-13.
von Schwedler UK, Stuchell M, Müller B, Ward DM, Chung HY, Morita E, Wang HE, Davis T, He GP, Cimbora DM, Scott A, Kräusslich HG, Kaplan J, Morham SG, Sundquist WI.

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Commentary by Dr. Wesley Sundquist

I wish to dedicate this feature to Uta von Schwedler, who passed away very recently and unexpectedly.  Uta was the first author and the driving force behind the paper that was reviewed here, and her death is a great loss for all of us.

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Dr. Wesley Sundquist
Our paper was part of a “second wave” of publications showing that HIV usurps the cellular ESCRT pathway to bud from cells, and I think its importance was in showing that cellular proteins are much more extensively involved in HIV budding than we had previously imagined.

Studies of HIV budding began with a seminal 1991 paper from Göttlinger and colleagues, who showed that sequences within the p6 region of the viral Gag protein are required for efficient virus release (1). In retrospect, that paper did not receive the attention it deserved, particularly as it is arguably just as important to understand how viruses get out of cells as how they get in. Nevertheless, the paper did help stimulate some nice work in several other labs, particularly those of Freed and Wills.

As a result, it was clear by the end of the 1990’s that retroviral Gag proteins contain different types of short sequence motifs, termed “late assembly” domains, which appeared to function by recruiting host factors that somehow facilitated the final stage of virus budding. Indeed, as early as 1996 Wills and colleagues proposed that cellular proteins containing WW domains might be binding partners for one class of late assembly domains found in Rous Sarcoma Virus and many other retroviruses (though not HIV) (2). It was difficult to identify the key players, however, owing to the large number of cellular WW domain proteins. 

In 2000, a flurry of papers showed that ubiquitin was involved in retrovirus budding (3-5). Unlike the earlier work, those papers did generate considerable excitement because they revealed that virus budding likely involved a significant cellular pathway(s), and because they appeared “back-to-back” in PNAS, together with an accompanying commentary (6). The actual function of ubiquitin in virus budding was murky, however, as captured in the title of Volker Vogt’s commentary, “Ubiquitin in retrovirus assembly: actor or bystander?”

The following year ushered in a significant breakthrough, when four different labs, including our own, published papers showing that the cellular protein TSG101 (Tumor Susceptibility Gene 101) was the functional binding partner for the primary late assembly domain of HIV-1 (7-10). This discovery dovetailed beautifully with concurrent genetic and biochemical analyses from the Emr lab showing that the yeast homolog of TSG101 functioned within a multi-protein complex that they named ESCRT-I (for Endosomal Protein Sorting Complex Required for Transport-I) (11). The yeast studies showed that ESCRT factors are required for formation of vesicles that bud into the lumen of the late endosome (12). This convergence of different research areas suggested that endosomal vesicle formation and virus budding might be analogous processes because both involve the formation of enveloped vesicles that bud away from the cytoplasm. This similarity was first noted in a remarkably prescient insight from Wills and colleagues (4), and was strongly supported by the discovery that TSG101 is required for both processes.

At that point, we began working in earnest to test the model that the human ESCRT pathway might be the machinery that catalyzes the membrane fission step of HIV budding. This required identifying human ESCRT factors (often by sequence comparisons with the known yeast ESCRT proteins), cloning and expressing the factors, mapping their protein-protein interactions, and testing whether their functions were required for HIV budding. We were fortunate to have excellent collaborators in the Kräusslich, Kaplan and Myriad Genetics labs, and we pulled in a number of folks from our own lab to do different experiments. The work was spearheaded by Uta von Schwedler, with strong support from Melissa Stuchell, a new graduate student who was baptized by fire! It was exciting, but also stressful because we were not experienced in proteomics-style analyses and because we knew we were racing with other labs. Ultimately, we identified 22 human ESCRT proteins (some of which were already known), mapped 25 new protein-protein interactions that defined the human ESCRT pathway, confirmed its similarity to the yeast pathway, and demonstrated that dominant negative versions of two different classes of late-acting proteins potently inhibited HIV-1 budding (13). The Göttlinger and Bieniasz labs published similar analyses at about the same time (14, 15) and the agreement between our independent studies, together with the Göttlinger lab’s discovery that another ESCRT protein, ALIX, was the binding partner for a different class of late assembly domains used by HIV-1 and EIAV (14), really solidified the idea that retroviruses employ an extensive, ESCRT-based machinery to bud from cells.

Our papers have generally stood the test of time, although subsequent studies have identified a surprising number of additional human ESCRT proteins (now >30) and have defined the subset required for HIV budding more precisely. In some ways, I think our 2003 paper can be justifiably described as a cataloging exercise, albeit a necessary one. That sentiment was nicely captured by a leading scientist in the field, who commented to me at the time that “your paper isn’t really that interesting, but I think it will serve as a great resource going forward”. What we all found remarkable, however, was the complexity and extent to which HIV uses host cell machinery during budding, particularly as compared to viral entry, where host factors play important but limited roles and most of the heavy lifting is done by the viral envelope protein.

Given the complexity of virus budding, it is not surprising that significant questions still remain a decade after the “TSG101” papers. Most importantly, we still don’t know for certain how the ESCRT machinery helps to pinch off the membrane neck at the base of the budding virus, although attractive models have been proposed (16, 17). Ironically, several areas where early progress was made remain enigmatic, as neither the provenance of the abundant free ubiquitin in virions (18) nor the precise role of ubiquitin in virus budding are known. Similarly, the interactions that link oncoretroviral late assembly domains and their WW domain-containing partners to the core ESCRT machinery remain to be elucidated. Perhaps the most satisfying aspect of the story so far is that studies of virus budding helped stimulate an intense focus on the mammalian ESCRT pathway, which in turn led to the discovery that the pathway plays critical roles in cell division, both in mediating the final stages of cytokinesis (19-21) and in helping to ensure the ordered completion of mitosis (22). In that sense, the ESCRT pathway has become another in the long line of examples that validate the maxim that “viruses are the window on the cell”.

About the Author: Dr. Wesley Sundquist is Benning Professor and Co-Chair of Biochemistry at the University of Utah.

References

  1. Gottlinger, H. G., T. Dorfman, J. G. Sodroski, and W. A. Haseltine. 1991. Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release. Proc Natl Acad Sci USA 88:3195-3199.
  2. Garnier, L., J. W. Wills, M. F. Verderame, and M. Sudol. 1996. WW domains and retrovirus budding. Nature 381:744-745.
  3. Strack, B., A. Calistri, M. A. Accola, G. Palu, and H. G. Gottlinger. 2000. A role for ubiquitin ligase recruitment in retrovirus release. Proc Natl Acad Sci USA 97:13063-13068.
  4. Patnaik, A., V. Chau, and J. W. Wills. 2000. Ubiquitin is part of the retrovirus budding machinery. Proc Natl Acad Sci USA 97:13069-13074.
  5. Schubert, U., D. E. Ott, E. N. Chertova, R. Welker, U. Tessmer, M. F. Princiotta, J. R. Bennink, H. G. Krausslich, and J. W. Yewdell. 2000. Proteasome inhibition interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2. Proc Natl Acad Sci USA 97:13057-13062.
  6. Vogt, V. M. 2000. Ubiquitin in retrovirus assembly: actor or bystander? Proc Natl Acad Sci USA 97:12945-12947.
  7. VerPlank, L., F. Bouamr, T. J. LaGrassa, B. Agresta, A. Kikonyogo, J. Leis, and C. A. Carter. 2001. Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55Gag. Proc Natl Acad Sci USA 98:7724-7729.
  8. Garrus, J. E., U. K. von Schwedler, O. W. Pornillos, S. G. Morham, K. H. Zavitz, H. E. Wang, D. A. Wettstein, K. M. Stray, M. Cote, R. L. Rich, D. G. Myszka, and W. I. Sundquist. 2001. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 107:55-65.
  9. Martin-Serrano, J., T. Zang, and P. D. Bieniasz. 2001. 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.
  10. Demirov, D. G., A. Ono, J. M. Orenstein, and E. O. Freed. 2002. 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.
  11. Katzmann, D. J., M. Babst, and S. D. Emr. 2001. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106:145-155.
  12. Coonrod, E. M., and T. H. Stevens. 2010. The yeast vps class E mutants: the beginning of the molecular genetic analysis of multivesicular body biogenesis. Mol Biol Cell 21:4057-4060.
  13. von Schwedler, U. K., M. Stuchell, B. Muller, D. M. Ward, H. Y. Chung, E. Morita, H. E. Wang, T. Davis, G. P. He, D. M. Cimbora, A. Scott, H. G. Krausslich, J. Kaplan, S. G. Morham, and W. I. Sundquist. 2003. The protein network of HIV budding. Cell 114:701-713.
  14. Strack, B., A. Calistri, S. Craig, E. Popova, and H. G. Gottlinger. 2003. AIP1/ALIX Is a Binding Partner for HIV-1 p6 and EIAV p9 Functioning in Virus Budding. Cell 114:689-699.
  15. Martin-Serrano, J., T. Zang, and P. D. Bieniasz. 2003. Role of ESCRT-I in Retroviral Budding. J Virol 77:4794-4804.
  16. Fabrikant, G., S. Lata, J. D. Riches, J. A. Briggs, W. Weissenhorn, and M. M. Kozlov. 2009. Computational model of membrane fission catalyzed by ESCRT-III. PLoS Comput Biol 5:e1000575.
  17. Hurley, J. H., and P. I. Hanson. 2010. Membrane budding and scission by the ESCRT machinery: it's all in the neck. Nat Rev Mol Cell Biol 11:556-566.
  18. Putterman, D., R. B. Pepinsky, and V. M. Vogt. 1990. Ubiquitin in avian leukosis virus particles. Virology 176:633-637.
  19. Carlton, J. G., and J. Martin-Serrano. 2007. Parallels between cytokinesis and retroviral budding: a role for the ESCRT machinery. Science 316:1908-1912.
  20. Elia, N., R. Sougrat, T. A. Spurlin, J. H. Hurley, and J. Lippincott-Schwartz. 2011. Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission. Proc Natl Acad Sci USA 108:4846-4851.
  21. Guizetti, J., L. Schermelleh, J. Mantler, S. Maar, I. Poser, H. Leonhardt, T. Muller-Reichert, and D. W. Gerlich. 2011. Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments. Science 331:1616-1620.
  22. Morita, E., L. A. Colf, M. A. Karren, V. Sandrin, C. K. Rodesch, and W. I. Sundquist. 2010. Human ESCRT-III and VPS4 proteins are required for centrosome and spindle maintenance. Proc Natl Acad Sci USA 107:12889-12894.


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