Wesley I Sundquist

University of Utah, Salt Lake City, Utah, United States

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Publications (111)1286.2 Total impact

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    ABSTRACT: The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 angstrom resolution cryogenic electron microscopy reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, charged multivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1). The inner strand comprises “open” CHMP1B subunits that interlock in an elaborate domain-swapped architecture and is encircled by an outer strand of “closed” IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively curved membranes in vitro and in vivo. Our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature.
    Full-text · Article · Dec 2015 · Science
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    ABSTRACT: The US research enterprise is under significant strain due to stagnant funding, an expanding workforce, and complex regulations that increase costs and slow the pace of research. In response, a number of groups have analyzed the problems and offered recommendations for resolving these issues. However, many of these recommendations lacked follow-up implementation, allowing the damage of stagnant funding and outdated policies to persist. Here, we analyze nine reports published since the beginning of 2012 and consolidate over 250 suggestions into eight consensus recommendations made by the majority of the reports. We then propose how to implement these consensus recommendations, and we identify critical issues, such as improving workforce diversity and stakeholder interactions, on which the community has yet to achieve consensus.
    Preview · Article · Jul 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: TRIM5α is an antiviral, cytoplasmic, E3 ubiquitin (Ub) ligase that assembles on incoming retroviral capsids and induces their premature dissociation. It inhibits reverse transcription of the viral genome and can also synthesize unanchored polyubiquitin (polyUb) chains to stimulate innate immune responses. Here, we show that TRIM5α employs the E2 Ub-conjugating enzyme Ube2W to anchor the Lys63-linked polyUb chains in a process of TRIM5α auto-ubiquitination. Chain anchoring is initiated, in cells and in vitro, through Ube2W-catalyzed monoubiquitination of TRIM5α. This modification serves as a substrate for the elongation of anchored Lys63-linked polyUb chains, catalyzed by the heterodimeric E2 enzyme Ube2N/Ube2V2. Ube2W targets multiple TRIM5α internal lysines with Ub especially lysines 45 and 50, rather than modifying the N-terminal amino group, which is instead αN-acetylated in cells. E2 depletion or Ub mutation inhibits TRIM5α ubiquitination in cells and restores restricted viral reverse transcription, but not infection. Our data indicate that the stepwise formation of anchored Lys63-linked polyUb is a critical early step in the TRIM5α restriction mechanism and identify the E2 Ub-conjugating cofactors involved. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.
    Preview · Article · Jun 2015 · The EMBO Journal
  • Wesley I Sundquist · Katharine S Ullman

    No preview · Article · Jun 2015 · Science
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    ABSTRACT: The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. Here we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains and thereby delays abscission in response to lagging chromosomes, nuclear pore defects and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations.
    Full-text · Article · May 2015 · eLife Sciences
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    ABSTRACT: The Endosomal Sorting Complexes Required for Transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body (MVB) biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 Microtubule Interacting and Trafficking (MIT) domains initially bind the C-terminal MIT Interacting Motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain, and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    No preview · Article · Apr 2015 · Journal of Biological Chemistry
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    Gaelle Mercenne · Steven L Alam · Jun Arii · Matthew S Lalonde · Wesley I Sundquist
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    ABSTRACT: eLife digest To multiply and spread infections, viruses must enter and exit cells. Once inside a cell, many viruses conscript the cell's machinery to produce new viral particles and release them into the surroundings. Some viruses—like HIV-1—exit the cell in a way that leads to them being wrapped (or ‘enveloped’) in membrane from the host cell. A virus protein called Gag is required for the release of HIV-1 and other enveloped viruses. In some cases, Gag proteins bind directly to members of the NEDD4 protein family to enable the viruses to be released. However, the Gag protein from HIV-1 does not appear to be able to interact directly with NEDD4 proteins, so it was not clear how Gag works in this case. Mercenne et al. studied how HIV-1 is released from human cells grown in the laboratory. The experiments show that members of a human protein family called the Angiomotins bind to both Gag and NEDD4L (a member of the NEDD4 family) and are required for the efficient release of viruses. Using a technique called electron microscopy, Mercenne et al. observed that when Angiomotins are present, Gag proteins assemble in spheres at the cell membrane and viruses are able to exit the cell. However, when Angiomotins are depleted or absent, incomplete spheres of Gag proteins accumulate on the inner membrane surface and viruses are not released. These findings show that Angiomotins act as a link between Gag and NEDD4L to promote the release of HIV-1 from human cells. The next step will be to learn precisely how this works. There are indications that the Angiomotins may also be involved in the release of other enveloped viruses, so the findings may be useful for the development of treatments for a variety of viral infections. DOI: http://dx.doi.org/10.7554/eLife.03778.002
    Full-text · Article · Jan 2015 · eLife Sciences
  • John McCullough · Wesley I Sundquist

    No preview · Article · Dec 2014 · Nature Structural & Molecular Biology
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    ABSTRACT: Tripartite motif (TRIM) proteins make up a large family of coiled-coil-containing RING E3 ligases that function in many cellular processes, particularly innate antiviral response pathways. Both dimerization and higher-order assembly are important elements of TRIM protein function, but the atomic details of TRIM tertiary and quaternary structure have not been fully understood. Here, we present crystallographic and biochemical analyses of the TRIM coiled-coil and show that TRIM proteins dimerize by forming interdigitating antiparallel helical hairpins that position the N-terminal catalytic RING domains at opposite ends of the dimer and the C-terminal substrate-binding domains at the center. The dimer core comprises an antiparallel coiled-coil with a distinctive, symmetric pattern of flanking heptad and central hendecad repeats that appear to be conserved across the entire TRIM family. Our studies reveal how the coiled-coil organizes TRIM25 to polyubiquitylate the RIG-I/viral RNA recognition complex and how dimers of the TRIM5α protein are arranged within hexagonal arrays that recognize the HIV-1 capsid lattice and restrict retroviral replication.
    Full-text · Article · Feb 2014 · Proceedings of the National Academy of Sciences
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    ABSTRACT: The cellular ESCRT pathway drives membrane constriction toward the cytosol and effects membrane fission during cytokinesis, endosomal sorting, and the release of many enveloped viruses, including HIV. A component of this pathway, the AAA ATPase Vps4, provides energy for pathway progression. Although it is established that Vps4 functions as an oligomer, subunit stoichiometry and other fundamental features of the functional enzyme are unclear. Higher-order oligomers have thus far only been characterized for a Walker B mutant of Vps4 in the presence of ATP. Here, we report that although some mutant Vps4 proteins form dodecameric assemblies, active wild-type S. cerevisiae and S. solfataricus Vps4 enzymes can form hexamers in the presence of ATP and ADP, as assayed by size exclusion chromatography and equilibrium analytical ultracentifugation. The Vta1p activator binds hexameric yeast Vps4p without changing the oligomeric state of Vps4p, implying that the active Vta1p:Vps4p complex also contains a single hexameric ring. Additionally, we report crystal structures of two different archaeal Vps4 homologs, whose structures and lattice interactions suggest a conserved mode of oligomerization. Disruption of the proposed hexamerization interface by mutagenesis abolished the ATPase activity of archaeal Vps4 proteins and blocked Vps4p function in S. cerevisiae. These data challenge the prevailing model that active Vps4 is a double ring dodecamer, and argue that, like other type I AAA ATPases, Vps4 functions as a single ring with six subunits.
    Full-text · Article · Oct 2013 · Journal of Molecular Biology
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    Virginie Sandrin · Wesley I Sundquist
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    ABSTRACT: Retroviruses and many other enveloped viruses usurp the cellular ESCRT pathway to bud from cells. However, the stepwise process of ESCRT-mediated virus budding can be challenging to analyze in retroviruses like HIV-1 that recruit multiple different ESCRT factors to initiate budding. In this study, we characterized the ESCRT factor requirements for budding of Equine Infectious Anemia Virus (EIAV), whose only known direct ESCRT protein interaction is with ALIX. siRNA depletion of endogenous ESCRT proteins and "rescue" experiments with exogenous siRNA-resistant wild type and mutant constructs revealed budding requirements for the following ESCRT proteins: ALIX, CHMP4B, CHMP2A and VPS4A or VPS4B. EIAV budding was inhibited by point mutations that abrogate the direct interactions between ALIX:CHMP4B, CHMP4B:CHMP2A, and CHMP2A:VPS4A/B, indicating that each of these interactions is required for EIAV budding. Unexpectedly, CHMP4B depletion led to formation of multi-lobed and long tubular EIAV virions. We conclude that EIAV budding requires an ESCRT protein network that comprises EIAV Gag-ALIX-CHMP4B-CHMP2A-VPS4 interactions. Our experiments also suggest that CHMP4B recruitment/polymerization helps control Gag polymerization and/or processing to ensure that ESCRT factor assembly and membrane fission occur at the proper stage of virion assembly. These studies help establish EIAV as a streamlined model system for dissecting the stepwise processes of lentivirus assembly and ESCRT-mediated budding.
    Preview · Article · Oct 2013 · Retrovirology
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    Jörg Votteler · Wesley I Sundquist
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    ABSTRACT: Enveloped viruses escape infected cells by budding through limiting membranes. In the decade since the discovery that HIV recruits cellular ESCRT (endosomal sorting complexes required for transport) machinery to facilitate viral budding, this pathway has emerged as the major escape route for enveloped viruses. In cells, the ESCRT pathway catalyzes analogous membrane fission events required for the abscission stage of cytokinesis and for a series of "reverse topology" vesiculation events. Studies of enveloped virus budding are therefore providing insights into the complex cellular mechanisms of cell division and membrane protein trafficking (and vice versa). Here, we review how viruses mimic cellular recruiting signals to usurp the ESCRT pathway, discuss mechanistic models for ESCRT pathway functions, and highlight important research frontiers.
    Preview · Article · Sep 2013 · Cell host & microbe
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    ABSTRACT: The identification of novel antiretroviral agents is required to provide alternative treatment options for HIV-1-infected patients. The screening of a phenotypic cell-based viral replication assay led to the identification of a novel class of 4,5-dihydro-1H-pyrrolo[3,4-c]pyrazol-6-one (pyrrolopyrazolone) HIV-1 inhibitors, exemplified by two compounds: BI-1 and BI-2. These compounds inhibited early postentry stages of viral replication at a step(s) following reverse transcription but prior to 2 long terminal repeat (2-LTR) circle formation, suggesting that they may block nuclear targeting of the preintegration complex. Selection of viruses resistant to BI-2 revealed that substitutions at residues A105 and T107 within the capsid (CA) amino-terminal domain (CANTD) conferred high-level resistance to both compounds, implicating CA as the antiviral target. Direct binding of BI-1 and/or BI-2 to CANTD was demonstrated using isothermal titration calorimetry and nuclear magnetic resonance (NMR) chemical shift titration analyses. A high-resolution crystal structure of the BI-1:CANTD complex revealed that the inhibitor bound within a recently identified inhibitor binding pocket (CANTD site 2) between CA helices 4, 5, and 7, on the surface of the CANTD, that also corresponds to the binding site for the host factor CPSF-6. The functional consequences of BI-1 and BI-2 binding differ from previously characterized inhibitors that bind the same site since the BI compounds did not inhibit reverse transcription but stabilized preassembled CA complexes. Hence, this new class of antiviral compounds binds CA and may inhibit viral replication by stabilizing the viral capsid.
    Full-text · Article · Jul 2013 · Antimicrobial Agents and Chemotherapy
  • Felix A. Rey · Wesley I. Sundquist

    No preview · Article · Apr 2013 · Current Opinion in Structural Biology
  • John McCullough · Leremy A Colf · Wesley I Sundquist
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    ABSTRACT: The endosomal sorting complexes required for transport (ESCRT) pathway was initially defined in yeast genetic screens that identified the factors necessary to sort membrane proteins into intraluminal endosomal vesicles. Subsequent studies have revealed that the mammalian ESCRT pathway also functions in a series of other key cellular processes, including formation of extracellular microvesicles, enveloped virus budding, and the abscission stage of cytokinesis. The core ESCRT machinery comprises Bro1 family proteins and ESCRT-I, ESCRT-II, ESCRT-III, and VPS4. Site-specific adaptors recruit these soluble factors to assemble on different cellular membranes, where they carry out membrane fission reactions. ESCRT-III proteins form filaments that draw membranes together from the cytoplasmic face, and mechanistic models have been advanced to explain how ESCRT-III filaments and the VPS4 ATPase can work together to catalyze membrane fission. Expected final online publication date for the Annual Review of Biochemistry Volume 82 is June 02, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
    No preview · Article · Mar 2013 · Annual review of biochemistry
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    Christopher P Hill · Wesley I Sundquist
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    ABSTRACT: A better understanding of the host cell protein complex that helps HIV replicate inside cells offers the possibility of new therapeutic targets.
    Preview · Article · Mar 2013 · eLife Sciences
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    Wesley I. Sundquist
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    ABSTRACT: The endosomal sorting complexes required for transport (ESCRT) pathway mediates membrane fission reactions during intraluminal endosomal vesicle formation, budding of HIV-1 and other enveloped viruses, and the final abscission step of cytokinesis in mammals and archaea. Current models hold that ubiquitin-binding ESCRT factors act early in the pathway to regulate factor recruitment and assembly, whereas the late acting ESCRT-III proteins form filaments that draw the membranes together and mediate fission, possibly, in collaboration with VPS4-ATPases. I will discuss our current understanding of the structures and functions of the different ESCRT factors in HIV budding and abscission with a particular focus on our studies aimed at understanding: (1) how ubiquitin regulates ESCRT recruitment during HIV-1 budding and (2) the structures and membrane-binding properties of ESCRT-III subunits and filaments.
    Preview · Article · Jan 2013 · Biophysical Journal
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    Preview · Article · Jan 2013 · Biophysical Journal
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    ABSTRACT: The diversity of ubiquitin (Ub)-dependent signaling is attributed to the ability of this small protein to form different types of covalently linked polyUb chains and to the existence of Ub binding proteins that interpret this molecular syntax. We used affinity capture/mass spectrometry to identify ALIX, a component of the ESCRT pathway, as a Ub binding protein. We report that the V domain of ALIX binds directly and selectively to K63-linked polyUb chains, exhibiting a strong preference for chains composed of more than three Ub. Sequence analysis identified two potential Ub binding sites on a single α-helical surface within the coiled-coil region of the V domain. Mutation of these putative Ub binding sites inhibited polyUb binding to the isolated V domain in vitro and impaired budding of lentiviruses. These data reveal an important role for K63 polyUb binding by ALIX in retroviral release.
    Preview · Article · Nov 2012 · Developmental Cell
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    Matthew Scott Lalonde · Wesley I Sundquist

    Full-text · Article · Nov 2012 · Proceedings of the National Academy of Sciences

Publication Stats

14k Citations
1,286.20 Total Impact Points

Institutions

  • 1993-2015
    • University of Utah
      • • Department of Biochemistry
      • • Department of Medicinal Chemistry
      Salt Lake City, Utah, United States
  • 2011
    • University of Sydney
      • School of Molecular Bioscience
      Sydney, New South Wales, Australia
  • 2004
    • Vanderbilt University
      • Department of Pathology, Microbiology and Immunology
      Nashville, Michigan, United States
  • 1997
    • Memorial Sloan-Kettering Cancer Center
      New York, New York, United States
  • 1994
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States