Wesley I Sundquist

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

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Publications (104)1175.63 Total impact

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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.
    Journal of Biological Chemistry 04/2015; DOI:10.1074/jbc.M115.642355 · 4.60 Impact Factor
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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
    eLife Sciences 01/2015; 4. DOI:10.7554/eLife.03778 · 8.52 Impact Factor
  • John McCullough, Wesley I Sundquist
    Nature Structural & Molecular Biology 12/2014; 21(12):1025-7. DOI:10.1038/nsmb.2928 · 11.63 Impact Factor
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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.
    Proceedings of the National Academy of Sciences 02/2014; 111(7):2494-9. DOI:10.1073/pnas.1318962111 · 9.81 Impact Factor
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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.
    Journal of Molecular Biology 10/2013; 426(3). DOI:10.1016/j.jmb.2013.09.043 · 3.96 Impact Factor
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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.
    Retrovirology 10/2013; 10(1):104. DOI:10.1186/1742-4690-10-104 · 4.77 Impact Factor
  • 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.
    Cell host & microbe 09/2013; 14(3):232-41. DOI:10.1016/j.chom.2013.08.012 · 12.19 Impact Factor
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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 (or pyrrolopyrazolone) HIV-1 inhibitors, exemplified by two compounds: BI-1 and BI-2. These compounds inhibited early post-entry stages of viral replication at a step(s) following reverse transcription but prior to 2-LTR circle formation, suggesting that they may block nuclear targeting of the pre-integration complex. Selection of viruses resistant to BI-2 revealed that substitutions at residues A105 and T107 within the 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 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.
    Antimicrobial Agents and Chemotherapy 07/2013; DOI:10.1128/AAC.00985-13 · 4.45 Impact Factor
  • 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.
    Annual review of biochemistry 03/2013; DOI:10.1146/annurev-biochem-072909-101058 · 26.53 Impact Factor
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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.
    eLife Sciences 03/2013; 2:e00577. DOI:10.7554/eLife.00577 · 8.52 Impact Factor
  • 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.
    Biophysical Journal 01/2013; 104(2):12-. DOI:10.1016/j.bpj.2012.11.097 · 3.83 Impact Factor
  • Biophysical Journal 01/2013; 104(2):12-. DOI:10.1016/j.bpj.2012.11.096 · 3.83 Impact Factor
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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.
    Developmental Cell 11/2012; DOI:10.1016/j.devcel.2012.10.023 · 10.37 Impact Factor
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    Matthew Scott Lalonde, Wesley I Sundquist
    Proceedings of the National Academy of Sciences 11/2012; 109(46). DOI:10.1073/pnas.1215940109 · 9.81 Impact Factor
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    ABSTRACT: The ESCRT pathway remodels membranes during multivesicular body biogenesis, the abscission stage of cytokinesis, and enveloped virus budding. The ESCRT-III and VPS4 ATPase complexes catalyze the membrane fission events associated with these processes, and the LIP5 protein helps regulate their interactions by binding directly to a subset of ESCRT-III proteins and to VPS4. We have investigated the biochemical and structural basis for different LIP5 ligand interactions and show that the first MIT (Microtubule Interacting and Trafficking) module of the tandem LIP5 MIT domain binds CHMP1B (and other ESCRT-III proteins) through canonical Type 1 MIT Interacting Motif (MIM1) interactions. In contrast, the second LIP5 MIT module binds with unusually high affinity to a novel MIM element within the ESCRT-III protein, CHMP5. A solution structure of the relevant LIP5-CHMP5 complex reveals that CHMP5 helices 5 and 6 and adjacent linkers form an amphipathic "Leucine Collar" that wraps almost completely around the second LIP5 MIT module but makes only limited contacts with the first MIT module. LIP5 binds MIM1-containing ESCRT-III proteins, CHMP5 and VPS4 ligands independently in vitro, but these interactions are coupled within cells because formation of stable VPS4 complexes with both LIP5 and CHMP5 requires LIP5 to bind both a MIM1-containing ESCRT-III protein and CHMP5. Our studies thus reveal how the tandem MIT domain of LIP5 binds different types of ESCRT-III proteins, promoting assembly of active VPS4 enzymes on the polymeric ESCRT-III substrate.
    Journal of Biological Chemistry 10/2012; DOI:10.1074/jbc.M112.417899 · 4.60 Impact Factor
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    ABSTRACT: The endosomal sorting complex required for transport-III (ESCRT-III) proteins are essential for budding of some enveloped viruses, the formation of intraluminal vesicles at the endosome and for the abscission step of cytokinesis. ESCRT-III proteins form polymers that constrict membrane tubes, leading to fission. We have used electron cryomicroscopy (cryo-EM) to determine the molecular organization of pleiomorphic ESCRT-III CHMP2A-CHMP3 polymers. The three-dimensional reconstruction at 22 Å resolution reveals a helical organization of filaments of CHMP molecules organized in a head-to-tail fashion. Protease susceptibility experiments indicate that polymerization is achieved via conformational changes that increase the protomer stability. Combinatorial siRNA knockdown experiments indicate that CHMP3 contributes synergistically to HIV-1 budding, and the CHMP3 contribution is ∼10-fold more pronounced in concert with CHMP2A than with CHMP2B. This is consistent with SPR affinity measurements that suggest sequential CHMP4B-CHMP3-CHMP2A recruitment while showing that both CHMP2A and CHMP2B interact with CHMP4B, in agreement with their redundant functions in HIV-1 budding. Our data thus indicate that the CHMP2A-CHMP3 polymer observed in vitro contributes to HIV-1 budding by assembling on CHMP4B polymers.
    Cellular Microbiology 10/2012; 15(2). DOI:10.1111/cmi.12041 · 4.82 Impact Factor
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    ABSTRACT: Transient transfection of small interfering RNA (siRNA) provides a powerful approach for studying cellular protein functions, particularly when the target protein can be re-expressed from an exogenous siRNA-resistant construct in order to rescue the knockdown phenotype, confirm siRNA target specificity, and support mutational analyses. Rescue experiments often fail, however, when siRNA-resistant constructs are expressed at suboptimal levels. Here, we describe an ensemble of mammalian protein expression vectors with CMV promoters of differing strengths. Using CHMP2A rescue of HIV-1 budding, we show that these vectors can combine high-transfection efficiencies with tunable protein expression levels to optimize the rescue of cellular phenotypes induced by siRNA transfection.
    BioTechniques 08/2012; DOI:10.2144/000113909 · 2.75 Impact Factor
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    Wesley I Sundquist, Hans-Georg Kräusslich
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    ABSTRACT: A defining property of retroviruses is their ability to assemble into particles that can leave producer cells and spread infection to susceptible cells and hosts. Virion morphogenesis can be divided into three stages: assembly, wherein the virion is created and essential components are packaged; budding, wherein the virion crosses the plasma membrane and obtains its lipid envelope; and maturation, wherein the virion changes structure and becomes infectious. All of these stages are coordinated by the Gag polyprotein and its proteolytic maturation products, which function as the major structural proteins of the virus. Here, we review our current understanding of the mechanisms of HIV-1 assembly, budding, and maturation, starting with a general overview and then providing detailed descriptions of each of the different stages of virion morphogenesis.
    Cold Spring Harbor Perspectives in Medicine 07/2012; 2(7):a006924. DOI:10.1101/cshperspect.a006924 · 7.56 Impact Factor
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    ABSTRACT: The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action.
    Journal of Virology 04/2012; 86(12):6643-55. DOI:10.1128/JVI.00493-12 · 4.65 Impact Factor
  • Wesley I Sundquist, Robert M Krug
    04/2012; 2(2):111-4. DOI:10.1016/j.coviro.2012.02.008

Publication Stats

12k Citations
1,175.63 Total Impact Points

Institutions

  • 1993–2015
    • University of Utah
      • • Department of Biochemistry
      • • Department of Medicinal Chemistry
      Salt Lake City, Utah, United States
  • 2013
    • Technion - Israel Institute of Technology
      H̱efa, Haifa, Israel
  • 2011
    • University of Sydney
      • School of Molecular Bioscience
      Sydney, New South Wales, Australia
  • 2004–2008
    • Vanderbilt University
      • Department of Pathology, Microbiology and Immunology
      Nashville, Michigan, United States
  • 1997
    • Memorial Sloan-Kettering Cancer Center
      New York, New York, United States
  • 1996
    • University of Maryland, Baltimore County
      • Department of Chemistry and Biochemistry
      Baltimore, MD, United States
  • 1994
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States