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ESCRT-III protein requirements for HIV-1 budding.

ABSTRACT: Two early-acting components of the cellular ESCRT pathway, ESCRT-I and ALIX, participate directly in HIV-1 budding. The membrane fission activities of ESCRT-III subunits are also presumably required, but humans express 11 different CHMP/ESCRT-III proteins whose functional contributions are not yet clear. We therefore depleted cells of each of the different CHMP proteins and protein families and examined the effects on HIV-1 budding. Virus release was profoundly inhibited by codepletion of either CHMP2 or CHMP4 family members, resulting in ?100-fold titer reductions. CHMP2A and CHMP4B proteins bound one another, and this interaction was required for budding. By contrast, virus release was reduced only modestly by depletion of CHMP3 and CHMP1 proteins (2- to 8-fold titer reductions) and was unaffected by depletion of other human ESCRT-III proteins. HIV-1 budding therefore requires only a subset of the known human ESCRT-III proteins, with the CHMP2 and CHMP4 families playing key functional roles.

SUBMITTER: Morita E

PROVIDER: S-EPMC3070458 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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ESCRT-III protein requirements for HIV-1 budding.

Morita Eiji E Sandrin Virginie V McCullough John J Katsuyama Angela A Baci Hamilton Ira I Sundquist Wesley I WI

Cell host & microbe 20110301 3

Two early-acting components of the cellular ESCRT pathway, ESCRT-I and ALIX, participate directly in HIV-1 budding. The membrane fission activities of ESCRT-III subunits are also presumably required, but humans express 11 different CHMP/ESCRT-III proteins whose functional contributions are not yet clear. We therefore depleted cells of each of the different CHMP proteins and protein families and examined the effects on HIV-1 budding. Virus release was profoundly inhibited by codepletion of either ...[more]

PMID: 21396898

Similar Datasets

ESCRT requirements for EIAV budding.

Project description:BackgroundRetroviruses 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.ResultsIn 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.ConclusionsWe 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.

| S-EPMC3907061 | biostudies-literature

An Inducible ESCRT-III Inhibition Tool to Control HIV-1 Budding.

Project description:HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites is limited due to spatial resolution and transient ESCRT-III recruitment. Here, we developed a drug-inducible transient HIV-1 budding inhibitory tool to enhance the ESCRT-III lifetime at budding sites. We generated autocleavable CHMP2A, CHMP3, and CHMP4B fusion proteins with the hepatitis C virus NS3 protease. We characterized the CHMP-NS3 fusion proteins in the absence and presence of protease inhibitor Glecaprevir with regard to expression, stability, localization, and HIV-1 Gag VLP budding. Immunoblotting experiments revealed rapid and stable accumulation of CHMP-NS3 fusion proteins. Notably, upon drug administration, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted no effect but synergized with CHMP2A-NS3. Localization studies demonstrated the relocalization of CHMP-NS3 fusion proteins to the plasma membrane, endosomes, and Gag VLP budding sites. Through the combined use of transmission electron microscopy and video-microscopy, we unveiled drug-dependent accumulation of CHMP2A-NS3 and CHMP4B-NS3, causing a delay in HIV-1 Gag-VLP release. Our findings provide novel insight into the functional consequences of inhibiting ESCRT-III during HIV-1 budding and establish new tools to decipher the role of ESCRT-III at HIV-1 budding sites and other ESCRT-catalyzed cellular processes.

| S-EPMC10748027 | biostudies-literature

Differential requirements for Alix and ESCRT-III in cytokinesis and HIV-1 release.

Project description:The ESCRT machinery functions in topologically equivalent membrane fission events, namely multivesicular body formation, the terminal stages of cytokinesis and HIV-1 release. Here, we show that the ESCRT-III-binding protein Alix is recruited to the midbody of dividing cells through binding Cep55 via an evolutionarily conserved peptide. Disruption of Cep55/Alix/ESCRT-III interactions causes formation of aberrant midbodies and cytokinetic failure, demonstrating an essential role for these proteins in midbody morphology and cell division. We also show that the C terminus of Alix encodes a multimerization activity that is essential for its function in Alix-dependent HIV-1 release and for interaction with Tsg101. Last, we demonstrate that overexpression of Chmp4b and Chmp4c differentially inhibits HIV-1 release and cytokinesis, suggesting possible reasons for gene expansion within the mammalian Class E VPS pathway.

| S-EPMC2492449 | biostudies-literature

Release of autoinhibition converts ESCRT-III components into potent inhibitors of HIV-1 budding.

Project description:The endosomal sorting complex ESCRT-III, which is formed by the structurally related CHMP proteins, is engaged by HIV-1 to promote viral budding. Here we show that progressive truncations into the C-terminal acidic domains of CHMP proteins trigger an increasingly robust anti-HIV budding activity. Together with biochemical evidence for specific intramolecular interactions between the basic and acidic halves of CHMP3 and CHMP4B, these results suggest that the acidic domains are autoinhibitory. The acidic half of CHMP3 also interacts with the endosome-associated ubiquitin isopeptidase AMSH, and the coexpression of AMSH or its CHMP3-binding domain converts wild-type CHMP3 into a potent inhibitor of HIV-1 release. Point mutations in CHMP3 that prevent binding to AMSH abrogate this effect, suggesting that binding to AMSH relieves the autoinhibition of CHMP3. Collectively, our results indicate that CHMP proteins are regulated through an autoinhibitory switch mechanism that allows tight control of ESCRT-III assembly.

| S-EPMC1748189 | biostudies-literature

Temporal and spatial organization of ESCRT protein recruitment during HIV-1 budding.

Project description:HIV-1 virions assemble at the plasma membrane of mammalian cells and recruit the endosomal sorting complex required for transport (ESCRT) machinery to enable particle release. However, little is known about the temporal and spatial organization of ESCRT protein recruitment. Using multiple-color live-cell total internal reflection fluorescence microscopy, we observed that the ESCRT-I protein Tsg101 is recruited together with Gag to the sites of HIV-1 assembly, whereas later-acting ESCRT proteins (Chmp4b and Vps4A) are recruited sequentially, once Gag assembly is completed. Chmp4b, a protein that is required to mediate particle scission, is recruited to HIV-1 assembly sites ∼10 s before the ATPase Vps4A. Using two-color superresolution imaging, we observed that the ESCRT machinery (Tsg101, Alix, and Chmp4b/c proteins) is positioned at the periphery of the nascent virions, with the Tsg101 assemblages positioned closer to the Gag assemblages than Alix, Chmp4b, or Chmp4c. These results are consistent with the notion that the ESCRT machinery is recruited transiently to the neck of the assembling particle and is thus present at the appropriate time and place to mediate fission between the nascent virus and the plasma membrane.

| S-EPMC4142993 | biostudies-literature

Two distinct modes of ESCRT-III recognition are required for VPS4 functions in lysosomal protein targeting and HIV-1 budding.

Project description:The ESCRT pathway mediates membrane remodeling during enveloped virus budding, cytokinesis, and intralumenal endosomal vesicle formation. Late in the pathway, a subset of membrane-associated ESCRT-III proteins display terminal amphipathic "MIM1" helices that bind and recruit VPS4 ATPases via their MIT domains. We now report that VPS4 MIT domains also bind a second, "MIM2" motif found in a different subset of ESCRT-III subunits. The solution structure of the VPS4 MIT-CHMP6 MIM2 complex revealed that MIM2 elements bind in extended conformations along the groove between the first and third helices of the MIT domain. Mutations that block VPS4 MIT-MIM2 interactions inhibit VPS4 recruitment, lysosomal protein targeting, and HIV-1 budding. MIT-MIM2 interactions appear to be common throughout the ESCRT pathway and possibly elsewhere, and we suggest how these interactions could contribute to a mechanism in which VPS4 and ESCRT-III proteins function together to constrict the necks of budding vesicles.

| S-EPMC2586299 | biostudies-literature

ESCRT-III CHMP2A and CHMP3 form variable helical polymers in vitro and act synergistically during HIV-1 budding.

Project description:The endosomal sorting complex required for transport-III (ESCRT-III) proteins are essential for budding of some enveloped viruses, for 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 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 surface plasmon resonance 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.

| S-EPMC3783020 | biostudies-literature

Dynamics of upstream ESCRT organization at the HIV-1 budding site.

Project description:In the late stages of the HIV-1 life cycle, membrane localization and self-assembly of Gag polyproteins induce membrane deformation and budding. Release of the virion requires direct interaction between immature Gag lattice and upstream ESCRT machinery at the viral budding site, followed by assembly of downstream ESCRT-III factors, culminating in membrane scission. However, molecular details of upstream ESCRT assembly dynamics at the viral budding site remain unclear. In this work, using coarse-grained (CG) molecular dynamics (MD) simulations, we investigated the interactions between Gag, ESCRT-I, ESCRT-II, and membrane to delineate the dynamical mechanisms by which upstream ESCRTs assemble templated by late-stage immature Gag lattice. We first systematically derived "bottom-up" CG molecular models and interactions of upstream ESCRT proteins from experimental structural data and extensive all-atom MD simulations. Using these molecular models, we performed CG MD simulations of ESCRT-I oligomerization and ESCRT-I/II supercomplex formation at the neck of the budding virion. Our simulations demonstrate that ESCRT-I can effectively oligomerize to higher-order complexes templated by the immature Gag lattice both in the absence of ESCRT-II and when multiple copies of ESCRT-II are localized at the bud neck. The ESCRT-I/II supercomplexes formed in our simulations exhibit predominantly columnar structures, which has important implications for the nucleation pathway of downstream ESCRT-III polymers. Importantly, ESCRT-I/II supercomplexes bound to Gag initiate membrane neck constriction by pulling the inner edge of the bud neck closer to the ESCRT-I headpiece ring. Our findings serve to elucidate a network of interactions between upstream ESCRT machinery, immature Gag lattice, and membrane neck that regulate protein assembly dynamics at the HIV-1 budding site.

| S-EPMC10397573 | biostudies-literature

ESCRT-III mediates budding across the inner nuclear membrane and regulates its integrity.

Project description:Vesicle-mediated nucleocytoplasmic transport is a nuclear pore-independent mechanism for the nuclear export of macromolecular complexes, but the molecular basis for this transport remains largely unknown. Here we show that endosomal sorting complex required for transport-III (ESCRT-III) is recruited to the inner nuclear membrane (INM) during the nuclear export of herpes simplex virus 1 (HSV-1). Scission during HSV-1 budding through the INM is prevented by depletion of ESCRT-III proteins. Interestingly, in uninfected human cells, the depletion of ESCRT-III proteins induces aberrant INM proliferation. Our results show that HSV-1 expropriates the ESCRT-III machinery in infected cells for scission of the INM to produce vesicles containing progeny virus nucleocapsids. In uninfected cells, ESCRT-III regulates INM integrity by downregulating excess INM.

| S-EPMC6107581 | biostudies-literature

Timing of ESCRT-III protein recruitment and membrane scission during HIV-1 assembly.

Project description:The Endosomal Sorting Complexes Required for Transport III (ESCRT-III) proteins are critical for cellular membrane scission processes with topologies inverted relative to clathrin-mediated endocytosis. Some viruses appropriate ESCRT-IIIs for their release. By imaging single assembling viral-like particles of HIV-1, we observed that ESCRT-IIIs and the ATPase VPS4 arrive after most of the virion membrane is bent, linger for tens of seconds, and depart ~20 s before scission. These observations suggest that ESCRT-IIIs are recruited by a combination of membrane curvature and the late domains of the HIV-1 Gag protein. ESCRT-IIIs may pull the neck into a narrower form but must leave to allow scission. If scission does not occur within minutes of ESCRT departure, ESCRT-IIIs and VPS4 are recruited again. This mechanistic insight is likely relevant for other ESCRT-dependent scission processes including cell division, endosome tubulation, multivesicular body and nuclear envelope formation, and secretion of exosomes and ectosomes.

| S-EPMC6080951 | biostudies-literature

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