Dynamics of ESCRT protein recruitment during retroviral assembly

Jouvenet, Nolwenn; Zhadina, Maria; Bieniasz, Paul D.; Simon, Sanford M.

doi:10.1038/ncb2207

Cited by 203 publications

(270 citation statements)

References 43 publications

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“…The ability of ESCRT-I and -II to form a supercomplex and the lack of a clear necessary role for Ub binding by ESCRT-II suggests ESCRT-I and -II function together rather than individually in sequence, as a secondary Ub-sorting receptor that receives cargo from ESCRT-0 (Amerik et al 2006;Shields et al 2009;Boura et al 2012). The idea that these work in sequence is supported by immunolocalization experiments showing a differential distribution of ESCRT-0 and ESCRT-I, live cell imaging that shows sequential assembly of ESCRTs at the sites of viral budding and cytokinesis, and biochemical studies in yeast mutants where assembly intermediates can be accumulated (Babst et al 2002;Bache et al 2003;Elia et al 2011;Guizetti et al 2011;Jouvenet et al 2011). In addition, several other proteins that are structurally related to ESCRT-0 have been proposed to work in parallel to ESCRT-0 as alternative early Ub-cargo receptors.…”

Section: Sequential Orchestration Of Escrt Functionmentioning

confidence: 51%

Ubiquitin-Dependent Sorting in Endocytosis

Piper

Đikić

Lukacs

2014

Cold Spring Harbor Perspectives in Biology

193

176

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When ubiquitin (Ub) is attached to membrane proteins on the plasma membrane, it directs them through a series of sorting steps that culminate in their delivery to the lumen of the lysosome where they undergo complete proteolysis. Ubiquitin is recognized by a series of complexes that operate at a number of vesicle transport steps. Ubiquitin serves as a sorting signal for internalization at the plasma membrane and is the major signal for incorporation into intraluminal vesicles of multivesicular late endosomes. The sorting machineries that catalyze these steps can bind Ub via a variety of Ub-binding domains. At the same time, many of these complexes are themselves ubiquitinated, thus providing a plethora of potential mechanisms to regulate their activity. Here we provide an overview of how membrane proteins are selected for ubiquitination and deubiquitination within the endocytic pathway and how that ubiquitin signal is interpreted by endocytic sorting machineries. HOW PROTEINS ARE SELECTED FOR UBIQUITINATIONU biquitin (Ub) is covalently attached to substrate proteins by the concerted action of E2-conjugating enzymes and E3 ligases (Varshavsky 2012). Most of the specificity and regulation of ubiquitination is at the level of the E3 ligases, which vastly outnumber the E2-conjugating enzymes. Ubiquitination results from the transfer of Ub, held either by the E2 or in some cases by the E3 as a high-energy thioester bond, onto a substrate protein, typically on the terminal amide of a substrate acceptor lysine side chain. Owing to the intense interest in the ubiquitination system, many of the simple rules and distinctions concerning different types of ligases, their specificity for forming particular polyUb chains, and even the kinds of residues in substrate proteins that can be ubiquitin modified, have been blurred with the discovery of mixed poly-Ub chains, new enzymatic mechanisms for Ub transfer, and ubiquitination of nonlysine residues (Kulathu and Komander 2012;Wenzel and Klevit 2012;McDowell and Philpott 2013). Nonetheless, in basic terms, Ub ligases function as platforms that coordinate substrate recognition with Ub transfer as well as configuring poly-Ub chain topology by the E2-conjugating enzyme. Substrates can be bound directly by the E3 ligase or by adaptor proteins that in turn bind to ligases, and selecEditors:

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Section: Sequential Orchestration Of Escrt Functionmentioning

confidence: 51%

Ubiquitin-Dependent Sorting in Endocytosis

Piper

Đikić

Lukacs

2014

Cold Spring Harbor Perspectives in Biology

193

176

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“…Less consistent with the yeast-based mechanism, CHMP6, CHMP3, and CHMP1A/B knockdowns have little or no HIV-1 release phenotype (26,32). These differences are hard to reconcile with live-cell imaging detecting CHMP1B at bud sites (33) and with the finding that CHMP2A and CHMP3 coassemble in vitro into tubules but do not do so on their own (34). The differences in the results with yeast ESCRT-III proteins and the knockdown, imaging, and tubule formation data for human proteins called out for further investigation.…”

mentioning

confidence: 95%

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

Carlson

Hurley

2012

Proc. Natl. Acad. Sci. U.S.A.

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Most membrane-enveloped viruses depend on host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release. HIV-1 is the prototypic ESCRT-dependent virus. The direct interactions between HIV-1 and the early ESCRT factors TSG101 and ALIX have been mapped in detail. However, the full pathway of ESCRT recruitment to HIV-1 budding sites, which culminates with the assembly of the late-acting CHMP4, CHMP3, CHMP2, and CHMP1 subunits, is less completely understood. Here, we report the biochemical reconstitution of ESCRT recruitment to viral assembly sites, using purified proteins and giant unilamellar vesicles. The myristylated full-length Gag protein of HIV-1 was purified to monodispersity. Myr-Gag forms clusters on giant unilamellar vesicle membranes containing the plasma membrane lipid PIð4,5ÞP 2 . These Gag clusters package a fluorescent oligonucleotide, and recruit early ESCRT complexes ESCRT-I or ALIX with the appropriate dependence on the Gag PTAP and LYPðXÞ n L motifs. ALIX directly recruits the key ESCRT-III subunit CHMP4. ESCRT-I can only recruit CHMP4 when ESCRT-II and CHMP6 are present as intermediary factors. Downstream of CHMP4, CHMP3 and CHMP2 assemble synergistically, with the presence of both subunits required for efficient recruitment. The very late-acting factor CHMP1 is not recruited unless the pathway is completed through CHMP3 and CHMP2. These findings define the minimal sets of components needed to complete ESCRT assembly at HIV-1 budding sites, and provide a starting point for in vitro structural and biophysical dissection of the system. confocal microscopy | host-pathogen interaction | membrane traffic | virus budding M ost membrane-enveloped viruses utilize host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release (1-3). This dependence was first described for HIV-1, where mutation of an ESCRTbinding motif (late domain) in the viral protein Gag was shown to stall virus bud release at a late stage in virus assembly (4, 5). Late domains have subsequently been found in other retroviruses and numerous other virus families including, e.g., flavi-, filo-and rhabdoviruses (6). Indeed, the only well-characterized case of ESCRT-independent release of a membrane-enveloped virus is influenza virus (7). In normal cell physiology, ESCRTs function in cytokinesis, formation of intralumenal vesicles in multivesicular endosomes, and vesicle release from the plasma membrane (8-10). These seemingly disparate processes all involve membrane budding away from the cytoplasm, and ESCRTs are the only described protein machinery to perform vesicle formation with this topology, which is analogous to virus release.The initial events in HIV-1 ESCRT recruitment are well characterized. The structural protein of HIV-1, Gag, binds earlyacting ESCRT factors through two late-domain motifs in its C-terminal p6 domain: a PTAP sequence that interacts with the TSG101 subunit of ESCRT-I (11-16) and a LYPðXÞ n L motif that interacts with the ESCRT...

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“…Additionally, this allows for the system to be perturbed at various levels of the assembly process to assess the effects in a spatiotemporal manner. For instance, expression of dominant-negative VPS4A stalled ESCRT III recycling and prevented HIV Gag VLP scission, suggesting that VPS4A plays a direct role in viral membrane scission [39]. These technologies and experimental methodologies show promise for elucidating the spatiotemporal timeline of EBOV VP40 assembly and egress.…”

Section: Timeframe Of Vp40-mediated Assembly and Buddingmentioning

confidence: 99%

“…This has been shown to provide robust spatiotemporal analysis of HIV virion assembly, allowing the identification of markers for different stages of the assembly process and the characterization of host cell participation using dual-fluorescence experimental systems. For instance, it has been revealed in equine infectious anemia virus (EIAV) that ESCRT components are recruited differentially [39]: Alix is gradually incorporated into the EIAV Gag VLP throughout assembly, while ESCRT III components as well as the ATPase VPS4A are recruited transiently in a pulse-like manner at the end of assembly; after which, scission occurs and ESCRT III components dissociate from the assembly site. Additionally, this allows for the system to be perturbed at various levels of the assembly process to assess the effects in a spatiotemporal manner.…”

Section: Timeframe Of Vp40-mediated Assembly and Buddingmentioning

confidence: 99%

“…However, advances in live-cell fluorescence microscopy are overcoming these obstacles and have already been utilized to study the assembly kinetics of human immunodeficiency virus (HIV) [16•, 32-39] and other retroviruses [39]. These investigators have established an experimental framework with which to study the assembly events of viruses as well as host cell interactions.…”

Section: Timeframe Of Vp40-mediated Assembly and Buddingmentioning

confidence: 99%

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The Ebola Virus Matrix Protein VP40 Interacts With Several Host Protein Networks to Facilitate Viral Replication

Frick

Stahelin

2015

Curr Clin Micro Rpt

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The Ebola virus (EBOV) is a lipid-enveloped virus that is filamentous in shape and belongs to the Filoviridae family. EBOV can cause viral hemorrhagic fever in humans and non-human primates with high rates of fatality. Using a negative sense RNA genome that encodes just eight proteins, the lipid-enveloped EBOV can enter mammalian cells and undergo efficient replication to produce more virions to sustain and spread the infection. Throughout the replication cycle, EBOV inhibits or hijacks several host protein networks to suppress the immune response and to enhance the efficiency of assembly and budding from the host cell plasma membrane. Assembly and budding of EBOV is regulated by its matrix protein VP40, which can form different structures with distinct functions in the viral life cycle. VP40 has been shown to interact with several important host proteins, and although the conformational regulation of these interactions is mostly unknown, the sites of interaction have been somewhat resolved and will serve as the basis of this review article.

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Dynamics of ESCRT protein recruitment during retroviral assembly

Cited by 203 publications

References 43 publications

Ubiquitin-Dependent Sorting in Endocytosis

Ubiquitin-Dependent Sorting in Endocytosis

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

The Ebola Virus Matrix Protein VP40 Interacts With Several Host Protein Networks to Facilitate Viral Replication

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