No strings attached: the ESCRT machinery in viral budding and cytokinesis

McDonald, Bethan; Martín-Serrano, Juan

doi:10.1242/jcs.028308

Cited by 103 publications

(105 citation statements)

References 153 publications

Supporting

Mentioning

104

Contrasting

Order By: Relevance

“…Accordingly, HIV-1 viral particle production is significantly reduced when Tsg101 is disrupted, whilst disrupting Alix function can be compensated for by Tsg101 interactions (McDonald & Martin-Serrano, 2009). …”

Section: Inhibition Of Vps4 Function Blocks Hcv Particle Productionmentioning

confidence: 99%

“…In this regard, it has been well documented that many enveloped viruses utilize aspects of the host endosomal pathway to acquire a membrane and effect assembly and release following membrane scission. Central to the endosomal pathway is the multivesicular body (MVB), which is generated by inward budding of numerous intraluminal vesicles, a process that is analogous to the mechanism whereby viruses acquire their envelope (for review see McDonald & Martin-Serrano, 2009). The sorting of cargo into MVBs is co-ordinated by several multi-protein complexes collectively termed the endosomal sorting complexes required for transport (ESCRTs).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles

Corless

Crump

Griffin

et al. 2009

Journal of General Virology

101

View full text Add to dashboard Cite

The mechanisms by which infectious hepatitis C virus (HCV) particles are assembled and released from infected cells remain poorly characterized. In this regard, many other enveloped viruses, notably human immunodeficiency virus type 1, have been shown to utilize the host vacuolar protein sorting machinery (also known as the endosomal sorting complex required for transport; ESCRT) to traffic through the cell and effect the membrane rearrangements required for the formation of enveloped particles. We postulated that this might also apply to HCV. To test this hypothesis, we established a method of conditional virus-like particle assembly involving transcomplementation of an envelope-deleted JFH-1 genome using plasmid transfection. This system reliably produced virus particles that were infectious and could be enumerated easily by focusforming assay in Huh7 cells. Following co-transfection with plasmids expressing various dominant-negative forms of either components of the ESCRT-III complex or Vps4 (the AAA ATPase that recycles the ESCRT complexes), a reduction in particle production was seen. No significant effect was observed after co-transfection of dominant-negative ESCRT-I or Alix, an ESCRT associated protein. Dominant-negative Vps4 or ESCRT-III components had no effect on either virus genome replication or the accumulation of intracellular infectious particles. These data were confirmed using cell culture infectious HCV and we conclude that HCV requires late components of the ESCRT pathway for release of infectious virus particles. INTRODUCTIONHepatitis C virus (HCV) represents a major global health burden. An estimated 170 million people are chronically infected worldwide and a significant proportion of these will go on to develop end stage liver disease. Current drug treatments are poorly tolerated and are only effective in approximately 50 % of individuals. There is, therefore, a pressing need for a greater understanding of the molecular mechanisms involved in HCV infection in order for novel drug targets to be identified.HCV is the prototype member of the genus Hepacivirus in the family Flaviviridae. It is a single-stranded, positive-sense RNA virus whose genome encodes a single ORF. This is translated in a cap-independent manner from an internal ribosome entry site (IRES) located in the 59 untranslated region, into a single polyprotein of approximately 3000 aa. The polyprotein is proteolytically cleaved by host and viral proteases to form 10 mature proteins; the structural proteins (core, E1 and E2), a viroporin p7 and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B).Although recent advances in the development of tissue culture systems for the study of the HCV life cycle (Lindenbach et al., 2005;Wakita et al., 2005;Zhong et al., 2005) have defined a critical role for lipid droplets in virus assembly (Miyanari et al., 2007), the precise mechanism by which infectious HCV particles are assembled and released remains poorly characterized. In particular, it has not been established how nascent...

show abstract

Section: Inhibition Of Vps4 Function Blocks Hcv Particle Productionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles

Corless

Crump

Griffin

et al. 2009

Journal of General Virology

101

View full text Add to dashboard Cite

show abstract

“…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)(9)(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.…”

mentioning

confidence: 99%

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.

View full text Add to dashboard Cite

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...

show abstract

“…ESCRT complexes are responsible for the endosomal sorting of ubiquitinated membrane proteins into multivesicular bodies (MVBs) en route to their lysosomal degradation (6,7). ESCRT-I in particular is also involved in plasma membrane functions in cytokinesis and the release of HIV-1 and other viruses (8). MABP domains are also found in many membrane-associated bacterial proteins and in the N-terminal region of human DENND4 isoforms.…”

mentioning

confidence: 99%

Structural basis for membrane targeting by the MVB12-associated β-prism domain of the human ESCRT-I MVB12 subunit

Bouřa

Hurley

2012

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

View full text Add to dashboard Cite

MVB12-associated β-prism (MABP) domains are predicted to occur in a diverse set of membrane-associated bacterial and eukaryotic proteins, but their existence, structure, and biochemical properties have not been characterized experimentally. Here, we find that the MABP domains of the MVB12A and B subunits of ESCRT-I are functional modules that bind in vitro to liposomes containing acidic lipids depending on negative charge density. The MABP domain is capable of autonomously localizing to subcellular puncta and to the plasma membrane. The 1.3-Å atomic resolution crystal structure of the MVB12B MABP domain reveals a β-prism fold, a hydrophobic membrane-anchoring loop, and an electropositive phosphoinositide-binding patch. The basic patch is open, which explains how it senses negative charge density but lacks stereoselectivity. These observations show how ESCRT-I could act as a coincidence detector for acidic phospholipids and protein ligands, enabling it to function both in protein transport at endosomes and in cytokinesis and viral budding at the plasma membrane.HIV | protein structure | protein-membrane interactions | X-ray crystallography | subcellular localization T he concept of modular membrane-targeting domains is an important theme in the architecture of regulatory proteins and central to our current understanding of the subcellular localization of proteins (1-3). The best known of these modules include the PH, PX, FYVE, C1, C2, and ENTH domains (1-3). Each of these domains was initially proposed on the basis of bioinformatics analysis and subsequently confirmed by structural, biochemical, and cell biological approaches.A recent bioinformatics analysis (4) led to the identification of a putative membrane-targeted domain, termed the "MVB12-associated β-prism" (MABP) domain (4). MABP domains are found at the N termini of MVB12A and B, which are subunits of the human ESCRT-I complex (5). ESCRT complexes are responsible for the endosomal sorting of ubiquitinated membrane proteins into multivesicular bodies (MVBs) en route to their lysosomal degradation (6, 7). ESCRT-I in particular is also involved in plasma membrane functions in cytokinesis and the release of HIV-1 and other viruses (8). MABP domains are also found in many membrane-associated bacterial proteins and in the N-terminal region of human DENND4 isoforms. DENND4 proteins are Rab10 guanine nucleotide exchange factors that localize to a tubular Golgi-proximal membrane compartment (9).We set out to test the inference that MABP domains interact with membranes and found that the MVB12A and B MABP domains did. The crystal structure, solved at atomic resolution, showed that the MABP domain has a β-prism fold, consistent with the bioinformatics analysis. The structure identified a potential membrane-binding site, which we confirmed experimentally in vitro and in vivo. We found that the domain bound to liposomes according to the negative charge density of the membrane but with little specificity for particular lipid head groups. We went on to characterize the...

show abstract

No strings attached: the ESCRT machinery in viral budding and cytokinesis

Cited by 103 publications

References 153 publications

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles

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

Structural basis for membrane targeting by the MVB12-associated β-prism domain of the human ESCRT-I MVB12 subunit

Contact Info

Product

Resources

About