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1.
2.
Heterologous expression of HIV-1 Gag in a variety of host cells results in its packaging into virus-like particles (VLPs) that are subsequently released into the extracellular milieu. This phenomenon represents a useful tool for probing cellular factors required for viral budding and has contributed to the discovery of roles for ubiquitin ligases and the endosomal sorting complexes required for transport (ESCRTs) in viral budding. These factors are highly conserved throughout eukaryotes and have been studied extensively in the yeast Saccharomyces cerevisiae, a model eukaryote previously utilized as a host for the production of VLPs. We used heterologous expression of HIV Gag in yeast spheroplasts to examine the role of ESCRTs and associated factors (Rsp5, a HECT ubiquitin ligase of the Nedd4 family; Bro1, a homolog of Alix; and Vps4, the AAA-ATPase required for ESCRT function in all contexts/organisms investigated) in the generation of VLPs. Our data reveal: 1) characterized Gag-ESCRT interaction motifs (late domains) are not required for VLP budding, 2) loss of function alleles of the essential HECT ubiquitin ligase Rsp5 do not display defects in VLP formation, and 3) ESCRT function is not required for VLP formation from spheroplasts. These results suggest that the egress of HIV Gag from yeast cells is distinct from the most commonly described mode of exit from mammalian cells, instead mimicking ESCRT-independent VLP formation observed in a subset of mammalian cells. As such, budding of Gag from yeast cells appears to represent ESCRT-independent budding relevant to viral replication in at least some situations. Thus the myriad of genetic and biochemical tools available in the yeast system may be of utility in the study of this aspect of viral budding.  相似文献   

3.
The three endosomal sorting complexes required for transport (ESCRTs) are integral to the degradation of endocytosed membrane proteins and multivesicular body (MVB) biogenesis. Here, we review evidence that ESCRTs have evolved as a specialized machinery for the degradative sorting of ubiquitinated membrane proteins and we highlight recent studies that have shed light on the mechanisms by which these complexes mediate protein sorting, MVB biogenesis, tumour suppression and viral budding. We also discuss evidence that some ESCRT subunits have evolved additional functions that are unrelated to membrane trafficking.  相似文献   

4.
The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L-) domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e.g. WWP1). It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV), where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed.  相似文献   

5.
As a mechanism of signal attenuation, receptors for growth factors, peptide hormones and cytokines are internalized in response to ligand binding, followed by degradation in lysosomes. Receptor ubiquitination is a key signal for such downregulation, and four protein complexes known as endosomal sorting complex required for transport (ESCRT)-0, -I, -II and -III have been identified as the machinery required for degradative endosomal sorting of ubiquitinated membrane proteins in yeast and metazoans. Three of these complexes contain ubiquitin-binding domains whereas ESCRT-III instead recruits deubiquitinating enzymes. The concerted action of the ESCRTs not only serves to sort ubiquitinated cargo but is also thought to cause inward vesiculation of endosomal membranes, thereby mediating biogenesis of multivesicular endosomes (MVEs). Because ligand-mediated receptor downregulation plays an important role in signal attenuation, it is not surprising that dysfunction of ESCRT components is associated with disease. In this review we discuss the possible roles of ESCRTs in protection against cancer, neurodegenerative diseases and bacterial infections, and we highlight the fact that many RNA viruses exploit the ESCRT machinery for the final abscission step of their budding from cells. We also review the additional functions of ESCRT proteins in cytokinesis and discuss how these may be related to ESCRT-associated pathologies.  相似文献   

6.
代军  仇旭升  丁铲 《生物工程学报》2023,39(10):3948-3965
内吞体分选转运复合体(endosomal sorting complex required for transport,ESCRT)系统驱动细胞的不同生命进程,包括内体分选、细胞器生物发生、囊泡运输、维持质膜完整性、细胞质分裂期间的膜裂变、有丝分裂后的核膜重组、自噬过程中吞噬孔的封闭以及包膜病毒出芽等。越来越多的证据表明,ESCRT系统能够被不同家族病毒劫持用于自身增殖。在病毒生命周期的不同阶段,病毒可以通过各种方式干扰或利用ESCRT系统介导的生理过程,最大限度地提高感染宿主的机会。此外,许多逆转录病毒和RNA病毒蛋白具有“晚期结构域”基序,可招募宿主ESCRT亚基蛋白帮助病毒内吞、运输、复制、出芽以及外排。因此,病毒“晚期结构域”基序和ESCRT亚基蛋白可能是病毒感染治疗中具有广泛应用前景的药物靶点。本文重点综述了ESCRT系统的组成及功能,ESCRT亚基和病毒“晚期结构域”基序对病毒复制的影响以及ESCRT介导的抗病毒作用,以期为抗病毒药物的开发和利用提供参考。  相似文献   

7.
The p6 region of HIV-1 Gag contains two late (L) domains, PTAP and LYPXnL, that bind the cellular proteins Tsg101 and Alix, respectively. These interactions are thought to recruit members of the host fission machinery (ESCRT) to facilitate HIV-1 release. Here we report a new role for the p6-adjacent nucleocapsid (NC) domain in HIV-1 release. The mutation of basic residues in NC caused a pronounced decrease in virus release from 293T cells, although NC mutant Gag proteins retained the ability to interact with cellular membranes and RNAs. Remarkably, electron microscopy analyses of these mutants revealed arrested budding particles at the plasma membrane, analogous to those seen following the disruption of the PTAP motif. This result indicated that the basic residues in NC are important for virus budding. When analyzed in physiologically more relevant T-cell lines (Jurkat and CEM), NC mutant viruses remained tethered to the plasma membrane or to each other by a membranous stalk, suggesting membrane fission impairment. Remarkably, NC mutant release defects were alleviated by the coexpression of a Gag protein carrying a wild-type (WT) NC domain but devoid of all L domain motifs and by providing alternative access to the ESCRT pathway, through the in trans expression of the ubiquitin ligase Nedd4.2s. Since NC mutant Gag proteins retained the interaction with Tsg101, we concluded that NC mutant budding arrests might have resulted from the inability of Gag to recruit or utilize members of the host ESCRT machinery that act downstream of Tsg101. Together, these data support a model in which NC plays a critical role in HIV-1 budding.  相似文献   

8.
The endosomal sorting complexes required for transport (ESCRT) proteins help in the recognition, sorting and degradation of ubiquitinated cargoes from the cell surface, long‐lived proteins or aggregates, and aged organelles present in the cytosol. These proteins take part in the endo‐lysosomal system of degradation. The ESCRT proteins also play an integral role in cytokinesis, viral budding and mRNA transport. Many neurodegenerative diseases are caused by toxic accumulation of cargo in the cell, which causes stress and ultimately leads to neuronal death. This accumulation of cargo occurs because of defects in the endo‐lysosomal degradative pathway—loss of function of ESCRTs has been implicated in this mechanism. ESCRTs also take part in many survival processes, lack of which can culminate in neuronal cell death. While the role played by the ESCRT proteins in maintaining healthy neurons is known, their role in neurodegenerative diseases is still poorly understood. In this review, we highlight the importance of ESCRTs in maintaining healthy neurons and then suggest how perturbations in many of the survival mechanisms governed by these proteins could eventually lead to cell death; quite often these correlations are not so obviously laid out. Extensive neuronal death eventually culminates in neurodegeneration.   相似文献   

9.
Late domains are short peptide sequences encoded by enveloped viruses to promote the final separation of the nascent virus from the infected cell. These amino acid motifs facilitate viral egress by interacting with components of the ESCRT (endosomal sorting complex required for transport) machinery, ultimately leading to membrane scission by recruiting ESCRT-III to the site of viral budding. PPXY late (L) domains present in viruses such as murine leukemia virus (MLV) or human T-cell leukemia virus type 1 (HTLV-1) access the ESCRT pathway via interaction with HECT ubiquitin ligases (WWP1, WWP2, and Itch). However, the mechanism of ESCRT-III recruitment in this context remains elusive. In this study, we tested the arrestin-related trafficking (ART) proteins, namely, ARRDC1 (arrestin domain-containing protein 1) to ARRDC4 and TXNIP (thioredoxin-interacting protein), for their ability to function as adaptors between HECT ubiquitin ligases and the core ESCRT machinery in PPXY-dependent budding. We present several lines of evidence in support of such a role: ARTs interact with HECT ubiquitin ligases, and they also exhibit multiple interactions with components of the ESCRT pathway, namely, ALIX and Tsg101, and perhaps with an as yet unidentified factor. Additionally, the ARTs can be recruited to the site of viral budding, and their overexpression results in a PPXY-specific inhibition of MLV budding. Lastly, we show that WWP1 changes the ubiquitination status of ARRDC1, suggesting that the ARTs may provide a platform for ubiquitination in PPXY-dependent budding. Taken together, our results support a model whereby ARTs are involved in PPXY-mediated budding by interacting with HECT ubiquitin ligases and providing several alternative routes for ESCRT-III recruitment.  相似文献   

10.
ESCRT & Co     
Components of the ESCRT (endosomal sorting complex required for transport) machinery mediate endosomal sorting of ubiquitinated membrane proteins. They are key regulators of biological processes important for cell growth and survival, such as growth‐factor‐mediated signalling and cytokinesis. In addition, enveloped viruses, such as HIV‐1, hijack and utilize the ESCRTs for budding during virus release and infection. Obviously, the ESCRT‐facilitated pathways require tight regulation, which is partly mediated by a group of interacting proteins, for which our knowledge is growing. In this review we discuss the different ESCRT‐modulating proteins and how they influence ESCRT‐dependent processes, for example, by acting as positive or negative regulators or by providing temporal and spatial control. A number of the interactors influence the classical ESCRT‐mediated process of endosomal cargo sorting, for example, by modulating the interaction between ubiquitinated cargo and the ESCRTs. Certain accessory proteins have been implicated in regulating the activity or steady‐state expression levels of the ESCRT components, whereas other interactors control the cellular localization of the ESCRTs, for example, by inducing shuttling between cytosol and nucleus or endosomes. In conclusion, the discovery of novel interactors has and will extend our knowledge of the biological roles of ESCRTs.  相似文献   

11.
Morita E 《The FEBS journal》2012,279(8):1399-1406
The endosomal sorting complexes required for transport (ESCRTs) mediate membrane fission from the cytoplasmic face of the bud neck. ESCRTs were originally identified as factors involved in multivesicular body vesicle biogenesis in yeast but have since been shown to function in other membrane fission events in mammalian cells, including enveloped virus budding and the abscission step of cytokinesis. Several recent studies have revealed that not all ESCRT factors are required for each of these biological processes, and this review summarizes our current understanding of the different requirements for ESCRT factors in these three different ESCRT-mediated mammalian membrane fission processes.  相似文献   

12.
The endosomal sorting complexes required for transport (ESCRTs) impact multiple cellular processes including multivesicular body sorting, abscission, and viral budding. The AAA-ATPase Vps4 is required for ESCRT function, and its full activity is dependent upon the co-factor Vta1. The Vta1 carboxyl-terminal Vta1 SBP1 Lip5 (VSL) domain stimulates Vps4 function by facilitating oligomerization of Vps4 into its active state. Here we report the identification of the Vps4 stimulatory element (VSE) within Vta1 that is required for additional stimulation of Vps4 activity in vitro and in vivo. VSE activity is autoinhibited in a manner dependent upon the unstructured linker region joining the amino-terminal microtubule interacting and trafficking domains and the carboxyl-terminal VSL domain. The VSE is also required for Vta1-mediated Vps4 stimulation by ESCRT-III subunits Vps60 and Did2. These results suggest that ESCRT-III binding to the Vta1 microtubule interacting and trafficking domains relieves linker region autoinhibition of the VSE to produce maximal activation of Vps4 during ESCRT function.  相似文献   

13.
A concentric circle model of multivesicular body cargo sorting   总被引:4,自引:0,他引:4  
Targeting of ubiquitylated transmembrane proteins into luminal vesicles of endosomal multivesicular bodies (MVBs) depends on their recognition by endosomal sorting complexes required for transport (ESCRTs), which are also required for MVB vesicle formation. The model originally proposed for how ESCRTs function succinctly summarizes much of the protein-protein interaction and genetic data but oversimplifies the coordination of cargo recognition and cannot explain why ESCRTs are required for the budding of MVB vesicles. Recent structural and functional studies of ESCRT complexes suggest an alternative model that might direct the next series of breakthroughs in understanding protein sorting through the MVB pathway.  相似文献   

14.
ESCRTs (endosomal sorting complexes required for transport) were first discovered in yeast and are known to be required in the biogenesis of the MVB (multivesicular body). Most ESCRT research has been carried out in vitro using models such as yeast and mammalian cells in culture. The role of the ESCRTs genes in endosome maturation is conserved from yeast to mammals, but little is known about their function during development in multicellular organisms. Since ESCRTs play a leading role in regulating some cell signalling pathways by addressing receptors to the lysosome, it appears important to monitor ESCRT functions in multicellular models. The present review summarizes recent research on the developmental and cellular functions of the ESCRT in Caenorhabditis elegans, Drosophila melanogaster, Mus musculus or Arabidopsis thaliana.  相似文献   

15.
HIV (human immunodeficiency virus) diverts the cellular ESCRT (endosomal sorting complex required for transport) machinery to promote virion release from infected cells. The ESCRT consists of four heteromeric complexes (ESCRT-0 to ESCRT-III), which mediate different membrane abscission processes, most importantly formation of intralumenal vesicles at multivesicular bodies. The ATPase VPS4 (vacuolar protein sorting 4) acts at a late stage of ESCRT function, providing energy for ESCRT dissociation. Recruitment of ESCRT by late-domain motifs in the viral Gag polyprotein and a role of ESCRT in HIV release are firmly established, but the order of events, their kinetics and the mechanism of action of individual ESCRT components in HIV budding are unclear at present. Using live-cell imaging, we show late-domain-dependent recruitment of VPS4A to nascent HIV particles at the host cell plasma membrane. Recruitment of VPS4A was transient, resulting in a single or a few bursts of at least two to five VPS4 dodecamers assembling at HIV budding sites. Bursts lasted for ~35 s and appeared with variable delay before particle release. These results indicate that VPS4A has a direct role in membrane scission leading to HIV-1 release.  相似文献   

16.
The p6 domain of human immunodeficiency virus type 1 (HIV-1) Gag has long been known to be monoubiquitinated. We have previously shown that the MA, CA, and NC domains are also monoubiquitinated at low levels (E. Gottwein and H. G. Krausslich, J. Virol. 79:9134-9144, 2005). While several lines of evidence support a role for ubiquitin in virus release, the relevance of Gag ubiquitination is unclear. To directly address the function of Gag ubiquitination, we constructed Gag variants in which lysine residues in the NC, SP2, and p6 domains were mutated to arginine either in individual domains or in combination. Using these mutants, we showed that in addition to MA, CA, NC, and p6, SP2 is also mono- or di-ubiquitinated at levels comparable to those of the other domains. Replacement of all lysine residues in only one of the domains had minor effects on virus release, while cumulative mutations in NC and SP2 or in NC and p6 resulted in an accumulation of late budding structures, as observed by electron microscopy analysis. Strikingly, replacement of all lysine residues downstream of CA led to a significant reduction in virus release kinetics and a fivefold accumulation of late viral budding structures compared to wild-type levels. These results indicate that ubiquitination of lysine residues in Gag in the vicinity of the viral late domain is important for HIV-1 budding, while no specific lysine residue may be needed and individual domains can functionally substitute. This is consistent with Gag ubiquitination being functionally involved in a transient protein interaction network at the virus budding site.  相似文献   

17.
The cellular ALIX protein functions within the ESCRT pathway to facilitate intralumenal endosomal vesicle formation, the abscission stage of cytokinesis, and enveloped virus budding. Here, we report that the C-terminal proline-rich region (PRR) of ALIX folds back against the upstream domains and auto-inhibits V domain binding to viral late domains. Mutations designed to destabilize the closed conformation of the V domain opened the V domain, increased ALIX membrane association, and enhanced virus budding. These observations support a model in which ALIX activation requires dissociation of the autoinhibitory PRR and opening of the V domain arms.  相似文献   

18.
Ubiquitin (Ub) sorting receptors facilitate the targeting of ubiquitinated membrane proteins into multivesicular bodies (MVBs). Ub-binding domains (UBDs) have been described in several endosomal sorting complexes required for transport (ESCRT). Using available structural information, we have investigated the role of the multiple UBDs within ESCRTs during MVB cargo selection. We found a novel UBD within ESCRT-I and show that it contributes to MVB sorting in concert with the known UBDs within the ESCRT complexes. These experiments reveal an unexpected level of coordination among the ESCRT UBDs, suggesting that they collectively recognize a diverse set of cargo rather than act sequentially at discrete steps.  相似文献   

19.
Membrane budding is essential for the egress of many enveloped viruses, and this process shares similarities with the biogenesis of multivesicular bodies (MVBs). In eukaryotic cells, the budding of intraluminal vesicles (IVLs) is mediated by the endosomal sorting complex required for transport (ESCRT) machinery and some viruses require ESCRT machinery components or functions to bud from host cells. Baculoviruses, such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV), enter host cells by clathrin-mediated endocytosis. Viral DNA replication and nucleocapsid assembly occur within the nucleus. Some progeny nucleocapsids are subsequently trafficked to, and bud from, the plasma membrane, forming budded virions (BV). To determine whether the host ESCRT machinery is important or necessary for AcMNPV replication, we cloned a cDNA of Spodoptera frugiperda VPS4, a key regulator for disassembly and recycling of ESCRT III. We then examined viral infection and budding in the presence of wild-type (WT) or dominant negative (DN) forms of VPS4. First, we used a viral complementation system, in combination with fluorescent tags, to examine the effects of transiently expressed WT or DN VPS4 on viral entry. We found that dominant negative VPS4 substantially inhibited virus entry. Entering virus was observed within aberrant compartments containing the DN VPS4 protein. We next used recombinant bacmids expressing WT or DN VPS4 proteins to examine virus egress. We found that production of infectious AcMNPV BV was substantially reduced by expression of DN VPS4 but not by WT VPS4. Together, these results indicate that a functional VPS4 is necessary for efficient AcMNPV BV entry into, and egress from, insect cells.  相似文献   

20.
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