After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs—an adaptor-like protein that binds membrane PtdIns3P via a FYVE motif—and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.
The cell's genetic program is modulated by extracellular signals that activate cell surface receptors and, in turn, intracellular effectors, to regulate transcription. For cells to function normally, these signals must be turned off to avoid permanent activation—a situation often associated with cancer. For many receptors, signaling is repressed, or down-regulated, in a process that first internalizes and then degrades the receptors. After receptors are removed from the cell surface into structures called early endosomes, they are selectively incorporated within vesicles that form inside the endosome. During this process, endosomal membranes are pulled away from the cytoplasm towards the endosome lumen, against the flow of intracellular membrane traffic, eventually resulting in the formation of a “multivesicular body” (vesicles within vesicles). The common view is that these intralumenal vesicles are then delivered to lysosomes, where they are degraded along with their receptor cargo. We have investigated the mechanisms responsible for the biogenesis of intralumenal vesicles in multivesicular bodies. We find that the small protein SNX3, which binds the signaling lipid phosphatidyl inositol-3-phosphate, is necessary for the formation of intralumenal vesicles, but is not involved in the degradation of the cell surface receptor for EGF. Conversely, we find that Hrs, which also binds phosphatidyl inositol-3-phosphate and mediates receptor sorting into intralumenal vesicles, is essential for lysosomal targeting but dispensable for multivesicular body biogenesis. Phosphatidyl inositol-3-phosphate thus controls the complementary functions of Hrs and SNX3 in the sorting of signaling receptors and multivesicular body biogenesis.
SNX3 plays a direct role in the formation of intralumenal vesicles of multivesicular bodies (MVBs) but is not involved in EGF receptor degradation, whereas Hrs is essential for lysosomal targeting but dispensable for MVB biogenesis. Hence, intralumenal vesicle formation in MVB biogenesis can be uncoupled from lysosomal targeting.
Herpes simplex virus type 1 (HSV-1) acquires its mature virus envelope by budding into the lumen of cytoplasmic membranous compartments carrying the viral glycoproteins. In a cellular context, a budding process with identical topology occurs during the formation of intraluminal vesicles in multivesicular bodies. The cellular machinery that mediates this budding process is composed of four protein complexes termed endosomal sorting complexes required for transport (ESCRTs) and several associated proteins, including the ATPase VPS4. We have recently shown that functional VPS4 is specifically required for the cytoplasmic envelopment of HSV-1. We now demonstrate that, consistent with a role of VPS4 in virus envelopment, dominant-negative ESCRT-III proteins potently block HSV-1 production. Retroviruses are known to recruit the ESCRT machinery by small peptide motifs termed late domains. These late domains interact with various ESCRT components and thereby promote ESCRT recruitment. The best-characterized late-domain interacting ESCRT proteins are ALIX and TSG101. The presence of potential ALIX and TSG101 binding sequence motifs in various structural HSV-1 proteins suggested a functional role of these proteins in HSV-1 envelopment. We therefore used a set of dominant-negative proteins, as well as RNA interference, to characterize the contribution of ALIX and TSG101 to HSV-1 production. Interestingly, despite the strict requirement for a functional ESCRT-III complex, our data suggest that HSV-1 production is independent of ALIX and TSG101 expression. In line with these data, we also find that ESCRT-III proteins and VPS4A/B are specifically incorporated into mature HSV-1 virions.
The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum) between 45 and 60 nm or a diameter (determined using a Ripley’s L-function analysis) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual molecules surrounding the central clusters was often observed, which we attribute to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Experiments performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein.
Viruses hijack the cellular machinery to complete their life cycle. In the case of HIV-1, the endosomal sorting complex required for transport (ESCRT) is recruited by nascent viruses to release themselves from infected cells. Currently, there has been an intense amount of research on how the ESCRT machinery induces viral release. Using super-resolution imaging with endogenous ESCRT proteins or ESCRT proteins containing a small tag, we are able to provide insight into how ESCRT leads to budding of HIV-1. Super resolution imaging of the early ESCRT factors Tsg101 and ALIX, as well as later factors CHMP4B and CHMP2A, also showed condensed, circular structures with diameters of roughly 60 to 90 nm. The cluster sizes were significantly smaller than that of the HIV-1 bud and the distribution of cluster sizes that colocalized with nascent HIV-1 assembly sites were narrower than for non-colocalizing structures. This indicates that the point of interaction between the ESCRT machinery and the HIV-1 assembly site is in the bud neck.
The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.
The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.
Old World arenaviruses include the prototypic lymphocytic choriomeningitis virus (LCMV) and the highly pathogenic Lassa virus (LASV) that causes a severe hemorrhagic fever in humans and infects several thousand individuals per year in Western Africa. Cell entry of a virus is the first step of every virus infection and represents a promising target for therapeutic intervention. We and others had shown that LCMV and LASV attach to a cellular receptor, α-dystroglycan, followed by internalization by endocytosis via a novel and unusual pathway. Here we investigated the largely unknown molecular mechanisms of cell entry of LASV and LCMV with the goal to identify host cell factors involved. We discovered that during cell entry LASV and LCMV pass through a particular intracellular compartment, the multivesicular body (MVB)/late endosome, which is implicated in the internalization and degradation of cellular membrane receptors. Productive infection of LASV and LCMV critically depended on cellular factors involved in the membrane dynamics and sorting processes in the MVB. Based on our studies, we propose a model for Old World arenavirus entry in which the viruses hijack a pathway that may be linked to the cellular trafficking and degradation of their cellular receptor.
Alix and ESCRT proteins are required for membrane fission during viral budding and egress and during the abscission stage of cytokinesis. These common roles have suggested that Alix functions as an ESCRT protein, a conclusion challenged by the finding that unlike ESCRTs, which control the formation of multivesicular endosomes, Alix does not influence the degradation of the EGF receptor. We previously showed that Alix controls neuronal death by an unknown mechanism, but dependent on its interaction with ESCRT proteins. Since then, numerous reports have shown that ESCRTs participate in macroautophagy. Given the direct interaction between ESCRTs and Alix, together with the known contribution of autophagy to cell death, it was hypothesized that Alix controls autophagy and thereby cell death. Our recent published results show that this is not the case. ESCRT protein activity therefore needs Alix for viral budding and cytokinesis but not for autophagy. The function of ESCRT can thus be clearly be disconnected from that of Alix.
Animals; Autophagy; Biological Transport; Calcium-Binding Proteins; metabolism; Caspases; metabolism; Endosomes; metabolism; Humans; Multiprotein Complexes; metabolism; Alix; Autophagy; ESCRT; endocytosis; MVB
HIV-1 relies on the host ESCRTs for release from cells. HIV-1 Gag engages ESCRTs by directly binding TSG101 or Alix. ESCRTs also sort ubiquitinated membrane proteins through endosomes to facilitate their lysosomal degradation. The ability of ESCRTs to recognize and process ubiquitinated proteins suggests that ESCRT-dependent viral release may also be controlled by ubiquitination. Although both Gag and ESCRTs undergo some level of ubiquitination, definitive demonstration that ubiquitin is required for viral release is lacking. Here we suppress ubiquitination at viral budding sites by fusing the catalytic domain of the Herpes Simplex UL36 deubiquitinating enzyme (DUb) onto TSG101, Alix, or Gag.
Expressing DUb-TSG101 suppressed Alix-independent HIV-1 release and viral particles remained tethered to the cell surface. DUb-TSG101 had no effect on budding of MoMLV or EIAV, two retroviruses that rely on the ESCRT machinery for exit. Alix-dependent virus release such as EIAV’s, and HIV-1 lacking access to TSG101, was instead dramatically blocked by co-expressing DUb-Alix. Finally, Gag-DUb was unable to support virus release and dominantly interfered with release of wild type HIV-1. Fusion of UL36 did not effect interactions with Alix, TSG101, or Gag and all of the inhibitory effects of UL36 fusion were abolished when its catalytic activity was ablated. Accordingly, Alix, TSG101 and Gag fused to inactive UL36 functionally replaced their unfused counterparts. Interestingly, coexpression of the Nedd4-2s ubiquitin ligase suppressed the ability of DUb-TSG101 to inhibit HIV-1 release while also restoring detectable Gag ubiquitination at the membrane. Similarly, incorporation of Gag-Ub fusion proteins into virions lifted DUb-ESCRT inhibitory effect. In contrast, Nedd4-2s did not suppress the inhibition mediated by Gag-DUb despite restoring robust ubiquitination of TSG101/ESCRT-I at virus budding sites.
These studies demonstrate a necessary and natural role for ubiquitin in ESCRT-dependent viral release and indicate a critical role for ubiquitination of Gag rather than ubiquitination of ESCRTs themselves.
Ubiquitin; HIV budding; ESCRT; Deubiquitination; Gag
HIV-1 release is mediated through two motifs in the p6 region of Gag, PTAP and LYPXnL, which recruit cellular proteins Tsg101 and Alix, respectively. The Nucleocapsid region of Gag (NC), which binds the Bro1 domain of Alix, also plays an important role in HIV-1 release, but the underlying mechanism remains unclear. Here we show that the first 202 residues of the Bro1 domain (Broi) are sufficient to bind Gag. Broi interferes with HIV-1 release in an NC–dependent manner and arrests viral budding at the plasma membrane. Similar interrupted budding structures are seen following over-expression of a fragment containing Bro1 with the adjacent V domain (Bro1-V). Although only Bro1-V contains binding determinants for CHMP4, both Broi and Bro1-V inhibited release via both the PTAP/Tsg101 and the LYPXnL/Alix pathways, suggesting that they interfere with a key step in HIV-1 release. Remarkably, we found that over-expression of Bro1 rescued the release of HIV-1 lacking both L domains. This rescue required the N-terminal region of the NC domain in Gag and the CHMP4 binding site in Bro1. Interestingly, release defects due to mutations in NC that prevented Bro1 mediated rescue of virus egress were rescued by providing a link to the ESCRT machinery via Nedd4.2s over-expression. Our data support a model in which NC cooperates with PTAP in the recruitment of cellular proteins necessary for its L domain activity and binds the Bro1–CHMP4 complex required for LYPXnL–mediated budding.
Human immunodeficiency virus type 1 (HIV-1) assembles its structural protein Gag into a viral shell at the plasma membrane. Gag is divided into several regions, each with its own distinct function(s). Within the p6 region of Gag, there are two short peptide sequences, called Late (L) domains, that serve to recruit cellular proteins Tsg101 and Alix. In an uninfected cell, these proteins facilitate membrane dynamics during vesicle budding into cellular compartments called endosomes. Upon infection, HIV-1 hijacks these proteins and makes use of the machinery to facilitate viral budding at the plasma membrane. Our study shows that, in addition to binding the p6 region, Alix also interacts with the Nucleocapsid (NC) region of Gag. Importantly, we show that when HIV-1 buds via the Alix-driven pathway, this interaction with NC is essential for recruiting host proteins necessary for HIV-1 release. Moreover, we show that a non-functional fragment of Alix inhibits Tsg101-mediated HIV-1 release in ways similar to those caused by mutations in the NC domain of Gag. Collectively, our findings favor a model in which the p6-located L domain motifs require cooperation with NC to facilitate HIV-1 release.
Hrs and the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are involved in the endosomal sorting of membrane proteins into multivesicular bodies and lysosomes or vacuoles. The ESCRT complexes are also required for formation of intraluminal endosomal vesicles and for budding of certain enveloped RNA viruses such as HIV. Here, we show that Hrs binds to the ESCRT-I subunit Tsg101 via a PSAP motif that is conserved in Tsg101-binding viral proteins. Depletion of Hrs causes a reduction in membrane-associated ESCRT-I subunits, a decreased number of multivesicular bodies and an increased size of late endosomes. Even though Hrs mainly localizes to early endosomes and Tsg101 to late endosomes, the two proteins colocalize on a subpopulation of endosomes that contain lyso-bisphosphatidic acid. Overexpression of Hrs causes accumulation of Tsg101 on early endosomes and prevents its localization to late endosomes. We conclude that Hrs mediates the initial recruitment of ESCRT-I to endosomes and, thereby, indirectly regulates multivesicular body formation.
endocytosis; lysosome; membrane traffic; Tsg101; protein sorting
When internalized receptors and other cargo are destined for lysosomal degradation, they are ubiquitinated and sorted by the ESCRT complexes 0, I, II, and III into multivesicular bodies. Multivesicular bodies are formed when cargo-rich patches of the limiting membrane of endosomes bud inward by an unknown mechanism and are then cleaved to yield cargo-bearing intralumenal vesicles. The biogenesis of multivesicular bodies was reconstituted and visualized using giant unilamellar vesicles, fluorescent ESCRT-0, I, II, and III complexes, and a membrane-tethered fluorescent ubiquitin fusion as a model cargo. ESCRT-0 forms domains of clustered cargo but does not deform membranes. ESCRT-I and II in combination deform the membrane into buds, in which cargo is confined. ESCRT-I and II localize to the bud necks, and recruit ESCRT-0-ubiquitin domains to the buds. ESCRT-III subunits localize to the bud neck and efficiently cleave the buds to form intralumenal vesicles. Intralumenal vesicles produced in this reaction contain the model cargo but are devoid of ESCRTs. The observations explain how the ESCRTs direct membrane budding and scission from the cytoplasmic side of the bud without being consumed in the reaction.
After ligand binding and endocytosis, cell surface receptors can continue to signal from endosomal compartments until sequestered from the cytoplasm. An important mechanism for receptor downregulation in vivo is via the inward budding of receptors into intralumenal vesicles to form specialized endosomes called multivesicular bodies (MVBs) that subsequently fuse with lysosomes, degrading their cargo. This process requires four heterooligomeric protein complexes collectively termed the ESCRT machinery. In yeast, ESCRT-I is a heterotetrameric complex comprised of three conserved subunits and a fourth subunit for which identifiable metazoan homologs were lacking. Using C. elegans, we identify MVB-12, a fourth metazoan ESCRT-I subunit. Depletion of MVB-12 slows the kinetics of receptor downregulation in vivo, but to a lesser extent than inhibition of other ESCRT-I subunits. Consistent with these findings, targeting of MVB-12 to membranes requires the other ESCRT-I subunits, but MVB-12 is not required to target the remaining ESCRT-I components. Both endogenous and recombinant ESCRT-I are stable complexes with a 1:1:1:1 subunit stoichiometry. MVB-12 has two human homologs that co-localize and co-immunoprecipitate with the ESCRT-I component TSG101. Thus, MVB-12 is a conserved core component of metazoan ESCRT-I that regulates its activity during MVB biogenesis.
The biogenesis of multivesicular bodies and endosomal sorting of membrane cargo are driven forward by the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III. ESCRT-I is characterized in yeast as a complex consisting of Vps23, Vps28, and Vps37. Whereas mammalian homologues of Vps23 and Vps28 (named Tsg101 and hVps28, respectively) have been identified and characterized, a mammalian counterpart of Vps37 has not yet been identified. Here, we show that a regulator of proliferation, hepatocellular carcinoma related protein 1 (HCRP1), interacts with Tsg101, hVps28, and their upstream regulator Hrs. The ability of HCRP1 (which we assign the alternative name hVps37A) to interact with Tsg101 is conferred by its mod(r) domain and is shared with hVps37B and hVps37C, two other mod(r) domain-containing proteins. HCRP1 cofractionates with Tsg101 and hVps28 by size exclusion chromatography and colocalizes with hVps28 on LAMP1-positive endosomes. Whereas depletion of Tsg101 by siRNA reduces cellular levels of both hVps28 and HCRP1, depletion of HCRP1 has no effect on Tsg101 or hVps28. Nevertheless, HCRP1 depletion strongly retards epidermal growth factor (EGF) receptor degradation. Together, these results indicate that HCRP1 is a subunit of mammalian ESCRT-I and that its function is essential for lysosomal sorting of EGF receptors.
The cellular endosomal sorting complex required for transport (ESCRT) machinery participates in membrane scission and cytoplasmic budding of many RNA viruses. Here, we found that expression of dominant negative ESCRT proteins caused a blockade of Epstein-Barr virus (EBV) release and retention of viral BFRF1 at the nuclear envelope. The ESCRT adaptor protein Alix was redistributed and partially colocalized with BFRF1 at the nuclear rim of virus replicating cells. Following transient transfection, BFRF1 associated with ESCRT proteins, reorganized the nuclear membrane and induced perinuclear vesicle formation. Multiple domains within BFRF1 mediated vesicle formation and Alix recruitment, whereas both Bro and PRR domains of Alix interacted with BFRF1. Inhibition of ESCRT machinery abolished BFRF1-induced vesicle formation, leading to the accumulation of viral DNA and capsid proteins in the nucleus of EBV-replicating cells. Overall, data here suggest that BFRF1 recruits the ESCRT components to modulate nuclear envelope for the nuclear egress of EBV.
Herpesviruses are large DNA viruses associated with human and animal diseases. After viral DNA replication, the herpesviral nucleocapsids egress through the nuclear membrane for subsequent cytoplasmic virion maturation. However, the mechanism by which the virus regulates the nuclear membrane and cellular machinery involved in this process remained elusive. The cellular endosomal sorting complex required for transport (ESCRT) machinery is known to participate in the biogenesis of multivesicular bodies, cytokinesis and the release of enveloped viruses from cytoplasmic membranes. Here, we show that functional ESCRT machinery is required for the maturation of Epstein-Barr virus (EBV). ESCRT proteins are redistributed close to the nucleus-associated membrane through interaction with the viral BFRF1 protein, leading to vesicle formation and structural changes of the nuclear membrane. Remarkably, inhibition of ESCRT machinery abolishes BFRF1-induced vesicle formation, and leads to the accumulation of viral DNA and capsid proteins in the nucleus. Specific interactions between BFRF1 and Alix are required for BFRF1-derived vesicle formation and crucial for the nuclear egress of EBV.
In mammalian cells, epidermal growth factor (EGF) stimulation promotes multivesicular body (MVB) formation and inward vesiculation within MVB. Annexin 1 is required for EGF-stimulated inward vesiculation but not MVB formation, demonstrating that MVB formation (the number of MVBs/unit cytoplasm) and inward vesiculation (the number of internal vesicles/MVB) are regulated by different mechanisms. Here, we show that EGF-stimulated MVB formation requires the tumor susceptibility gene, Tsg101, a component of the ESCRT (endosomal sorting complex required for transport) machinery. Depletion of Tsg101 potently inhibits EGF degradation and MVB formation and causes the vacuolar domains of the early endosome to tubulate. Although Tsg101 depletion inhibits MVB formation and alters the morphology of the early endosome in unstimulated cells, these effects are much greater after EGF stimulation. In contrast, depletion of hepatocyte growth factor receptor substrate (Hrs) only modestly inhibits EGF degradation, does not induce tubulation of the early endosome, and causes the generation of enlarged MVBs that retain the ability to fuse with the lysosome. Together, these results indicate that Tsg101 is required for the formation of stable vacuolar domains within the early endosome that develop into MVBs and Hrs is required for the accumulation of internal vesicles within MVBs and that both these processes are up-regulated by EGF stimulation.
The cellular ESCRT (endosomal sorting complex required for transport) system functions in cargo-sorting, in the formation of intraluminal vesicles that comprise multivesicular bodies (MVB), and in cytokinesis, and this system can be hijacked by a number of enveloped viruses to promote budding. The respiratory pathogen human parainfluenza virus type I (HPIV1) encodes a nested set of accessory C proteins that play important roles in down-regulating viral transcription and replication, in suppressing the type I interferon (IFN) response, and in suppressing apoptosis. Deletion or mutation of the C proteins attenuates HPIV1 in vivo, and such mutants are being evaluated preclinically and clinically as vaccines. We show here that the C proteins interact and co-localize with the cellular protein Alix, which is a member of the class E vacuolar protein sorting (Vps) proteins that assemble at endosomal membranes into ESCRT complexes. The HPIV1 C proteins interact with the Bro1 domain of Alix at a site that is also required for the interaction between Alix and Chmp4b, a subunit of ESCRT-III. The C proteins are ubiquitinated and subjected to proteasome-mediated degradation, but the interaction with AlixBro1 protects the C proteins from degradation. Neither over-expression nor knock-down of Alix expression had an effect on HPIV1 replication, although this might be due to the large redundancy of Alix-like proteins. In contrast, knocking down the expression of Chmp4 led to an approximately 100-fold reduction in viral titer during infection with wild-type (WT) HPIV1. This level of reduction was similar to that observed for the viral mutant, P(C-) HPIV1, in which expression of the C proteins were knocked out. Chmp4 is capable of out-competing the HPIV1 C proteins for binding Alix. Together, this suggests a possible model in which Chmp4, through Alix, recruits the C proteins to a common site on intracellular membranes and facilitates budding.
The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the “late domain”) binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs; residues 222–777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222–777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.
virus budding; virions; ubiquitin; vacuolar protein sorting; multivesicular body
Kaposi’s sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitination of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human microvascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and–III, play a central role in clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress. We have recently shown that ESCRT-0 component Hrs protein associates with the plasma membrane during macropinocytosis and mediates KSHV entry via ROCK1 mediated phosphorylation of NHE1 and local membrane pH change. Here, we demonstrate that the ESCRT-I complex Tsg101 protein also participates in the macropinocytosis of KSHV and plays a role in KSHV trafficking. Knockdown of Tsg101 did not affect virus entry in HMVEC-d and human umbilical vein endothelial (HUVEC) cells but significantly inhibited the KSHV genome entry into the nucleus and consequently viral gene expression in these cells. Double and triple immunofluorescence, proximity ligation immunofluorescence and co-immuoprecipitation studies revealed the association of Tsg101 with the KSHV containing macropinosomes, and increased levels of Tsg101 association/interactions with EphA2R, c-Cbl, p130Cas and Crk signal molecules, as well as with upstream and downstream ESCRT components such as Hrs (ESCRT-0), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells. Tsg101 was also associated with early (Rab5) and late endosomal (Rab7) stages of KSHV intracellular trafficking, and CHMP5 (ESCRT-III) was also associated with Rab 5 and Rab 7. Knockdown of Tsg101 significantly inhibited the transition of virus from early to late endosomes. Collectively, our studies reveal that Tsg101 plays a role in the trafficking of macropinocytosed KSHV in the endothelial cells which is essential for the successful viral genome delivery into the nucleus, viral gene expression and infection. Thus, ESCRT molecules could serve as therapeutic targets to combat KSHV infection.
KSHV is etiologically associated with human endothelial Kaposi’s sarcoma, and understanding of endothelial infection is essential to design methods to block infection. KSHV infection of endothelial cells is initiated by its interaction with cell surface heparan sulfate, various integrins and the Ephrin A2 receptor tyrosine kinase (EphA2R) molecule which results in the induction of integrin-c-Cbl mediated signaling, leading to KSHV entry by the macropinocytic mode of endocytosis. Host ESCRT complex proteins are involved in the cargo trafficking and play roles in viral egress. We have shown that ESCRT-0 Hrs protein facilitates the assembly of signaling molecules in KSHV macropinocytosis. Studies here demonstrate for the first time that the ESCRT-I Tsg101 protein, known to contribute to clathrin-mediated endocytosis, participates in macropinocytosis and plays a role in a post-macropinocytic step of KSHV infection. Tsg101 associates with macropinosomes containing KSHV, receptor (EphA2R), signal molecules (c-Cbl, p130Cas and Crk), and with upstream and downstream ESCRT proteins. Tsg101 is important for the virus to transition from early to late endosomes. These studies reveal that ESCRT proteins can be an important target for therapeutic interventions against KSHV infection.
The ubiquitin hydrolase activating factor Bro1 enhances ESCRT-III stability by inhibiting Vps4-mediated disassembly.
Endosomal sorting complexes required for transport (ESCRTs) promote the invagination of vesicles into the lumen of endosomes, the budding of enveloped viruses, and the separation of cells during cytokinesis. These processes share a topologically similar membrane scission event facilitated by ESCRT-III assembly at the cytosolic surface of the membrane. The Snf7 subunit of ESCRT-III in yeast binds directly to an auxiliary protein, Bro1. Like ESCRT-III, Bro1 is required for the formation of intralumenal vesicles at endosomes, but its role in membrane scission is unknown. We show that overexpression of Bro1 or its N-terminal Bro1 domain that binds Snf7 enhances the stability of ESCRT-III by inhibiting Vps4-mediated disassembly in vivo and in vitro. This stabilization effect correlates with a reduced frequency in the detachment of intralumenal vesicles as observed by electron tomography, implicating Bro1 as a regulator of ESCRT-III disassembly and membrane scission activity.
HIV virions assemble on the plasma membrane and bud out of infected cells using interactions with endosomal sorting complexes required for transport (ESCRTs). HIV protease activation is essential for maturation and infectivity of progeny virions, however, the precise timing of protease activation and its relationship to budding has not been well defined. We show that compromised interactions with ESCRTs result in delayed budding of virions from host cells. Specifically, we show that Gag mutants with compromised interactions with ALIX and Tsg101, two early ESCRT factors, have an average budding delay of ~75 minutes and ~10 hours, respectively. Virions with inactive proteases incorporated the full Gag-Pol and had ~60 minutes delay in budding. We demonstrate that during budding delay, activated proteases release critical HIV enzymes back to host cytosol leading to production of non-infectious progeny virions. To explain the molecular mechanism of the observed budding delay, we modulated the Pol size artificially and show that virion release delays are size-dependent and also show size-dependency in requirements for Tsg101 and ALIX. We highlight the sensitivity of HIV to budding “on-time” and suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release.
ESCRTs are implicated in cellular processes which require fission of budding membranes. Likely the most studied of these processes is the HIV-ESCRT interactions. The canonical view is that interference with ESCRT recruitment results in a late budding arrest of virions at the plasma membrane and this mechanistic view of ESCRTs has shaped our understanding of their function in almost all cell biology. In this manuscript, we present a full kinetic analysis of HIV virion release under all known mutations in Gag that affect HIV-ESCRT interactions. Our data show that contrary to the canonical view, a defect in ESCRT recruitment does not inhibit virion budding, however it creates a delay. We further show that during budding delay, activated proteases release critical HIV enzymes back to host cytosol, leading to budding of non-infectious progeny virions. We suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release and can be the basis for developing antiviral treatments which slow the budding process and therefore disproportionally affect infectious retroviral release. We also suggest that such budding delay may be one of the mechanisms underlying cellular innate immune responses which inhibit the spread of retroviral infection.
Multivesicular body (MVB) formation is the result of invagination and budding of the endosomal limiting membrane into its intralumenal space. These intralumenal vesicles (ILVs) contain a subset of endosomal transmembrane cargoes destined for degradation within the lysosome, the result of active selection during MVB sorting. Membrane bending and scission during ILV formation is topologically similar to cytokinesis in that both events require the abscission of a membrane neck that is oriented away from the cytoplasm. The endosomal sorting machinery required for transport (ESCRTs) represents cellular machinery whose function makes essential contributions to both of these processes. In particular the AAA-ATPase Vps4 and its substrate ESCRT-III are key components that seem to execute the membrane abscission reaction. This review summarizes current knowledge about the Vps4-ESCRT-III system and discusses a model how the recruitment of Vps4 to the different sites of function might be regulated.
ESCRT; MVB pathway; cytokinesis; endocytosis; protein trafficking
The modular adaptor protein ALIX is critically involved in endosomal sorting complexes required for transport (ESCRT)-mediated multivesicular body (MVB) sorting of activated epidermal growth factor receptor (EGFR); however, ALIX contains a default intramolecular interaction that renders ALIX unable to perform this ESCRT function. The ALIX partner protein ALG-2 is a calcium-binding protein that belongs to the calmodulin superfamily. Prompted by a defined biological function of calmodulin, we determined the role of ALG-2 in regulating ALIX involvement in MVB sorting of activated EGFR. Our results show that calcium-dependent ALG-2 interaction with ALIX completely relieves the intramolecular interaction of ALIX and promotes CHMP4-dependent ALIX association with the membrane. EGFR activation induces increased ALG-2 interaction with ALIX, and this increased interaction is responsible for increased ALIX association with the membrane. Functionally, inhibition of ALIX activation by ALG-2 inhibits MVB sorting of activated EGFR as effectively as inhibition of ALIX interaction with CHMP4 does; however, inhibition of ALIX activation by ALG-2 does not affect cytokinetic abscission or equine infectious anemia virus (EIAV) budding. These findings indicate that calcium-dependent ALG-2 interaction with ALIX is specifically responsible for generating functional ALIX that supports MVB sorting of ubiquitinated membrane receptors.
ALIX; ALG-2; MVB sorting; EGFR; CHMP4
The release of human immunodeficiency virus type 1 (HIV-1) and of other retroviruses from certain cells requires the presence of distinct regions in Gag that have been termed late assembly (L) domains. HIV-1 harbors a PTAP-type L domain in the p6 region of Gag that engages an endosomal budding machinery through Tsg101. In addition, an auxiliary L domain near the C terminus of p6 binds to ALIX/AIP1, which functions in the same endosomal sorting pathway as Tsg101. In the present study, we show that the profound release defect of HIV-1 L domain mutants can be completely rescued by increasing the cellular expression levels of ALIX and that this rescue depends on an intact ALIX binding site in p6. Furthermore, the ability of ALIX to rescue viral budding in this system depended on two putative surface-exposed hydrophobic patches on its N-terminal Bro1 domain. One of these patches mediates the interaction between ALIX and the ESCRT-III component CHMP4B, and mutations which disrupt the interaction also abolish the activity of ALIX in viral budding. The ability of ALIX to rescue a PTAP mutant also depends on its C-terminal proline-rich domain (PRD), but not on the binding sites for Tsg101, endophilin, CIN85, or for the newly identified binding partner, CMS, within the PRD. Our data establish that ALIX can have a dramatic effect on HIV-1 release and suggest that the ability to use ALIX may allow HIV-1 to replicate in cells that express only low levels of Tsg101.
HIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and −2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants.
Whereas budding normally results in formation of single spherical particles ~100 nm in diameter and containing a characteristic electron-dense conical core, the substitution of Phe for S40, a change that does not alter the amino acids encoded in the overlapping pol reading frame, resulted in defective CA-SP1 cleavage, formation of strings of tethered particles or filopodia-like membrane protrusions containing Gag, and diminished infectious particle formation. The S40F-mediated release defects were exacerbated when the viral-encoded protease (PR) was inactivated or when L domain-1 function was disrupted or when budding was almost completely obliterated by the disruption of both L domain-1 and −2. S40F mutation also resulted in stronger Gag-Alix interaction, as detected by yeast 2-hybrid assay. Reducing Alix binding by mutational disruption of contact residues restored single particle release, implicating the perturbed Gag-Alix interaction in the aberrant budding events. Interestingly, introduction of S40F partially rescued the negative effects on budding of CA NTD mutations EE75,76AA and P99A, which both prevent membrane curvature and therefore block budding at an early stage.
The results indicate that the S40 residue is a novel determinant of HIV-1 egress that is most likely involved in regulation of a critical assembly event required for budding in the Tsg101-, Alix-, Nedd4- and CA N-terminal domain affected pathways.
HIV-1; Alix; Tsg101; Nedd4; CA NTD; Viral particle maturation; Protease; Viral budding
Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. Here we show that ALIX and the ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC) division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. We demonstrate that ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, we show that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. We conclude that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo.
Cytokinesis, the final step of cell division, concludes with a process termed abscission, during which the two daughter cells physically separate. In spite of their importance, the molecular machineries controlling abscission are poorly characterized especially in the context of living metazoan tissues. Here we provide molecular insight into the mechanism of abscission using the fruit fly Drosophila melanogaster as a model organism. We show that the scaffold protein ALIX and the ESCRT-III component Shrub are required for completion of abscission in Drosophila female germline stem cells (fGSCs). ESCRT-III has been implicated in topologically similar membrane scission events as abscission, namely intraluminal vesicle formation at endosomes and virus budding. Here we demonstrate that ALIX and Shrub co-localize and interact to promote abscission with correct timing in Drosophila fGSCs. We thus show that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs cells and report an evolutionarily conserved function of the ALIX/ESCRT-III pathway during cytokinesis in a multi-cellular organism.
Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition Alix is associated with the actin cytoskeleton and may regulate cytoskeletal dynamics.
Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane called RME-1. Analysis of alx-1 mutants indicates that ALX-1 is required for endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by analysis of rme-1 mutants. Expression of truncated human Alix in HeLa cells disrupts recycling of MHCI, a known Ehd1/RME-1 dependent transport step, suggesting phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears dispensable for ALX-1 function in MVEs/late endosomes.
This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1.