Endosomal Sorting Complex Required For Transport I Research Articles

Foot-and-mouth disease virus downregulates vacuolar protein sorting 28 to promote viral replication.

Vacuolar protein sorting 28 (Vps28), a component of the ESCRT-I (endosomal sorting complex required for transport I), plays an important role in the pathogen life cycle. Here, we investigated the reciprocal regulation between Vps28 and the foot-and-mouth disease virus (FMDV). Overexpression of Vps28 decreased FMDV replication. On the contrary, the knockdown of Vps28 increased viral replication. Subsequently, the mechanistic study showed that Vps28 destabilized the replication complex (RC) by associating with 3A rather than 2C protein. In addition, Vps28 targeted FMDV VP0, VP1, and VP3 for degradation to inhibit viral replication. To counteract this, FMDV utilized tactics to restrict Vps28 to promote viral replication. FMDV degraded Vps28 mainly through the ubiquitin-proteasome pathway. Additional data demonstrated that 2B and 3A proteins recruited E3 ubiquitin ligase tripartite motif-containing protein 21 to degrade Vps28 at Lys58 and Lys25, respectively, and FMDV 3Cpro degraded Vps28 through autophagy and its protease activity. Meantime, the 3Cpro-mediated Vps28 degradation principally alleviated the ability to inhibit viral propagation. Intriguingly, we also demonstrated that the N-terminal and C-terminal domains of Vps28 were responsible for the suppression of FMDV replication, which suggested the elaborated counteraction between FMDV and Vps28. Collectively, our results first investigate the role of ESCRTs in host defense against picornavirus and unveil underlying strategies utilized by FMDV to evade degradation machinery for triumphant propagation. IMPORTANCE ESCRT machinery plays positive roles in virus entry, replication, and budding. However, little has been reported on its negative regulation effects during viral infection. Here, we uncovered the novel roles of ESCRT-I subunit Vps28 on FMDV replication. The data indicated that Vps28 destabilized the RC and impaired viral structural proteins VP0, VP1, and VP3 to inhibit viral replication. To counteract this, FMDV hijacked intracellular protein degradation pathways to downregulate Vps28 expression and thus promoted viral replication. Our findings provide insights into how ESCRT regulates pathogen life cycles and elucidate additional information regarding FMDV counteraction of host antiviral activity.

VPS37C facilitates erythroid differentiation by promoting EKLF stability

The process of erythroid differentiation is orchestrated at the molecular level by a complex network of transcription factors. Erythroid Krüppel-like factor (EKLF/KLF1) is a master erythroid gene regulator that directly regulates most aspects of terminal erythroid differentiation. However, the underlying regulatory mechanisms of EKLF protein stability are still largely unknown. In this study, we identified Vacuolar protein sorting 37 C (VPS37C), a core subunit of the Endosomal sorting complex required for transport-I (ESCRT-I) complex, as an essential regulator of EKLF stability. Our study showed that VPS37C interacts with EKLF and prevents K48-linked polyubiquitination of EKLF and proteasome-mediated EKLF degradation, thus enhancing EKLF protein stability and transcriptional activity. VPS37C overexpression in murine erythroleukemia (MEL) cells promotes hexamethylene bisacetamide (HMBA)-induced erythroid differentiation manifested by up-regulating erythroid-specific EKLF target genes and increasing benzidine-positive cells. In contrast, VPS37C knockdown inhibits HMBA-induced MEL cell erythroid differentiation. Particularly, the restoration of EKLF expression in VPS37C-knockdown MEL cells reverses erythroid-specific gene expression and hemoglobin production. Collectively, our study demonstrated VPS37C is a novel regulator of EKLF ubiquitination and degradation, which plays a positive role in erythroid differentiation of MEL cells by enhancing EKLF protein stability.

Late domain dependent E-cadherin recruitment into extracellular vesicles.

E-cadherin, a transmembrane protein involved in epithelial cell-cell adhesion and signaling, is found in exosomal fractions isolated from human body fluids. A cellular mechanism for recruitment of E-cadherin into extracellular vesicles (EVs) has not yet been defined. Here, we show that E-cadherin is incorporated into the membrane of EVs with the extracellular domain exposed at the vesicle surface. This recruitment depends on the endosomal sorting complex required for transport I (ESCRT-I) component Tsg101 and a highly conserved tetrapeptide P(S/T)AP late domain motif in the cytoplasmic tail of E-cadherin that mediates interaction with Tsg101. Mutation of this motif results in a loss of interaction and a dramatic decrease in exosomal E-cadherin secretion. We conclude, that the process of late domain mediated exosomal recruitment is exerted by this endogenous non-ESCRT transmembrane protein.

The deubiquitinases UBP12 and UBP13 integrate with the E3 ubiquitin ligase XBAT35.2 to modulate VPS23A stability in ABA signaling.

Ubiquitination-mediated protein degradation in both the 26S proteasome and vacuole is an important process in abscisic acid (ABA) signaling. However, the role of deubiquitination in this process remains elusive. Here, we demonstrate that two deubiquitinating enzymes (DUBs), ubiquitin-specific protease 12 (UBP12) and UBP13, modulate ABA signaling and drought tolerance by deubiquitinating and stabilizing the endosomal sorting complex required for transport-I (ESCRT-I) component vacuolar protein sorting 23A (VPS23A) and thereby affect the stability of ABA receptors in Arabidopsis thaliana. Genetic analysis showed that VPS23A overexpression could rescue the ABA hypersensitive and drought tolerance phenotypes of ubp12-2w or ubp13-1. In addition to the direct regulation of VPS23A, we found that UBP12 and UBP13 also stabilized the E3 ligase XB3 ortholog 5 in A. thaliana (XBAT35.2) in response to ABA treatment. Hence, we demonstrated that UBP12 and UBP13 are previously unidentified rheostatic regulators of ABA signaling and revealed a mechanism by which deubiquitination precisely monitors the XBAT35/VPS23A ubiquitination module in the ABA response.

The Novel ALG-2 Target Protein CDIP1 Promotes Cell Death by Interacting with ESCRT-I and VAPA/B.

Apoptosis-linked gene 2 (ALG-2, also known as PDCD6) is a member of the penta-EF-hand (PEF) family of Ca2+-binding proteins. The murine gene encoding ALG-2 was originally reported to be an essential gene for apoptosis. However, the role of ALG-2 in cell death pathways has remained elusive. In the present study, we found that cell death-inducing p53 target protein 1 (CDIP1), a pro-apoptotic protein, interacts with ALG-2 in a Ca2+-dependent manner. Co-immunoprecipitation analysis of GFP-fused CDIP1 (GFP-CDIP1) revealed that GFP-CDIP1 associates with tumor susceptibility gene 101 (TSG101), a known target of ALG-2 and a subunit of endosomal sorting complex required for transport-I (ESCRT-I). ESCRT-I is a heterotetrameric complex composed of TSG101, VPS28, VPS37 and MVB12/UBAP1. Of diverse ESCRT-I species originating from four VPS37 isoforms (A, B, C, and D), CDIP1 preferentially associates with ESCRT-I containing VPS37B or VPS37C in part through the adaptor function of ALG-2. Overexpression of GFP-CDIP1 in HEK293 cells caused caspase-3/7-mediated cell death. In addition, the cell death was enhanced by co-expression of ALG-2 and ESCRT-I, indicating that ALG-2 likely promotes CDIP1-induced cell death by promoting the association between CDIP1 and ESCRT-I. We also found that CDIP1 binds to vesicle-associated membrane protein-associated protein (VAP)A and VAPB through the two phenylalanines in an acidic tract (FFAT)-like motif in the C-terminal region of CDIP1, mutations of which resulted in reduction of CDIP1-induced cell death. Therefore, our findings suggest that different expression levels of ALG-2, ESCRT-I subunits, VAPA and VAPB may have an impact on sensitivity of anticancer drugs associated with CDIP1 expression.

The Late Domain of Prototype Foamy Virus Gag Facilitates Autophagic Clearance of Stress Granules by Promoting Amphisome Formation.

Prototype foamy virus (PFV), a complex retrovirus belonging to Spumaretrovirinae, maintains lifelong latent infection. The maintenance of lifelong latent infection by viruses relies on the repression of the type I interferon (IFN) response. However, the mechanism involving PFV latency, especially regarding the suppression of the IFN response, is poorly understood. Our previous study showed that PFV promotes autophagic flux. However, the underlying mechanism and the role of PFV-induced autophagy in latent infection have not been clarified. Here, we report that the PFV viral structural protein Gag induced amphisome formation and triggered autophagic clearance of stress granules (SGs) to attenuate type I IFN production. Moreover, the late domain (L-domain) of Gag played a central role in Alix recruitment, which promoted endosomal sorting complex required for transport I (ESCRT-I) formation and amphisome accumulation by facilitating late endosome formation. Our data suggest that PFV Gag represses the host IFN response through autophagic clearance of SGs by activating the endosome-autophagy pathway. More importantly, we found a novel mechanism by which a retrovirus inhibits the SG response to repress the type I IFN response.IMPORTANCE Maintenance of lifelong latent infection for viruses relies on repression of the type I IFN response. Autophagy plays a double-edged sword in antiviral immunity. However, the role of autophagy in the regulation of the type I IFN response and the mechanism involving virus-promoted autophagy have not been fully elucidated. SGs are an immune complex associated with the antiviral immune response and are critical for type I IFN production. Autophagic clearance of SGs is one means of degradation of SGs and is associated with regulation of immunity, but the detailed mechanism remains unclear. In this article, we demonstrate that PFV Gag recruits ESCRT-I to facilitate amphisome formation. Our data also suggest that amphisome formation is a critical event for autophagic clearance of SGs and repression of the type I IFN response. More importantly, we found a novel mechanism by which a retrovirus inhibits the SG response to repress the type I IFN response.

Arabidopsis ALIX Regulates Stomatal Aperture and Turnover of Abscisic Acid Receptors.

The plant endosomal trafficking pathway controls the abundance of membrane-associated soluble proteins, as shown for abscisic acid (ABA) receptors of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) family. ABA receptor targeting for vacuolar degradation occurs through the late endosome route and depends on FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FYVE1) and VACUOLAR PROTEIN SORTING23A (VPS23A), components of the ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT-I (ESCRT-I) complexes. FYVE1 and VPS23A interact with ALG-2 INTERACTING PROTEIN-X (ALIX), an ESCRT-III-associated protein, although the functional relevance of such interactions and their consequences in cargo sorting are unknown. In this study we show that Arabidopsis (Arabidopsis thaliana) ALIX directly binds to ABA receptors in late endosomes, promoting their degradation. Impaired ALIX function leads to altered endosomal localization and increased accumulation of ABA receptors. In line with this activity, partial loss-of-function alix-1 mutants display ABA hypersensitivity during growth and stomatal closure, unveiling a role for the ESCRT machinery in the control of water loss through stomata. ABA-hypersensitive responses are suppressed in alix-1 plants impaired in PYR/PYL/RCAR activity, in accordance with ALIX affecting ABA responses primarily by controlling ABA receptor stability. ALIX-1 mutant protein displays reduced interaction with VPS23A and ABA receptors, providing a molecular basis for ABA hypersensitivity in alix-1 mutants. Our findings unveil a negative feedback mechanism triggered by ABA that acts via ALIX to control the accumulation of specific PYR/PYL/RCAR receptors.

Leptin Modulates Exosome Biogenesis in Breast Cancer Cells: An Additional Mechanism in Cell-to-Cell Communication.

Exosomes—small membrane vesicles secreted by both normal and malignant cells upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane—play an important role in cell-to-cell communication. During the last decade, several reports have highlighted the involvement of these nanovesicles in many aspects of breast cancer development and progression, but the extracellular signals governing their generation in breast cancer cells have not been completely unraveled. Here, we investigated the role of the obesity hormone leptin, a well-known adipokine implicated in mammary tumorigenesis, on the mechanisms regulating exosome biogenesis and release in both estrogen receptor α (ERα)—positive MCF-7 and triple-negative MDA-MB-231 breast cancer cells. We found that leptin treatment enhanced the number of MVBs in the cytoplasm of breast cancer cells and increased the amount of exosomes released in cell conditioned media. At molecular level, leptin increased the protein expression of Tsg101—a key component of the endosomal sorting complex required for transport I (ESCRT-I)—by a post-transcriptional mechanism involving its direct interaction with the chaperone protein Hsp90. Targeting leptin signaling, by a selective leptin receptor antagonist the peptide LDFI (Leu-Asp-Phe-Ile), abrogated leptin effects on Tsg101 expression and on exosome secretion in breast cancer cells. In conclusion, our findings, identifying for the first time leptin/leptin receptor/Hsp90 axis as an important regulator of exosome generation in mammary carcinoma cells, suggest that targeting this signaling pathway might represent a novel therapeutic strategy to impair exosome secretion and interrupt the dangerous cell-to-cell communication in breast cancer.

HCRP-1 regulates EGFR\u2013AKT\u2013BIM-mediated anoikis resistance and serves as a prognostic marker in human colon cancer

Hepatocellular carcinoma-related protein-1 (HCRP-1), a subunit of mammalian endosomal sorting complex required for transport-I (ESCRT-I), is frequently downregulated in various kinds of malignant tumors. The role of HCRP-1 in colorectal cancer (CRC) remains unknown. We investigate the clinical value of HCRP-1 and its impact on anoikis in CRC. The negative expression of HCRP-1 was significantly correlated with tumor size (P = 0.033), PT status (P = 0.001), TNM stage (P = 0.039), and histological grade (P = 0.01). Univariate and multivariate analyses revealed that HCRP-1 was an independent prognostic factor for CRC (hazard ratio (HR) = 0.237, P < 0.001 for 5-year overall survival). In the in vitro assay, we found that HCRP-1 depletion resulted in cell anoikis resistance. Knockdown of HCRP-1 suppressed Bcl-2 interacting mediator of cell death (BIM) expression, with phosphorylation of AKT and p-FoxO3a, which was reversed by AKT siRNA or AKT inhibitor. Further analysis showed that loss of HCRP-1 obviously increased the activation of EGFR. Inhibition of EGFR blocked si-HCRP1-mediated phosphorylation of EGFR, AKT, FoxO3a, and BIM expression. Moreover, the in vivo results revealed that loss of HCRP-1 promoted cancer metastasis. Our findings implied that reduced HCRP-1 expression in CRC resulted in anoikis resistance and contributed to CRC metastasis and poor prognosis. These data may help design effective therapy targeting HCRP-1 pathway to control colon cancer growth and metastasis.

Disruption of the plant-specific CFS1 gene impairs autophagosome turnover and triggers EDS1-dependent cell death

Cell death, autophagy and endosomal sorting contribute to many physiological, developmental and immunological processes in plants. They are mechanistically interconnected and interdependent, but the molecular basis of their mutual regulation has only begun to emerge in plants. Here, we describe the identification and molecular characterization of CELL DEATH RELATED ENDOSOMAL FYVE/SYLF PROTEIN 1 (CFS1). The CFS1 protein interacts with the ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT I (ESCRT-I) component ELCH (ELC) and is localized at ESCRT-I-positive late endosomes likely through its PI3P and actin binding SH3YL1 Ysc84/Lsb4p Lsb3p plant FYVE (SYLF) domain. Mutant alleles of cfs1 exhibit auto-immune phenotypes including spontaneous lesions that show characteristics of hypersensitive response (HR). Autoimmunity in cfs1 is dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)-mediated effector-triggered immunity (ETI) but independent from salicylic acid. Additionally, cfs1 mutants accumulate the autophagy markers ATG8 and NBR1 independently from EDS1. We hypothesize that CFS1 acts at the intersection of autophagosomes and endosomes and contributes to cellular homeostasis by mediating autophagosome turnover.

A Pseudomonas aeruginosaTIR effector mediates immune evasion by targetingUBAP1 and TLR adaptors.

Bacterial pathogens often subvert the innate immune system to establish a successful infection. The direct inhibition of downstream components of innate immune pathways is particularly well documented but how bacteria interfere with receptor proximal events is far less well understood. Here, we describe a Toll/interleukin 1 receptor (TIR) domain‐containing protein (PumA) of the multi‐drug resistant Pseudomonas aeruginosa PA7 strain. We found that PumA is essential for virulence and inhibits NF‐κB, a property transferable to non‐PumA strain PA14, suggesting no additional factors are needed for PumA function. The TIR domain is able to interact with the Toll‐like receptor (TLR) adaptors TIRAP and MyD88, as well as the ubiquitin‐associated protein 1 (UBAP1), a component of the endosomal‐sorting complex required for transport I (ESCRT‐I). These interactions are not spatially exclusive as we show UBAP1 can associate with MyD88, enhancing its plasma membrane localization. Combined targeting of UBAP1 and TLR adaptors by PumA impedes both cytokine and TLR receptor signalling, highlighting a novel strategy for innate immune evasion.

Identification of the endosomal sorting complex required for transport-I (ESCRT-I) as an important modulator of anti-miR uptake by cancer cells.

Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells.

Biogenesis of Lysosome-related Organelles Complex-1 Subunit 1 (BLOS1) Interacts with Sorting Nexin 2 and the Endosomal Sorting Complex Required for Transport-I (ESCRT-I) Component TSG101 to Mediate the Sorting of Epidermal Growth Factor Receptor into Endosomal Compartments

Biogenesis of lysosome-related organelles complex-1 (BLOC-1) is a component of the molecular machinery required for the biogenesis of specialized organelles and lysosomal targeting of cargoes via the endosomal to lysosomal trafficking pathway. BLOS1, one subunit of BLOC-1, is implicated in lysosomal trafficking of membrane proteins. We found that the degradation and trafficking of epidermal growth factor receptor (EGFR) were delayed in BLOS1 knockdown cells, which were rescued through BLOS1 overexpression. A key feature to the delayed EGFR degradation is the accumulation of endolysosomes in BLOS1 knockdown cells or BLOS1 knock-out mouse embryonic fibroblasts. BLOS1 interacted with SNX2 (a retromer subunit) and TSG101 (an endosomal sorting complex required for transport subunit-I) to mediate EGFR lysosomal trafficking. These results suggest that coordination of the endolysosomal trafficking proteins is important for proper targeting of EGFR to lysosomes.

Backbone and side-chain NMR assignments for the C-terminal domain of mammalian Vps28

Vps28 is one of four cytosolic proteins comprising the endosomal sorting complex required for transport I (ESCRT-I). ESCRT-I is involved in sorting ubiquitinated proteins to multivesicular bodies as well as in mediating budding of retroviruses. Here, we report the backbone and side-chain assignments of the mammalian C-terminal domain of Vps28 (mVps28(CTD)), which is involved in interactions with other ESCRT components. We also compare the predicted secondary structures of mVps28(CTD) with those of the published X-ray crystal structures of Saccharomyces cerevisiae and Xenopus laevis Vps28(CTD). These NMR resonance assignments will facilitate chemical shift mapping and structural determination of mammalian Vps28 interactions with other components of the endosomal sorting machinery that sorts ubiquitinated proteins for lysosomal degradation.

TANK-Binding Kinase 1 Attenuates PTAP-Dependent Retroviral Budding through Targeting Endosomal Sorting Complex Required for Transport-I

Retroviruses need to bud from producer cells to spread infection. To facilitate its budding, some virus hijacks the multivesicular body (MVB) pathway that is normally used to cargo and degrade ubiquitylated cellular proteins, through interaction between the late domain of Gag polyproteins and the components of MVB machinery. In this study, we demonstrated that TANK-binding kinase 1 (TBK1) directly interacted with VPS37C, a subunit of endosomal sorting complex required for transport-I (ESCRT-I) in the MVB pathway, without affecting the ultrastructure or general function of MVB. Interestingly, overexpression of TBK1 attenuated, whereas short hairpin RNA interference of TBK1 enhanced HIV-1 pseudovirus release from Vero cells in type I IFN (IFN-I)-independent manner. Down-regulation of TBK1 by short hairpin RNA in TZM-bl cells also enhanced live HIV-1 NL4-3 or JR-CSF virus budding without involvement of IFN-I induction. Furthermore, infection of TBK1-deficient mouse embryonic fibroblast cells with a chimeric murine leukemia virus/p6, whose PPPY motif was replaced by PTAP motif of HIV-1, showed that lack of TBK1 significantly enhanced PTAP-dependent, but not PPPY-dependent retrovirus budding. Finally, phosphorylation of VPS37C by TBK1 might regulate the viral budding efficiency, because overexpression of the kinase-inactive mutant of TBK1 (TBK1-K38A) in Vero cells accelerated HIV-1 pseudovirus budding. Therefore, through tethering to VPS37C of the ESCRT-I complex, TBK1 controlled the speed of PTAP-dependent retroviral budding through phosphorylation of VPS37C, which would serve as a novel mechanism of host cell defense independent of IFN-I signaling.

Abnormal regulation of TSG101 in mice with spongiform neurodegeneration

Spongiform neurodegeneration is characterized by the appearance of vacuoles throughout the central nervous system. It has many potential causes, but the underlying cellular mechanisms are not well understood. Mice lacking the E3 ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1) develop age-dependent spongiform encephalopathy. We identified an interaction between a “PSAP” motif in MGRN1 and the ubiquitin E2 variant (UEV) domain of TSG101, a component of the endosomal sorting complex required for transport I (ESCRT-I), and demonstrate that MGRN1 multimonoubiquitinates TSG101. We examined the in vivo consequences of loss of MGRN1 on TSG101 expression and function in the mouse brain. The pattern of TSG101 ubiquitination differed in the brains of wild-type mice and Mgrn1 null mutant mice: at 1 month of age, null mutant mice had less ubiquitinated TSG101, while in adults, mutant mice had more ubiquitinated, insoluble TSG101 than wild-type mice. There was an associated increase in epidermal growth factor receptor (EGFR) levels in mutant brains. These results suggest that loss of MGRN1 promotes ubiquitination of TSG101 by other E3s and may prevent its disassociation from endosomal membranes or cause it to form insoluble aggregates. Our data implicate loss of normal TSG101 function in endo-lysosomal trafficking in the pathogenesis of spongiform neurodegeneration in Mgrn1 null mutant mice.

Peptide Inhibitors of HIV-1 Egress

HIV-1 release requires a direct interaction between the p6 domain of the Gag protein and Tsg101, a component of the cellular endosomal sorting complex required for transport I (ESCRT-I). Disruption of the binding between Gag and Tsg101 is highly detrimental to particle release, making this viral-host cell interaction a potential target for the development of novel anti-HIV-1 agents. An article in this issue reports on the application of a bacterial reverse two-hybrid strategy to identify a cyclic peptide that disrupts Gag-Tsg101 binding and suppresses HIV-1 particle release.

Molecular Characterization of Feline Immunodeficiency Virus Budding

Infection of domestic cats with feline immunodeficiency virus (FIV) is an important model system for studying human immunodeficiency virus type 1 (HIV-1) infection due to numerous similarities in pathogenesis induced by these two lentiviruses. However, many molecular aspects of FIV replication remain poorly understood. It is well established that retroviruses use short peptide motifs in Gag, known as late domains, to usurp cellular endosomal sorting machinery and promote virus release from infected cells. For example, the Pro-Thr/Ser-Ala-Pro [P(T/S)AP] motif of HIV-1 Gag interacts directly with Tsg101, a component of the endosomal sorting complex required for transport I (ESCRT-I). A Tyr-Pro-Asp-Leu (YPDL) motif in equine infectious anemia virus (EIAV), and a related sequence in HIV-1, bind the endosomal sorting factor Alix. In this study we sought to identify and characterize FIV late domain(s) and elucidate cellular machinery involved in FIV release. We determined that mutagenesis of a PSAP motif in FIV Gag, small interfering RNA-mediated knockdown of Tsg101 expression, and overexpression of a P(T/S)AP-binding fragment of Tsg101 (TSG-5') each inhibited FIV release. We also observed direct binding of FIV Gag peptides to Tsg101. In contrast, mutagenesis of a potential Alix-binding motif in FIV Gag did not affect FIV release. Similarly, expression of the HIV-1/EIAV Gag-binding domain of Alix (Alix-V) did not disrupt FIV budding, and FIV Gag peptides showed no affinity for Alix-V. Our data demonstrate that FIV relies predominantly on a Tsg101-binding PSAP motif in the C terminus of Gag to promote virus release in HeLa cells, and this budding mechanism is highly conserved in feline cells.

Parallels Between Cytokinesis and Retroviral Budding: A Role for the ESCRT Machinery

During cytokinesis, as dividing animal cells pull apart into two daughter cells, the final stage, termed abscission, requires breakage of the midbody, a thin membranous stalk connecting the daughter cells. This membrane fission event topologically resembles the budding of viruses, such as HIV-1, from infected cells. We found that two proteins involved in HIV-1 budding-tumor susceptibility gene 101 (Tsg101), a subunit of the endosomal sorting complex required for transport I (ESCRT-I), and Alix, an ESCRT-associated protein-were recruited to the midbody during cytokinesis by interaction with centrosome protein 55 (Cep55), a centrosome and midbody protein essential for abscission. Tsg101, Alix, and possibly other components of ESCRT-I were required for the completion of cytokinesis. Thus, HIV-1 budding and cytokinesis use a similar subset of cellular components to carry out topologically similar membrane fission events.

Molecular Architecture and Functional Model of the Complete Yeast ESCRT-I Heterotetramer

The endosomal sorting complex required for transport-I (ESCRT-I) complex, which is conserved from yeast to humans, directs the lysosomal degradation of ubiquitinated transmembrane proteins and the budding of the HIV virus. Yeast ESCRT-I contains four subunits, Vps23, Vps28, Vps37, and Mvb12. The crystal structure of the heterotetrameric ESCRT-I complex reveals a highly asymmetric complex of 1:1:1:1 subunit stoichiometry. The core complex is nearly 18 nm long and consists of a headpiece attached to a 13 nm stalk. The stalk is important for cargo sorting by ESCRT-I and is proposed to serve as a spacer regulating the correct disposition of cargo and other ESCRT components. Hydrodynamic constraints and crystallographic structures were used to generate a model of intact ESCRT-I in solution. The results show how ESCRT-I uses a combination of a rigid stalk and flexible tethers to interact with lipids, cargo, and other ESCRT complexes over a span of approximately 25 nm.