Correspondence to Paul D. Bieniasz: pbienias{at}adarc.org
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Abbreviations used in this paper: EbVP40, Ebola virus VP40; ENaC, epithelial Na+ channel; ESCRT, endosomal sorting complex required for transport; HECT, homologous to E6AP COOH terminus; MLV, murine leukemia virus; MVB, multivesicular body; RSV, Rous sarcoma virus; VPS, vacuolar protein-sorting.
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Introduction |
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Three viral L-domain types with distinguishable but overlapping requirements for the various VPS factors are defined by PT/SAP (Gottlinger et al., 1991; Huang et al., 1995), YPXL/LXXLF (Puffer et al., 1997; Strack et al., 2003), or PPXY (Wills et al., 1994) peptide motifs. PTAP motifs recruit ESCRT-I by binding to Tsg101 (Garrus et al., 2001; Martin-Serrano et al., 2001; VerPlank et al., 2001; Demirov et al., 2002), whereas YPXL and LXXLF motifs recruit AIP-1/ALIX, a class E VPS factor that binds to both ESCRT-I and -III (Martin-Serrano et al., 2003a; Strack et al., 2003; von Schwedler et al., 2003). All L-domain types require a subset of class E VPS factors and their function is blocked by dominant negative forms of ESCRT-III components (Martin-Serrano et al., 2003a; Strack et al., 2003; von Schwedler et al., 2003) or VPS4 (Garrus et al., 2001; Martin-Serrano et al., 2003b; Tanzi et al., 2003).
Thus, PTAP- and YPDL-type L-domains bind directly to class E VPS factors but how PPXY motifs access the class E VPS pathway is uncertain. PPXY is a consensus sequence for interaction with WW-domains, which are present in homologous to E6AP COOH terminus (HECT) ubiquitin ligases. Although yeast has a single HECT ubiquitin ligase (Rsp5; Huibregtse et al., 1995), mammals have elaborated this family of proteins to 10 members (Rotin et al., 2000). Rsp5-mediated ubiquitination of cargo or transacting factors is required for the endocytosis of at least some transmembrane proteins and/or for the sorting of endocytic and biosynthetic cargo into the yeast vacuole (Galan et al., 1996; Dunn and Hicke, 2001; Katzmann et al., 2004). In mammals, the most widely studied member of this family of proteins, Nedd4, is recruited by PPXY motifs in, for example, the cytoplasmic domains of the amiloride-sensitive epithelial Na+ channel (ENaC) and induces its down-regulation (Staub et al., 1996, 2000). Heretofore, the WW and membrane binding (C2) domains are thought to be responsible for directing the localization and substrate recognition of Nedd4/Rsp5 like proteins, whereas the role of the HECT ubiquitin ligase domain has been thought to be confined to modifying cargo or transacting factors with ubiquitin (Dunn and Hicke, 2001; Hicke, 2001).
Several studies suggest that Nedd4-like ubiquitin ligases play roles in viral budding. Overexpression of various HECT ubiquitin ligase-derived WW-domains can block viral budding, and the PPXY motifs in vesicular stomatitis virus (Harty et al., 1999), Ebola virus (Harty et al., 2000; Yasuda et al., 2003), Rous sarcoma virus (RSV; Kikonyogo et al., 2001), human T cell leukemia virus (Bouamr et al., 2003; Blot et al., 2004; Heidecker et al., 2004; Sakurai et al., 2004), and Mason Pfizer monkey virus (Yasuda et al., 2002) have been reported bind to Nedd4, LDI-1, LDI-2, BUL1, or WWP1 HECT ubiquitin ligases. PPXY motifs can also cause retroviral Gag proteins to become ubiquitinated and an ENaC-derived peptide sequence exhibits L-domain activity in the context of a retroviral Gag protein (Strack et al., 2000, 2002). Other observations suggest that ubiquitin itself plays a general role in viral budding, although the details of its participation are not understood. Proteasome inhibitors block the release of certain rhabdoviruses (Harty et al., 2001) and some retroviruses (Patnaik et al., 2000, 2002; Schubert et al., 2000; Strack et al., 2000; Ott et al., 2002, 2003), perhaps due to depletion of free ubiquitin. Importantly, these inhibitors induce a defective viral assembly phenotype that resembles that of L-domain mutants.
Because it remained uncertain as to precisely which HECT ubiquitin ligases mediate PPXY motifdependent viral budding and how the class E VPS pathway is accessed by these proteins, we surveyed an array of HECT ubiquitin ligases and found that fragments of the HECT ubiquitin ligases WWP1 and WWP2 are unusually potent and specific inhibitors of viral PPXY motif function. The sequence requirements for the binding of the murine leukemia virus (MLV) L-domain motif to WWP1, WWP2, and the closely related ligase, Itch, but not Nedd4 precisely recapitulate those required for virus release. Moreover, WWP1 can be recruited by PPXY motifs to sites of MLV and Ebola virus particle budding from cells. Importantly, we show that these ligases can actively promote PPXY-dependent virus particle release, and that this function requires an enzymatically active HECT domain. Finally, we also show that several HECT ubiquitin ligases display characteristics of class E VPS factors in that they are recruited to endosomal compartments induced by dominant negative VPS4 and, surprisingly, that the HECT domain is largely responsible for this property in WWP1. Together, these data indicate that the enzymatic activity of particular HECT ubiquitin ligases and a physical association with the class E VPS pathway promotes PPXY motifdependent viral budding.
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Results |
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Using yeast 2-hybrid assays, we next tested the ability of each WW-domain to bind to MLV Gag proteins containing the various PPXY motifs. The isolated WW-domains from WWP1, WWP2, and Itch could not be used in this assay, due to toxicity in yeast and constitutive transcriptional activation activity. Therefore, the full-length proteins were used in these cases. As can be seen in Fig. 1 E, the full-length Nedd4 protein and the Nedd4WW fragment exhibited the same specificity for binding to the various PPXY motifs. The intact MLV Gag protein bound to Nedd4, Nedd4WW Nedd4-LWW, WWP1, WWP2, and Itch, and these interactions required the PPXY motif. The MLV/ENaC Gag protein bound to all the proteins tested except Smurf1 and Smurf2. Conversely, MLV/RSVp2 and MLV/Ebola Gag proteins bound only to WWP1, WWP2, and Itch. Overall, the ability of the WW-domains to bind to PPXY motifs in the yeast 2-hybrid assay correlated quite well with the ability of the corresponding YFP-WW-domain fusion proteins to act as dominant inhibitors in the viral budding assay, although there were some WW-domains (Nedd4, Nedd4-L, Itch) that appeared capable of binding to the intact MLV Gag protein that had relatively modest, albeit significant, activity as MLV budding inhibitors (threefold inhibition). That MLV, ENaC, RSVp2, and Ebola PPXY motifs could bind to endogenous WWP1, WWP2, and Itch ubiquitin ligases, was determined using co-precipitation assays. Each of these ubiquitin ligases was co-precipitated with GST-p12 fusion proteins containing MLV, ENaC, RSV, and Ebola PPXY motifs, but not with GST-p12(dPY) (Fig. 1 F). Other ubiquitin ligases were not tested in this assay, either because antibodies were not available or because we were unable to detect them in 293T cell lysates using commercially available antisera.
Sequence requirements for MLV L-domain function and HECT ubiquitin ligase binding
If PPXY motifs indeed mediate viral budding by recruiting specific HECT ubiquitin ligases, then the sequence requirements for viral budding activity should closely match those for binding to the ligases. We tested a series of six, single amino acid to alanine, point mutants scanning through MLV Gag(p12) residues 161166 (DPPPYR). Each mutation was tested in the context of an MLV proviral plasmid for effects on virus release, and in the context of Gag and GST-p12 proteins for effects on binding to HECT ubiquitin ligases. As can be seen in Fig. 2 A, MLV proviral plasmids carrying the APPPYR, DPPAYR, and DPPPYA mutations generated virions with similar efficiency to wild-type MLV, whereas release of the DAPPYR, DPAPYR, and DPPPAR mutant virions was attenuated. Loss of viral budding was accompanied by a Gag processing defect with accumulation of unprocessed Gag and aberrant Gag fragments in cell lysates and little mature p30 capsid formation (Fig. 2 A). Virion release by the panel of MLV mutants correlated with the ability of Gag to bind to WWP1, WWP2, and Itch, but not to Nedd4 or Nedd4L (Fig. 2 B). In particular, the DPPAYR Gag mutant was devoid of Nedd4 or Nedd4L binding activity (Fig. 2 B), but supported efficient virion release (Fig. 2 A). The DAPPYR, DPAPYR, and DPPPAR mutations inhibited co-precipitation of WWP1, WWP2, and Itch from 293T cell lysates by GST-p12 fusion proteins, whereas the APPPYR, DPPAYR, and DPPPYA mutations had no effect (Fig. 2 C), concordant with the findings in the virus release and yeast 2-hybrid assays (Fig. 2, A and B). A small amount of WWP2 co-precipitation was detected with the DAPPYR mutant (Fig. 2 C) and this mutant supported a low residual level of virion formation (Fig. 2 A). Because the WWP1 and Itch antisera were less efficacious than the WWP2 antiserum, it was unclear whether our inability to detect WWP1 and Itch co-precipitation by GST-p12(DAPPYR) was due to real differences in their binding specificity as compared with WWP2. Similar GST-p12 mutant coprecipitation experiments could not be done with Nedd4. This was because Nedd4 did not bind efficiently to wild-type GST-p12, even when overexpressed, despite interaction with MLV Gag in the yeast 2-hybrid assay. In fact, Nedd4 was only efficiently coprecipitated by the GST-p12(ENaC) protein (unpublished data).
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Recruitment of HECT ubiquitin ligases to VPS4-induced endosomes
These data demonstrate that certain HECT ubiquitin ligases can mediate PPXY-dependent viral budding and that their enzymatic activity is required for this function. However, because WWP1 fragments completely lacking the HECT domain were substantially more potent dominant inhibitors of budding that those containing a catalytically inactive HECT domain (Fig. 4), we investigated the possibility that this protein domain may serve some additional function, perhaps class E VPS factor recruitment. Using yeast 2-hybrid and/or co-precipitation assays (Martin-Serrano et al., 2003a) we tested whether WWP1 or WWP2 could bind to an array of class E VPS factors, including all known components of mammalian ESCRT-I, -II, and -III as well as Hrs, HBP, AIP1/ALIX, LIP-5, VPS4, and the AIP1/ALIX binding proteins CIN85 and CMS. These results were uniformly negative (unpublished data). However, a characteristic property of class E VPS pathway components is that they are often relocalized to aberrant endosomes induced by VPS4 ablation in yeast (Katzmann et al., 2001; Babst et al., 2002a, b) or by expression of catalytically inactive VPS4(DN) mutants in mammalian cells (Bishop and Woodman, 2001). This is probably because most class E VPS factors, including VPS4 itself, cycle on and off the limiting membrane of late endosomes and their disassembly and release requires VPS4 activity. In the absence of VPS4 overexpression (Fig. 3) or in the presence of wild-type CFP-VPS4, which is distributed diffusely in the cytoplasm at steady state (Fig. 7 A), YFP-WWP1 localized primarily at the plasma membrane. However, upon coexpression with CFP-VPS4(DN), YFP-WWP1 accumulated on aberrant CFP-VPS4(DN)-induced endosomes (Fig. 7 A). Surprisingly, this property of WWP1 appeared, at least in part, to be conferred by the HECT domain, because YFP-WWP1HECT also accumulated on VPS4(DN)-induced endosomes, whereas YFP-WWP1C2 remained at the plasma membrane and YFP-WWP1WW exhibited only a marginal tendency to relocalize (Fig. 7 A). Although the YFP-WWP1C2-WW protein exhibited some recruitment to CFP-VPS4(DN)-induced endosomes (unpublished data), this tendency was clearly reduced as compared with full-length YFP-WWP1. Moreover, full-length YFP-WWP1 but not YFP-WWP1C2-WW appeared to enhance the formation of CFP-VPS4(DN)-labeled endosomes (unpublished data).
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Discussion |
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Although our analyses suggest that HECT domains have unanticipated additional functions, they also clearly demonstrate that the HECT active site, and presumably therefore, ubiquitination of a substrate protein, is required for PPXY-dependent viral budding. What the functional target of ubiquitination is in this context is unclear. Several studies have shown that retroviral Gag proteins are ubiquitinated (Ott et al., 2000; Strack et al., 2000; Blot et al., 2004; Martin-Serrano et al., 2004), and this may stabilize the localization of ubiquitin-binding class E VPS factors at sites of viral budding. Alternatively, other factors, including the class E VPS factor Hrs, are also ubiquitinated during cellular cargo trafficking (Polo et al., 2002) in at least some instances by HECT ubiquitin ligases (Marchese et al., 2003). In addition, there is clear evidence that Rsp5-dependent trafficking in yeast can require ubiquitination of substrates other than the cargo protein (Dunn and Hicke, 2001). In principle, therefore, ubiquitination of factors other than the viral protein could influence their function in viral budding.
Which HECT ubiquitin ligase is actually used during PPXY-dependent viral budding and receptor trafficking? Overexpressed YFP-fusions of WWP1, WWP2, and Itch exhibited distinct patterns of localization, but each of these ligases bound to the MLV L-domain with the sequence specificity expected of a genuine PPXY-type L-domain cofactor. In addition, each of these proteins was recruited to sites of viral budding by MLV Gag and restored the release of a partly defective MLV Gag mutant. All three of these ligases are ubiquitously expressed (Wood et al., 1998) suggesting that some degree of functional redundancy exists, at least for PPXY-dependent viral budding. In addition, these data do not necessarily exclude a role for other HECT ligases such as Nedd4, which bound to a more limited subset of viral PPXY motifs, as viral budding cofactors. However, Nedd4 was not relocalized by MLV Gag or Ebola VP40 (unpublished data) and Nedd4 binding activity was not required for MLV L-domain function. Nonetheless, like other HECT ubiquitin ligases, Nedd4 exhibited evidence of interaction with the class E pathway, because it was recruited to VPS4(DN)-induced compartments. We suppose that multiple HECT ubiquitin ligases can recruit class E VPS factors and precisely which one is used during viral budding likely depends on expression levels in infected tissues, perhaps subcellular localization, and the particular sequence context of the viral PPXY motif. However, at least three PPXY-type L-domains from widely divergent enveloped viruses, as well as the cellular ENaC PPXY motif, proved capable of recruiting WWP1, WWP2, and Itch. Interestingly, based on sequence comparison, these three proteins form a readily distinguishable subgroup of the mammalian HECT ubiquitin ligases.
Although this study focused on the use of HECT ubiquitin ligases and VPS factors during PPXY motifdependent viral budding, similar considerations likely apply to the recruitment of HECT ubiquitin ligases by cellular proteins to facilitate receptor internalization and entry into the MVB sorting pathway. The elaboration of this family of proteins by mammals as compared with yeast might be to cope with a wider range of substrates. Alternatively, it may be that different ligases act at different points in mammalian trafficking pathways. The cellular PPXY motif from the cellular protein ENaC appeared particularly promiscuous with respect to WW-domain binding, and it may be that multiple ligases are exploited by a single motif at different locations during the trafficking of a protein within a cell. In the absence of PPXY motifcontaining viral proteins, the localization of overexpressed WWP1 was governed by the C2-domain that directed the protein to the plasma membrane, and the differential localization of the overexpressed YFP-HECT ubiquitin ligase fusion proteins suggests that endogenous proteins may be located on distinct cellular membranes. In the context of viral budding, constitutive membrane localization of overexpressed HECT ubiquitin ligases appears not to be essential to support viral budding, perhaps because the localized concentration of hundreds or thousands of Gag molecules containing PPXY motifs acts as a potent recruiting influence for WW domains. Nonetheless, the C2 domain potentiates the activity of dominant inhibitory WWP1 fragments, presumably because it would limit the diffusion of the protein to the confines of the plasma membrane, increasing the likelihood of encountering membrane associated viral proteins. Similarly, the C2 domain of Rsp5 has been shown to facilitate the sorting of CPS into the yeast MVB pathway, but mutants lacking this domain retain residual activity (Dunn et al., 2004; Katzmann et al., 2004).
Overall, this study indicates that WWP1, WWP2, and Itch are likely the most frequently used HECT ubiquitin ligases during PPXY motifdependent viral budding and that their HECT ubiquitin ligase activity is required for this activity. Moreover, these findings also suggest that, in addition to acting as ubiquitin ligases, HECT domains have a previously unappreciated association with the class E pathway. Further work will clarify the relationship between ubiquitin ligases, the sorting of proteins at MVBs and the budding of enveloped viruses.
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Materials and methods |
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MLV and EbVP40 viral particle formation assays
293T cells were transfected with MLV proviral plasmids or myc-tagged EbVP40 expression vectors using Lipofectamine Plus (Invitrogen) and supernatants were harvested at 24 h and 48 h after transfection for experiments involving EbVP40 and MLV, respectively. Where the ability of HECT ubiquitin ligases (or fragments thereof) to enhance or inhibit particle release was measured, various amounts of YFP-fusion protein expression plasmid were cotransfected. For both MLV and EbVP40, the culture supernatants were clarified by low speed centrifugation and viral particles were harvested by centrifugation through a 20% sucrose cushion at 100,000 g for 1.5 h. Viral proteins in cell and viral lysates were analyzed by Western blotting.
Proteinprotein interaction assays
For two-hybrid assays, yeast cells (Y190) were transformed with Gal4 and VP16 fusion protein expression plasmids and proteinprotein interactions were measured by ß-galactosidase reporter activity as described previously (Martin-Serrano et al., 2001). For GST-fusion protein co-precipitation assays, 293T cells were transfected with pCAGGS/GST-p12 expression plasmids. 48 h later, GST-p12 and proteins bound to it were precipitated using glutathione-Sepharose beads, eluted, and analyzed by Western blotting as described previously (Martin-Serrano et al., 2003a).
Western blot analyses
Virion and cell lysates or bead eluates were separated on 10 or 12% acrylamide gels and transferred to nitrocellulose membranes. The blots were probed with primary antibodies against WWP1, WWP2, and Itch (Santa Cruz Biotechnology, Inc.) MLV CA, the Myc epitope tag (9E10), or GFP (Roche), followed by peroxidase-conjugated secondary antibodies, and developed with chemiluminescent substrate reagents (Pierce Chemical Co.).
Microscopy
293T cells were plated in 35-mm coverslip dishes (Mattek) and transfected with a variable amount of a pCR3.1/YFP derivative expressing a YFP-HECT ubiquitin ligase fusion protein along with 800 ng of a wild-type or mutant pCAGGS/MLVGag-CFP or pCR3.1/CFP-EbVP40 plasmid. Alternatively, the YFP-fusion protein expression plasmids were cotransfected with 300 ng of pCR3.1/CFP/VPS4. The cells were fixed with PFA 24 h after transfection and images were collected and processed as described in the online supplemental material.
MLV infectivity assays
293T cells were transfected with wild-type, chimeric or mutant MLV proviral plasmids, a vesicular stomatitis virus-G envelope expression plasmid and pMSCV/Tat. HeLa P4/R5 cells, which carry a Tat responsive HIV-1 LTR-LacZ reporter gene, were used as targets to measure infectious MLV production, as described previously (Martin-Serrano et al., 2004). In some experiments, where the ability of ubiquitin ligase fragments to inhibit particle formation was measured, various amounts of YFP-fusion protein expression plasmid were cotransfected with the viral constructs.
Online supplemental material
Details of the plasmid construction, microscopy, and image analysis are contained herein. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200408155/DC1.
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Acknowledgments |
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This work was supported by the National Institutes of Health (RO1AI52774, RO1AI50111) and amFAR (02865-31). P.D. Bieniasz is an Elizabeth Glaser Scientist of the Elizabeth Glaser Pediatric AIDS Foundation.
Submitted: 26 August 2004
Accepted: 12 November 2004
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