The cellular VPS pathway normally sorts endosomal membrane proteins to a late endosomal compartment, the MVB, for transport to the lysosome and degradation. After trafficking to the late endosome, membrane proteins are sorted from the limiting membrane of the endosome into membrane vesicles within the lumen of the organelle. Vesicles, and their included membrane proteins, are released into the lumen to create MVBs by a membrane fission event that is topologically analogous to viral budding. Sorting of proteins into the region of the endosomal membrane from which the vesicles derive and vesicular budding require specific cis-
acting signals. In yeast and mammalian cells monoubiquitination is a key signal for VPS-mediated sorting. A currently accepted model hypothesizes that cellular VPS proteins, which normally carry out this membrane fission reaction or assemble factors that catalyze the reaction, are recruited to the site of viral particle release through interaction with signals encoded in Gag late domains where these cellular factors mediate the late stages of viral budding (17
EIAV is unique among retroviruses studied to date in that it utilizes a YXXL motif in Gag to direct the late stage of viral release and not a PTAP or PPPY late-domain motif commonly found either alone or in tandem in other retroviral Gag proteins. Although different late-domain motifs are used by these retroviruses, based on the data presented here for EIAV Gag and previously for M-MuLV and HIV-1, it appears that all of the identified late domains require cellular VPS machinery for efficient particle release (17
). Here we utilized a dn form of VPS4 to demonstrate that the YXXL late domain in EIAV Gag p9 requires an intact VPS pathway for efficient budding. Moreover, we have shown that the dnVPS4-mediated inhibition of EIAV Gag release is specific to a late viral budding event (Fig. ).
Distinct retroviral late domains recruit VPS factors in different ways. PTAP late domains bind to the VPS ESCRT-1 complex protein TSG101 (17
). PPPY-encoding Gag polyproteins enter the VPS pathway in a less defined manner. Gag polyproteins utilizing the PPPY late-domain motif have been shown to interact with the WW domain-containing Nedd4 family of ubiquitin ligases (25
). Nedd4 and Nedd4-like proteins are implicated in the ubiquitination of plasma membrane proteins destined for lysosomal degradation via the MVB (14
). Monoubiquitination of target proteins appears to be a prerequisite for their entry into the VPS pathway (35
). Indeed, numerous VPS proteins (including TSG101) contain ubiquitin binding domains (35
EIAV Gag, like all other retroviral Gag proteins studied to date, is modified by ubiquitin (31
). The relationship between Gag and ubiquitin remains to be fully elucidated. Gag polyproteins from a number of retroviruses are modified by ubiquitin (30
). Furthermore, investigators have reported that release of retroviruses utilizing either the PPPY (i.e., Rous sarcoma virus and MuLV) or PTAP (HIV-1) late domain are potently blocked by agents that inhibit ubiquitin modification (34
). Interestingly, EIAV Gag release is relatively resistant to ubiquitin-blocking agents (31
). The reason for this disparity has not been determined but may be due to a region of EIAV Gag with homology to ubiquitin and which has been speculated to enable direct binding of EIAV Gag to ubiquitin binding factors (33
The mechanism of EIAV Gag recruitment of VPS factors remains to be determined. However, data indicating that EIAV Gag does not require ubiquitination or TSG101 for particle egress suggest that Gag enters the VPS pathway at a point distinct from Gag polyproteins that utilize PTAP or PPPY late domains. EIAV Gag has been shown to bind to AP-2 via the YDPL motif that constitutes its late domain (39
); however, it remains unclear if AP-2 is functionally required for EIAV budding. Unlike TSG101 or Nedd4, AP-2 has not yet been implicated as a player in the VPS pathway, and thus, it is unclear how AP-2 binding could promote entry into the VPS pathway. Further research into EIAV budding will likely reveal new information regarding associations between known VPS factors and other cellular proteins and trafficking pathways.
In yeast, protein sorting onto MVB vesicles requires the sequential action of a series of protein complexes called ESCRT-1, ESCRT-2, and ESCRT-3 (1
). VPS23, the TSG101 ortholog, is an essential component, along with VPS28 and VPS37, of the ESCRT-1 complex (24
). Unlike HIV-1, M-MuLV and EIAV Gag particle release is insensitive to TSG101 depletion, implying that the late domains in these retroviruses function independently of TSG101. The ability of EIAV and M-MuLV Gag to bud in a TSG101-independent manner may suggest that these Gag polyproteins are able to bind other ESCRT-1 components. However, this hypothesis would require either that TSG101 is not essential for ESCRT-1 function or, alternately, that another protein may substitute for TSG101 in the ESCRT-1 complex to facilitate M-MuLV and EIAV virion budding. Another hypothesis for the observed lack of TSG101 dependence is that EIAV accesses the VPS pathway downstream of ESCRT-1 possibly via ESCRT-2, ESCRT-3, or other factors. Our data demonstrating efficient release of EIAV ld(−)Gag-VPS28 suggest that entry into the VPS pathway is flexible and that viruses may therefore utilize numerous mechanisms to usurp the VPS machinery.
One possibility is Eps15, an adaptor complex binding protein that is implicated in trafficking of ubiquitinated proteins to the MVB (35
). Another alternative is Hrs, an endosomal adaptor protein required for epidermal growth factor receptor sorting to the MVB (46
) and a mammalian ortholog of the yeast class E protein VPS27p. Hrs associates with a range of adaptor proteins including Eps15 (reviewed in reference 10
) and may contain a ubiquitin interaction motif (35
). An enticing feature of invoking Eps15 and/or Hrs involvement in viral budding is their physical proximity to other known sorting proteins (ESCRT) and their potential ability to bind ubiquitinated cargo. The complete set of factors involved in MVB formation in mammalian cells is not clearly elucidated. Our results with a chimeric EIAV Gag suggest a unique method of identifying factors involved in MVB formation in higher eukaryotes. Although EIAV does not normally require the MVB ESCRT-1 complex component TSG101 for efficient virus budding, our data demonstrate that fusion to another protein from this complex, VPS28, can rescue budding of a late-domain-defective EIAV Gag. Previously it was shown that fusion of Gag to ubiquitin, a signal for sorting to the MVB, also restores efficient viral budding to a late-domain mutant (34
). The work herein extends this finding to demonstrate that proteins of the VPS pathway can be appended to Gag and function as de facto late domains. Together these results imply that fusion to a late-domain-defective Gag may be a general method for confirming the identity of proteins that are thought to be involved in MVB formation and for mapping functional domains within these proteins. Additionally, our data demonstrating coincorporation of TSG101 into the chimeric VPS28-Gag particles indicate that this strategy might be used to identify novel MVB factors by employing viral budding to enrich and purify those proteins tightly associated with the chimeric protein. For example, the yeast ESCRT-1 complex contains three tightly associated proteins, TSG101, VPS28, and VPS37. However, the mammalian ortholog of VPS37 remains unidentified. Comparison of the proteins in chimeric VPS28-Gag VLPs with those in wt Gag VLPs may help to identify mammalian VPS37 and other proteins specifically associated with VPS28.
A number of retroviral envelope proteins contain a highly conserved YXX
motif in their cytoplasmic tail (5
) that, like the EIAV Gag late-domain motif, is thought to interact with AP-2. Although several studies have demonstrated that for some retroviruses these motifs can function to direct endocytosis, in other viral glycoproteins the function of this motif was not clear (29
), and the role that these cytoplasmic tail motifs play in viral replication is vague. One intriguing possibility is that, similarly to the YPDL motif in EIAV Gag, these motifs in retroviral envelope proteins serve as signals that help to recruit the cellular machinery required for viral budding or to help concentrate the viral envelope proteins at sites of budding. In this regard, it is interesting that the effect of mutation in the EIAV Gag p9 YPDL motif is partially overcome when the complete viral genome is expressed (9
), perhaps suggesting that the EIAV envelope protein is compensating for the defective assembly signal in Gag.
Nonretroviral enveloped viruses such as rhabdoviruses and filoviruses also encode PTAP and PPPY late-domain motifs (12
). It thus seems reasonable to presume that these viruses also recruit VPS factors during particle release. Indeed, TSG101 has been shown to be required for Ebola virus VP40 release (28
). Thus, highly divergent enveloped viruses may utilize a common membrane fission mechanism during particle release. A conserved enveloped virus release mechanism is likely an excellent target for antiviral drugs. A further understanding of the mechanisms whereby viruses recruit these cellular factors should aid greatly in development of this class of therapeutics.