Role of ESCRT-I in Retroviral Budding

The final step in the life cycle of enveloped viruses is a membrane fusion event that releases the nascent virion from the host cell. Studies on the morphogenesis of human immunodeficiency virus type 1 (HIV-1) were the first to suggest that a virus-encoded function within the Gag protein was necessary for this late budding step to proceed efficiently (15). Subsequent investigations have shown that all retroviruses examined encode a so-called late-budding (L) domain within Gag whose disruption results in a phenotype characterized by normal virion assembly, with the exception of particle release (27, 37-39, 41). Moreover, at least some otherwise unrelated rhabdo-, filo-, and possibly reoviruses also encode sequences with proven or putative (based on sequence homology) L-domain activity (11, 22, 33, 34). Viral L domains fall into one of three classes, based on characteristic sequence motifs. The majority contain either PT/SAP or PPXY tetrapeptide motifs (15, 18, 38, 39, 41). In some, both motifs are present within a relatively short linear sequence. Conversely, equine infectious anemia virus (EIAV) encodes an unusual L-domain sequence that does not conform to the PT/SAP or PPXY consensus sequences but instead contains a critical YPDL motif that is essential for function (27). In several cases, viral L domains are functionally interchangeable and can exert their activity when positioned at different locations within a retroviral Gag protein (22, 24, 40). In fact, an HIV-1 strain that is noninfectious due to inactivating mutations in the PTAP-type L domain can be complemented in trans by coexpression of a Gag protein fused to either a homologous (PTAP) or heterologous (YPDL) L domain (22). These observations strongly suggest that L domains act by recruiting host cell factors to facilitate egress, rather than by directly influencing virion morphogenesis. In fact, specific host cell factors have been implicated in mediating the activity of each class of viral L domain. For EIAV, the YPDL motif conforms to the YXXL consensus sequence that mediates endocytosis of certain transmembrane proteins via interaction with the AP2 clathrin adaptor (10). Accordingly, EIAV p9 was shown to bind to the AP50 subunit of the AP-2 complex in vitro and induce AP-2 relocalization to sites of viral assembly in infected cells (28). The PPXY L-domain motif found in some retroviruses, rhabdoviruses, and filoviruses is similar to the consensus motif that binds WW domains (13). In fact, the L-domain sequences of Rous sarcoma virus, Ebola virus, and vesicular stomatitis virus have each been reported to bind to a particular class of WW domain-containing proteins, namely, the Nedd4-like E2 ubiquitin ligases (16, 17, 20), and a physiological Nedd4 binding site has been reported to exhibit viral L-domain activity (33). The most compelling evidence that a specific cellular protein mediates the activity of viral L domains exists for Tsg101. A variety of physiological activities have been attributed to this protein, but it appears likely that its primary function is in endosomal protein sorting (3, 7, 8, 19). Tsg101 has been demonstrated to interact with the HIV-1 PTAP-type L domain by using various protein-protein interaction assays (14, 22, 35), and we and others have established that Tsg101 recruitment is indeed the mechanism by which the HIV-1 L domain mediates particle budding. Specifically, there is an almost perfect correlation between the abilities of HIV-1 p6 mutants to bind Tsg101 and to mediate virion production (14, 18, 22, 35). In addition, depletion of Tsg101 by using small interfering RNA results in an HIV-1 budding defect that recapitulates the phenotype of a PTAP-defective virus, and reintroduction of Tsg101 restores viral budding (14). Moreover, tethering Tsg101 to sites of HIV-1 particle assembly by fusion to Gag reverses the HIV-1 budding defect that results from mutational inactivation of the PTAP L domain (22). It is also evident that the PTAP-type L domain encoded by Ebola virus, a filovirus, recruits Tsg101 to particle assembly sites to facilitate budding (22). Tsg101 is a member of a family of mammalian proteins whose orthologs in yeast are required for the formation of multivesicular bodies. In yeast, the Tsg101 ortholog is termed Vps23, and genetic elimination of this factor, or any one of approximately 15 other factors, results in the so-called class E phenotype, in which aberrant enlarged endosomes lacking internal vesicles accumulate (29). Several interactions between members of this group of proteins have been documented, and class E factors participate in the formation of at least three protein complexes, termed ESCRT-I, -II and -III (1, 2, 19). These complexes select a subset of transmembrane proteins for delivery to the endosomal lumen and mediate intraluminal vesicle budding. Tsg101 is one subunit of mammalian ESCRT-I (3), which also includes VPS28 (8) and, possibly, an as-yet-unidentified mammalian ortholog of Vps37, an additional component of the yeast ESCRT-I complex (19). The precise mechanisms by which class E Vps factors mediate vesicle budding are not well understood, but ESCRT-I, -II, and -III may act sequentially. Ultimately, an ATPase, Vps4, binds to components of ESCRT-III and mediates dissociation of this and perhaps other class E factors from the endosome (4, 5). Catalytically inactive VPS4 mutants have a dominant negative (DN) phenotype and induce the formation of aberrant endosomes lacking internal vesicles on which class E factors accumulate in both yeast and mammalian cells (4, 5, 8). Despite the fact that Tsg101 depletion does not affect the activity of the PPXY-type L domain encoded by murine leukemia virus (MuLV), evidence that both PTAP- and PPXY-type L domains utilize class E vacuolar protein sorting (VPS) proteins derives from the observation that the budding of both HIV-1 and MuLV is arrested at late stage by DN VPS4 (14). Thus, the processes of vesicle budding at endosomes and retroviral budding at the plasma membrane are topologically equivalent and can both be blocked by DN VPS4, and at least one viral L-domain type recruits a known component of the ESCRT-I complex. Because of this, an attractive but as-yet-unproven hypothesis is that the same set of factors mediates both processes in a mechanistically analogous manner. We have previously shown that a C-terminal Tsg101 domain (residues 250 to 390) is largely sufficient to mediate HIV-1 budding, provided that it is artificially recruited to sites of particle assembly (22). Thus, it appears that the N-terminal 250 amino acids are dispensable and serve only to mediate Tsg101 recruitment to particle budding sites. In this study, we present evidence that HIV-1 particle budding is highly dependent on the integrity of the Tsg101 C-terminal domain. Sequences that mediate VPS28 binding and a putative leucine zipper are each required. The integrity of other C-terminal Tsg101 sequences that mediate a newly described homomultimerization activity is also important for HIV-1 budding. Together, these findings suggest that an intact ESCRT-I complex is necessary for PTAP-type L-domain function. Moreover, minimal Gag binding and multimerizing subdomains of Tsg101 act as DN inhibitors of the HIV-1 p6 (PTAP-type) late budding domain but do not affect budding mediated by EIAV (YPDL-type) or MuLV (PPXY-type) L domains. However, because the EIAV L domain is inhibited by DN VPS4, these data suggest that each of the three known classes of L domains accesses a common pathway involving class E VPS factors but that the PTAP type is unique in requiring an intact ESCRT-I complex to exploit this cellular function during viral budding.