An LYPSL Late Domain in the Gag Protein Contributes to the Efficient Release and Replication of Rous Sarcoma Virus

The efficient release of newly assembled retrovirus particles from the plasma membrane requires the recruitment of a network of cellular proteins (ESCRT machinery) normally involved in the biogenesis of multivesicular bodies and in cytokinesis. Retroviruses and other enveloped viruses recruit the ESCRT machinery through three classes of short amino acid consensus sequences termed late domains: PT/SAP, PPXY, and LYPX_nL. The major late domain of Rous sarcoma virus (RSV) has been mapped to a PPPY motif in Gag that binds members of the Nedd4 family of ubiquitin ligases. RSV Gag also contains a second putative late domain motif, LYPSL, positioned 5 amino acids downstream of PPPY. LYPX_nL motifs have been shown to support budding in other retroviruses by binding the ESCRT adaptor protein Alix. To investigate a possible role of the LYPSL motif in RSV budding, we constructed PPPY and LYPSL mutants in the context of an infectious virus and then analyzed the budding rates, spreading profiles, and budding morphology. The data imply that the LYPSL motif acts as a secondary late domain and that its role in budding is amplified in the absence of a fully functional PPPY motif. The LYPXL motif proved to be a stronger late domain when an aspartic acid was substituted for the native serine, recapitulating the properties of the LYPDL late domain of equine infectious anemia virus. The overexpression of human Alix in the absence of a fully functional PPPY late domain partially rescued both the viral budding rate and viral replication, supporting a model in which the RSV LYPSL motif mediates budding through an interaction with the ESCRT adaptor protein Alix.Retroviruses acquire their lipid envelopes from the plasma membrane as they bud from the cell. Although the structural protein Gag is both necessary and sufficient for the assembly of virus-like particles (VLPs), the membrane scission step of virus egress requires the recruitment of a network of cellular proteins normally involved in two analogous cellular membrane fission events, the budding of cargo-containing vesicles into multivesicular bodies (MVBs) (for review, see references 1, 5, 11, and 50) and the separation of two daughter cells during cytokinesis (3, 4). This cellular network of proteins, collectively called the ESCRT (endosomal sorting complex required for transport) machinery, includes four sequentially recruited high-molecular-weight protein complexes (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) and is essential for the transport of transmembrane cargo proteins to the lysosome for degradation via an MVB intermediate.In addition to the multiprotein ESCRT complexes, other proteins are required to promote the budding of vesicles into the MVB. Ubiquitin ligases (such as Nedd4) monoubiquitinate both ESCRT components and transmembrane cargo proteins, tagging them for the MVB pathway. Adaptor proteins connect cargo proteins to ESCRT complexes or ESCRT complexes to each other. Ultimately, the final membrane fission event of vesicle budding is mediated by an AAA ATPase (Vps4).Retroviruses as well as other enveloped viruses use three amino acid consensus sequences, PPXY, PT/SAP, and LYPX_nL, as docking sites for the components of the cellular ESCRT machinery. The deletion or mutation of these sequences, termed late domains, results in the failure of the virus to recruit the budding machinery to the site of assembly and thereby results in a block at the late stage of virus release in which fully assembled but immature virus particles remain attached to the plasma membrane. The PPXY late domain interacts with the WW domains of the Nedd4 family of ubiquitin ligases. Multiple ESCRT components bind to monoubiquitin tags on both cargo and ESCRT proteins. The PT/SAP late domain binds the ESCRT-I complex component, Tsg101 (tumor susceptibility gene 101). The LYPX_nL late domain interacts with an adaptor protein of the ESCRT pathway, Alix (ALG-2-interacting protein X; also called AIP1) (reviewed in reference 12). Alix interacts with both Tsg101 of the ESCRT-I complex and CMHP4 of the ESCRT-III complex. A possible fourth class of late domains for the paramyxovirus SV5 was reported previously (47). The late domain function in this case has been mapped to an FPIV sequence in the M (matrix) protein. To date, this motif has yet to be shown to be important for the budding of any other virus, and an FPIV-interacting cellular protein has yet to be identified.Often, retroviruses rely on multiple late domains for efficient budding (2, 13, 16, 29, 30). For example, in addition to its PT/SAP motif in human immunodeficiency virus type 1 (HIV-1) p6, which binds Tsg101 (6, 14, 34, 52), HIV-1 also harbors an Alix-binding LYPX_nL motif that functions in budding (13, 33, 34, 48, 52). Mutation of this LYPX_nL motif results in only a modest reduction in HIV-1 budding (10). However, the effects of mutations in the LYPX_nL motif become more obvious in the context of a minimal Gag in which the globular domain of MA and the N-terminal domain of CA are absent (48). Furthermore, the role of this motif also seems to vary among cell types. For example, the deletion of this motif decreases HIV-1 particle production 2- to 3-fold in COS-7 cells (15) but has no consequence for HeLa cells (7). The relationship of the two viral late domains to each other is unknown. It is possible that they are partially redundant, are cooperative (since they act at slightly different steps in the ESCRT pathway), or are cell type specific. It has been observed that the mutation of one late domain has a larger effect on budding than the mutation of the other, implying a hierarchy of function. For example, in HIV-1, PTAP acts as the dominant late domain and LYPX_nL acts as a secondary late domain. Equine infectious anemia virus (EIAV) seems to be an exception in that it relies only on a single LYPDL motif for late domain function.Like other retroviruses, the avian alpharetrovirus Rous sarcoma virus (RSV) requires the ESCRT pathway for release, as evidenced by the observation that a dominant-negative mutant of the ATPase Vps4, which is required for the final step of the ESCRT pathway that releases the ESCRT-III complex, inhibits RSV budding in a dose-dependent manner (37). Mutational analysis mapped the RSV late domain to the PPPY motif in the small spacer peptide p2b of Gag (41, 54, 56). This PPPY motif was previously shown to interact with chicken members of the Nedd4 family of ubiquitin ligases (21, 51). RSV Gag also harbors an LYPSL late domain consensus motif 5 amino acids downstream from PPPY in the p10 domain, which could potentially promote budding via an interaction with Alix.Alix, a 97-kDa adaptor protein with diverse functions, is composed of an N-terminal Bro1 domain, a central V domain, and a C-terminal proline-rich region (10, 22, 26, 58). The proline-rich region is assumed to be unstructured and binds Tsg101 and endophilins. The Bro1 domain, which binds CHMP4, is curved and resembles a banana shape. CHMP4 binding is functionally important for promoting HIV-1 budding (10). It was suggested previously that its convex face may allow Alix to sense negative curvatures in membranes (17, 22). At least for HIV-1, the Alix Bro1 domain also interacts with the Gag NC domain (42, 43). The central V domain of Alix, which is named for its shape, has a conserved hydrophobic pocket on the second arm near the apex of the V that is responsible for the binding of the LYPX_nL late domains of HIV-1 and EIAV (10, 26, 58).In the present study, we investigated the role of the LYPSL motif in RSV budding and replication. We report here that not only the PPPY motif but also the LYPSL motif act as late domains. The contribution of the LYPSL motif to the budding rate and spreading rate is secondary to that of the PPPY motif but increases in the absence of a fully functional PPPY motif. The Alix overexpression-mediated rescue of PPPY mutants supports a model in which the LYPSL late domain functions through an interaction with Alix.