Here, we present a detailed analysis of the relative contributions of the closely spaced MPMV PPPY and PSAP motifs to MPMV particle production. In agreement with previous reports (38
), a block to viral release was observed for MPMV lacking the PPPY motif. Procapsids accumulated underneath the plasma membrane or on intracellular vesicles, displaying an apparent defect mostly in the early stages of budding. Surprisingly, release of particles from cells cotransfected with equal amounts of wt and PPPY mutant constructs was inefficient as well, and a twofold excess of PPPY mutant Gag protein over wt Gag completely abolished virus release in a trans
-dominant-negative way. Thus, at least one-half of the MPMV Gag molecules needs to carry an intact PPPY motif to allow efficient virus release. This is in contrast to findings which showed that one-fifth of HIV-1 Gag molecules carrying a PTAP L domain were sufficient to allow efficient particle release (17
). Conceivably, there is a certain threshold for factor recruitment which may be different depending on the virus and/or the host cell.
While the MPMV PSAP motif cannot replace a functional PPPY motif (38
; this study), our results clearly showed that the PSAP motif functions as a second L domain and promotes virus release in the context of an intact PPPY motif. The phenotype of the PSAP mutation was less pronounced than that of the PPPY mutation, since virus release was reduced by only a factor of two when PSAP was changed. The release defect was much more evident on thin-section EM analysis, however, which revealed a late budding arrest, with most procapsids in immature budding structures or budding chains containing multiple procapsids. These budding chains were apparently released from transfected cells, thus contributing to the extracellular virus detected. The phenotype of a construct carrying both the PPPY and PSAP mutations was indistinguishable from that of the PPPY mutant. Thus, in the context of MPMV Gag, PPPY and PSAP motifs are not redundant. This is different from results observed in substitution experiments, where the PPPY L domains of RSV and MLV were artificially exchanged for a HIV-1-derived PTAP motif. In these studies, the first 12 or 18 residues of HIV-1 p6 were used to replace the respective PPPY L-domain peptide, thereby reconstituting particle release (21
Like MPMV, HTLV-1, vesicular stomatitis virus, and Ebola virus contain closely spaced or overlapping L-domain sequences. The Ebola virus VP40 protein harbors the peptide P7
. Martin-Serrano et al. showed that substitution of P7
, which disrupts the PTAP but not the PPXY motif, severely reduced the release of VP40 (17
). This result indicated that the Ebola virus PTAP motif contributes to virus release as well, similarly to the result observed for MPMV in this study. One may speculate that the combined function of two different L-domain motifs is also required for efficient release of the other viruses that contain more than one L domain. Furthermore, the Ebola virus VP40 L-domain peptide has been shown to functionally replace the HIV-1 PSAP motif of the HIV-1 Gag polyprotein (17
). Like in the context of VP40, the P7
mutation, which leaves the PPPY motif intact, resulted in a complete loss of HIV-like particle release (17
). Similarly, Strack et al. replaced the entire p6 domain of HIV-1 Gag with a VP40-derived peptide (31
). Consistent with the observation by Martin-Serrano et al., substituting Y13
induced no phenotype. When the same Ebola virus-derived peptide was used in the context of a minimal Gag protein, which lacked the globular domain of MA and the N-terminal part of CA and in which the NC-sp2 region was replaced by a leucine zipper, the PT/SAP motif did not suffice to promote particle release. Instead, as was observed in this study for MPMV, a PPPY motif was required to achieve release of particles. Strack et al. suggested that in the context of HIV-1, the PT/SAP motif cooperates with NC-p1 or the remainder of p6 (31
). Thus, it seems likely that whether or not a PT/SAP motif can function as an L domain depends on the context of the Gag protein.
The EM results obtained in this study suggest that the defects of the PPPY and the PSAP variants of MPMV are both at the stage of virus release but are not identical. Most PPPY variant budding structures appeared to be much closer to the plane of the plasma membrane than was observed for the PSAP variant, indicating an earlier budding defect for the PPPY variant. Some late budding structures connected to the plasma membrane or to each other by only a thin membrane stalk were observed for the PPPY variant as well, but this was not common. In most cases, procapsids completely failed to initiate budding and did not induce membrane curvature at all. Le Blanc et al. reported a similar finding for HTLV-1, indicating that the intact PPPY motif may be required at a relatively early stage for the initiation of budding (16
). If the PPPY motif acts upstream of the PSAP motif, this could explain why an intact PPPY motif is needed for the action of PSAP as an L domain (see also below).
In several cells, we observed intracellular vesicles decorated with PPPY mutant viral procapsids that did not initiate budding. Conceivably, some wt MPMV procapsids may also be transported to intracellular vesicles, but this may be normally overlooked, because it constitutes a minor pathway which becomes visible only under conditions of budding arrest. It is interesting in this regard that budding of HIV-1 into intracellular major histocompatibility complex class II vesicles has recently been reported, and this may be an important route of release at least in some cell types (27
). Alternatively, these decorated vesicles may be the result of a partial defect in viral assembly even earlier than budding, i.e., during procapsid trafficking, or may be due to the expression of large amounts of viral Gag proteins.
TSG101 has been shown to bind to the HIV-1 PTAP L domain, and this interaction is essential for virus release (8
). We find readily detectable amounts of endogenous TSG101 present within MPMV particles, suggesting that the protein is recruited to the budding site and incorporated via its interaction with the PSAP motif. This incorporation is likely to be specific, because Tsg101 was not detected in preparations from mock-transfected cells, there was no incorporation of a highly expressed cytosolic protein into particles, and the virion levels of TSG101 were strongly reduced in the case of the PSAP variant. The finding that TSG101 incorporation is not abolished in particles lacking the PSAP motif raises the question whether TSG101 packaging is also specific in this case. Furthermore, it is unclear if the amount of TSG101 still detected accounts for the intermediate phenotype caused by PSAP mutation. Based on published data, TSG101 should not be able to bind to the inactivated PSAP motif (12
). Since TSG101 shows weak binding to ubiquitin alone (8
), it could be recruited by binding to ubiquitinated Gag protein. The fact that an L-domain phenotype was observed, even though some TSG101 was present in virus preparations, may indicate that a relatively high level of TSG101 is required at the budding site.
Besides TSG101, we also detected the E3 ubiquitin ligase Nedd-4 in MPMV particles, albeit at low levels. Nedd-4 incorporation was unaltered by mutation of the PSAP motif, as expected. Recently, a Nedd-4-like protein named Bul-1 has been reported to interact with MPMV Gag in a PPPY motif-dependent way, and Bul-1 overexpression was shown to stimulate MPMV release (39
). Accordingly, Nedd-4 itself may be only weakly recruited by the MPMV Gag polyprotein, explaining its low concentration inside viral particles. It should be noted, however, that the major Nedd-4 immunoreactive protein in viral preparations migrated with an apparent mobility of ca. 50 kDa, which suggests that significantly more Nedd-4 may be incorporated into viral particles and cleaved by the viral PR. The 50-kDa protein was not detectable in cytoplasmic extracts, and the signal could not be assigned to any of the major viral proteins. It seems unlikely that a cellular protein is incorporated into virions at levels high enough to be detectable by unspecific binding of the antibody, and the possibility of PR-mediated cleavage of Nedd-4 is currently under investigation.
As observed for the release of HIV-1 and MLV (8
), MPMV particle production was completely abolished by overexpression of dominant-negative variants of the cellular ATPase VPS4A as well. Virus release was also blocked in the case of the PSAP variant of MPMV, indicating that VPS4A promotes virus release independently of the PT/SAP motif and that the two L-domain motifs target the same pathway. This supports and extends previous findings for MLV, which also contains an essential PPPY motif but carries an additional PSAP motif in its MA domain, which may also contribute to virus release.
In summary, our results indicate that both L-domain motifs of MPMV contribute to virus release, with the PPPY motif being essential and a PPPY mutation causing budding arrest at an earlier stage than a PSAP mutation. In the case of the VPS machinery-dependent sorting of endosomal proteins into MVBs, initial monoubiquitination of cargo and adapter proteins is followed by recruitment of the TSG101-containing ESCRT-1 complex and further complexes of the Vps pathway (14
). By analogy, one may speculate that PPPY-dependent recruitment of Nedd-4 (or a Nedd-4 like protein) serves to monoubiquitinate Gag or a Gag-associated protein with subsequent binding of TSG101, both through the PSAP motif within Gag and through the ubiquitin moiety. Since TSG101 was incorporated into virions even in the absence of a PSAP motif, this motif in the case of MPMV may mainly serve to recruit sufficient amounts of TSG101 for efficient virus release, e.g., in cell types where TSG101 concentrations are limiting. This hypothesis predicts that TSG101 would also be required for the release of an MPMV variant lacking the PSAP motif, and this is under investigation. In the case of MLV, however, virus release was not severely altered following depletion of cellular TSG101 (8
), indicating that the PPPY motif can function independently of TSG101 at least in this case.