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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Virology. Author manuscript; available in PMC Jul 20, 2009.
Published in final edited form as:
PMCID: PMC2528022
NIHMSID: NIHMS57248

Tsg101 can replace Nedd4 function in ASV Gag release but not membrane targeting

SUMMARY

The Late (L) domain of the avian sarcoma virus (ASV) Gag protein binds Nedd4 ubiquitin ligase E3 family members and is the determinant of efficient virus release in avian and mammalian cells. We previously demonstrated that Nedd4 and Tsg101 constitutively interact raising the possibility that Nedd4 links ASV Gag to the ESCRT machinery. We now demonstrate that covalently linking Tsg101 to ASV Gag lacking the Nedd4 binding site (Δp2b-Tsg101) ablates the requirement for Nedd4, but the rescue of budding occurs by use of a different budding mechanism than that used by wild type ASV Gag. The evidence that Tsg101 and Nedd4 direct release by different pathways is: (i) Release of the virus-like particles (VLPs) assembled from Gag in DF-1, an avian cell line, was resistant to dominant-negative interference by a Tsg101 mutant previously shown to inhibit release of both HIV and Mo-MLV. (ii) Release of VLPs from DF-1 cells was resistant to siRNA-mediated depletion of the endogenous pool of Tsg101 in these cells. (iii) VLPs assembled from wild-type ASV Gag exhibited highly efficient release from endosome-like membrane domains enriched in the tetraspanin protein CD63 or a fluorescent analogue of the phospholipid phosphatidylethanolamine. However, the VLPs assembled from the L domain mutant Δp2b or a chimeric Δp2b-Tsg101 Gag lacked these domain markers even though the chimeric Gag was released efficiently compared to the Δp2b mutant. These results suggest that Tsg101 and Nedd4 facilitate Gag release through functionally exchangeable but independent routes and that Tsg101 can replace Nedd4 function in facilitating budding but not directing through the same membranes.

INTRODUCTION

Expression of the retroviral structural precursor polyprotein, Gag, is sufficient for the production of non-infectious viral-like particles (VLPs; Gottlinger, 2001). Gag contains 3 distinct domains necessary for virus assembly and budding (Wills and Craven, 1991). The membrane-binding (M) domain mediates the association of Gag to the plasma membrane via a cluster of basic residues and, in some retroviruses, a myristic acid moiety co-translationally added to the N-terminus of the Gag precursor (Spearman et al., 1994; Zhou and Resh, 1996). The interaction (I) domain promotes Gag-Gag multimerization crucial for particle assembly (Burniston et al., 1999; Cimarelli et al., 2000; Sandefur et al., 1998) and the late assembly (L) domain facilitates the pinching off of viral particles from the plasma membrane (Gottlinger et al., 1991; Xiang et al., 1996; reviewed in Bieniasz, 2006). L domains have been identified in all retroviruses studied to date, as well as in other families of enveloped viruses, such as the Rhabdoviruses, Paramyxoviruses, and Filoviruses (Bieniasz, 2006). Retroviral L domains are categorized into three classes, defined by their conserved amino acid core sequence: the Pro-Thr-X-Pro (usually PTAP) motif (Gottlinger et al., 1991); the Pro-Pro-X-Tyr (PY) motif (Wills et al., 1994); and the Tyr-X-X-Leu (YXXL) motif (Puffer et al., 1997). Acting as docking sites for cellular proteins, L domains recruit components of the endocytic trafficking pathway to execute the fission of viral from cellular membranes during the budding process (Garrus et al., 2001; Kikonyogo et al., 2001; Martin-Serrano et al., 2001, 2003; Strack et al., 2003; VerPlank et al., 2001; von Schwedler et al., 2003).

Through the PTAP motif, HIV-1 Gag binds directly to Tsg101 (Garrus et al., 2001; Martin-Serrano et al., 2001; VerPlank et al., 2001). Genetic screening utilizing the yeast two-hybrid assay identified ~20 proteins involved in vacuolar protein sorting that function in conjunction with Tsg101 [von Schwedler et al., 2003]. Disrupting the normal function of these vacuolar protein sorting (vps) proteins, termed ‘class E’ Vps proteins, induces formation of abnormally enlarged endosomes (class E compartments) in which Vps proteins become trapped (Raymond et al., 1992) and VLP release is consequently blocked. The class E Vps proteins function in complexes called endosomal sorting complex required for transport (ESCRT)-0, -1, -2, and –3, which may normally be sequentially recruited from the cytosol to deliver cargo to the late endosome/multivesicular body (LE/MVB) compartment where it is sorted for delivery to its ultimate destination. In yeast, the destination is most often vacuoles (equivalent to lysosomes). The ESCRT complexes associated with the limiting membrane of late endosomes are released back into the cytosol through the action of the AAA ATPase Vps4 (Babst et al., 1998). Vps4 containing mutations that disrupt its ATPase activity are dominant negative inhibitors of VLP release (Garrus et al., 2001; Medina et al., 2005).

Tsg101 is a component of ESCRT-I that recognizes cargo proteins modified by ubiquitin, triggering the ESCRT machinery to induce MVB biogenesis (Bishop et al., 2002; Katzmann et al, 2001). PY motifs bind directly to members of the Nedd4 family of HECT ubiquitin (E3) ligases, which ubiquitinate cargo destined for delivery to degradative compartments. Nedd4 may therefore function upstream of Tsg101 and the other ESCRT proteins in the assembly and release pathway of ASV. Tsg101 and Nedd4 associate constitutively in the cytoplasm (Medina et al., 2005), however, Tsg101 also has been found to associate with several other E3 enzymes (Amit et al., 2004; Kim et al., 2007; Li et al., 2001). Here, we demonstrate that release of ASV Gag from avian cells is insensitive to dominant negative-interference or depletion of Tsg101, indicating that ASV Gag release may not require Tsg101 for budding. Also, although release of chimeric ASV Gag lacking the Nedd4 binding site was rescued by translational fusion to Tsg101, the particles containing Tsg101 lacked the markers of transit through endosome-like membrane domains that characterized wild-type ASV Gag release. Interestingly, the particles assembled from another ESCRT-1 component, Vps37C, behaved similarly while chimeric VLPs formed with an ESCRT-2 or an ESCRT-3 factor behaved like the wild-type. Together, these findings suggest that although the Tsg101- and Nedd4-directed pathways are functionally exchangeable in the release process, the cellular proteins facilitate release through independent mechanisms and different membrane domains.

MATERIALS AND METHODS

Constructs and Reagents

The following plasmids were previously described: pCMV-HIV-1gag or pCMV-ASVgag encoding HIV-1 and ASV Gag C-terminally tagged with green fluorescent protein (Gag-GFP; Medina et al., 2005); DNA encoding HA-Tsg101 (Lu et al., 2003); and DNA encoding myc-tagged fragments of Tsg101 (Li et al., 2001). DNA encoding ASV Gag Δp2b-Tsg101 was constructed as follows: p2036 (Kikonyogo et al., 2001) was doubly digested with KpnI and XbaI to remove the gfp coding region. Using PCR-based methods, a 5’ KpnI site was introduced upstream of the start codon of ASV Gag-Δp2b. Additionally, a 3’ HpaI site was introduced downstream of the ASV Gag-Δp2b coding sequence. Similarly, a 5’ HpaI and a 3’XbaI site were introduced upstream and downstream, respectively, of the full-length coding sequence of tsg101. Both PCR products were then ligated to the KpnI and XbaI doubly digested p2036 vector DNA to produce p2036 ASVgag-Δp2b-tsg101, in which Tsg101 is translationally fused in the correct reading frame to the C-terminus of ASV Gag-Δ p2b. An identical strategy was followed to construct the HIVgag P7L-tsg101 fusion construct and the ASVgagp2b-vps37C, -Δp2b-eap20, and-chmp4B constructs. The following probes were purchased as indicated: Antibodies recognizing: actin, CD63, Tsg101, myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA); GFP (Clontech Laboratories, Mountain View, CA, USA); influenza virus HA (Covance, Berkeley, Calif.); TRITC-tagged secondary antibody (Molecular Probes); and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[lissamine rhodamine B sulfonyl] (N-Rh-PE; Molecular Probes). Anti-AMV MA (p19) monoclonal antibody, which recognizes ASV MA, was developed by David Boettiger and was obtained from the Developmental Studies Hybridoma Bank under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences. (Iowa City, IA, USA).

Cell Culture, Transfection, Preparation of Cytoplasmic Extracts, and Virus Isolation

COS-1, 293/E, DF-1, or DF-1/RCAS/BP(A) cells were cultured in DMEM supplemented with fetal bovine serum (5%, 10%, 10%, and 10%, respectively) and antibiotics to 60% confluency at 37°C. The relatively high ratio of cytoplasm to nucleus and ability to spread on tissue culture plates and coverslips make COS-1 cells advantageous for use in imaging studies. Expression of plasmids in the p2036 background was higher in 293/E cells (described in reference 16) because 293/E cells stably express the EBNA1 protein of EBV and the p2036 constructs contain the EBV FR plasmid maintenance element that EBNA 1 binds. Therefore 293/E cells were used when proteins expressed from p2036 were to be detected by Western analysis. Unless otherwise indicated, the cells were transfected by using the FuGene 6 reagent (Roche, Indianapolis, IN, USA) according to the instructions of the manufacturer. At 48 hr post-transfection, the cells were washed with phosphate-buffered saline (PBS) and lysed with RIPA buffer (1% lgepal CA-630, 0.5% sodium deoxychlolate, and 0.1% SDS in 1X PBS) containing protease inhibitor cocktail tablets (Roche) for 15 min at 4°C. The lysate was then passaged through a 21-gauge needle and incubated on ice for 60 min. Cellular debris was pelleted at 10,000 x g for 10 min at 4°C (lysate fraction). ASV Gag was identified by Western blotting using specific anti-MA (ASV) monoclonal antibody. To isolate VLPs, the cell culture media was filtered (0.25 μm), applied to a cushion of 20% sucrose in a centrifuge tube and then spun at 30,000 rpm for 80 min at 4°C (Beckman SW41 rotor). The pelleted particles were suspended by gentle shaking at 4°C in 50 μl of PBS or RIPA buffer containing protease inhibitor cocktail. Samples were analyzed by SDS-PAGE and Western analysis. Equivalent amounts of cell lysates and of media fractions were used for all samples. Semi-quantitative determinations of VLP release (VLP/cell lysate + VLP ratio) were made using a phosphorimager and NIH Image.

RNA Interference

We obtained 21 nt RNA duplexes with symmetric 3’-UU overhangs corresponding to coding nucleotides 326–344 of avian Tsg101 (GUACUGUCCCGGUGAAAUA; Dharmacon, Lafayette, CO, USA). 20 or 50 nM of Tsg101 siRNA or a non-targeting control siRNA were transfected into DF-1 cells in six-well plates using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA). 48 h post-siRNA transfection, DF-1 cells were transfected with p2036 encoding WT ASV Gag using FuGene 6. 24 h later, VLPs were pelleted through a 20% sucrose cushion at 100,000xg for 1 h. ASV Gag expression and VLP release were analyzed by Western blotting with anti-AMV p19 monoclonal antibody. The endogenous level of avian Tsg101 was analyzed by Western blotting with anti-Tsg101 antibody raised against full-length Tsg101 of mouse origin, which cross-reacts with mammalian and avian Tsg101.

Fluorescence Microscopy

Fluorescent microscopy images were captured with an inverted fluorescent/dic Zeiss Axiovert 200M microscope equipped with an AxioCam HRm camera (Zeiss, Thornwood, NY, USA) and mercury arc lamp light source using a 63X Plan-Apochromat (NA 1.40) oil objective and operated by AxioVision Version 4.5 (Zeiss) software.

Electron Microscopy

Sixty-millimeter dishes of DF1/RCASBP(A) cells were washed with PBS at room temperature and fixed in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) at 4 °C for 30 min. Cells were scraped from the tissue culture dish and pelleted at 1000 × g for 10 min at 4 °C. The cell pellet was fixed for an additional 2 h in 2.5% glutaraldehyde and post-fixed for 1 h with osmium tetroxide. The cell pellet was dehydrated in a series of alcohol washes and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a Zeiss 900 electron microscope.

Protein detection

Proteins were separated by electrophoresis through 12.5% SDS-polyacrylamide gels. Following electrophoresis, the gels were transferred to nitrocellulose and analyzed by Western blotting with the antibodies specified in the text. Proteins were visualized by chemiluminescence using Lumi-Light reagents (Roche, Indianapolis, IN, USA).

RESULTS

Co-expression of ASV Gag with a Dominant Negative Interference Fragment of Tsg101 (Tsg-DN) in Avian Cells Did Not Reduce ASV Release Efficiency

Our previous findings showed that fragments of Tsg101 that inhibit release of HIV-1 and Mo-MLV Gag in a dominant-negative manner failed to appreciably inhibit ASV Gag release from 293E or COS-1 cells [Medina et al., 2005 and data not shown]. In this study, we examined ASV release from avian DF-1 cells (Himly et al., 1998; Schaefer-Klein et al., 1998). The same Tsg101 fragment used in the previous study (Tsg-DN; Medina et al., 2005) was transfected into the DF-1 cells. The fragment induced the formation of large, aberrant structures in the cytoplasm Figure 1, panel A), as was previously observed in both avian and mammalian cells (Goila-Gaur et al., 2003; Johnson et al., 2005; Medina et al., 2005). The Tsg101 fragment was then co-transfected with GFP-tagged ASV Gag (containing a D37S mutation to inactive PR), and viral particles were harvested from the media fraction forty-eight hours later. As shown in the Western blot in Figure 1B and the semi-quantitative analysis of VLP release in Figure 1C, virus release from DF-1 cells was resistant to dominant negative interference by the Tsg101 fragment. In a separate experiment, DF-1 cells constitutively expressing ASV (DF1/RCASPB(A) (Himly et al., 1998; Schaefer-Klein et al., 1998) were mock-transfected or transfected with DNA encoding Tsg-DN and examined by electron microscopy after thin sectioning (panel D, left and right, respectively). Fully assembled particles were observed outside of the mock-transfected and the Tsg-DN-transfected cells (enlarged in insets D1and D2, respectively). The effect of expression of the Tsg-DN in DF-1/RCASPB(A) cells can be seen in the right panel in D, where aggregates of particles are detected inside of cells (enlarged in inset D3). These aggregates were not observed in the mock transfected cells (left panel). Taken together with the results in 293E and COS-1 cells (Medina et al., 2005), these observations suggest that ASV Gag assembly and release is resistant to any disruption of the endocytic machinery that expression of the Tsg101 fragment may cause in these cells.

FIG. 1FIG. 1
Co-expression of ASV Gag and Tsg-DN in Avian Cells Does Not Reduce ASV Release Efficiency

Depletion of Endogenous Tsg101 Did Not Inhibit ASV Gag Release

To further examine whether VLP release requires Tsg101 function, we determined the effect of siRNA-mediated depletion of the endogenous pool of Tsg101 in DF-1 cells on ASV Gag release. We used a siRNA sequence designed to specifically target avian Tsg101 expression. Gag proteins in lysates prepared from gag-transfected cells and VLP in media were harvested at forty-eight hours post-transfection as described in Materials and Methods analyzed by SDS-PAGE and Western blotting. As shown in Figure 2 (panel A), treatment with Tsg101-specific siRNA (20 or 50 nM) was sufficient to deplete cells of ~90% of the Tsg101 protein compared to the same amount of control, non-targeting siRNA (compare lanes 1 and 2 to lanes 3 and 4, respectively). Under these conditions, the steady-state level of actin and the amount of Gag detected in the lysate were not detectably reduced. The significant depletion of endogenous Tsg101 also failed to inhibit ASV Gag release. A semi-quantitative analysis (VLP/Cell lysate + VLP) panel B) indicated that the budding efficiency of ASV VLPs was not significantly different from control levels. These results indicate that WT ASV Gag release is not dependent on the steady-state level of Tsg101 in its natural host cell and suggest that budding may occur independently of Tsg101 and therefore, perhaps, the ESCRT-I complex.

Fig. 2
Depletion of endogenous Tsg101 in DF-1 cells by siRNA-targeting

Tsg101 Replaced Nedd4 Function in ASV Gag Budding

Taken together, the observed resistance of ASV Gag budding to co-expression with Tsg-DN and to siRNA-mediated Tsg101 depletion indicates that ASV Gag is not strongly dependent on Tsg101 for release. To determine whether Tsg101 could substitute for Nedd4 in mediating the release function, the protein was translationally fused to the C-terminus of Gag-Δp2b, an L domain mutant that lacks the Nedd4 docking site, to form a chimeric Gag protein, Δp2b-Tsg101 (described in Materials and Methods). As avian Tsg101 has not been isolated, human (293E), rather than avian, cells were used for these experiments so that the Tsg101 in the chimeric Gag protein, the Tsg101 being tested for its effect in trans (i.e., adventitiously expressed Tsg101), and the Tsg101 in the cytoplasm would all be identical sequences. As shown in Figure 3, VLPs assembled from wild-type (WT) ASV Gag were released into the media with high efficiency (panel A) in the absence (lane 1) or presence (lane 2) of adventitious Tsg101 expression, consistent with previous findings (Medina et al., 2005). The L domain mutant Δp2b was released at <10% of WT efficiency (Kikonyogo et al., 2001; Medina et al., 2006) and this level was the same whether Tsg101 was expressed or not (lanes 3 and 4). Translational fusion of Tsg101 to the GagΔp2b mutant to form Gag Δp2b-Tsg101 resulted in production of significantly more VLPs (lane 5), indicating that the release function was rescued, however, the wild-type level was never achieved. An estimate of VLP release efficiency indicated that VLPs containing the chimeric ASV Gag protein budded at a level of ~50% compared to WT protein (panel B) (n = 4). Consistent with previous findings (Martin-Serrano and Bieniasz, 2003), we observed that translational fusion of Tsg101 to an L domain mutant of HIV-1 Gag (P7L-Tsg101) resulted in efficient rescue (panel B inset). Although the level of rescue obtained for ASV Gag-Δp2b was lower than that obtained for HIV-1 Gag P7L under similar conditions, the results nevertheless indicate that Tsg101 can replace the Nedd4 function required for ASV Gag budding.

Fig. 3
Translational fusion of Tsg101 to the C-terminus of ASV Gag Δp2b rescues VLP release

Tsg101 and Nedd4 Direct VLP Release from Different Membrane Microdomains

Tsg101 and Nedd4 may direct release through functionally unlinked pathways. If so, a switch from the Nedd4-dependent mechanism to a Tsg101-dependent mechanism might be accompanied by changes in Gag trafficking. We therefore tested for changes in Gag delivery to the budding site using as membrane markers the endosomal lipid tracer, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[lissamine rhodamine B sulfonyl] (N-Rh-PE), a fluorescent phosphatidyl ethanolamine analogue (Vidal et al., 1997; Willem et al., 1990) or the tetraspanin protein CD63, a marker of late endosome (LE) membranes found at steady-state mostly on LE/multivesicular body (LE/MVB) compartments but also on the plasma membrane (Nydegger et al., 2003; 2006). To do these experiments, 24 hr post-transfection N-Rh-PE was added to the media for 1 hr at 4°C, the cells were then washed, and incubated an additional 24 hr at 37°C. In all cases, VLPs isolated from the media were purified through sucrose cushions, spun onto poly-lysine-coated coverslips, and examined by deconvolution confocal microscopy. As shown in Figure 4A, only small amounts of the markers were detected in the VLPs formed when HIV-1 Gag was expressed alone. VLPs assembled from P7L-GFP or P7L-Tsg101 also contained little N-Rh-PE (panels B and C) or CD63 (not shown).

Fig. 4
Examination of WT and chimeric HIV-1 VLPs for N-Rh-PE

To examine the VLPs assembled from ASV Gag, COS-1 cells were transfected with DNA encoding the WT Gag, the L domain mutant (Δp2b), or the chimeric Gag protein (Δp2b-Tsg101) and labeled with N-Rh-PE in the same manner as the cells described above expressing HIV-1 Gag. Figure 5 shows images representative of the samples in at least three independent experiments. In contrast to HIV-1 Gag, most of the particles assembled from WT ASV Gag-GFP contained the lipid marker (82%, n = 300), as indicated by the co-localization of the Gag-GFP and the rhodamine (red) fluorescence of N-Rh-PE in yellow particles (Figure 5, panel C). The fluorescent signal was specific, as no signal was detected in mock-treated control samples derived from untransfected cells similarly treated with N-Rh-PE (panel 5A). In contrast to WT ASV particles, few VLPs assembled from the Δp2b-GFP mutant (panel B) contained the tracer (7%; n = 375), indicating that these particles were not released from the same membrane region as WT VLPs and suggesting that Nedd4 binding is necessary for association with such membranes. Consistent with this supposition, WT but not Δp2b co-localized with N-Rh-PE-positive membranes in the cytoplasm (not shown). VLPs released from cells expressing the chimeric Δp2b-Tsg101 protein were detected by indirect immunofluorescence using antibody against the MA domain in Gag (panel D). Like the ASVΔp2b VLPs, few particles assembled from the chimeric Δp2b-Tsg101 Gag protein contained the N-Rh-PE tracer (1%, n = 650). Similar results were obtained by fusing Vps37C (panel E). Interestingly, N-Rh-PE was detected in VLPs released from cells expressing Δp2b fused to Eap20 (panel F; 55%, n = 180) or Chmp4B (panel F, inset; 40%, n = 20). These results suggest that the WT and the chimeric VLPs containing Tsg101 or its binding partner Vps37C were released from different membrane regions. Apparently, Nedd4 was necessary for association with the membrane domains containing N-Rh-PE. Eap20 or Chmp4B could substitute for Nedd4 in this regard.

Fig. 5Fig. 5
Examination of WT and chimeric ASV VLPs for N-Rh-PE and CD63

If Nedd4, but not Tsg101, indeed directs ASV VLP release from endosome-like membrane domains, CD63 might also be present in the particles. VLPs, isolated as described above, were therefore examined for CD63 by indirect immunofluorescence. As shown in panel G, VLPs assembled from WT ASV Gag contained the tetraspanin protein in amounts ranging from 10% (n = 150) to 40% (n =150), supporting the conclusion that the particles budded through endosome-like membrane domains. In contrast, no CD63 was detected in VLPs assembled from Δp2b (not shown) or Δp2b-Tsg101 (panel H). The absence of both N-Rh-PE and CD63 from these particles supports the notion that Nedd4 directs Gag to endosome-like membrane domains while in its absence, either by deletion of the Nedd4 docking site or by substitution of Tsg101, Gag associates with a different membrane region.

DISCUSSION

Unlike PTAP- and YPDL-type L domains, which bind Tsg101 (Vps23) and Alix (Vps31), respectively, and therefore directly link Gag to class E Vps proteins, the PY-type L domains, which bind the Nedd4 family of HECT ubiquitin ligases (Kikonyogo et al.; 2001; Vana et al., 2004), must access the MVB machinery in a different way. Previously, we established that Nedd4-like proteins bind to Tsg101 and that this interaction allows ASV Gag to co-localize with Tsg101 (Medina et al., 2005). This might provide ASV Gag with access to the Tsg101-directed pathway and to ESCRT factors that it requires for budding, such as Eap20 (Pincetic, Medina, Carter, and Leis, submitted).

We showed here that ASV Gag release from avian cells was not blocked following co-expression of Gag with Tsg-DN or by depletion of the endogenous pool of Tsg101. Similar interventions have been shown to inhibit release of HIV-1 Gag from primate cells (Garrus et al., 2001; Goila-Gaur et al., 2003; Medina et al., 2005 and data not shown). Tsg-DN has been shown to induce formation of aberrant endosomes that disrupt endocytic sorting (Goila-Gaur et al., 2003; Johnson et al., 2005). We detected similar aberrant structures in DF-1 cells. Nevertheless, we observed no inhibition of ASV Gag release. We demonstrated that Tsg101 can replace Nedd4 and rescue Δp2b release (Figure 3). This observation suggests that the function of Nedd4 in ASV release can be replaced by Tsg101 and most likely explains the well-documented functional exchangeability of the HIV-1 and ASV L domains (Parent et al., 1995). However, our studies now provide evidence that the L domain contributes to membrane targeting as we found that different outcomes result from Nedd4-, Eap20-, or Chmp4B- vs Tsg101- or Vps37C-facilitated budding. It is interesting that neither Tsg101 nor Vps37C, which are linked in ESCRT-1, rescued the membrane targeting of WT ASV Gag. In contrast, this function was rescued by Eap20 and Chmp4B, which are linked through Chmp6 (Yorikawa et al., 2005).

Although the translational fusion of Tsg101 to Gag-Δp2b restored Gag release, it placed Tsg101 on every Gag molecule, raising the possibility that potential steric effects hindered an association between the chimeric Gag proteins and membranes containing N-Rh-PE or CD63. This possibility is unlikely because the chimeric Gag proteins were found to associate with N-Rh-PE- and CD63-positive membranes in the cytoplasm (data not shown). Moreover, we showed that the translational fusion of Eap20 or Chmp4B to GagΔp2b restored both release and WT targeting. Most likely, Tsg101 and Nedd4 do not direct ASV trafficking via the same route as we observed that depletion of Tsg101 was not effective in blocking release of the WT ASV Gag compared to the previously described effect (Garrus et al., 2001) of Tsg101 depletion on HIV Gag release. As already noted, Nedd4 function is apparently not required when Tsg101 is provided in cis (Pincetic, Medina, Carter, and Leis, submitted). These considerations lead us to conclude that Nedd4 family members direct release through a pathway that functionally parallels that directed by Tsg101. The fact that neither Δp2b-Tsg101 nor -Eap20 form a stable complex with Nedd4 (Pincetic, Medina, Carter, Leis) suggests that Nedd4 may not be needed to enter the pathways they target. Perhaps shared binding partners normally provide a conduit to Tsg101 and ESCRT-regulated trafficking pathways. It is interesting to note that release of HIV with L domain mutations has been demonstrated to be rescued by over-expression of a Nedd4 variant, Nedd4L (Chung et al., 2008; Usami et al., 2008). Over-expression of Nedd4L in cells expressing HIV-1 Gag with Tsg101 and AIP-1 binding site mutations was found to rescue VLP release. Over-expression or depletion of Nedd4L had no detectable effect on WT HIV-1, suggesting that rescue represents an alternative mechanism that the virus uses to release from cells. As noted above, Tsg101 is known to bind several E3 proteins in the cytoplasm, including avian Nedd4. As we found no requirement for Tsg101, it is unlikely that the interaction plays a critical role in the release function. Although the precise site of interaction between Tsg101 and Nedd4-like proteins has not been mapped, we showed previously that the interaction utilized the C-terminal region of Tsg101 and the N-terminal portion of the Nedd4 protein (Medina et al., 2005). This indicates that the interaction is not a canonical E2-E3 interaction, which would require the N-terminal ubiquitin-E2-like (UEV) domain in Tsg101 and the C-terminal catalytic (HECT) domain in Nedd4. In contrast, the rescue of the L domain defect of HIV by Nedd4L cited above requires the ubiquitin-ligase activity of the enzyme (Chung et al., 2008; Usami et al., 2008). Even if Nedd4 recognizes Tsg101 as an E2 homologue, Nedd4-directed ASV Gag release need not be dependent on Tsg101 if Nedd4, like Tsg101, interacts with other ESCRT proteins.

The differences in use of membrane sites for budding by ASV and HIV-1 Gag may be related to the fact that the M domain membrane transport signals of ASV and HIV-1 Gag differ. The former is found in basic residues in the MA coding sequence and in the fourth alpha helix (Scheifele et al., 2003) while the latter is found in a bipartite motif comprised of basic residues and an N-terminal myristic acid moiety (Zhou et al., 1994). Moreover, Nedd4, which is recruited by the L domain located downstream of MA in the Gag precursor (Kikonyogo et al., 2001), contains a membrane-binding C2 domain (Dunn et al., 2004) that may direct ASV Gag to specific areas of the plasma membrane. This notion is supported by the observation that Gag, which binds the WW domain in Nedd4 (Kikonyogo et al., 2001), co-localized with the protein in different subcellular locations, depending on the presence or absence of the Nedd4 C2 domain (data not shown). We speculate that the combination of trafficking signals in both Nedd4 and ASV Gag transports these proteins to different membrane sites for budding than the signals that direct HIV-1 Gag transport. It will be of interest to determine specifically how ESCRT factors versus Nedd4 family members facilitate trafficking and release of ASV Gag.

Acknowledgments

This work was supported by National Institutes of Health grants AI068463 (to CC) and AI054143 (to JL). GM was supported in part by a W. Burghardt Turner Pre-doctoral Fellowship, the NSF-HRD funded SUNY AGEP Program at Stony Brook University, Grant #35583 and by NIH Pre-Doctoral Training grant 5T32 CA-09176. AP is supported by the NIH pre-doctoral Training Grant T32 AI060523.

Footnotes

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