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Journal of Virology, October 2008, p. 9770-9775, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00741-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Basal Budding and Replication of the Murine Leukemia Virus Are Independent of the Gag L Domains

Yosef Sabo, Nihay Laham-Karam, and Eran Bacharach^*

Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv 69978, Israel

Received 4 April 2008/ Accepted 20 July 2008

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Moloney murine leukemia virus (MMuLV) Gag protein contains three identified late (L) domains, PPPY, YPAL, and PSAP, which are thought to interact with the endosomal sorting machinery to assist budding. We created single and combined L-domain mutants in all permutations and tested the resulting clones for budding and replication. Budding and replication of all viruses with mutated PPPY were greatly reduced; however, the basal replication level was retained, demonstrated by the slow spread of the viruses in culture. Mutations in PSAP or YPAL did not affect budding or spreading, demonstrating that these two motifs are dispensable for efficient MMuLV replication. Furthermore, the basal budding level was maintained following inhibition of endosomal sorting machinery, emphasizing that the basal budding of MMuLV is independent of this machinery.

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Late (L) domains are short motifs, first identified for the Gag protein of human immunodeficiency virus type 1 (HIV-1) (10, 14, 34), which assist in the late stage of virion release; indeed, mutations that disrupt the L domains result in accumulation of the mutant virions at the plasma membrane (7, 23, 25). Several functional L domains have been identified to date and include the P(T/S)AP, YP(X)_nL, and PPXY motifs (5, 23). These sequences function in various budding viruses, including HIV (10, 14, 28), Moloney murine leukemia virus (MMuLV) (27, 35), and other enveloped RNA viruses (3, 11-13, 18, 22, 26, 32, 33).

L domains serve as docking sites for proteins that are part of the endosomal sorting complex required for transport (ESCRT), or for the Nedd4 family of E3 ubiquitin ligases, which are also thought to be involved in ESCRT function (8, 23). The ESCRT complexes, which are part of the class E vacuolar protein sorting (Vps) pathway (15, 16, 24), are involved in the trafficking and sorting of cargo proteins in the endosomes, whereby the cargo is processed through microvesicles that bud into late endosomal compartments forming multivesicular bodies (16, 24). ESCRT components also function in the plasma membrane fission step that separates the two daughter cells during cytokinesis (4). These events that involve membrane separation resemble the "pinching-off" step in viral budding. Indeed, viral L domains recruit the above-mentioned cellular machinery to facilitate viral budding at the plasma membrane. Specifically, the P(T/S)AP, YP(X)_nL, and PPXY L domains were shown to bind Tsg101 (9, 31), Alix (21, 28), and Nedd4 (17, 27), respectively. These interactions allow the viruses to "hijack" the cellular sorting machinery to facilitate pinching and budding.

In some retroviruses, two functional L domains exist in the Gag precursor. Examples of these dual domains include the PTAP and YPDL motifs in HIV Gag (9, 28) and the PSAP and PPPY motifs in Mason-Pfizer monkey virus Gag (11) and porcine endogenous retrovirus Gag (19). Recently, it has been proposed that MMuLV Gag is unique in that it possesses three L domains (27): PPPY (nested in the p12 domain), first identified by Yuan et al. (35); PSAP (in the MA domain); and YPAL (at the border between the MA and p12 domains). Accordingly, the PPPY, PSAP, and YPAL motifs were shown to interact with Nedd4, Tsg101, and Alix, respectively (27). Furthermore, Gag with point mutations that disrupt these interactions showed reduced virion-like particle (VLP) production (27). However, these experiments were conducted only with the Gag and Gag-yellow fluorescent protein (YFP) fusions, not with replicating viruses.

To evaluate the relative contributions of each of these motifs to the production and replication of MMuLV, we mutated each of the three L domains of the gag gene of an infectious MMuLV clone (1, 6), generating mutant clones L1 (PSAP to PSAA), L2 (YPAL to YPAA), and L3 (PPPY to AAAA) (Fig. 1A). These mutations were chosen because they render the cognate wild-type L domains inactive (27, 35). In addition, we generated all combinations of these mutations, creating MMuLV clones L1/2, L1/3, L2/3, and L1/2/3. Importantly, we confirmed that the YPAA mutation that overlaps the MA-p12 cleavage site (Fig. 1A) did not affect the processing of this site (data not shown).

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FIG. 1. Gag expression and virion production of L-domain mutants. (A) Schematic presentation of MMuLV Gag organization (MA, matrix; CA, capsid; NC, nucleocapsid), as well as the wild-type (wt) and mutant L domain (L1 to L3) sequences. Arrowheads indicate the MA-p12 cleavage site. (B) Western blot analysis of Gag expression (using goat anti-MuLV CA polyclonal antibody; National Cancer Institute product no. 81S-263) of wild-type and L-domain mutants in 293T cells, which were transfected with equal amounts of plasmid DNA (15 µg/60-mm plate) using the calcium phosphate method. Mock indicates cells transfected with no DNA. The Gag precursor and its cleavage products are indicated by an arrow and asterisks, respectively. (C) Exogenous RT activity of MMuLV virions in the supernatants of transfected cells described for panel B. (D) Presentation of phosphorimager (Fujifilm FLA 2000) quantification of RT signals (gray bars) and densitometric analysis of Gag levels (black bars) for three independent experiments (means ± SD). The data for the indicated mutants were normalized to wild-type levels, which were set as 100%.

We next tested Gag expression and virion production of the wild-type and mutant clones (Fig. 1B and C). Equal amounts of plasmid DNA encoding the above clones were transfected into 293T cells; at 2 days posttransfection, the cells and culture supernatants were harvested for further analysis. An analysis of cell lysates by Western blotting, using anti-capsid antibodies, demonstrated that the L1, L2, and L1/2 clones showed wild-type levels of Gag precursor (Pr65^gag), whereas all clones harboring the L3 mutation showed higher levels of the MMuLV Gag (Fig. 1B, compare lanes 2 to 5 to lanes 6 to 9). Culture supernatants, tested for virion content using exogenous reverse transcriptase (RT) assays (30), revealed lower RT levels for all L3-inclusive mutants than for the wild type and L1, L2, and L1/2 mutants (Fig. 1C, compare lanes 2 to 5 to lanes 6 to 9). The reduced RT levels correlated to a reduction in virion-associated capsid levels in the supernatant (data not shown). To better assess the RT and Western blot results of the represented experiment, additional experiments were performed and quantified by phosphorimaging (RT signals) and densitometry (Western blotting), the data were normalized to those for the wild type, and the means ± standard deviations (SD) (n = 3) are represented in Fig. 1D. Overall, these results suggest that the cellular accumulation of Gag seen in the L3-associated mutants is due to reduced viral release. These results suggest that in the context of a replication-competent virus, the PPPY motif, but not the PSAP or YPAL motif, is the crucial L domain for particle release.

We next tested the spreading kinetics of the above viruses in infected cultures. Supernatants of transfected 293T cells were harvested and filtered through a 0.45-µm filter. Equal amounts of virions, normalized by RT activity, were then used to infect naïve NIH 3T3 cells. These cells were maintained under subconfluent conditions, and aliquots from the supernatants were harvested daily for RT assays to determine viral spreading (Fig. 2). Clear RT signals could be detected for the wild-type, L1, L2, and L1/2 clones as early as 2 days postinfection (Fig. 2A, lanes 2 to 5). In contrast, only on day 5 could any RT signal be detected for clones harboring the L3 mutation (Fig. 2A, lanes 6 to 9). The RT levels of all L3 mutant clones did not reach that of the wild type even after 15 days of culture. The RT signals were also quantified by phosphorimaging, the results of which are represented in Fig. 2B. The L1 or L2 mutations did not have any effect on viral spreading; at most, there was a slight and transient effect when these mutations were combined, in contrast to the L3 mutants, for which viral spreading was greatly reduced (Fig. 2B). The results of the spreading assay correlate to those for the release of MMuLV, shown above, and emphasize the importance of the Gag PPPY motif, but not the PSAP or YPAL motif, in the replication of MMuLV. Likewise, the PPPY motif was recently identified as the dominant L domain in the release of porcine endogenous retrovirus in a functional hierarchy of L-domain activity (19). Yet our results contrast in part with those of a previous report (27) demonstrating a three- to fivefold reduction in VLP levels of MuMLV Gag-YFP harboring the L1 and/or L2 mutations, which suggested a role for PSAP and YPAL motifs in MMuLV budding. However, only one mutation out of two (YPAA but not APAL) that disrupted Alix binding reduced VLP levels (27), putting into question the necessity for this motif in the budding process. Furthermore, the depletion of Tsg101 by small interfering RNA had an effect only on the VLP production of Gag-YFP, not of wild-type Gag (27). Actually, in the context of Gag without the YFP fusion, the L1 and/or L2 mutations did not show any affect on VLP production 38 h posttransfection, and a mild effect, at most, was observed at an earlier time (27). Thus, in the context of wild-type virus, the YPAL and PSAP motifs in Gag are dispensable for viral spreading.

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FIG. 2. Spreading kinetics of MMuLV and L-domain mutants. Naïve NIH 3T3 cells infected with equivalent virion amounts (normalized by RT activity) of (A) wild-type MMuLV (wt) or with the indicated L-domain mutants or (C) with a deletion mutant of PPPY (PPPY). Samples of the supernatants were collected daily following infection, and 10% of the cells were recultured on the days indicated by the arrows. (B) Phosphorimaging quantification of the RT radioactive signals shown in panel A, in arbitrary units of pixels/microliter of culture supernatant. (D) Virion production of the wild type and of PPPY and L3 mutants at day 50 of cell culture. The infections were carried out as described above; over the 50 days of the experiment, the cells were passaged every 3 days; every 10 days, equal volumes (1 µl) of the supernatants were used to initiate infection in naïve cultures. Mock indicates cells infected with media lacking virions. Panels A, C, and D represent independent experiments.

The clones that harbor the L3 mutation, including L1/2/3, in which all three possible L domains were inactivated, showed low but clear basal budding and replication activity (Fig. 1 and 2). This basal activity was unexpected, since previously it was reported that no infectivity was observed for the L3 mutation following 14 days of infection (35). This discrepancy is probably due to the difference in infection protocols; whereas in the previous study (35) equal volumes of supernatant from wild-type and mutant transfectants were used to infect NIH 3T3, in this study we used equal amounts of virions to initiate the spreading assay. This is important since there exists a large difference in levels of virions released from the transfected cells of wild-type and L3 mutants, as observed in both studies (35) (Fig. 1C). Thus, the replication of viruses with low levels of virion release and slow spreading, like the L3 virus, may be overlooked if a small number of particles are used to initiate infection. To confirm that a PPPY mutant virus can slowly replicate in culture, we made an additional mutant virus which lacked the entire PPPY sequence (PPPY) (35). This deletion mutant showed the same basal replication as the L3 mutant virus (Fig. 2C). Importantly, during the 50-day course of repeated cycles of infection, which were carried out by a passage of the cells every 3 days and a passage of the infectious supernatants to naïve cells every 10 days, both the L3 and the PPPY mutations were maintained in the viruses (data not shown), and these viruses retained their characteristic slow replication (Fig. 2D). The stability of these mutations is unpredicted, since mouse cells contain many retrovirus-like elements which can easily recombine with replicating viruses to generate revertants with the wild-type phenotype. Such stability if applied to other viruses may aid the design of attenuated strains with mutated L domains that may serve as efficient vaccines.

To further characterize the particles produced by Gag proteins with L-domain mutations, we compared the L1/2/3 virions to that of the wild type. We transfected 293T cells with wild-type or L1/2/3 clones and harvested and filtered the culture supernatant 2 days posttransfection. Supernatants with equal amounts of RT activity were layered on 25% sucrose cushions and centrifuged at 107,000 x g for 2 h. The resulting pellets were analyzed by Western blotting with anti-capsid antibody, which revealed comparable amounts of Gag precursor (Pr65^gag) and processed capsid proteins in the L1/2/3 and wild-type pellets (Fig. 3A). Thus, the L1/2/3 mutant can produce particles that transverse the sucrose cushion as efficiently as the wild type and show normal Gag processing. In addition, we analyzed the presence of genomic RNA in these particles by RT-PCR using primers derived from the gag sequence. We observed comparable PCR products for the L1/2/3 clone and the wild type (Fig. 3B); this result was confirmed by quantitative PCR using Sybr green reagent and the following primers: PNCS qPCR (MA) FW, 5'-AGCCCTTTGTACACCCTAAGC-3'; PNCS qPCR (MA) REV, 5'-GAGGTTCAAGGGGGAGAGAC-3'. The mean result for triplicate experiments ± SD for L1/2/3 was 0.023 ± 0.007 ng, compared to that for the wild type, which was 0.021 ± 0.001 ng. In addition, we tested the specific infectivity of the L1/2/3 and wild-type particles in a single cycle of infection. We transfected L1/2/3 or wild-type clones (10 µg) into 293T cells in 60-mm plates, together with pQCXIP-GFP (10 µg) encoding an MLV-based, self-inactivating vector, which expresses enhanced green fluorescent protein (GFP) as a reporter (20). After confirmation of equivalent GFP expression in the transfected cells by fluorescence activated cell sorter (FACS) analysis, the supernatants were filtered and assayed for RT activity. Volumes of media with equal RT activity were used to infect NIH 3T3 cells, and 48 h later, they were analyzed for GFP expression by FACS analysis. Importantly, although virus replication can occur within 48 h of culture, the QCXIP-GFP self-inactivating vector is immobilized immediately at its integration. Figure 3C shows the average results of three independent experiments and demonstrates that the specific infectivity of the L1/2/3 mutant is similar to that of the wild type. Altogether, these results demonstrate that the L1/2/3 clone, like the wild type, can release genuine particles into the culture supernatant with comparable Gag and RNA assembly processes as well as maturation and infectivity levels.

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FIG. 3. Properties of the L1/2/3 particles. Virions of the wild type (wt) and the L1/2/3 mutant were pelleted through 25% sucrose cushions using ultracentrifugation. Pellets were resuspended and processed by Western blot analysis (A) as described for Fig. 1B, or RNA was extracted using EZ-RNA (Biological Industries Co., Beit Haemek, Israel), treated with DNase, and then reverse transcribed (+RT) using random primers and AMV-RT (Promega). Gag sequences were amplified using pNCS BsrgI FW (5'-CCCAGGTTAAGATCAAGG-3') and pNCS XhoI REV (5'-CTTGGCCAAATTGGTGGG-3') primers to generate an 875-bp PCR fragment (B). To control for plasmid contamination, reactions with no AMV-RT but with PCR amplification were included (–RT). (C) Single-cycle infection assays. NIH 3T3 cells were infected with the wild type (wt) or with L1/2/3 clones carrying the QCXIP-GFP vector and normalized by RT. The means ± SD (n = 3) of the percentage of GFP-positive cells as detected by FACS analysis at 2 days postinfection are represented.

The basal replication level observed for the L3 mutants, which correlates to their inefficient budding, would indicate that the release of these viruses is independent of the action of the cellular class E Vps machinery. If that is true, then inhibition of this machinery should reduce the budding of the wild-type virus to the basal budding level of the L3 mutant viruses, while the release of the latter should remain unchanged. To test this, we overexpressed Vps4_E228Q, an ATPase-defective, dominant negative form of Vps4, which inhibits the class E Vps machinery and consequently suppresses retroviral budding (2, 9). We cloned Vps4_E228Q or wild-type Vps4 as fusion proteins with GFP (GFP-Vps4_E228Q and GFP-Vps4, respectively) (9) into pcDNA4/TO (Invitrogen)_. The resulting plasmids were transfected into T-Rex-293 cells, and colonies stably expressing either the GFP-Vps4_E228Q (Vps4EQ-Col4) or GFP-Vps4 (Vps4-Col6) proteins under tight doxycycline regulation were selected according to the manufacturer's instructions (Invitrogen). During the course of this study, a similar system was reported (29). Wild-type or L1/2/3 clones were transfected into Vps4EQ-Col4 and Vps4-Col6, after which the expression of GFP-Vps4_E228Q and GFP-Vps4 was induced. The induction conditions (Fig. 4A) were optimized to minimize Vps4_E228Q-induced toxicity while achieving strong expression of the fusion proteins in >95% of cells (Fig. 4B). Transfected cells (uninduced and induced with doxycycline) expressed similar levels of Gag protein as determined by Western blot analysis (Fig. 4C). The induction of GFP-Vps4 did not result in any change in the levels of either the wild-type or the L1/2/3 virions in culture supernatants (Fig. 4D and E). Importantly, upon induction of GFP-Vps4_E228Q, the amount of wild-type virus was reduced to the basal levels of the L1/2/3 clone, while the virion release of L1/2/3 remained unchanged (Fig. 4D and E). These data confirm that the basal budding of MMuLV is independent of the class E Vps machinery and, accordingly, no L domains in Gag are required for this basal activity. The exact mechanism of the basal budding is unknown; however, it may be similar to the mechanism of budding of other enveloped viruses which have also been shown to be insensitive to the action of the dominant negative Vps4 (5, 29).

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FIG. 4. Budding of the wild type (wt) and the L-domain mutant (L1/2/3) following inhibition of the class E Vps machinery. (A) Timeline of the experimental protocol. Cells were transfected with either the wild type or L1/2/3 clones (20 µg of plasmid DNA/60-mm plate) with calcium phosphate; these were then divided into two separate cultures, one of which was induced with doxycycline (0.1 µg/ml) at 48 h posttransfection, while the other remained uninduced (no doxycycline). The exchange of media, following 6 h of induction, insured that virions produced prior to the induction of the GFP-Vps4 or GFP-Vps4E228Q (Vps4/EQ) were removed. The induction was maintained for another 18 h, after which the cells and culture supernatant were harvested and analyzed. Mock indicates cells transfected with no DNA. (B) The indicated cells lines were transfected with either the wild type or L1/2/3 clones; these were either induced with doxycycline (solid lines) or remained uninduced (dashed lines). FACS analysis was performed to measure the induction of GFP fusion proteins. (C) Western blot analysis, using anti-capsid polyclonal antibodies, of wild-type and L1/2/3 Gag expression in the above transfected cells with and without induction. The Gag precursor and its cleavage products are indicated by an arrow and asterisks, respectively. (D) Exogenous RT activity of supernatants from the cultures described above. (E) RT activity was measured by a phosphorimager and quantified using the program Tina. The results obtained for the wild-type virus in uninduced Vps4-Col6 or Vps4EQ-Col4 were set as 100%, and all other results were normalized accordingly. The graph represents the means ± SD (n = 3).

Overall, these data suggest that in the context of the replicating MMuLV, only the PPPY motif serves as an efficient L domain. Our findings highlight that MMuLV is still capable of slow replication in the absence of any functional L domain in Gag; perhaps slower-replicating viruses with no L domains were the predecessors of the current MLVs, which evolved to harbor L domains to take advantage of the cellular class E Vps machinery to facilitate their budding and spreading. The cell lines developed in this study, in which inhibition of the Vps machinery can be achieved, should be of benefit to assess the involvement of this cellular machinery in the replication of other viruses. Furthermore, if the stability of the L-domain mutants observed here holds for other viruses that use L domains for efficient replication, then inactivating their L domains may serve as a useful means for the generation of attenuated, replication-competent vaccine strains.

 
We gratefully acknowledge W. I. Sundquist (University of Utah) and S. P. Goff (Columbia University) for providing various plasmids.

This work was supported by the Israel Science Foundation (grant 1184/05) and by the Ela Kodesz Institute for Research on Cancer Development and Prevention.

 
* Corresponding author. Mailing address: Department of Cell Research and Immunology, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Phone: 972-3-640-7692. Fax: 972-3-642-2046. E-mail: eranbac{at}post.tau.ac.il

Published ahead of print on 30 July 2008.

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Journal of Virology, October 2008, p. 9770-9775, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00741-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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