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Journal of Virology, September 2007, p. 9193-9201, Vol. 81, No. 17
0022-538X/07/$08.00+0     doi:10.1128/JVI.00044-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Ubiquitination of Human Immunodeficiency Virus Type 1 Gag Is Highly Dependent on Gag Membrane Association{triangledown}

Stefanie Jäger, Eva Gottwein,{dagger} and Hans-Georg Kräusslich*

Abteilung Virologie, Universitätsklinikum Heidelberg, D-69120 Heidelberg, Germany

Received 8 January 2007/ Accepted 16 June 2007


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ABSTRACT
 
Ubiquitin is important for the release of human immunodeficiency virus 1 (HIV-1) and several other retroviruses. All major domains of the HIV-1 Gag protein are monoubiquitinated, but the modifying machinery and the function of HIV-1 Gag ubiquitination remain unclear. Here, we show that the induction of a late budding arrest by mutation of the HIV-1 PTAP motif or by specific inhibition of selected ESCRT components leads to an increase of Gag-ubiquitin conjugates in cells, which coincides with an accumulation of detergent-insoluble, multimerized Gag at the plasma membrane. Membrane flotation experiments revealed that ubiquitinated Gag is highly enriched in membrane-bound fractions. Based on these findings, we propose that a blocking of virus release results in increased Gag ubiquitination as a consequence of its prolonged membrane association. Consistent with this, ubiquitination of a membrane-binding-defective (G2A)Gag mutant was dramatically reduced and the ubiquitination levels of truncated Gag proteins correlated with their abilities to bind to membranes. We therefore propose that membrane association and multimerization of HIV-1 Gag proteins, rather than a specific motif within Gag, trigger recognition by the cellular ubiquitination machinery.


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INTRODUCTION
 
The internal structure of human immunodeficiency virus type 1 (HIV-1) is formed by its Gag polyprotein, which is proteolytically processed into the proteins matrix (MA), capsid (CA), nucleocapsid (NC), and p6 as well as spacer peptides 1 and 2 (SP1 and SP2) upon virion maturation. HIV-1 Gag polyproteins are targeted to membranes by their N-terminal myristoylated MA domain, and Gag membrane association is stabilized by multimerization (29, 34, 36). Gag oligomerization starts in the cytoplasm (19, 20, 26, 39, 44), but assembly of multimers occurs mainly on membranes (14, 15, 44). Accordingly, Gag membrane association and assembly are likely to promote each other, thereby driving the budding process.

Release of retroviruses depends on the interaction of viral late domains, short peptide sequences within their Gag proteins, with cellular factors. Mutations of the HIV-1 PTAP late domain or the PPxY motif of Rous sarcoma virus and many other retroviruses lead to a budding arrest with assembled immature particles that fail to be severed from the cell surface. PTAP motifs were identified in several viruses (reviewed in reference 4) and have been shown to bind TSG101, a component of the cellular endosomal sorting complex required for transport (ESCRT) (8, 27, 40). The ESCRT system consists of several subcomplexes, of which ESCRT-I (including TSG101), ESCRT-III (consisting of various CHMP proteins), and the ESCRT-associated ATPase VPS4 are thought to act sequentially in HIV-1 release and formation of the multivesicular body (MVB). Knockdown of TSG101 or expression of dominant-negative CHMP or VPS4 proteins blocks the budding process at a late stage, similar to late-domain mutations (8, 11, 46).

PPxY late-domain motifs were shown to interact with ubiquitin (Ub) E3 ligases of the Nedd4 family (17, 47). Monoubiquitination of cellular cargo proteins serves as a signal for endocytosis and ESCRT-mediated sorting into the MVB (16), but it is currently not clear whether monoubiquitination of Gag and/or cellular proteins at the viral budding site plays a similar role in retrovirus budding. Although HIV-1 Gag contains no PPxY motif, all major HIV-1 Gag domains were shown to be monoubiquitinated and HIV-1 particles incorporate significant levels of free Ub (13, 33). Several lines of evidence suggest a functional contribution of ubiquitination to HIV-1 budding. In vitro, fusion of Ub to the C terminus of HIV-1 Gag enhanced its interaction with TSG101 (8). More importantly, interference with the cellular Ub pool and cumulative mutations of Ub acceptor sites in the C-terminal region of Gag both impaired virus release from Gag-producing cells (12, 38).

It is currently not clear which E3 Ub ligase is responsible for HIV-1 Gag ubiquitination. Although some evidence points to an influence of the PTAP late motif on Gag ubiquitination, the current data are not conclusive: in the context of a minimal Gag protein, the presence of the PTAP motif increased Gag ubiquitination and the level of Gag ubiquitination correlated with the efficiency of virus-like particle release (42). In contrast, we and others observed that mutation of the PTAP motif in the context of full-length Gag led to increased Gag ubiquitination, suggesting the possible recruitment of a Ub hydrolase and not of a Ub ligase by the PTAP motif (13, 23). In the current study, we addressed the relationship between HIV-1 Gag ubiquitination and virus morphogenesis by investigating the extents of Gag ubiquitination in different cellular fractions. We found that membrane-bound Gag was monoubiquitinated to a much higher extent than soluble Gag. Accordingly, induction of a budding arrest by various means invariably led to an increase in the ratio of ubiquitinated to nonubiquitinated Gag, independently of the presence of any specific domain or motif. Based on these results, we propose that assembly of HIV-1 Gag polyproteins at cellular membranes, rather than a specific motif within Gag, triggers recognition by the cellular ubiquitination machinery.


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MATERIALS AND METHODS
 
Expression constructs. The plasmids p{Delta}R/PR(-), p{Delta}R(PTAP-)/PR(-), and pHA-Ub were described in references 12 and 13. Plasmids encoding green fluorescent protein (GFP)-wild-type VPS4A, GFP-VPS4A-E228Q, GFP-VPS4A-K183Q, CHMP3-YFP, and cyan fluorescent protein CFP-CHMP4C (8, 46) were kindly provided by W. Sundquist. The pNL4-3 plasmid and its G2A mutant [pNL4-3(G2A)] (7) were kindly provided by E. Freed. The pcDNA3-based, Rev-independent Gag expression constructs were described in reference 13. The Gly->Val mutation encoded by codon 2 of the gag open reading frame in pcDNA3-Gag was introduced by mutagenic PCR. In MACANTD, pcDNA3-Gag is truncated after the N-terminal CA domain (amino acid 151).

Cell culture, transfections, analysis of virion release, and pulse-chase. 293T cells were maintained and transfected using the calcium phosphate precipitation method as described previously (13). Medium was changed 6 h after transfection, and cells were further processed 24 h after transfection. Virion preparations and the pulse-chase experiment were performed as described in reference 12.

ELISA, Western blotting, and antibodies. An enzyme-linked immunosorbent assay (ELISA) for detecting CA was carried out as described previously (18). For improved detection of uncleaved Gag, samples were boiled in protein sample buffer containing 1% sodium dodecyl sulfate (SDS) before ELISA. For Western blotting, protein samples were resolved on 17.5% low-cross-linking polyacrylamide gels and probed with the indicated antibody dilutions. Rabbit anti-GFP (1:5,000), rabbit anti-MA (1:5,000), and sheep anti-CA (used for immunoprecipitation) were raised against recombinant proteins. HIV-1 CA-specific monoclonal antibody (183-H12-5C) was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, from Bruce Chesebro (5). Rat anti-hemagglutinin (anti-HA; clone 3F10; 1:500) was purchased from Roche, mouse anti-transferrin receptor (anti-TfR; 1:1,500) from Zymed, and mouse anti-actin (C2; 1:500) from Santa Cruz.

Gag immunoprecipitation. 293T cells were lysed in 150 µl boiling SDS lysis buffer (1% SDS, 150 mM NaCl, 50 mM Tris, pH 8.0) and boiled for 10 min at 95°C. The lysate was diluted to 1x radioimmunoprecipitation assay (RIPA) buffer concentrations (0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM Tris, pH 8.0). After sonification, the lysate was cleared by centrifugation for 10 min at 17,000 x g, 30 µl protein A-Sepharose beads (Amersham Biosciences) and 5 µl sheep anti-CA serum were added, and immunoprecipitation was performed for 2 h at 4°C. The amounts of immunoprecipitated Gag from different samples were thoroughly normalized by multiple rounds of ELISA.

Consecutive immunoprecipitations. 293T cells were lysed in 0.5 ml cold 1x RIPA buffer containing 5 mM N-ethylmaleimide (NEM) and protease inhibitors. The lysate was cleared by centrifugation for 3 min at 1,000 x g at 4°C, and Gag was immunoprecipitated for 1 to 2 h at 4°C. Sepharose beads were then pelleted (1 min, 80 x g); the supernatant was adjusted to a 1% final SDS concentration, boiled for 10 min at 95°C, and diluted to a 1x RIPA concentration; and the second Gag immunoprecipitation was performed.

Membrane flotation assay. 293T cells swollen on ice for 20 min in cold hypotonic buffer (10 mM Tris, pH 8.0, 1 mM Mg2Cl, protease inhibitors, and 5 mM NEM) were disrupted by Dounce homogenization and adjusted to 40% OptiPrep (Progen). The mixture was overlaid with 28% OptiPrep and buffer and centrifuged at 165,000 x g for 3 h at 4°C. Eight fractions were collected from the top of the gradient.


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RESULTS
 
A late budding arrest leads to a specific increase in detergent-resistant, membrane-associated Gag. We initially sought to test whether arresting HIV-1 budding at different stages might reveal differences in the ubiquitination status of Gag during the budding process. Therefore, 293T cells were transfected with the full-length Gag expression plasmid p{Delta}R/PR(-) or a PTAP-mutated variant [p{Delta}R(PTAP-)/PR(-)] or were cotransfected with p{Delta}R/PR(-) and an expression plasmid for wild-type or dominant-negative VPS4A. Cells were lysed in cold RIPA buffer 24 h after transfection, and lysates were cleared by centrifugation at 17,000 x g. Surprisingly the amounts of Gag detected in the lysates were approximately equal for all samples (white bars in Fig. 1A), although a blocking of virus release should lead to an accumulation of intracellular Gag. To test whether the insoluble material obtained after clearing the lysates contained residual Gag, the pellets were solubilized by boiling them in SDS buffer and their Gag content was determined after dilution to RIPA conditions (Fig. 1A, black bars). In the case of wild-type Gag, more than 50% of total intracellular Gag was recovered in the RIPA-insoluble fraction. The amount of RIPA-insoluble Gag was further increased by a factor of 3 to 4 when budding was arrested by PTAP mutation or cotransfection of dominant-negative VPS4A, whereas the amount of Gag in the soluble fraction remained largely constant. The same effect was observed when virus release was blocked by dominant-negative CHMP4C or small interfering RNA-mediated knockdown of TSG101 (not shown). This suggests that RIPA-insoluble Gag mainly represents protein accumulating in virus buds when release is inhibited.


Figure 1
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  		FIG. 1. Analysis of HIV-1 Gag solubility and immunoprecipitation. (A) 293T cells transfected with wild-type or PTAP-defective p{Delta}R/PR(-) or cotransfected with p{Delta}R(WT)/PR(-) and VPS4A expression constructs were lysed in RIPA buffer, and the insoluble fraction was removed by centrifugation. The pellet was dissolved by boiling it in SDS buffer and subsequently diluted to the same volume as the supernatant. The Gag contents in both fractions were determined by ELISA. Error bars represent standard deviations for three independent experiments. (B) Cells transfected with the same plasmids as those for panel A were lysed in RIPA buffer, and Gag was immunoprecipitated (IP) directly from the lysate (white bars) or following denaturation of the unbound supernatant (black bars). The amounts of precipitated Gag were determined by ELISA. Error bars represent standard deviations for three independent experiments. (C) p{Delta}R/PR(-)-transfected cells were labeled with [35S]methionine for 7 min and lysed in cold RIPA buffer after the indicated chase periods. Two successive Gag immunoprecipitations with an in-between denaturation step were performed as described for panel B. In parallel, virions were purified from the cell culture supernatant by centrifugation through sucrose cushions and Gag was immunoprecipitated after denaturation. All samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography, and the Gag signal of each sample was quantified using Quantity One software (Bio-Rad). A representative example from two independent experiments is shown. (D) Cells transfected with p{Delta}R/PR(-) were subjected to membrane flotation by density gradient centrifugation. The membrane-bound and soluble fractions were divided into two aliquots, and one was left at 4°C and adjusted to 1x RIPA buffer (white bars), while the other was adjusted to 1% SDS, incubated for 10 min at 95°C, and then diluted to 1x RIPA buffer (black bars). Gag was immunoprecipitated from the four samples, and the amounts of precipitated Gag were determined by ELISA. Error bars represent standard deviations for three independent experiments.

This hypothesis was consistent with a previous study by Ono et al. (32), who showed that immunoprecipitation of membrane-bound HIV-1 Gag requires denaturation, presumably due to epitope masking in higher-order Gag multimers. We therefore performed consecutive Gag immunoprecipitations from cell lysates: Gag was first immunoprecipitated under nondenaturating conditions, followed by denaturation of the supernatant and a second Gag immunoprecipitation. A considerable fraction of Gag was resistant to immunoprecipitation under native conditions and could be immunoprecipitated only after denaturation (Fig. 1B). Consistent with the result presented in Fig. 1A, induction of a budding arrest by either PTAP mutation or coexpression of dominant-negative VPS4A led to a two- to threefold increase in the amount of Gag in the fraction requiring denaturation for immunoprecipitation, whereas wild-type VPS4A had no effect. This result supported the suggestion that multimerized Gag in the process of assembly and budding requires denaturation to become accessible to CA antibodies.

To test whether the Gag portion that requires denaturation for immunoprecipitation takes part in virus formation, we performed a pulse-chase experiment. Cells transfected with p{Delta}R/PR(-) were pulse labeled for 7 min, and Gag amounts in the soluble, denaturation-dependent, and virus-associated fractions were quantified after different chase periods (Fig. 1C). Soluble Gag displayed the highest value at the earliest time point measured (10 min), while epitope-masked Gag, requiring denaturation for immunoprecipitation, reached its maximum after 25 min of chase. Subsequently, both fractions showed similar rates of decline concomitant with an increase of extracellular particulate Gag. This result suggests that the detergent-resistant fraction consists, at least in part, of Gag multimers that serve as assembly intermediates in the process of virus release.

To further validate our conclusion that the Gag fraction requiring denaturation for immunoprecipitation represents assembling molecules and is not due to cytoplasmic, nonproductive Gag aggregates, we analyzed whether it is membrane bound. Lysates from transfected cells were subjected to membrane flotation, and the top (membrane) and bottom (soluble) fractions were collected and divided into two aliquots. One of these was kept at 4°C, while the other was denatured, and Gag was subsequently immunoprecipitated from both samples (Fig. 1D). We observed that denaturation of the membrane-bound fraction increased Gag recovery approximately fivefold, while there was no effect on Gag recovery from the soluble fraction. Taken together, our results and those reported by Ono et al. (32) revealed that the epitope-masked fraction of Gag, requiring denaturation for immunoprecipitation, most likely represents assembly intermediates and assembled structures. Collectively, the results indicate that immunoprecipitation of immature HIV-1 Gag is strongly affected by loss of assembly intermediates following centrifugation (Fig. 1A) and by inefficient binding of antibodies to multimerized Gag (Fig. 1B). Both obstacles can be overcome by vigorous lysing conditions. An additional advantage of this procedure is the rapid inactivation of potential Gag-modifying enzymes, such as Ub hydrolases.

A late budding arrest leads to increased HIV-1 Gag ubiquitination. Having optimized the experimental conditions for HIV-1 Gag recovery by immunoprecipitation, we now investigated the influence of the ESCRT pathway on the ubiquitination status of Gag. According to the model of ESCRT action, PTAP mutation should impair the entry of Gag into the ESCRT pathway, while dominant-negative CHMP proteins should arrest virus budding at an intermediate stage, and dominant-negative VPS4A should block the disassembly of the ESCRT complex. 293T cells were cotransfected with p{Delta}R/PR(-), pHA-Ub, and a construct expressing either wild-type or dominant-negative VPS4A, dominant-negative CHMP3-YFP, or CFP-CHMP4C (8, 46). Gag was immunoprecipitated from cell lysates obtained under denaturing conditions, and equal amounts of Gag (Fig. 2A, lower) were then subjected to Western blotting with an anti-HA antibody to determine the extents of Gag ubiquitination (Fig. 2A, upper). Typically, different isoforms of mono- and diubiquitinated Gag appeared as ladders of bands as previously described (13).


Figure 2
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  		FIG. 2. Analysis of Gag-Ub levels for budding-arrested HIV-1. (A) 293T cells were cotransfected with pHA-Ub together with either p{Delta}R(PTAP-)/PR(-) or wild-type p{Delta}R(WT)/PR(-) and expression plasmids for wild-type (WT) or dominant-negative ESCRT proteins, as indicated above each lane. As a control, HA-Ub was coexpressed with p{Delta}R(WT)/PR(-) alone (lane 1). Cells were lysed by boiling them in SDS lysis buffer, diluted into RIPA buffer, and subjected to Gag immunoprecipitation. Comparable amounts of Gag were analyzed by Western blotting using antisera directed against HA (top) or CA (bottom). Please note that minor differences in the HA-Ub signal intensities (lanes 2 and 4 to 7) are below the sensitivity of the method and were not reproducible. Gag and Gag-HA-Ub conjugates are identified at the right, and molecular mass standards are shown on the left. (B) Lysates from the same transfection as that for panel A were tested for Gag (top) or GFP (middle) expression, and particle preparations from the same transfections were analyzed by anti-CA immunoblot (bottom). Molecular mass standards (in kDa) are indicated on the left. YFP, yellow fluorescent protein.

Consistent with earlier studies, the ubiquitination level of PTAP mutant Gag (Fig. 2A, lane 2) was strongly increased compared to that of wild-type Gag (lane 1). Induction of a budding arrest by coexpression of dominant-negative VPS4A variants (lanes 4 and 5) or CHMP fusion proteins (lanes 6 and 7) led to similar increases in the amounts of Gag-Ub conjugates. In contrast, coexpression of wild-type VPS4A had only a slight effect (lane 3), which is consistent with its minor effect on virus release (8). Virus release was significantly inhibited upon late-domain mutation and coexpression of dominant-negative ESCRT proteins (Fig. 2B, bottom panel). As a control for expression, cell lysates were also probed with antibodies against CA or GFP (Fig. 2B, upper panels). In conclusion, induction of a late budding arrest at different stages consistently led to an increase in Gag-Ub levels, independent of the presence or absence of an intact late-domain motif.

Multimerized, detergent-insoluble Gag is ubiquitinated at higher levels than soluble Gag. Next, we investigated the ubiquitination levels of multimerized, membrane-bound Gag and soluble Gag. HA-tagged Ub was coexpressed with Gag, and consecutive Gag immunoprecipitations, with denaturation of the remaining protein after the first immunoprecipitation, were performed as described above. Similar amounts of Gag from both immunoprecipitations were subsequently analyzed by anti-HA blotting (Fig. 3A, left). At the conditions used, ubiquitination of soluble Gag was undetectable, while significant amounts of ubiquitinated Gag were found in the denaturation-dependent fraction. This still represented only a minor fraction of total Gag, since Ub-modified Gag products were not seen on the anti-CA blot (Fig. 3A, lower-left).


Figure 3
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  		FIG. 3. Comparison of Gag ubiquitination levels in different cellular fractions. (A) 293T cells cotransfected with p{Delta}R/PR(-) and pHA-Ub expression plasmids were lysed in cold RIPA buffer at 24 h posttransfection. Detergent-resistant Gag was separated from soluble Gag either by two consecutive Gag immunoprecipitations (IP) as described for Fig. 1B (left) or by pelleting the RIPA-insoluble fraction for 10 min at 17,000 x g (right). Equal amounts of Gag were analyzed by Western blotting using antisera against HA (top) or CA (bottom). Gag and Gag-HA-Ub conjugates are identified on the right, and molecular mass standards (in kDa) are shown on the left. (B) Cells were cotransfected with p{Delta}R(WT)/PR(-) and pHA-Ub, lysed by Dounce homogenization, and subjected to membrane flotation by density gradient centrifugation. Fractions were collected successively from top to bottom, and equal volumes of each fraction were subjected to SDS-PAGE. Each fraction was analyzed for Gag content, membrane-bound TfR, and soluble actin by immunoblot analysis (lower panels). Fractions 1 (membrane associated) and 6 (soluble) were denatured, followed by Gag immunoprecipitation. Equal amounts of Gag were analyzed by Western blotting using antisera against HA and CA (top panels). (C) Cells were cotransfected with pHA-Ub and either the wild-type (WT) HIV-1 proviral plasmid pNL4-3, the membrane-binding-deficient variant pNL4-3(G2A), the variant pNL4-3{Delta}p17 targeted to the endoplasmic reticulum (6), or the pNL43/78VE mutant targeted to the MVB (7, 31). Transfections were performed in the presence of 5 µM of the HIV protease inhibitor indinavir (45) to prevent Gag processing. Cells were lysed under denaturing conditions, Gag was immunoprecipitated, and equal amounts of Gag were analyzed by Western blotting using antisera against HA (top) and CA (bottom).

We also analyzed the level of Gag ubiquitination in the RIPA-soluble and RIPA-insoluble fractions (Fig. 3A, right). This experiment demonstrated that the Gag protein recovered from the RIPA-insoluble fraction was specifically ubiquitinated, while no Gag ubiquitination was detectable for RIPA-soluble Gag. Taken together, the results obtained for denatured and RIPA-insoluble HIV-1 Gag suggest that ubiquitination of Gag occurs during the process of multimerization and assembly, thus explaining why arresting the budding process at any stage would result in elevated levels of ubiquitinated Gag.

HIV-1 Gag ubiquitination is dependent on membrane association. Our group previously compared the levels of Gag ubiquitination in the membrane-bound and soluble fractions by membrane flotation experiments and subsequent Gag immunoprecipitation under nondenaturing conditions. In these experiments, we did not detect any difference in the relative levels of Gag ubiquitination (13). Based on the observation that ubiquitinated Gag is mainly found in the antibody-inaccessible fraction, we repeated this experiment but denatured the samples prior to immunoprecipitation. Cell lysates of 293T cells expressing HA-Ub and Gag were subjected to membrane flotation, and individual fractions were analyzed by Western blotting. Gag was detected in the top and bottom fractions of the gradient, while control proteins were distributed as expected (Fig. 3B, lower panels). Subsequently, samples from the top and bottom factions were denatured, Gag was immunoprecipitated, and equal amounts of Gag were analyzed for ubiquitination by anti-HA immunoblotting. This experiment revealed virtually no ubiquitination in the bottom fraction of the gradient, while a strong HA signal was detected for Gag polyproteins recovered from the membrane fraction (Fig. 3B, upper panel).

If Gag ubiquitination depends on membrane association, a membrane-binding-deficient Gag polyprotein should be ubiquitinated to a lesser extent than wild-type Gag. To test this hypothesis, we made use of a nonmyristoylated (G2A)Gag variant whose membrane binding is reduced to 2% compared to that for wild-type Gag (30). Total Gag was immunoprecipitated from denatured cell lysates of transfected 293T cells and analyzed for ubiquitination by HA immunoblot analysis. This experiment showed a strongly reduced level of ubiquitination for membrane-binding-deficient (G2A)Gag compared to that for wild-type Gag (Fig. 3C).

To test whether ubiquitination of Gag takes place specifically at the plasma membrane, where assembly and budding of HIV Gag normally occur, we tested two Gag variants with altered membrane targeting properties. Deletion of the globular head of MA (pNL43{Delta}p17) and a point mutation within the HIV-1 MA domain (pNL43/87VE) had previously been shown to retarget Gag to intracellular membranes of the endoplasmic reticulum and the MVB, respectively (6, 7, 31). Both mutant polyproteins exhibited highly elevated levels of ubiquitinated Gag but also showed different patterns of ubiquitination (Fig. 3C). While wild-type Gag and budding-arrested wild-type Gag underwent predominant mono- and diubiquitination, a strong signal for polyubiquitinated Gag was observed for the retargeted Gag variants, precluding direct comparison (Fig. 3C). Taken together, the results of these experiments clearly show that HIV-1 Gag ubiquitination depends on membrane association. Due to the dramatic increase of Gag polyubiquitination for the targeting mutants, however, it is currently not clear whether the relevant machinery is confined to the plasma membrane or is present at intracellular membranes as well.

Relative ubiquitination levels of truncated HIV-1 Gag proteins correlate with their membrane-binding capacities. Previously, we have shown that all lysine-containing domains of HIV-1 Gag are monoubiquitinated (13). We now reinvestigated the ubiquitination of MA, CA, MACA (i.e., Gag truncated after the CA domain), and MACANTD (i.e., Gag truncated after the N-terminal CA domain) under denaturing conditions. The different proteins were immunoprecipitated from denatured lysates of transfected 293T by cells by using antibodies against CA or MA. The precipitates were adjusted to give approximately equal signal intensities in anti-MA and anti-CA blots (Fig. 4A, right) and were analyzed for ubiquitination by anti-HA immunoblotting (Fig. 4A, left). In agreement with previous data (13), equal ubiquitination signals were observed for full-length Gag and MACA, with much weaker signals for MA and MACANTD and virtually no detectable ubiquitination of CA (Fig. 4A, left). The background signal was derived from the rabbit antibody heavy chain used for immunoprecipitation of MA and MACANTD.


Figure 4
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  		FIG. 4. Ubiquitination and membrane association of truncated Gag proteins. 293T cells were transfected with pcDNA3-based expression vectors for Rev-independent production of HIV-1 Gag or Gag subdomains as indicated above each lane. (A) Cells were lysed by boiling them in SDS lysis buffer and diluted to RIPA buffer conditions, and HIV-1 specific proteins were immunoprecipitated (IP) with rabbit antiserum against MA (lanes 1 and 3) or sheep antiserum against CA (lanes 2, 4, and 5). Precipitated proteins were normalized to yield comparable signal intensities and analyzed by Western blotting (WB) using a mixture of antisera against MA and CA (right). Ubiquitination was analyzed by Western blotting using antiserum against HA (left). The asterisk marks the background signal of the heavy chain of the rabbit antibody used for the immunoprecipitation of MA and MACANTD, which lack the C-terminal CA domain recognized by the sheep anti-CA antiserum. Please note that the ubiquitinated forms of MACANTD migrate very close to the heavy chain. (B) The same lysates as those for panel A were subjected to membrane flotation by density gradient centrifugation. Fractions were collected from top to bottom, and equal volumes were analyzed by Western blotting using antisera against MA (panels MA and MACANTD) or CA (all other panels). Antibodies against membrane-bound TfR and soluble actin were used as controls. Molecular mass standards are given in kDa. (C) Top: the pixel densities of the HA-Ub Western blot signals of Gag and the different Gag subdomains (A, left panel) were measured (Quantity One software; Bio-Rad) and divided by the signal intensities of the loading control (A, right panel). Bottom: the membrane association levels of the different Gag subdomains were displayed relative to that for wild-type Gag by measuring the pixel intensities of the Western blots shown in Fig. 4B. The band intensity in the floating fraction (fraction 2) was divided through the sum of the signal intensities of the bands in the bottom fractions (fractions 6 to 8).

It appears unlikely that the different number of Ub acceptor lysines accounts for the observed difference, since MACA yielded a much stronger signal than the combined signals for MA and CA. We therefore investigated whether the ubiquitination levels of truncated Gag proteins correlated with their respective membrane-binding capacities. Previous studies had shown much weaker membrane binding for MA than for Gag with approximately 40% of full-length Gag, but only 3% of MA recovered in the membrane fraction (29). This difference in membrane binding is believed to be due to a myristoyl switch masking the hydrophobic properties of MA (41, 49). To correlate the ubiquitination levels of truncated Gag proteins with their respective membrane associations, membrane flotations were performed in parallel to the experiment described above. All MA-containing Gag derivatives were detected in the membrane fraction, albeit to various extents, while CA was completely absent from the membrane fraction (Fig. 4B). Membrane association levels were approximately equal for full-length Gag and MACA, while MA and MACANTD showed only weak membrane association. Comparing the relative intensities of the HA-Ub immunoblot signals from Fig. 4A (Fig. 4C, top) with the ratio of membrane-bound to soluble Gag-derived proteins in Fig. 4B (Fig. 4C, bottom) revealed a very similar pattern. These results strongly support our conclusion that Gag ubiquitination is directly linked to membrane association.

The effect of a late-domain mutation on HIV-1 Gag ubiquitination depends on membrane binding. Our collective results indicate that the increased ubiquitination of HIV-1 carrying a mutation in the PTAP motif is due to the increased amount of Gag arrested in the budding process. This conclusion predicts that a PTAP mutation would have no effect on ubiquitination if introduced into a membrane-binding-defective Gag variant. To test this, the PTAP motif was mutated in the context of a myristoylation-deficient Gag protein [(G2V)Gag(PTAP-)] and the effects of the PTAP mutation on Gag ubiquitination were compared for myristoylated and nonmyristoylated Gag. Figure 5A shows that the membrane association of Gag is strongly enhanced upon introduction of a late-domain mutation into wild-type Gag. Very weak membrane association was observed for (G2V)Gag, and this was not affected by the late-domain mutation. A pronounced increase in the ubiquitination of Gag was observed for a PTAP-defective variant of Gag (Fig. 5B, upper panels). Importantly, this effect was completely lost in the context of the G2V variant, which exhibited weak ubiquitination levels, irrespective of a functional late domain. As expected, both PTAP mutation and lack of myristoylation led to severe reductions of virus release (Fig. 5B, lower two panels). This result further strengthens the conclusion that mutation of the PTAP motif influences Gag ubiquitination through alteration of the ratio of membrane-bound to soluble Gag.


Figure 5
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  		FIG. 5. The effect of a PTAP mutation on HIV-1 Gag ubiquitination depends on membrane association. 293T cells were cotransfected with pHA-Ub and pcDNA3-based expression vectors coding for wild-type (WT) or budding-deficient (PTAP-), Rev-independent HIV-1 Gag, either with or without mutations in the myristoylation signal (G2V). (A) Half of the cells were lysed by homogenization and subjected to membrane flotation by density gradient centrifugation. Gradient fractions were collected from top to bottom, subjected to SDS-PAGE, and analyzed by anti-CA Western blotting. (B) The other half of the cells were lysed by boiling them in SDS lysis buffer and diluted to RIPA buffer, and Gag was immunoprecipitated (IP). Equal amounts of Gag were analyzed by Western blotting (WB) using antisera against HA (top panel) or CA (second panel). As a control for the efficiency of Gag release, virus particle preparations from the culture supernatants of the same transfections (third panel) and the Gag contents of the cell lysates (bottom panel) were directly analyzed by Western blotting with antiserum against CA. Gag and Gag-HA-Ub conjugates are identified on the right, and molecular mass standards (in kDa) are shown on the left.


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DISCUSSION
 
HIV-1 Gag ubiquitination has been shown to be important for virus formation (12, 38), while the identity of the responsible machinery and how it is recruited to the budding site are largely unknown. In this study, we have analyzed the relationship between HIV-1 Gag multimerization, membrane association, and ubiquitination. Our results indicate that ubiquitination is directly correlated with membrane binding and may not require a specific recruitment signal. Unprocessed Gag polyproteins were initially soluble in RIPA buffer but became rapidly insoluble due to multimerization and were then inaccessible to antiserum against CA under native conditions. These observations are in accordance with previous reports of detergent-resistant assembly intermediates (19-21, 29, 35) and especially with a recent publication by Ono et al., who showed that Gag epitopes within large complexes are masked and not recognized by several Gag-specific antisera unless the protein has been denatured (32). We further demonstrated that the membrane-bound Gag fraction is specifically monoubiquitinated, while the ubiquitination level of soluble Gag is low. This finding is in contrast to our previous experiments using immunoprecipitation under native conditions, where no enrichment of ubiquitinated Gag at membranes was detected (14). Based on the results of the present study, it is clear that the previous result was due to loss of assembled Gag during immunoprecipitation under native conditions. The fact that Gag ubiquitination in the membrane-bound, antibody-accessible fraction (recovered under native conditions) was much lower than that in the membrane-bound, multimerized fraction (recovered under denaturing conditions) suggests that formation of assembly intermediates is a prerequisite for HIV-1 Gag ubiquitination, in addition to membrane localization. This hypothesis is difficult to prove, however, since membrane binding and multimerization of HIV-1 Gag are tightly linked events. For example, we observed that MACA was ubiquitinated at higher levels than MACANTD, which lacks the C-terminal assembly domain of CA. However, MACA also had a higher membrane affinity than MACANTD, probably due to synergistic membrane-binding effects of multimerized Gag molecules.

The observation that PTAP mutations caused increases in Gag ubiquitination had led to the hypothesis that a Ub hydrolase may be recruited by the ESCRT machinery (4, 13, 23). This hypothesis is in line with the observation that in Saccharomyces cerevisiae, the Ub hydrolase Doa4 deubiquitinates MVB cargo prior to release from the ESCRT pathway (2). Doa4 is recruited by Bro1 (22), the yeast homologue of AIP1/ALIX, an ESCRT-associated protein that also interacts with HIV-1 Gag. As we observed no effect of small interfering RNA-mediated knockdown of AIP1/ALIX on HIV-1 Gag ubiquitination or release (not shown), we propose that if there is a Ub hydrolase recruited by HIV-1 Gag, it is not through AIP1/ALIX. Rather, our results suggest that increased ubiquitination of PTAP-deficient HIV-1 Gag is indirect, resulting from an elevated ratio of membrane-bound, multimerized Gag to soluble Gag. This conclusion is mainly based on the following arguments: (i) both PTAP mutation and induction of a budding arrest by other means yielded augmented Gag ubiquitination levels, (ii) the ubiquitination levels of truncated Gag proteins strongly correlated with their respective membrane affinities, and (iii) in the absence of membrane association, a PTAP mutation did not enhance Gag ubiquitination.

Binding of the Ub hydrolase AMSH to ESCRT-III relieves an autoinhibitory conformation of the CHMP proteins, and overexpressed AMSH can block HIV-1 release (1, 9, 25, 48). It was recently shown that cotransfection of a catalytically inactive AMSH variant increased ubiquitination of a PTAP-dependent MLV Gag protein and impaired particle release (1). AMSH was therefore suggested to be the mammalian counterpart of Doa4, responsible for the deubiquitination of MVB cargo proteins. In light of our results, we suggest an alternative interpretation: increased ubiquitination of the MLV Gag variant may be a consequence of the AMSH-induced budding arrest, similar to the effect of dominant-negative ESCRT components. Overexpression of TSG101 has also been shown to increase ubiquitination of HIV-1 and HIV-2 Gag proteins, and it has been proposed that TSG101 recruits a Gag-specific Ub ligase (3, 28). Since it is known that overexpression of TSG101 disturbs the stoichiometry and therefore the function of ESCRT-I (10, 24), an alternative explanation is that the increase in HIV Gag ubiquitination due to TSG101 overexpression may result from delayed budding as well.

The conclusion that increased ubiquitination results from budding delay may also explain why HIV-1 Gag expressed from the complete viral genome was ubiquitinated at higher levels than Gag produced from a Rev-independent Gag expression vector (13). We observed that Gag expressed from p{Delta}R/PR(-) (and other proviral expression constructs) exhibited a higher ratio of membrane-bound to soluble Gag than Gag expressed from pcDNA3 constructs (compare Fig. 3B with 4B or 5A). The reason for this difference in membrane association is currently unclear. Preliminary experiments argue against an influence of accessory proteins or different expression levels (data not shown). Recently, it has been suggested that the RNA trafficking pathway and Rev dependence of HIV-1 Gag mRNA influence particle formation (43), and this may also affect membrane association. The comparatively weak membrane association of pcDNA3-expressed Gag proteins also accounts for the much smaller difference in ubiquitination levels between wild-type and non-membrane-bound (G2V)Gag (Fig. 5B, lanes 1 and 3) than between wild-type and nonmyristoylated Gag expressed from the proviral plasmid (Fig. 3C).

An important question is how an E3 Ub ligase is recruited by Gag polyproteins lacking a PPxY motif, such as HIV-1. The C-terminal NC-p6 region of Gag is clearly dispensable since its deletion had little effect on overall levels of ubiquitination. An intriguing finding was that both MA and CA were weakly ubiquitinated, while MACA was ubiquitinated at levels similar to those for wild-type Gag. One explanation for this observation is the existence of a cooperatively acting recruitment site for a Ub ligase in MA and CA. An obvious candidate is the sequence around the MA-CA cleavage site. An FPIV late-domain motif has recently been reported to be in paramyxoviruses (37), and the HIV-1 MA-CA cleavage site consists of the closely related sequence YPIV. However, we observed no difference in HIV-1 Gag ubiquitination or particle release compared to that for wild-type Gag when a variant protein with a deletion of 12 codons spanning the MA-CA junction (codons 127 to 138) was analyzed (data not shown), excluding the possibility of an influence of the MA-CA cleavage site on HIV-1 Gag ubiquitination. In further experiments, we showed that MA (except for the N-terminal myristoylation signal required for membrane targeting) is also completely dispensable for ubiquitination since deletion of codons 8 to 126 had no influence on Gag modification (data not shown). Taken together, these results argue against a specific signal for recruitment of a Ub ligase in HIV-1 Gag, although the N-terminal domain of CA has not been analyzed so far.

In summary, our results show that Gag multimerization, membrane association, and ubiquitination are closely linked processes and provide a straightforward explanation for several previously unexplained phenomena. These include the effect of PTAP mutation on ubiquitination and the differences in ubiquitination levels of single Gag domains and Gag proteins expressed from viral or Rev-independent vectors. We propose that the amount of Ub-modified Gag in transfected or infected cells at steady state largely reflects the amount of membrane-bound, multimerized Gag in the process of budding. Since virtually all Gag domains are dispensable for ubiquitination, we further propose that membrane-bound, multimerized Gag is recognized by a membrane-associated cellular Ub ligase without the need for a specific recruitment sequence or motif. The existence of a Ub ligase with the observed localization and specificity for large protein assemblies has yet to be demonstrated, however.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB638, project A9).

We thank H. Göttlinger, E. Freed, and W. Sundquist for providing expression plasmids and S. Welsch for critical reading of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Abteilung Virologie, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany. Phone: 49-6221-565002. Fax: 49-6221-565003. E-mail: Hans-Georg.Kraeusslich{at}med.uni-heidelberg.de Back

{triangledown} Published ahead of print on 3 July 2007. Back

{dagger} Present address: Department of Microbiology and Genetics, Box 3025, Duke University Medical Center, Durham, NC 27710. Back


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Journal of Virology, September 2007, p. 9193-9201, Vol. 81, No. 17
0022-538X/07/$08.00+0     doi:10.1128/JVI.00044-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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