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J Virol. Feb 2004; 78(3): 1503–1512.
PMCID: PMC321366

Both the PPPY and PTAP Motifs Are Involved in Human T-Cell Leukemia Virus Type 1 Particle Release

Abstract

In retroviruses, the late (L) domain has been defined as a conserved motif in the Gag polyprotein precursor that, when mutated, leads to the emergence of virus particles that fail to pinch off from the plasma membrane. These domains have been observed to contain the PPXY, PTAP, or YXXL motifs. The deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2, have a conserved PPPY motif in the C-terminal region of the matrix (MA) domain of Gag, while HTLV-1 also encodes a PTAP motif in MA. In this study, we analyzed the roles of the PPPY and PTAP motifs in the C terminus of MA in HTLV-1 particle release. Mutation of either motif (i.e., PPPY changed to APPY or PTAP changed to PTRP) reduced budding efficiencies. Particle buds and electron-dense regions of plasma membrane were observed by electron microscopy. When the locations of PPPY and PTAP were switched, particle release was eliminated. Intriguingly, the replacement of the PTAP motif with either the PPPY or YPDL motifs did not influence the release of virus particles, but the replacement of the PPPY motif with either PTAP or YPDL eliminated particle production. This indicates that the role that PPPY plays in HTLV-1 budding cannot be replaced with either PTAP or YPDL. A similar observation was made with the BLV PPPY motif. Finally, HTLV-1 particle release was found to be sensitive to proteasome inhibitors, implicating a role for ubiquitin in HTLV-1 budding. In summary, our observations indicate that (i) the PPPY motif plays a crucial role in virus budding and (ii) the PTAP motif plays a more subtle role in HTLV-1 particle release. Each of these motifs may play an important role in virus release from specific cell types and therefore be important in efficient virus spread and transmission.

The assembly of retrovirus particles requires the expression of the Gag polyprotein precursor (Gag), which is used as a principal building scaffold for retrovirus assembly and budding from infected cells (40, 46). During or after the process of particle release, the action of the retroviral protease cleaves Gag into mature matrix (MA), capsid (CA), and nucleocapsid (NC) proteins (13, 40, 46). There are three functional assembly domains in Gag (29); each domain is responsible for a separate function in the assembly process: the membrane-binding domain (M domain), the late domain (L domain), and the interaction domain (I domain). The M domain for most retroviruses resides primarily in the MA domain and contains a myristylation signal (3, 37), while the I domain resides in the CA and NC domains of Gag.

L domains are critical for efficient pinching off of the virus particle from the cell membrane. L domains have been identified by extensive mutational analysis in many retroviruses (7, 9, 12, 32, 45, 47, 48, 50). Different retroviruses may utilize different viral proteins and structural motifs to accomplish the same late budding function. Rous sarcoma virus, murine leukemia virus, and Mason-Pfizer monkey virus (MPMV) have L domains that consist of a highly conserved PPPY motif as the core sequence that is located near the junction of the MA and CA domains in Gag (5, 29, 44, 47-49). A PPPY motif has been also found in the matrix protein of rhabdoviruses and can function as an L domain (5). In contrast, the L domains of lentiviruses are located at the C terminus of Gag and have distinct core motifs, PTAP in human immunodeficiency virus type 1 (HIV-1) and YXXL in equine infectious anemia virus (EIAV) (9, 32, 50). The retroviral L domains are protein interaction domains and function by binding to specific cellular proteins that facilitate the late stages of retroviral particle release (42). It has been shown that L domains can be functionally interchanged among several different viruses (29, 32, 49).

Ubiquitin has been found to play a role in virus particle release from infected cells (27, 31, 34, 42). Specifically, cellular factors in the ubiquitin pathway have been shown to interact with L domains (8, 15, 31, 33, 41). The Nedd4-like family of E3 ubiquitin protein ligases (specifically LDI-1) has been identified as the cellular protein that interacts with the PPPY motif (11, 15). TSG101, a putative ubiquitin regulator that is involved in trafficking of endosomal proteins, has been reported to interact with the PTAP motif and therefore be involved in L domain function (6, 8, 41). The EIAV L domain, YPDL, has been found to bind the medium chain (AP-50) subunit of the AP-2 complex (33). TSG101 and its yeast ortholog Vps23 are members of the class E family of vacuolar protein sorting proteins and are involved in the formation of the multivesicular body (MVB)/late endosome, as well as sorting cargo into the MVB/late endosome (1, 19, 36). Recent data suggest that the PTAP, PPPY, and YPDL motifs access a common pathway involving class E vacuolar protein sorting factors (8, 25).

Proteasome inhibitors, which deplete free ubiquitin in cells, have been found to inhibit release for many different retroviruses (28, 38, 39). In particular, it has been observed that retrovirus budding is reduced by proteasome inhibitors by viruses utilizing either a PPPY- or PTAP-based L domain and that the effect does not depend on the assembly site or the presence of monoubiquitinated Gag in the virion (28). Interestingly, EIAV and mouse mammary tumor virus have been observed to be insensitive to proteasome inhibitors (28, 30).

The deltaretroviruses include bovine leukemia virus (BLV), human T-cell leukemia virus type 1 (HTLV-1), and HTLV-2. These viruses replicate to low titers in their natural hosts and are poorly infectious in cell culture. Cocultivation is typically used to infect permissive host cells. Because of these difficulties, information regarding the molecular details of their life cycles, including virus assembly and release, is limited. To date, most studies of deltaretrovirus assembly have been focused on BLV RNA encapsidation, BLV and HTLV-1 Gag myristylation, and the role of basic residues in MA of HTLV-1 Gag membrane localization and virus production (2, 14, 18, 22-24).

Two recent studies have focused on deltaretrovirus particle release. First, the PPPY motif at the C terminus of MA was found to function as a BLV late domain. Mutations in the PPPY motif caused defects in particle budding characteristic of an L domain defect (43). The PPPY motif is conserved among all deltaretroviruses, including BLV, HTLV-1, and HTLV-2. Intriguingly, HTLV-1 encodes both the PPPY and PTAP motifs at the C terminus of MA. Second, the analysis of HTLV-1 PPPY-motif mutants revealed that particle release was abolished (17). An accumulation of Gag was observed at the plasma membrane by electron microscopy, but not the development of virus particle buds. These observations were interpreted to mean that the PPPY mutants caused a late assembly-early budding defect (17).

In this study, site-directed mutagenesis of the PPPY and PTAP motifs was used to study the role(s) of these motifs in HTLV-1 particle release. Mutation of either motif led to reductions of particle release and resulted in virus buds that did not release from the plasma membrane. The function of the PPPY and PTAP motifs were found to be positionally dependent, and PPPY was more sensitive to replacement by other motifs than was PTAP, indicating that PPPY plays a more important role than PTAP in virus budding. The BLV PPPY motif was also sensitive to replacement, extending observations made with HTLV-1 to BLV. HTLV-1 particle release was found to be sensitive to proteasome inhibitors, which suggests that ubiquitin is involved in the process of HTLV-1 particle release.

MATERIALS AND METHODS

Cell culture and transfection.

293T cells were maintained in Dulbecco's modified Eagle's medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% Fetal Clone III serum (HyClone, Logan, Utah). MT-2 cells (human T-cell leukemia cells chronically infected with HTLV-1) were maintained in RPMI 1640 (Gibco BRL) supplemented with 10% Fetal Clone III. Transfection of 293T cells was performed by the calcium phosphate precipitation method. Typically, 60-mm-diameter petri dishes were used unless otherwise noted. A total of 10 μg of purified plasmid DNA was mixed with 50 μl of 2.5 M CaCl2 and 2× HEPES-buffered saline and then added to cells that were split the day before transfection and at 50 to 70% confluence. At 12 to 15 h posttransfection, medium was replaced with medium containing 10 mM sodium butyrate and 20 mM HEPES, incubated for 8 h at 37°C, and then replaced with fresh Dulbecco's modified Eagle's medium containing 20 mM HEPES. The proteasome inhibitor clasto-lactacystin β-lactone was obtained from Boston Biochem (Cambridge, Mass.) and was dissolved in dimethyl sulfoxide (DMSO) and used at a final concentration of 10 μM.

Plasmids, mutagenesis, and molecular cloning.

The HTLV-1 gag gene was cloned into the pMH vector (Roche, Indianapolis, Ind.) to construct pMH-Gag. Substitutions within PPPY or PTAP motifs were introduced into pMH-Gag vector by using the QuikChange XL kit (Stratagene) as previously described (43). The mutagenic oligonucleotides used in this study are shown in Table Table1.1. The plasmid pCMV-HT1, graciously provided by David Derse (National Cancer Institute, Frederick, Md.), was derived from an infectious molecular clone of HTLV-1 where the 5′ long terminal repeat was replaced with a CMV immediate-early promoter joined to a small fragment from the R region of the long terminal repeat which contains the major splice donor site (4). Plasmid DNAs containing the HTLV-1 proviral sequence can readily be deleted during amplification in Escherichia coli. To limit the likelihood of deletions occurring during plasmid amplification, pCMV-HT1 was introduced into the SCS1 strain of E. coli by transformation and plated onto Luria-Bertani agar plates containing ampicillin and incubating at 37°C for 18 h. The smaller bacterial colonies formed on plates were randomly picked with sterile toothpicks and used to inoculate 250 ml of Superbroth, grown overnight, and then purified using a commercially available kit (Qiagen). To introduce the mutations into pCMV-HT1 expressing vector, a DraIII-NheI fragment (~366 bp) from pMH-Gag mutant constructs was cloned into the pCMV-HT1 wild-type (wt) vector. Nucleotide sequencing was done to verify the presence of the desired mutations. The BLV virus-like particle (VLP) vector PR+ has been previously described (43). Substitution of the BLV PPPY motif with either PTAP or YPDL was done using the QuikChange XL kit.

TABLE 1.
Mutagenic oligonucleotides used to create PPPY and PTAP mutants

Immunoprecipitation (IP) and immunoblot analysis.

Methods for preparing cell and VLP lysates have been detailed previously (43). Briefly, 3 days after transfection, cells were lysed in radioimmunoprecipitation assay buffer and immunoprecipitated with anti-HTLV1-p19 monoclonal antibody (Zeptometrix, Buffalo, N.Y.). Supernatant collected from transfected cells was subjected to ultracentrifugation at 40,000 × g for 1 h to obtain the VLP pellets. HTLV immunoblot analysis was done using an anti-HTLV1-p19 monoclonal antibody as primary antibody and a horseradish peroxidase-conjugated anti-mouse immunoglobulin (Ig) (Amersham, Arlington Heights, Ill.) as secondary antibody with the ECL Western analysis kit (Amersham). BLV immunoblot analysis was done using an anti-BLV CA monoclonal antibody (i.e., BLV3) (VMRD, Pullman, Wash.). The efficiency of VLP production was normalized for cell-associated Gag. Real-time quantitation of band intensities was done using the Quantity One software package with the Chemi Doc 2000 Documentation System (Bio-Rad, Richmond, Calif.).

Transmission electron microscopy.

Transmission electron microscopy was performed as previously described (43). For thin sectioning, transiently transfected 293T-cell pellets were fixed with 2.5% glutaraldehyde. After dehydration in a graded series of cold ethanol, the samples were embedded in Epon 812 resin. Ultrathin sections (90 nm thick) were then stained with uranyl acetate. The stained sections were observed with a Philips CM 12 electron microscope.

Confocal microscopy.

Transfected cells were grown on coverslips and fixed with 4% paraformaldehyde and permeabilized with Triton X-100, both diluted in phosphate-buffered saline. The cells were then either incubated with anti-HTLV-1 p24 polyclonal antibody (Zeptometrix) followed by incubation with Alexa Fluor 568-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, Oreg.) (for HTLV-1) or incubated with BLV3 (VMRD) by incubation with Alexa Fluor 488-conjugated anti-mouse IgG (Molecular Probes) (for BLV). Images were collected with a Bio-Rad MRC 600 or a Zeiss 510 META laser scanning confocal microscope.

Membrane-binding assay.

The membrane-binding assay used in this study was modified from a previously published protocol (26). HeLa cells transfected with either wt or mutant VLP vector (i.e., PTAP-PPPY) were collected 2 days posttransfection and resuspended in a 10 mM Tris-HCl (pH 7.5)-4 mM EDTA solution containing a complete protease inhibitor cocktail. The cell suspensions were sonicated and spun at 2,000 × g for 3 min to obtain postnuclear supernatants. The supernatants were then mixed with 85.5% (wt/vol) sucrose and placed on the bottom of a centrifuge tube. On top of this postnuclear supernatant-sucrose mixture was layered 65% (wt/vol) sucrose and then 10% (wt/vol) sucrose. The gradients were centrifuged at 33,200 rpm for 16 h in a Beckman SW60 rotor. Nine fractions were collected from the top of the centrifuge tube. Fractionated samples were analyzed by IP and Western blotting.

RESULTS

Both the PPPY and the PTAP motifs function in HTLV-1 particle release.

Unlike BLV and HTLV-2, HTLV-1 contains both PPPY and PTAP motifs at the C terminus of MA. Both motifs have been individually identified as late domains for many retroviruses (for a review, see reference 7), and in BLV we have shown that the PPPY motif functions in particle release (43). We hypothesized that both PPPY and PTAP motifs located at the C terminus of the HTLV-1 MA may function in the budding process. To test this hypothesis, mutations were introduced into the gag gene of pCMV-HT1 (Fig. (Fig.1).1). To test whether the PPPY and PTAP motifs individually functioned in HTLV-1 particle release, each motif was first mutated separately. In the APPY-PTAP and AAAA-PTAP mutants, the PPPY motif was mutated and the PTAP motif remained intact. In the mutants PPPY-PTRP or PPPY-LIRL, the PTAP motif was mutated while the PPPY motif remained intact.

FIG. 1.
Mutants constructed to test the role of the PPPY and PTAP motifs in HTLV-1 particle release. The wt and mutant amino acid sequences are shown for the C terminus of the HTLV-1 MA domain of Gag. The wt and mutated PPPY and PTAP motifs are indicated by letters ...

Immunoblot analysis was used to measure reductions in virus particle release. To determine the linear range of detection for the assay, a protein dilution series was analyzed. Based upon this analysis, the linear range of detection for the assay is at least 16-fold (Fig. (Fig.2A).2A). To ascertain how these mutations would influence particle release, mutants were transfected into 293T cells in parallel with the parental vector. Gag expression in cells was analyzed by IP-immunoblot analysis, and VLP production was analyzed by immunoblot analysis (Fig. (Fig.2B).2B). In general, only Pr46Gag was readily observed from cells, while only p19 was readily observed from VLPs. A single mutation of the PPPY motif with an intact PTAP motif (i.e., APPY-PTAP) reduced particle production to about 30% that of the parental vector, while the mutant with an intact PPPY and a single mutation in the PTAP motif (i.e., PPPY-PTRP) reduced particle production to about 60% that of the parental (Fig. (Fig.2C).2C). The phenotype of the PPPY-PTRP mutant is interesting because an HIV-1 mutant with the same substitution in PTAP led to a dramatic reduction in particle production (~5% of wt) (12). Mutating all four residues in the PPPY motif while maintaining an intact PTAP motif (i.e., AAAA-PTAP) eliminated VLP release. The mutant with the intact PPPY motif and all four residues of the PTAP motif mutated (i.e., PPPY-LIRL) was also found to produce no detectable VLPs. These observations indicate that each motif plays a role in HTLV-1 particle production and that they are not redundant in function.

FIG. 2.
Both the PPPY and PTAP motifs influence HTLV-1 particle release. (A) Linear range of detection for immunoblot analysis. Protein was diluted (1:1, 1:2, 1:4, 1:5, 1:8, 1:10, and 1:16) and was subjected to immunoblot analysis. The band intensity (arbitrary ...

To determine whether nascent buds accumulated on the plasma membrane, electron microscopy of cells transiently transfected with each of the mutants was done. When the parental vector was used, released particles with mature cores were readily observed (Fig. 3A and B). Interestingly, when cells producing the APPY-PTAP mutant were analyzed, distinct phenotypes were observed along the cell surface. In particular, electron microscopy revealed the appearance of either virus particles that were in the process of pinching off from the plasma membrane but did not release or electron-dense regions of plasma membrane that were in the early stages of forming virus particle buds (Fig. 3C and D). Similar observations were made for the PPPY-PTRP mutant (Fig. 3E and F). In addition, released particles were observed for both the APPY-PTAP and the PPPY-PTRP mutants. The analysis of the AAAA-PTAP and PPPY-LIRL mutants also led to interesting observations. For the AAAA-PTAP mutant, both particles in the process of pinching off from the plasma membrane and electron-dense regions of plasma membrane forming virus particle buds were observed (Fig. 3G and K). However, the PPPY-LIRL mutant did not reveal either particle buds that had not released or regions of plasma membrane where particle buds were forming (data not shown). This observation with the PPPY-LIRL mutant suggests that the complete mutation of the PTAP motif may have resulted in an assembly defect and/or a Gag membrane targeting defect perhaps by creating a conformational change in Gag. No released particles were observed for either the AAAA-PTAP and PPPY-LIRL mutants. Taken together, these data provide evidence for the hypothesis that both the PPPY and PTAP motifs function in virus particle release and mutation of either motif appeared to arrest VLPs at early or late stages in the budding process.

FIG. 3.
Electron microscopy of 293T cells expressing HTLV-1 PPPY and PTAP mutants. Transiently transfected 293T-cell pellets were fixed with 2.5% glutaraldehyde. After dehydration in a graded series of cold ethanol, samples were embedded, and ultrathin sections ...

Influence of PPPY and PTAP motif location for function in HTLV-1 particle release.

Analysis of the amino acid sequence of the HTLV-1 MA reveals that the PTAP motif lies closer to the C terminus than PPPY (Fig. (Fig.1).1). To determine whether the specific location of the PPPY and PTAP motifs in relationship to one another was important for function in particle release, a mutant was created in which their location was switched (PTAP-PPPY) (Fig. (Fig.1).1). This mutant was then transiently transfected into 293T cells and analyzed. Comparable levels of Gag were detected in cells expressing the mutant to that of the parental vector. However, no VLP production was detected for the mutant, which was likely due to a block in the particle assembly pathway (Fig. 4A and D). This observation indicates that the function of the PPPY and PTAP motifs in particle release may be positionally dependent. An alternative explanation is that the defect could be due to a conformational defect in Gag. However, no defects were observed in Gag-membrane binding of the PTAP-PPPY mutant compared to wt (Fig. (Fig.4E),4E), indicating that the introduced mutations did not interfere with the ability of Gag to bind membrane. Also, the cellular distribution of this Gag mutant was similar to that of wt (see below).

FIG. 4.
Switching location and replacing PPPY and PTAP motifs influence HTLV-1 particle release. (A through C) Analysis of VLP production. 293T cells were transfected with wt or mutant constructs, and VLP and cell-associated Gag was analyzed as in Fig. ...

The PTAP motif, but not the PPPY motif, can be functionally replaced in HTLV-1 particle release.

To determine whether the PPPY motif and/or the PTAP motif could be functionally replaced either by each other or with the YPDL motif, additional constructs were created (Fig. (Fig.1B).1B). First, to test whether the PPPY motif could be functionally exchanged, it was swapped with either PTAP or with YPDL (i.e., the EIAV L domain motif) creating the mutants PTAP-PTAP and YPDL-PTAP, respectively. Each construct expressed wt levels of Gag in cells, but did not produce VLPs (Fig. 4B and D). This indicates that the PPPY motif cannot be functionally replaced with either the PTAP or YPDL motifs. Second, we tested whether the PTAP motif could be functionally exchanged by creating the mutants PPPY-PPPY and PPPY-YPDL, where the PTAP motif was changed to either the PPPY motif or the YPDL motif, respectively (Fig. (Fig.1B).1B). Levels of Gag detected in cells expressing the mutants were similar to that of the parental VLP vector (Fig. (Fig.4C).4C). Intriguingly, each of the mutants led to VLP production that was close (PPPY-PPPY) or comparable (PPPY-YPDL) to that of the parental vector (Fig. (Fig.4,4, panels C and D). This observation indicates that the PTAP motif can be functionally exchanged with either the PPPY motif or the YPDL motif. These data also indicate that the PPPY motif is crucial for virus particle budding, while the function of PTAP can be replaced by other motifs.

Cellular distribution of Gag for mutants with defects in HTLV-1 particle release.

Several of the mutants analyzed in this study eliminated VLP production as determined by IP-immunoblot analysis. In one instance, with the PPPY-LIRL mutant, there was a suggestion of a defect in Gag distribution in cells because of the inability by electron microscopy to readily detect VLP buds or regions of plasma membrane that were in the process of forming virus buds. To further analyze the nature of why some of the mutants created in this study did not produce VLPs, we analyzed the cellular distribution of Gag in cells transiently transfected with each mutant vector by confocal microscopy. The main observation made from this analysis is that the cellular localization for the mutants was comparable to that seen with the wt. In particular, a distinctive punctate staining pattern was observed throughout the cytoplasm of cells (Fig. (Fig.5).5). This punctate staining has been previously observed for HTLV-1 Gag (16). Such a staining pattern throughout the cytoplasm was also observed with the analysis of MVB localization using antibody directed against LAMP-1 or CD63 (data not shown). This suggests a colocalization of HTLV-1 Gag with the MVB.

FIG. 5.
Cellular localization of Gag in cells. Transfected cells were grown on coverslips, fixed with 4% paraformaldehyde, and permeabilized with Triton X-100. Cells were then incubated with an anti-HTLV-1 p24 polyclonal antibody followed by incubation with Alexa ...

The BLV PPPY motif is crucial for virus particle release.

The data presented in Fig. Fig.4B4B indicate that when the HTLV-1 PPPY motif was replaced with either PTAP or YPDL, particle release was eliminated. Since BLV also contains a PPPY motif that was previously shown to function as an L domain (43), we next tested whether replacement of PPPY with either PTAP or YPDL would interfere with BLV particle release. As shown in Fig. Fig.6A,6A, mutation of the BLV PPPY to either PTAP or YPDL eliminated virus particle release, similar to that observed for the HTLV-1 PPPY motif. Microscopy analysis indicated that the BLV PTAP and YPDL mutants did not alter Gag localization compared to wt (Fig. (Fig.6B).6B). Together, these data indicate that the PTAP and YPDL motifs cannot replace the PPPY motif in BLV L domain function.

FIG. 6.
BLV PPPY motif is crucial for virus particle release. (A) VLP production of BLV PPPY mutants. 293T cells were transfected with wt or derivatives. Forty-eight hours posttransfection equal volumes of supernatant medium from each culture were collected and ...

Proteasome inhibitors decrease the efficiency of HTLV-1 particle release.

Proteasome inhibitors have been shown to inhibit particle release for some retroviruses (encoding either a PPPY or PTAP motif), but not for others (encoding a YPDL motif). Since HTLV-1 MA contains both the PPPY and PTAP motifs, we wanted to investigate whether the two motifs together would influence the susceptibility of HTLV-1 particle release to proteasome inhibitors. To test this, MT-2 cells (human T-cell leukemia cells chronically infected with HTLV-1) were treated with 10 μM lactacystin for 11 h, and then cells and virus particles were recovered, lysed, and analyzed by immunoblot analysis. Compared to MT-2 cells not treated with proteasome inhibitors (DMSO only), lactacystin-treated cells led to reductions in the level of virus particle production (Fig. (Fig.7).7). Treatment with lactacystin led to a fourfold reduction to that from virus released from untreated cells. This fourfold reduction is within the range reported for MPMV, HIV-1, and murine leukemia virus (28). Interestingly, a comparison of the ratio of p19 to total Gag protein expressed in cells for untreated and proteasome inhibitor-treated cells indicated that the inhibitor led to a mild reduction (~30% less than untreated cells) in Pr46Gag processing. MG-132, another proteasome inhibitor, led to mild reductions in virus release (data not shown). Overall, these observations indicate that HTLV-1 particle release is sensitive to proteasome inhibitors.

FIG. 7.
HTLV-1 particle release is sensitive to a proteasome inhibitor. MT-2 cells (human T-cell leukemia cells chronically infected with HTLV-1) were treated with 10 μM clasto-lactacystin β-lactone for 11 h, and then virus-and cell-associated ...

DISCUSSION

This study reports the analysis of a panel of mutants created to determine whether the PPPY and PTAP motifs in the carboxy terminus of the MA domain of Gag function in HTLV-1 particle release. We observed that (i) mutations into either the PPPY or PTAP motifs reduced the budding efficiency of HTLV-1 particles; (ii) the function of the PPPY and PTAP motifs was not maintained when their positions were switched; (iii) the replacement of the PTAP motif with either the PPPY or YPDL motifs did not influence virus particle release, but the replacement of the PPPY motif with either PTAP or YPDL eliminated particle release; (iv) replacement of the BLV PPPY with either PTAP or YPDL also eliminated particle release; and (v) HTLV-1 particle release was found to be sensitive to proteasome inhibitors, which is consistent with a role for ubiquitin in HTLV-1 budding.

A recent study reported that mutations of the HTLV-1 PPPY motif (i.e., LPPY, PLPY, PPLY, and PPPD) abolished particle release from 293-TSA cells (17), which is similar to what we have reported in our study with one of the mutants we had analyzed in 293T cells (i.e., AAAA-PTAP). In contrast, another PPPY mutant that we analyzed in our study (i.e., APPY-PTAP) reduced but did not eliminate virus particle release. The authors of the previous study found an accumulation of Gag in cells and by electron microscopy found an accumulation of electron-dense material at the plasma membrane but not the formation of virus buds and concluded that the PPPY mutants that they analyzed caused a late assembly-early budding defect. We observed by electron microscopy the accumulation of Gag at the plasma membrane but also found virus buds that had not released from the plasma membrane (both for the AAAA-PTAP and the APPY-PTAP mutants). In total, our PPPY mutants display some similarities to those in the previous study with the HTLV-1 PPPY motif but have phenotypic differences. These differences could be due in part to the different cells and the different virus constructs used. However, our observations provide evidence which strongly supports the conclusion that the PPPY motif functions in virus particle release and when mutated can cause L domain defects similar to those observed with other retroviruses.

We observed in this study for the first time that the function of the PPPY and PTAP motifs was eliminated when the locations of the motifs were swapped in HTLV-1 Gag, as a mutant with the motifs switched in location eliminated particle release. Previous studies have shown that L domains (consisting of single motifs) could function when located in distal positions in Gag, indicating that the L domain could function independently of its position (29, 32). In contrast, a more recent study with EIAV found evidence for positional dependence (in virus replication) when the YPDL motif in the p9 protein of Gag was replaced by YPDL or PTAP at the C terminus of the MA domain in Gag (20). It was suggested by these authors that full functionality of the EIAV L domain in virus replication was dependent on its location in the context of the p9 protein of Gag, which may suggest the influence of other p9 sequences on L domain functions.

Previous studies have indicated the interchangeable nature of the EIAV YPDL motif, the Rous sarcoma virus PPPY motif, and the HIV-1 PTAP motif in L domain function (20, 29, 32). It has been suggested that these observations may indicate that diverse motifs can utilize different entry points to common cellular machinery for viral budding and release from the plasma membrane (20). In our study, we found that the replacement of the PTAP motif with either the PPPY or YPDL motifs did not influence the release of virus particles, indicating that PTAP was functionally interchangeable with either PPPY or YPDL. In contrast, we also observed that the replacement of the PPPY motif with either PTAP or YPDL eliminated the detection of released particles. These data indicate that the PPPY motif was not interchangeable in function with either the PTAP or YPDL motifs. We interpret these findings as (i) PPPY is crucial for HTLV-1 particle release and (ii) PTAP has a less-prominent role in HTLV-1 particle release. We further observed that the BLV PPPY motif could not be replaced with either PTAP or YPDL, which extends the observations made with BLV to other deltaretroviruses. The overlapping Ebola virus VP40 protein PTAP and PPEY motifs have recently been reported to function independently as late budding domains (21). More recently, it was shown that both the MPMV PPPY and PSAP motifs contribute to virus release but that PSAP requires an intact PPPY motif for function (10). Taken together, these data suggest that the PPPY and PTAP motifs play distinct roles in HTLV-1 particle release. These observations also suggest that the functional differences in the PPPY and PTAP motifs will likely provide important clues for their function in HTLV-1 particle release, in particular, and in retrovirus particle release, in general.

The punctate staining pattern of Gag seen with wt or mutants that produced VLPs suggests colocalization with the MVB. A previous study with HTLV-1 suggested that this may not occur (16). However, the potential colocalization of HTLV-1 Gag with the MVB raises intriguing possibilities regarding HTLV-1 budding. For example, particle budding may occur in the MVB and then particles may ultimately be released from cells through exocytosis. Second, localization of Gag with the MVB/late endosome would allow for the sorting of Gag into the lumen. Budding of HIV-1 particles into the lumen of the MVB has been observed in macrophages and not from the plasma membrane (35). This indicates that HIV-1 particle release can occur via different modes in different cell types. Such possibilities have not been extensively investigated with HTLV-1.

We observed that HTLV-1 particle release was sensitive to a proteasome inhibitor. Based upon previous reports with other retroviruses that encode the PPPY or PTAP motifs, this may have been expected (28, 38). However, we have found that a proteasome inhibitor can influence virus budding and release of a retrovirus containing both the PPPY and PTAP motifs. The sensitivity of HTLV-1 to proteasome inhibitors strongly suggests a role for ubiquitin in HTLV-1 particle release.

In summary, we have described mutants that have allowed for the initial characterization of the PPPY and PTAP motifs and their roles in HTLV-1 particle release. Our observations indicate that both motifs are important for HTLV-1 budding. These mutants will be extremely useful for future studies directed at understanding the cellular proteins and pathways involved in virus particle release. Furthermore, the analysis of HTLV-1 particle release should provide new insight into the specific functions of the PPPY and PTAP motifs in retrovirus budding and release. We speculate that each motif plays an important role in virus release from specific cell types and is ultimately important in efficient virus spread and transmission. A greater understanding of how these viruses release from cells will likely provide new targets for antiretroviral drug development.

Acknowledgments

We thank E. Freed and J. Wills for critical review of the manuscript, K. Wolken for assistance with confocal and electron microscopy, A. Ono for advice on the membrane-binding assay, and D. Derse for providing reagents.

This work was supported by NIH grant AI053155.

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