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.

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 in boldface type and are boxed in the wt.

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 units) for each dilution was determined with the Quantity One software package of the Chemi Doc 2000 Documentation System (Bio-Rad) and plotted against the amount of protein used for immunoblot analysis (16, 8, 4, 3.2, 2, 1.6, or 1 μl) to determine if protein detection within the dilution range was linear. (B) Analysis of PPPY and PTAP mutants. 293T cells were transfected with wt or derivatives. Forty-eight hours posttransfection equal volumes of supernatant medium from each culture were collected and VLPs were concentrated by ultracentrifugation. VLP production was analyzed by immunoblot analysis using an anti-HTLV-1 p19 antibody. Cell-associated material was immunoprecipitated with anti-HTLV-1 p19 prior to immunoblot analysis (see Materials and Methods). The positions of Pr46Gag and p19 (MA) are indicated. Quantitation of band intensities was determined by real-time acquisition of signals. Representative data from at least three independent experiments are shown. (C) Summarized data from the experiments shown in panel B.

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 were then stained with uranyl acetate and viewed. (A and B) Parental wt HTLV-1; (C and D) APPY-PTAP; (E and F) PPPY-PTRP; (G to K) AAAA-PTAP. Arrows point to budding particles. Scale bar, 100 nm.

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. 2. The positions of Pr46Gag and p19 (MA) are indicated. Quantitation of band intensities was determined by real-time acquisition of signals. Representative data from at least three independent experiments are shown. (D) Summarized data from panels A through C. (E) Membrane-binding assay. Parental wt and PTAP-PPPY mutant were analyzed as described in Materials and Methods.

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 Fluor 568-conjugated goat anti-mouse IgG. Images were obtained with a confocal microscope. (A) Parental wt vector; (B) APPY-PTAP; (C) PPPY-PTRP; (D) AAAA-PTAP; (E) APPY-LIRL; (F) PTAP-PPPY; (G) PPPY-PPPY; (H) PPPY-YPDL; (I) PTAP-PTAP; (J) YPDL-PTAP.

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 VLPs were concentrated by ultracentrifugation. VLP production was analyzed by immunoblot analysis using anti-BLV CA antibodies. The positions Gag and p24 (CA) are indicated. Representative data from at least three independent experiments are shown. The PTAP mutant has the PPPY motif replaced with PTAP, and the YPDL mutant has the PPPY motif replaced with YPDL. (B) Cellular localization of BLV Gag mutants. Transfected cells were grown on coverslips, fixed, and permeabilized. Cells were then incubated with an anti-BLV p24 antibody followed by incubation with Alexa Fluor 488-conjugated anti-mouse IgG. Images were obtained with a confocal microscope.

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 Gag was analyzed by immunoblot analysis. The lane marked DMSO contains cells treated with DMSO only and no proteasome inhibitor. (A) Representative data; (B) summarized data from three independent experiments.